Apoptogenic peptides from Tityus discrepans scorpion venom acting against the SKBR3 breast cancer
tpo36三篇托福阅读TOEFL原文译文题目答案译文背景知识
tpo36三篇托福阅读TOEFL原文译文题目答案译文背景知识阅读-1 (2)原文 (2)译文 (3)题目 (5)答案 (10)背景知识 (11)阅读-2 (12)原文 (12)译文 (14)题目 (15)答案 (20)背景知识 (20)阅读-3 (24)原文 (24)译文 (25)题目 (27)答案 (32)背景知识 (33)阅读-1原文Soil Formation①Living organisms play an essential role in soil formation. The numerous plants and animals living in the soil release minerals from the parent material from which soil is formed, supply organic matter, aid in the translocation (movement) and aeration of the soil, and help protect the soil from erosion. The types of organisms growing or living in the soil greatly influence the soil's physical and chemical characteristics. In fact, for mature soils in many parts of the world, the predominant type of natural vegetation is considered the most important direct influence on soil characteristics. For this reason, a soil scientist can tell a great deal about the attributes of the soil in any given area simply from knowing what kind of flora the soil supports. Thus prairies and tundra regions, which have characteristic vegetations, also have characteristic soils.②The quantity and total weight of soil flora generally exceed that of soil fauna. By far the most numerous and smallest of the plants living in soil are bacteria. Under favorable conditions, a million or more of these tiny, single-celled plants can inhabit each cubic centimeter of soil. It is the bacteria, more than any other organisms, that enable rock or other parent material to undergo the gradual transformation to soil. Some bacteria produce organic acids that directly attack parent material, breaking it down and releasing plant nutrients. Others decompose organic litter (debris) to form humus (nutrient-rich organic matter). A third group of bacteria inhabits the root systems of plants called legumes. These include many important agricultural crops, such as alfalfa, clover, soybeans, peas, and peanuts. The bacteria that legumes host within their root nodules (small swellings on the root) change nitrogen gas from the atmosphere into nitrogen compounds that plants are able to metabolize, a process, known as nitrogen fixation, that makes the soil more fertile. Other microscopic plants also are important in soil development. For example, in highly acidic soils where few bacteria can survive, fungi frequently become the chief decomposers of organic matter.③More complex forms of vegetation play several vital roles with respect to the soil. Trees, grass, and other large plants supply the bulk of the soil's humus. The minerals released as these plants decompose on the surface constitute an important nutrient source for succeeding generations of plants as well as for other soil organisms. In addition, trees can extend their roots deep within the soil and bring up nutrients from far below the surface. These nutrients eventually enrich the surface soil when the tree drops its leaves or when it dies and decomposes. Finally, trees perform the vital function of slowing water runoff and holding the soil in place with their root systems, thus combating erosion. The increased erosion that often accompanies agricultural use of sloping land is principally caused by the removal of its protective cover of natural vegetation.④Animals also influence soil composition. The faunal counterparts of bacteria are protozoa. These single-celled organisms are the most numerous representatives of the animal kingdom, and, like bacteria, a million or more can sometimes inhabit each cubic centimeter of soil. Protozoa feed on organic matter and hasten its decomposition. Among other soil-dwelling animals, the earthworm is probably the most important. Under exceptionally favorable conditions, up to a million earthworms (with a total body weight exceeding 450 kilograms) may inhabit an acre of soil. Earthworms ingest large quantities of soil, chemically alter it, and excrete it as organic matter called casts. The casts form a high-quality natural fertilizer. In addition, earthworms mix of soil both vertically and horizontally, improving aeration and drainage.⑤Insects such as ants and termites also can be exceedingly numerous under favorable climatic and soil conditions. In addition, mammals such as moles, field mice, gophers, and prairie dogs sometimes are present in sufficient numbers to have significant impact on the soil. These animals primarily work the soil mechanically. As a result, the soil is aerated broken up, fertilized, and brought to the surface, hastening soil development.译文土壤形成①活生物体在土壤形成中起着重要作用。
APOPTOSIS.ppt
activated either by autocatalyic cleavage or by other proteases: cascade of casepases 4. Total 14 members (11 in human), and caspase-2, 3, 6, 7, 8, 9, and 10 are known to be involved in apoptosis
Pro-caspases Bcl-xL Bcl-2 BID
MEKK-1 PKC and PAK2/hPAK65 p21WAF1/CIP1 FAK
Transcription factors/regulators MDM2
RB Sp1 IkB-a
DNA/RNA damage and repair mechanisms
PARP与DNA修复、基因完整性监护有关
Important Substrates of Caspases
Structural/trafficking proteins Actin
Fodrin Gelsolin Nuclear lamins A, B, C
Regulatory/signaling proteins
Discovery of Bcl-2
p53 discovered as an apoptosis regulator
Role Cytochrome c and apoptosome confirmed
1887
1965 1972 19771982 1987 1989 1991 1993 1996-7 2000
统考版2024届高考英语二轮专项分层特训卷阅读七选五保分练三
阅读七选五保分练(三)Test 1[2023·新课标Ⅱ卷]As an artist who shares her journey on social media, I'm often asked by curious followers how to begin an art journey. Unfortunately, there is no magic list I can offer. I do remember, though, what it was like to be a complete beginner. So I've put together some good tips for starting an art journey.·Start small. I suggest using a sketchbook (素描本) for small studies. These small studies provide inspiration and may be a springboard for more complex works in the future. __1__ You'll want to look back on your journey to see how far you've come.·Paint often and paint from life. There's no better way to improve than to put in those brush miles. Whether you paint still lives, portraits, or landscapes, paint from life as much as possible. __2__·Continually chall enge yourself to try something new. __3__ Artistic growth can be a bit painful. Welcome to the club；we've all been there. I love taking on challenges. I once took up a challenge to create a painting every day for a month and post the works online.·__4__Seeking and accepting constructive feedback (反馈) is crucial to growth.I post my work on social media and, in turn, have met some of the kindest people. They make me feel valued and respected, no matter my level of artistic ability.The journey you're on won't follow a straight path. __5__ Push through, give it time and put in the effort. You will harvest the rewards of an artistic life.A.Get out of your comfort zone.B．Make career plans and set goals.C．Don't throw away your beginner art.D．Share your work if you feel comfortable doing so.E.You'll hit roadblocks, and you'll feel discouraged at times.F.Evaluate your performance and, if needed, redefine your role.G.You'll develop that painting muscle memory that only comes with repetition.[答题区]1．________ 2.________ 3.________ 4.________ 5.________Test 2[2023·武汉市高三模拟]Lake Baikal, the biggest body of fresh water on Earth near Russia's border with Mongolia, is home to several unusual animals, including the world's only species of freshwater seal (海豹).Seals exist in large quantities in Baikal, about 100，000 of them, though the lake is poor in nutrition. __1__. A study just published by Watanabe Yuuki of the National Institute of Polar Research in Tokyo suggests the answer is tiny organisms.Most seals eat fish. And Baikal seals do, indeed, have needlepointed teeth. But in 1982 researchers noted that they developed a second sort of specialized tooth behind those canines (犬齿). They have sharp teeth which look like combs (梳子). __2__. But Dr Watanabe guessed that they might be an adaptation for feeding on otherstrange creatures in the lake.Seals arrived in Baikal 2 million years ago, from the Arctic Ocean. So too did some much smaller sea creatures, known as amphipods. These have grown into more than 340 species.__3__. But Dr Watanabe wondered if the Baikal seals' comblike teeth might enable them to filter (过滤) these tiny creatures from the water in sufficient quantities to make them useful food sources. __4__.Records showed that the seals would dive in with their mouths open and collect amphipod groups that form at night. Dr Watanabe estimated that each seal catches an average of 57 amphipods per dive. __5__， for the seals do hunt fish as well. But they also compete with those fish for the amphipods, thus partially bypassing a link in the food chain and perhaps maintaining themselves in larger numbers than what would otherwise be possible.A．The needlelike canines are necessaryB．So how they do so well has been a puzzleC．This has led to their numbers increasing sharplyD．At the time, nobody knew what to make of themE．He therefore used waterproof cameras to observe a few sealsF．Cameras remained attached to some seals for between two and four daysG．Sea mammals of the size of seals would normally see amphipods as too small to hunt[答题区]1．________ 2.________ 3.________ 4.________ 5.________Test 3[2023·济南市高三模拟]“A ship in the harbor is safe, but that is not what ships are built for，” said John Augustus Shedd, an earl y 20th century author. Throughout COVID19, we've all become used to assessing risk in new ways. We've come to understand, though we can never get rid of risk altogether，we have great power to make choices both large and small to protect ourselves. __1__ It stops us from stepping outside of our comfort zones and trying new things.Developmental psychologists talk about “positive risks”—socially acceptable risks that our lives can benefit from. __2__ But what about those who develop into adults? Researchers have found that happiness for older adults is being able to choose how they spend their time, including ways that are adventurous, new and even mildly risky，like hiking or other outdoor activities.One guide to positive risk management lists ways that people can make sure their risks are on the “safe” side of the risk range. For example, to ensure a successful bike ride, you should in advance equip yourself with a fullycharged cell phone and a full water bottle. __3__ Or if you are concerned about your physical capacity, consult a certified trainer or a medical professional before departure.__4__ This means adapting to changing situations. In response to the deadly virus，people choose to step back into normal life only after they are vaccinated. In this way, people could be confident that the risk is tolerable.Nobody wants to be needlessly risky. But using our newlyacquiredriskassessment tools, we can once again learn and grow in our lives. __5__ A．Take risks in a positive way.B．But the selfprotective mind state carries its own risk.C．Overall happiness is one benefit of positive risktaking.D．Remember to tell one of your friends or families your destination.E．Besides taking preventive steps, flexible thinking is also encouraged.F．Like a ship sailing away from its harbor, that is what we were built to do.G．For teenagers, this means risks like running for a monitor or trying out for a team.[答题区]1．________ 2.________ 3.________ 4.________ 5.________Test 4[2023·武汉市高三调研]Now I live in Paris. Actually, I don't mind the food or the people, but I do care about the land that extraordinarily lacks characteristics of hiking. I enjoy being outdoors and hiking，but Paris's natural resources don't hold much potential for my bent. So when my dad asked if I wanted to go hiking with him, I was overjoyed. __1__. It turned out that I was too optimistic.We started our main hike up Flattop Mountain. As we hiked it was much more challenging than I had expected. __2__. The path is winding and we saw mountains towering over the green valleys and cliffs that seemed endless. What amazed me most was that thick clouds enveloped the mountainsides. __3__.I stared up at the top to see how much farther we had to go and I began to worry that I wouldn't finish it. __4__. It made me doubtful about my persistence (坚持). But when we looked back at the path that faded in the distance, I was filled with fulfillment with seeing how much we had done.As I was hiking, I started to reflect on how hiking was similar to other aspects of my life. __5__. It reminded me of my struggles in my classes and the difficulties I had to overcome. I spotted that the process of learning isn't always the most fun, but the fulfillment after seeing how much I had finished just increases the thirst for knowledge, causing me to long for more learning. Looking down at the mountain I was content but never fully satisfied. Having considered a lot, I was determined to pursue the summit (山顶).A．Hiking could make me flyB．Hiking gave time for my mind to wanderC．I thought I would finish the journey with easeD．But I was shocked at the beauty surrounding meE．I was satisfied with the landscape along the wayF．It was unlike anything I had ever witnessed beforeG．A recent ankle injury had left me unsure of my physical capabilities[答题区]1．________ 2.________ 3.________ 4.________ 5.________阅读七选五保分练（三）Test 1语篇类型：说明文主题语境：人与社会——生活方式——介绍艺术之旅的好建议【文章大意】文章介绍了一些开始艺术之旅的好建议。
2019-ted,寻找蛋白质的替品-我们为什么不食用昆虫呢-,的英语演讲稿-精选word文档 (8页)
本文部分内容来自网络整理，本司不为其真实性负责，如有异议或侵权请及时联系，本司将立即删除！== 本文为word格式，下载后可方便编辑和修改！ ==ted,寻找蛋白质的替品:我们为什么不食用昆虫呢?,的英语演讲稿篇一：TED英语演讲稿TED英语演讲稿TED英语演讲稿I was one of the only kids in college who had a reason to go to the P.O. box at the end of the day, and that was mainly because my mother has never believed in email, in Facebook, in texting or cell phonesin general. And so while other kids were BBM-ing their parents, I was literally waiting by the mailbox to get a letter from home to see how the weekend had gone, which was a little frustrating when Grandma was in the hospital, but I was just looking for some sort of scribble, some unkempt cursive from my mother.And so when I moved to New York City after college and got completely sucker-punched in the face by depression, I did the only thing I could think of at the time. I wrote those same kinds of letters that my mother had written me for strangers, and tucked them all throughout the city, dozens and dozens of them. I left them everywhere, in cafes and in libraries, at the U.N., everywhere. I blogged about those letters and the days when they were necessary, and I posed a kind of crazy promise to the Internet: that if you asked me for a hand-written letter, I would write you one, no questions asked. Overnight, my inbox morphed into this harbor of heartbreak -- a single mother in Sacramento, a girl being bullied in rural Kansas, all asking me, a 22-year-old girl who barely even knew her own coffee order, to write them a love letter and give them a reason to wait by the mailbox.Well, today I fuel a global organization that is fueled by thosetrips to the mailbox, fueled by the ways in which we can harness social media like never before to write and mail strangers letters when they need them most, but most of all, fueled by crates of maillike this one, my trusty mail crate, filled with the scriptings of ordinary people, strangers writing letters to other strangers not because they're ever going to meet and laugh over a cup of coffee,but because they have found one another by way of letter-writing.But, you know, the thing that always gets me about these letters is that most of them have been written by people that have never known themselves loved on a piece of paper. They could not tell you about the ink of their own love letters. They're the ones from my generation, the ones of us that have grown up into a world where everything is paperless, and where some of our best conversationshave happened upon a screen. We have learned to diary our pain onto Facebook, and we speak swiftly in 140 characters or less.But what if it's not about efficiency this time? I was on the subway yesterday with this mail crate, which is a conversation starter, let me tell you. If you ever need one, just carry one of these. (Laughter) And a man just stared at me, and he was like, "Well, why don't youuse the Internet?" And I thought, "Well, sir, I am not a strategist, nor am I specialist. I am merely a storyteller." And so I could tell you about a woman whose husband has just come home from Afghanistan, and she is having a hard time unearthing this thing called conversation, and so she tucks love letters throughout the house as away to say, "Come back to me. Find me when you can." Or a girl who decides that she is going to leave love letters around her campus in Dubuque, Iowa, only to find her efforts ripple-effected the next day when she walks out onto the quad and finds love letters hanging from the trees, tucked in the bushes and the benches. Or the man who decides that he is going to take his life, uses Facebook as a way to say goodbye to friends and family. Well, tonight he sleeps safelywith a stack of letters just like this one tucked beneath his pillow, scripted by strangers who were there for him when.These are the kinds of stories that convinced me that letter-writing will never again need to flip back her hair and talk about efficiency, because she is an art form now, all the parts of her, the signing,the scripting, the mailing, the doodles in the margins. The mere fact that somebody would even just sit down, pull out a piece of paper and think about someone the whole way through, with an intention that is so much harder to unearth when the browser is up and the iPhone is pinging and we've got six conversations rolling in at once, that isan art form that does not fall down to the Goliath of "get faster,"no matter how many social networks we might join. We still clutch close these letters to our chest, to the words that speak louder thanloud, when we turn pages into palettes to say the things that we have needed to say, the words that we have needed to write, to sisters and brothers and even to strangers, for far too long. Thank you. (Applause) (Applause)篇二：Ted视频点评-我们为什么不食用昆虫呢？Ted视频点评-我们为什么不食用昆虫呢？Ted演讲，《我们为什么不使用昆虫呢？》是由荷兰瓦赫宁根大学的一位教授Marcel Dicke演讲的，他从经济，营养，疾病环境等多个角度进行演讲，说服人们食用昆虫。
【高考生物】王玉梅托福词汇里的生物类单词总结
（生物科技行业）王玉梅托福词汇里的生物类单词总结adaptadapableadaptation--modification(以上三词都与动物进化有关) antibiotic抗菌的，抗生的antibiotics抗生素aquaticadj.水生的，水栖的aquariumn.水族馆arborealadj.树栖的，树的；乔木的arid--dryadj.干旱的semiaridadj.雨量特别少的aroman. 芳香，香气aromatic--fragrantadj.芳香的fragrant--aromatica,fragrance--scentn,香味perfumen,香味，香水backbone--spinen,脊椎，中枢baldeagle秃头鹰（美国国鸟）bardn, 鱼钩等的 - 倒勾，倒刺；bark--outercoveringn,树皮n/vi. 狗叫beakn, 鸟嘴polarbear北极熊grizzlybear灰熊biologistn.生物学家biologicaldiversity生物多样性bisonn. 美洲或欧洲的野牛bloomn/vi. 花/ 开花blossomn/vi. 花/ 开花boardern,寄生者baboonn,狒狒bouquetn,花簇breed--raise,hatch,matevt,养育，生殖n,品种crossbreedingn,异种杂交budn, 芽，蓓蕾vi, 萌芽bulbn ，球茎cannibalismn,同类相食carapacen,龟蟹等得-甲壳cardiacadj,心脏的，cardinaladj, 红衣凤头鸟 - 一种美洲鸟，雄性有深红色羽毛carnivorousadj,食肉的caterpillarn,毛虫chimpanzeen,黑猩猩gorillan,大猩猩calmn, 蚌肉clutchn, 一次所孵的蛋condorn,秃鹰conifern,松类针叶树coralreef珊瑚礁carbn, 蟹crawl--creepvi,爬行crown, 乌鸦crustaceann，甲壳动物culturevi,培养（微生物细胞组织等）daisyn, 雏菊deciduousadj,每年落叶的，decompositionn,腐化，分解decompose--decayv,分解，使腐化rodentn,edentaten,贫齿类动物dolphinn,海豚domesticate-cultivatevt,驯养驯化domesticated-tameadj. dormantadj,休眠的endotoxinn,内梅素endothermn,恒温动物draftanimal耕种动物（ horse之类）dragonflyn,蜻蜓ecologicaladj,生态学的，生态的ecologistnecologynecologistemn生态系统embryo-completelyundevelopedformn,胚胎；embryologicaladj,胚胎学的embryonicadj,萌芽期的，endanger-threaen,jeopardizevt,危及endangeredadj,有灭绝危险的，将要绝种的evergreenadj 常绿的， n,常绿植物extinctadj, 动物 -灭绝的；extinctionn.falconn, 猎鹰falconern,养猎鹰之人faunan, 动物群floran植物群floraladj,花的，植物的feedv, 饲养，靠。
a study of some physicochemical properties of high-crystalline tapioca starch
©WILEY-VCH Verlag GmbH, 69451 Weinheim, 20010038-9056/2001/1111-0577 $17.50+.50/0Starch/Stärke 53(2001)577–581577R e s e a r c h P a p e r1 IntroductionNative starch is composed of almost linear amylose, an α-1,4 polymer, and amylopectin, a branched polymer con-sisting of short linear α-1,4 polymer chains linked to each other by α-1,6 linkages [1]. These two components are amenable to form a semicrystalline structure in the starch granules, which consist of crystalline lamellae (ordered,tightly packed of parallel glucan chains) and amorphous lamellae (less-ordered regions, predominantly branch points) [2]. Starches of different origin have a different de-gree of crystallinity (range about 15–45%) [1].High-crystalline starch can be obtained by acid hydrolysis of native starch in a heterogeneous system [3]. The acid preferentially attacks the amorphous regions, thus % rel-ative crystallinity is increased in the starch granules.High-crystalline starches prepared from various origins have been applied in many branches of food industry so their structure and properties have been well studied [4–9].However, the properties of high-crystalline tapioca starch have not been studied up to now.Therefore, the objective of this study was to investigate the effect of crystallinity on the properties of tapioca starch. High-crystalline tapioca starches were produced by acid hydrolysis and their properties and relative crys-tallinities were studied by X-ray diffraction. Then the rela-tionship between crystallinity and starch properties was investigated. The results of this study will be used for elu-cidating the structure and properties of high-crystalline tapioca starch and help to promote its utilization.2 Materials and Methods2.1 MaterialsTapioca starch was the product of Choheng Co., Ltd.(Thailand). Iodine, potassium iodide, ethanol, sodium hy-droxide, acetic acid, hydrochloric acid and potato amy-lose were obtained from Sigma Chemical Co., Ltd, USA.2.2 Preparation of high-crystalline tapioca starch by acid hydrolysisTapioca starch (400 g, dry basis) was hydrolyzed by sus-pension in 600mL 6% (w/v) aqueous HCl solution at room temperature for a certain period of time, i.e., 12, 24,48, 96, 192, 384 and 768h. After the end of the hydroly-sis time, the suspension was neutralized with 10% (w/v)NaOH solution, and washed three times with distilled wa-ter. The washing water was removing by centrifuging (Sorvall RC 3B Plus, Du Pont Company, Delaware, USA)at 1000 ×g for 2min and decanting. The wet acid-modi-fied starch was either air-dried at room temperature (for granule size analysis) or spray-dried with a mobile minor spray dryer (Gea-Niro, Denmark) at an inlet temperature of 160°C and an outlet temperature of 60°C (for the oth-er experiments). The dried powder was sieved through a 100-mesh sifter to obtain acid-modified starch powder.Napaporn Atichokudom-chai a , Sujin Shobsngob b ,Pavinee Chinachoti c ,Saiyavit Varavinit aaDepartment of Biotechno-logy, Faculty of Science,Mahidol UniversitybDepartment of Chemistry,Faculty of Science, Mahidol University,Bangkok, Thailand cDepartment of Food Science, University of Massachusetts, Amherst,MA 01003, U.S.A.A Study of Some Physicochemical Properties of High-Crystalline Tapioca StarchTapioca starch was partially hydrolyzed in hydrochloric acid solution at room tempera-ture for various lengths of time to obtain high-crystalline starches. RVA viscoamylo-grams of acid-modified starches demonstrated a very low viscosity as compared to that of native tapioca starch. The relative crystallinity of native and acid-modified tapi-oca starches were measured by X-ray diffraction ranging from 39.53% to 57.75%. The native and acid-modified tapioca starches were compressed into tablets using various compression forces. The % relative crystallinity of starch increased with the increase in hydrolysis time and the crushing strength of the tablet was also increased in line with the crystallinity while the amylose content decreased when the crystallinity increased.These results suggested that the erosion of amylose might cause the rearrangement of starch structure into a new more tightly packed form, which provided the higher crushing strength for the tablets.Keywords:Tapioca starch; Acid-modified starch; X-ray diffraction; Amylose content;Crystallinity2.3 Proximate analysisProtein [10], fat [11], ash and moisture contents [12] were determined by the methods described in AOAC.2.4 Amylose contentThe amylose contents were measured by iodine affinity method [13] (using a spectrophotometer, model Pharma-cia LKB Novaspec II, USA).2.5 Granule size analysisThe granule size distribution of native and air-dried acid-modified tapioca starch were measured with a laser dif-fraction spectrometer (Malvern Instruments Ltd., Malvern, UK) using a range lens of 300RF mm and a beam length of 2.40mm.2.6 X-ray powder diffraction and percentage of relative crystallinity measurementsX-ray powder diffractograms were obtained with a JEOL, JDX-3530, Tokyo, Japan using the following conditions: Monochromatic Cu-Kαradiation 1.542 ÅX-ray generator power40kV, 30 mA Scanning , 2 θ4°to 30°Step interval0.02°Scanning rate2°/minDivergence slit1°Receiving slit1°Scattering slit0.15°Measurement temperature ambient temperature The percent relative crystallinity of starches were mea-sured following the method of Komiya and Nara [4].2.7 Viscosity analysisA Rapid Visco Analyser or RVA(Senes 4V, Newport Sci-entific Pty. Ltd, Australia) was employed to investigate the pasting properties of the starch samples. In this assay, 15g (dry basis) of an acid-modified tapioca starch was dispersed in 25mL distilled water. The heating and cool-ing cycles were programmed in the following manner: The sample was held at 50°C for 1min, heated to 95°C with-in 3min and then held at 95°C for 2min. It was subse-quently cooled to 50°C within 3min and then held at 50°C for 2min.2.8 Crushing strength (tablet press and hardness) measurementNative and acid-modified tapioca starches were com-pressed with a 8mm flat-face-beveled-edge punch on a tablet press instrument (Fette, Germany), which had been equipped with resistance strain gauges and strain amplifi-er (Kyowa, model DPM-712B, Kyowa Co., Ltd., Japan) according to Wray et al. [14] to monitor compression and ejection forces. The design of this machine was described in detail in [15]. Only a single station was used to help minimize tooling errors. The same circular, flat-faced punches with a die of 8mm diameter and 300mg target weight, were used throughout the study. A compression time of 1.6s per tablet was selected for this study. The tablet hardness was determined with an electronic hard-ness tester (Schleuniger Model 4M Dr. Schleuniger Co., Switzerland). All crushing strengths (tablet hardness) re-ported were based on the means of three determinations.3 Results and Discussion3.1 PropertiesThe compositions of native and acid-modified tapioca starches (moisture, protein, lipid, and ash) are presented in Tab.1. The protein and lipid contents decreased when the hydrolysis time increased, because both proteins and lipids were also slowly hydrolyzed by hydrochloric acid at ambient temperature.578Atichokudomchai et al.Starch/Stärke53(2001)577–581The acid-modified tapioca starch obtained after 768h of hydrolysis time showed a particle size distribution similar to that of native tapioca starch with average particle di-ameters about 14.28 (±0.34) and 14.29 (±0.59) µm, re-spectively. In other words, acid hydrolysis did not reduce the size of the starch granules. Therefore, the acid at-tacked the amorphous regions first at the surface and then progressed to the inside of the starch granules cre-ating porous structures in the granules [16]. This was sup-ported by the ash content presented in Tab.1. The longer the hydrolysis time, the higher the ash content. The high ash content resulted from NaCl, produced by neutraliza-tion of HCl by NaOH, NaCl penetrated the starch granules and was trapped inside.Though acid hydrolysis could not reduce the size of starch granules, the loss of starch on acid modification could be determined. It was found that at the early stage of acid modification a relatively fast loss of starch oc-curred (Tab.1). This was due to the attack of acid at the amorphous regions on the starch granules surface at the early stage. On prolonged treatment of starch with acid (768h) the loss of starch occurred again at a high rate,due to the attack of acid on both the amorphous and the crystalline regions to obtain smaller water soluble mole-cules.RVA characteristics of acid-modified tapioca starches are shown in Tab.2. In order to compare the RVA character-istics, the ratio of starch to water for the RVA measure-ment must be the same for all samples. The RVA viscogram of the acid-modified starches could be seen only at high concentration (15g starch per 25mL distilled water) while the viscosity of native tapioca starch was too high to be recorded. Only the viscograms of acid-modified starches after a hydrolysis time 12, 24, 48, and 96h could be detected. Higher acid hydrolysis time provided starch-es with a very low viscosity which could not be detected by the RVA at this concentration.From Tab.2, it could be seen that the peak viscosity of acid-modified starches decreased with increasing the hy-drolysis time. The peak viscosity had a strong correlation to crystallinity, the correlation factor being 0.98. As the peak viscosity decreased, the crystallinity increased. The drop in viscosity resulted from the partial hydrolysis of the amorphous regions [19], thus the crystallinity increased.X-ray diffraction patterns of native and acid-modified tapi-oca starches during the acid hydrolysis are summarized in Fig.1. The patterns showed the same typical feature ofStarch/Stärke 53(2001)577–581A Study of Some Physicochemical Properties 579Fig. 1. X-ray diffraction patterns of native and acid-modi-fied tapioca starches at various lengths of hydrolysis time:(A) 0h; (B) 12h; (C) 24h; (D); 48h; (E) 96h; (F) 192h;(G) 384h and (H) 768h.A-type starch with strong peaks at 2 θat about 15°, 17°,18°, and 23°[1, 17]. % Relative crystallinity of the starch-es was determined and presented in Fig.2. The effect ofmoisture content of starches on the % reIative crystallini-ty was neglected since the moisture contents of all starch-es were almost the same (~9%) (Tab.1). Fig.2 demon-strated that the % relative crystallinity increased with the increase in hydrolysis time in contrast to the consecutive decrease in amylose content. The highest % relative crys-tallinity was observed at 57.75%, where the amylose con-tent approached zero. This result indicated that the amy-lose embedded in the amorphous regions [1, 18] was preferentially hydrolyzed by the acid, resulting in a higher % relative crystalIinity.3.2 ApplicationIt was attempted to utilize the acid modified tapioca starch as tablet filler for the pharmaceutical industries. Since the price of tapioca starch is low, the modification process is simple. Fig.3 shows the relationship between % relative crystallinity and crushing strength of the tablets at various compressive forces. The results suggest that a strong re-lationship between % relative crystallinity and crushing strength of the tablets exists, with a correlation factor (r 2)of 0.95–0.99. As % relative crystallinity increased, the crushing strength of the tablets was also increased. The crystalline region is an ordered arrangement of double helical amylopectin structures. Embedded in the amor-phous regions, amylose has been proposed to disrupt thecrystalline packing of amylopectin [20]. The erosion of the amorphous regions by acid hydrolysis may result in a re-duced hindrance for the double helical chains to approach each other. Thus, when applying the compaction force to the starch granules, the crystalline regions could be forced to become closely packed and so the intermolecu-lar forces, i.e., van de Waals forces and hydrogen bond-ing, increased, leading to a more order rearrangement within the starch granules. The stronger packing structure resulted in an increase in the crushing strength of the tablet as observed.4 ConclusionAcid hydrolysis of tapioca starch could not reduce the size of the starch granules. The peak viscosity of acid modified starches decreased with an increase in the hydrolysis time. The peak viscosity was strongly correlated with crystallinity, as the peak viscosity decreased, the crys-tallinity increased. Acid hydrolysis also resulted in a decrease in amylose content and increase in crystallinity.The increasing crystallinity and the more ordered structure of acid-modified starches resulted in an in-crease in crushing strength of tablets. The % relative crystallinity and crushing strength were highly correlated580Atichokudomchai et al.Starch/Stärke 53(2001)577–581Fig. 2. % Relative crystallinity and % amylose content of native and acid-modified tapioca starches prepared by various hydrolysis times.Fig. 3. Relationship between % relative crystallinity and crushing strength (N) at various extent of compaction force (kN) (contacting time 1.6s).(r2= 0.95–0.99). The highly crystalline starches produced could potentially be employed as tablet filler in the phar-maceutical industry.AcknowledgementsThe authors would like to thank Thailand Research Found (TRF) for financial support via The Royal Golden Jubilee Ph.D. Program, the National Metal and Materials Technol-ogy Center (MTEC) for providing X-ray powder diffraction and the Cassava and Starch Technology Research Unit, Kasetsart Agricultural and Agro-Industrial Product Im-provement Institute (KAPI) for providing the Rapid Visco Analyser.Bibliography[1]H.F. Zobel: Molecules to granules: A comprehensive starchreview. Starch/Stärke 1988, 40,44–50.[2] C.G. Oates: Towards an Understanding of Starch GranuleStructure and Hydrolysis. Trend Food Sci. Technol. 1997, 8, 375–382.[3] D. French: Organization of Starch Granules, in StarchChemistry and Technology(Ed. R.L. Whistler), Academic Press New York, 1984,pp.183–247.[4]T. Komiya, S. Nara: Changes in Crystallinity and Gelatiniza-tion Phenomena of Potato Starch by Acid Treatment.Starch/Stärke1986,38,9–13.[5]T. Komiya, T. Yamada, S. Nara: Crystallinity of Acid TreatedCorn Starch. Starch/Stärke1987,39,308–311.[6]S. Vasudeva, A.S. Zakiuddin, S. Divakar: 13C CP/MASNMR Spectroscopy of Native and Acid Modified Starches.Starch/Stärke 1993,45,59–62.[7]J.L. Chun, J.Y. Takeda, M. Shoki: Properties of High-Crys-talline Rice Amylodextrins Prepared in Acid-Alcohol Media as Fat Replacers. Cereal Food World 1997,42,813–819.[8]P.J. Jenkins, A.M. Donald: The Effect of Acid Hydrolysis onNative Starch Granule Structure. Starch/Stärke1997,49, 262–267.[9]S. Varavinit, T. Nuyim, S. Shobsngob: Utilization of AcidModified Sago Starch for Pharmaceutical Industries. Sago Communication 1998,9,1–5.[10]AOAC, Official Method of Analysis: Protein, 15th edition, As-sociation of Official Analytical Chemistry, Arlington, USA, 1990, p.781.[11]AOAC, Official Method of Analysis: Fat, 15th edition, Associ-ation of Official Analytical Chemistry, Arlington, USA, 1990, p.780.[12]AOAC, Official Method of Analysis: Ash and moisture con-tent, 15th edition, Association of Official Analytical Chem-istry, Arlington, USA, 1990, p.777.[13] C.A. Knutson: A Simple Colorimetric Procedure for Deter-mination of Amylose in Maize Starches. Cereal Chem.1986, 63,89–92.[14]P.W. Wray, J.G. Vincent , F.W. Moller, G.J. Jackson: Pa-per presented at The Industrial Pharmaceutical Section, (A Ph A) Academy of Pharmaceutical Science, Dallas, meeting Apr. 1976, 25–29.[15] A.M. Salpekar, L.L. Augsburger: Magnesium Lauryl Sul-fate in Tableting: Effect on Ejection Force and Compress-ibility. J. Pharm. Sci. 1974,63,289.[16]N. Atichokudomchai, S. Shobsngob, S. Varavinit: Morpho-logical Properties of Acid-modified Tapioca starch.Starch/Stärke 2000, 52, 283–289.[17]R.P. Veregin, C.A. Fyfe, R.H. Marchessault, M.G. G. Tay-lor: Characterization of the Crystalline A and B Starch Poly-morphs and Investigation of Starch Crystallization by High Resolution 13C CP/MAS NMR. Macromolecules 1986, 19, 1030–1034.[18]N.W.H. Cheetam, L. Tao: Variation in Crystalline Type withAmylose Content in Maize Starch Granules: an X-ray Pow-der Diffraction Study. Carbohydr. Polym.1998, 36,277–284.[19]P.D. Cock: Functional Properties of Starch: Methods andApplications. Agro-Food-Industry Hi-Tech1996,Juli/August, 18–22.[20]P.J. Jenkins, A.M. Donald: The Influence of Amylose onStarch Granule Structure. Int. J. Biol. Macromol. 1995,17, 315–321.(Received: January 4, 2001)(Revision received: May 23, 2001)Starch/Stärke53(2001)577–581A Study of Some Physicochemical Properties 581。
tpo35三篇阅读原文译文题目答案译文背景知识
tpo35三篇阅读原文译文题目答案译文背景知识阅读-1 (1)原文 (2)译文 (5)题目 (8)答案 (17)背景知识 (18)阅读-2 (21)原文 (21)译文 (24)题目 (27)答案 (36)背景知识 (36)阅读-3 (39)原文 (39)译文 (43)题目 (46)答案 (54)背景知识 (55)阅读-1原文Earth’ s Age①One of the first recorded observers to surmise a long age for Earth was the Greek historian Herodotus, who lived from approximately 480 B.C. to 425 B.C. He observed that the Nile River Delta was in fact a series of sediment deposits built up in successive floods. By noting that individual floods deposit only thin layers of sediment, he was able to conclude that the Nile Delta had taken many thousands of years to build up. More important than the amount of time Herodotus computed, which turns out to be trivial compared with the age of Earth, was the notion that one could estimate ages of geologic features by determining rates of the processes responsible for such features, and then assuming the rates to be roughly constant over time. Similar applications of this concept were to be used again and again in later centuries to estimate the ages of rock formations and, in particular, of layers of sediment that had compacted and cemented to form sedimentary rocks.②It was not until the seventeenth century that attempts were madeagain to understand clues to Earth's history through the rock record. Nicolaus Steno (1638-1686) was the first to work out principles of the progressive depositing of sediment in Tuscany. However, James Hutton (1726-1797), known as the founder of modern geology, was the first to have the important insight that geologic processes are cyclic in nature. Forces associated with subterranean heat cause land to be uplifted into plateaus and mountain ranges. The effects of wind and water then break down the masses of uplifted rock, producing sediment that is transported by water downward to ultimately form layers in lakes, seashores, or even oceans. Over time, the layers become sedimentary rock. These rocks are then uplifted sometime in the future to form new mountain ranges, which exhibit the sedimentary layers (and the remains of life within those layers) of the earlier episodes of erosion and deposition.③Hutton's concept represented a remarkable insight because it unified many individual phenomena and observations into a conceptual picture of Earth’s history. With the further assumption that these geologic processes were generally no more or less vigorous than they are today, Hutton's examination of sedimentary layers led him to realize that Earth's history must be enormous, that geologic time is anabyss and human history a speck by comparison.④After Hutton, geologists tried to determine rates of sedimentation so as to estimate the age of Earth from the total length of the sedimentary or stratigraphic record. Typical numbers produced at the turn of the twentieth century were 100 million to 400 million years. These underestimated the actual age by factors of 10 to 50 because much of the sedimentary record is missing in various locations and because there is a long rock sequence that is older than half a billion years that is far less well defined in terms of fossils and less well preserved.⑤Various other techniques to estimate Earth's age fell short, and particularly noteworthy in this regard were flawed determinations of the Sun's age. It had been recognized by the German philosopher Immanuel Kant (1724-1804) that chemical reactions could not supply the tremendous amount of energy flowing from the Sun for more than about a millennium. Two physicists during the nineteenth century both came up with ages for the Sun based on the Sun's energy coming from gravitational contraction. Under the force of gravity, the compressionresulting from a collapse of the object must release energy. Ages for Earth were derived that were in the tens of millions of years, much less than the geologic estimates of the lime.⑥It was the discovery of radioactivity at the end of the nineteenth century that opened the door to determining both the Sun’s energy source and the age of Earth. From the initial work came a suite of discoveries leading to radio isotopic dating, which quickly led to the realization that Earth must be billions of years old, and to the discovery of nuclear fusion as an energy source capable of sustaining the Sun's luminosity for that amount of time. By the 1960s, both analysis of meteorites and refinements of solar evolution models converged on an age for the solar system, and hence for Earth, of 4.5 billion years.译文地球的年龄①希腊历史学家希罗多德是最早有记录的推测地球年龄的观察家之一，他生活在大约公元前480年到公元前425年。
葡萄糖脱氢酶法的英文
葡萄糖脱氢酶法的英文Glucose Dehydrogenase Method.The glucose dehydrogenase method is a spectrophotometric assay for determining the concentration of glucose in a sample. It is based on the enzymatic reaction catalyzed by glucose dehydrogenase (GDH), which oxidizes glucose to gluconic acid and reduces NAD+ to NADH. The amount of NADH produced is stoichiometrically equivalent to the amount of glucose in the sample, and can be measured by its absorbance at 340 nm.The reaction is carried out in a cuvette containing a buffer, GDH, NAD+, and the sample. The change in absorbance at 340 nm is monitored over time, and the rate of change is used to calculate the concentration of glucose in the sample.The glucose dehydrogenase method is a simple, rapid, and accurate method for determining the concentration ofglucose in a variety of samples, including blood, urine, and food products. It is commonly used in clinical chemistry laboratories and in food analysis.Principle of the Method.The glucose dehydrogenase method is based on the following enzymatic reaction:Glucose + NAD+ + H2O → Gluconic acid + NADH + H+。
2024高考英语新课标1卷阅读解析
阅读-B篇
25. Why did Farber decide to try acupuncture on pets?He was trained in it at university. He was inspired by another veterinarian. He benefited from it as a patient. He wanted to save money for pet owners.
D
【解析】词义猜测题。根据第二段前两句“When reading texts of several hundred words or more, learning is generally more successful when it's on paper than onscreen. A large amount of research confirms this finding."可知，大量研究证实，当阅读几百字或更多的文本时，纸质阅读比屏幕阅读的学习效果更好。根据画线短语所在句可知，当实验人员从简单的任务（如找出阅读文章的主旨）转变为需要抽象思维的任务（如从文本中得出推论）时，纸质阅读的好处尤其_______。结合该段第一句以及画线短语前的particularly可知，此处表示纸质阅读的好处在这种情况下尤为明显，即更容易被注意到，故画线部分意为“很明显”
阅读-B篇积累
acupuncture针灸ahead of time领先时代vet-veterinarian兽医holistic medicine整体医学chiropractic按摩疗法herbal medicine草药医学alternative可替代的practice习俗，习惯put sb. To sleep（手术麻醉）使失去知觉indication迹象-indicate
apoptosis细胞凋亡
13.3 Apoptosis Depends on an Intracellular Proteolytic Cascade That Is Mediated by Caspases
• The Intracellular machinery responsible for apoptosis is similar in all animal cells. It depends on a family of proteases that have a cysteine at their active site and cleave their target proteins at specific aspartic acids.
Normal Cell
Apoptotic Cell
The Procedure of Apoptosis
◆Early stage of apoptosis凋亡的起始: 细胞表面的特化结构如微绒 毛消失，细胞间接触的消失，但细胞膜依然完整；线粒体大体完整， 但核糖体逐渐从内质网上脱离，内质网囊腔膨胀，并逐渐与质膜融合； 染色质固缩，形成新月形帽状结构等形态，沿着核膜分布 ◆Forming apoptotic body凋亡小体的形成：核染色质断裂为大小不 等的片段，与某些细胞器如线粒体一起聚集，为反折的细胞质膜所包 围。细胞表面产生了许多泡状或芽状突起，逐渐形成单个的凋亡小体
• Caspases: c for cysterine and asp for aspartic acid.
• Caspases are synthesized in the cell as inactive precursors, or procaspases, which is typically activated by proteolytic cleavage.
微生物英文文献Qualitative and quantitative methodologies for determination
REVIEWQualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operationsRobert S.Dungan ÆApril B.LeytemReceived:12August 2008/Accepted:8April 2009/Published online:26April 2009ÓUS Government 2009Abstract The generation of airborne microorganisms from concentrated animal-feeding operations (CAFOs)is a concern from a human and animal health perspective.To better understand the airborne microorganisms found in these environments,a number of collection and analytical techniques have been utilized and will be discussed in this review.The most commonly used bioaerosol collection method is the liquid impingement format,which is suitable with a number of culture-based and non-culture molecular-based approaches,such as polymerase chain reaction.However,the vast majority of airborne microorganism studies conducted at CAFOs utilize culture-based analyses.Because of the limitations often associated with culture-based analyses,we focused our discussion on the applica-tion of molecular-based techniques to identify and/or quantify microorganisms,as they have promising applica-tion in bioaerosol research.The ability to rapidly charac-terize airborne microorganisms will help to ensure protection of public and environmental health.Keywords Airborne microorganisms ÁBioaerosol ÁConcentrated animal-feeding operations ÁImpaction ÁImpingement ÁNucleic acid ÁPolymerase chain reaction ÁReal-time PCRIntroductionModern animal husbandry has changed from one that was low density pasture-based to one that predominately employs confinement of animals at high stocking density.Confined or concentrated animal-feeding operations (CA-FOs)concentrate a large population of single species in one area to increase production and reduce costs.During recent decades,CAFOs have become common in many countries including The Netherlands,Denmark,France,USA,Can-ada,China,Germany,and Poland (Schulze et al.2006).A consequence of high stocking densities combined with enclosed rearing facilities,in some cases,is that the air may contain bioaerosol levels that are sufficiently high to cause adverse health effects in both animals and workers (Thorne et al.1992).Crook and Sherwood-Higham (1997)indicated that inhalation of airborne microorganisms and their constituents can be detrimental to health through infection,allergy,or toxicosis.As the environment within CAFOs can be potentially hazardous to both human and animal health at the facility as well as in surrounding areas,research is being pursued in order to quantify,characterize,and control the release of bioaerosols from CAFOs.Bioaerosols is a term commonly used to describe via-ble and non-viable airborne biological particles,such as fungal spores,bacteria,pollen,and viruses and their fragments and byproducts (Grinshpun et al.2007).Fungal spores,bacteria,and pollen are typically 1–30,0.25–8,and 17–58l m in diameter,respectively,while viruses generally have diameters \0.3l m (Jones and Harrison 2004).Matthais-Maser et al.(2000)suggested that up to 28%(by volume)of the particulate matter suspended over remote land surfaces is comprised of biological particles.Womiloju et al.(2003)concluded that fungal cells and pollen accounted for 4–11%of the total mass of airborneThe use or mention of any commercial products does not imply any endorsement of that product by either the authors or the US Department of Agriculture.R.S.Dungan (&)ÁA.B.LeytemUSDA-Agricultural Research Service,Northwest Irrigation and Soils Research Laboratory,3793North 3600East,Kimberly,ID 83341,USAe-mail:robert.dungan@123World J Microbiol Biotechnol (2009)25:1505–1518DOI 10.1007/s11274-009-0043-1particulate matter\2.5l m(PM2.5).Although microor-ganisms are ubiquitous in the ambient environment,pre-vious studies have shown higher airborne microorganism concentrations in animal houses than in industrial,resi-dential,or ambient settings(Clark et al.1983;Thorne et al.1992;Griffiths et al.1997).Bioaerosols are typically associated with particulate matter or surrounded by a thin layer of water,having an aerodynamic diameter range of0.5–100l m(Lighthart 1994;Cox1995).Bioaerosol particles1–5l m in diameter present the most concern since they are readily transported into the lung,with the greatest retention of the1–2l m particles in the alveoli(Salem and Gardner1994).The microbial component of respirable bioaerosols contributes significantly to the pulmonary diseases associated with inhalation of agricultural dusts(Merchant1987;Lacy and Crook1988).The allergenic,toxic,and inflammatory responses are caused by exposure to not only viable but also non-viable microorganisms present in bioaerosols (Robbins et al.2000;Gorny et al.2002).An estimation of occupational and residential risks from bioaerosol exposure have been addressed by Brooks et al.(2005a,b)and Tanner et al.(2008).As the generation of bioaerosols from CAFOs is a concern from a human and animal health perspective, the sampling and analysis of airborne microorganisms is of great interest.Protection of public and environmental health is dependent upon the ability to efficiently collect bioaerosol samples,then accurately identify and quantify the airborne microorganisms.In this concise review,we focus our discussion on bio-aerosol sampling and sample processing methods that are most suitable to quantitatively and qualitatively determine airborne microorganisms at CAFOs,although their appli-cation to other situations is not limited.The majorfindings of bioaerosol studies conducted at CAFOs are also dis-cussed.While this is not meant to be an exhaustive review of the literature,the reader willfind an excellent array of peer-reviewed articles on aerosol science and molecular biology and their application to studies of air quality.This review will be very useful to those interested in conducting bioaerosol research using both traditional microbiological and molecular techniques.Airborne microorganism samplingThe collection of airborne microorganisms is performed through active air sampling,which results in the efficient removal and collection of biological particles from the air in a manner that maximizes the ability to detect the organisms.Airborne microorganisms can be collected using a number of different techniques(Lundholm1982; Juozaitis et al.1994;Grinshpun et al.1996;Terzieva et al.1996;Duchaine et al.2001),but two inertial techniques,surface impaction and liquid impingement, are used in the majority of outdoor aerosol studies.Fil-tration is a non-inertial technique that separates particles from the airstream when air is passed through a porous medium,such asfibrousfilters,membranefilters,or etched membranes(Crook1995a).For airborne microor-ganisms,however,filtration poses two major disadvan-tages:(a)dehydration of cells and therefore loss of viability and/or culturability due to the large volume of air passing over the particle that is deposited on a dry medium,and(b)inconsistent and poor recovery of the deposited material from certainfilter types.Two addi-tional techniques,gravity sampling and electrostatic precipitation,have been employed for airborne microor-ganism collection but are not routinely used due to cali-bration errors and unknown performance characteristics (Pillai and Ricke2002).The most common bioaerosol sampling techniques uti-lized at cattle,poultry and swine CAFOs are presented in Table1.Direct impaction of airborne microorganisms on filters was used in*40%of the studies,while a combi-nation of liquid impingement and multistage or single stage impaction was used in*33%of the studies.Other sam-pling techniques included use of a personal slide sampler to measure fungi in a cattle shed(Adhikari et al.2004)and drag swab for determination of Salmonella in a poultry house(Endley et al.2001).The target organisms in these studies included Wallemia sebi,total bacteria and fungi, Gram-negative bacteria,heterotrophs,E.coli,enteric bac-teria,Salmonella,yeast,and molds.Impaction samplersThe surface impaction method separates particles from the airstream by utilizing the inertia of the particles to force their deposition onto a collection surface(Grinshpun et al. 2007).The collection surface is usually an agar medium for culture-based analysis or an adhesive-coated surface that can be analyzed microscopically.A commonly used impaction system is the multi-stage Andersen viable sam-pler(Thermo Scientific,Waltham,MA,USA)that con-centrates bioaerosols based on their size characteristics. Two-stage and six-stage Andersen models are available. The six-stage Andersen sampler is capable of concentrating particles in the size range of0.65–7.0l m in diameter (Grinshpun et al.2007).Air enters the sampler through an inlet nozzle and heavier particles are deposited on thefirst stage.Lighter particles not deposited on thefirst stage are carried by the airstream onto the successive stages.Single-stage impactors,which use an agar or adhesive-coated impacting surface,are available from a variety of1506World J Microbiol Biotechnol(2009)25:1505–1518 123T a b l e 1B i o a e r o s o l s t u d i e s c o n d u c t e d a t c o n c e n t r a t e d a n i m a l -f e e d i n g o p e r a t i o n s i n c l u d i n g t h e t y p e o f o p e r a t i o n ,t h e t a r g e t o r g a n i s m ,s a m p l i n g t e c h n i q u e s u t i l i z e d a n d t h e a n a l y t i c a l m e t h o d s u s e d f o r d e t e r m i n a t i o n o f m i c r o o r g a n i s m sO p e r a t i o n T a r g e t o r g a n i s m sS a m p l i n g t e c h n i q u e sA n a l y t i c a l m e t h o d s R e f e r e n c e sC o w h o u s e W a l l e m i a s e b iD i r e c t i m p a c t i o n o n fil t e r sC u l t u r e t e c h n i q u e s ,c o n v e n t i o n a l a n d r e a l t i m e P C R Z e n g e t a l .2004D u c k -f a t t e n i n g u n i tT o t a l a n d a e r o b i c G r a m -n e g a t i v e b a c t e r i a ,f u n g i ,e n d o t o x i n sL i q u i d i m p i n g e m e n t ,m u l t i -s t a g e i m p a c t i o n ,a n d d u s t s a m p l i n gC u l t u r e t e c h n i q u e s ,w h o l e b l o o d a s s a y ,E L I S A ,l i m u l u s a m e b o c y t e l y s a t e a s s a y Z u c k e r e t a l .2006C a t t l e f e e d l o t B a c t e r i a a n d f u n g iM u l t i -s t a g e i m p a c t i o nC u l t u r e t e c h n i q u e sW i l s o n e t a l .2002bC a t t l e s h e d F u n g iM u l t i -s t a g e i m p a c t i o n a n d P e r s o n a l s l i d e s a m p l e rC u l t u r e t e c h n i q u e s a n d m i c r o s c o p yA d h i k a r i e t a l .2004P i g g e r y s h e d s H e t e r o t r o p h s a n d E .c o l iL i q u i d i m p i n g e m e n t a n d m u l t i -s t a g e i m p a c t i o nC u l t u r e t e c h n i q u e sC h i n i v a s a g a m a n d B l a c k a l l 2005S w i n e b a r n sT o t a l a n d G r a m -n e g a t i v e e n t e r i c b a c t e r i a ,t o t a l f u n g iM u l t i -s t a g e i m p a c t i o n ,l i q u i d i m p i n g e m e n t ,d i r e c t i m p a c t i o n o n fil t e r sC u l t u r e t e c h n i q u e s a n d flu o r e s c e n c e m i c r o s c o p yT h o r n e e t a l .1992S w i n e b a r n sC u l t u r a l b a c t e r i a ,G r a m -n e g a t i v e b a c t e r i a ,f u n g iL i q u i d i m p i n g e m e n t ,m u l t i -s t a g e i m p a c t i o nC u l t u r e t e c h n i q u e sC h a n g e t a l .2001P o u l t r y H o u s e S a l m o n e l l aD r a g s w a b ,d i r e c t i m p a c t i o n o n fil t e r sC u l t u r e t e c h n i q u e s a n d P C RE n d l e y e t a l .2001S w i n e b a r n s H e t e r o t r o p h i c b a c t e r i aD i r e c t i m p a c t i o n o n fil t e r sC u l t u r e t e c h n i q u e sP r e d i c a l a e t a l .2001S w i n e b a r n sT o t a l a n d r e s p i r a b l e m i c r o o r g a n i s m sD i r e c t i m p a c t i o n o n fil t e r s ,m u l t i -s t a g e i m p a c t i o nC u l t u r e t e c h n i q u e sP r e d i c a l a e t a l .2002S w i n e b a r n s T o t a l b a c t e r i a a n d f u n g iS i n g l e s t a g e i m p a c t i o nC u l t u r e t e c h n i q u e sK i m e t a l .2006,2007P o u l t r y H o u s e T o t a l b a c t e r i aD i r e c t i m p a c t i o n o n fil t e r s ,l i q u i d i m p i n g e m e n tC u l t u r e t e c h n i q u e sW o o d w a r d e t a l .2004S w i n e C A F O B a c t e r i aM u l t i -s t a g e i m p a c t i o nC u l t u r e t e c h n i q u e sG r e e n e t a l .2006P o u l t r y h o u s e T o t a l a e r o b i c b a c t e r i aS i n g l e s t a g e i m p a c t i o nC u l t u r e t e c h n i q u e sV e n t e r e t a l .2004P o u l t r y ,c o w ,a n d s w i n e h o u s eA i r b o r n e m i c r o o r g a n i s m sD i r e c t i m p a c t i o n o n fil t e r sE p i flu o r e s c e n c e m i c r o s c o p yH e l d a l e t a l .1996S w i n e b a r n s T o t a l a n d c u l t u r a l b a c t e r i aL i q u i d i m p i n g e m e n t ,i m p a c t i o n o n g e l a t i n m e m b r a n e sC u l t u r e t e c h n i q u e s ,r e a l t i m e P C R ,d e n a t u r i n g g r a d i e n t g e l e l e c t r o p h o r e s i s ,p h y l o g e n e t i c a n a l y s i sN e h m e e t a l .2008D a i r y b a r n sY e a s t s ,m o l d s ,m e s o p h i l i c b a c t e r i a ,t h e r m o p h i l i c b a c t e r i aL i q u i d i m p i n g e m e n tC u l t u r e t e c h n i q u e sL a n g e e t a l .1997S w i n e C A F O V i a b l e b a c t e r i aL i q u i d i m p i n g e m e n tC u l t u r e t e c h n i q u e sR u l e e t a l .2005World J Microbiol Biotechnol (2009)25:1505–15181507123manufacturers.Adhesive-coated impacting surfaces are used for the detection of total fungal spores and pollen.In addition to the Andersen impactors,there are other impaction-based devices,such as the rotating impactor,slit sampler,and sieve-type sampler(Crook1995b).Disad-vantages associated with culture-based impactors are:(a) detection of microorganisms relies on their ability to grow after sampling and losses of culturability may occur due to sampling stress,(b)multiple particles each containing one or more organisms passing through a single impaction hole may be inaccurately counted as a single colony,and(c) culturable counts account for only0.0001–10%of the total population within environmental samples,which can severely underestimate the total population of microor-ganisms in the sample(Parkes and Taylor1985).This is also a problem when using culture-based techniques with impingement samplers.Impingement samplersImpingement samplers remove bioaerosols over a wide range of airborne particle concentrations(Grinshpun et al. 2007).The primary difference between impingement and impaction is that the bioaerosols are trapped in a liquid (e.g.,water,mineral oil,buffered solution,or dilute pep-tone solution).In theory,buffered or dilute peptone solu-tions are used to maintain the viability of the microbial cells.Most impingers are constructed from glass with a single collection chamber;though multi-stage glass liquid impinges are available(Crook1995b).The All-Glass Impinger(AGI)-30(Ace Glass,Inc,Vineland,NJ,USA)is a single chamber design that has been widely used to measure bioaerosols under various conditions(Pillai et al. 1996;Chang et al.2001;Rule et al.2005;Tanner et al. 2005;Taha et al.2006).The SKC BioSamplerÒ(SKC Inc, Eighty-Four,PA,USA)is an improved design over the AGI-30and can be operated for up to8h when mineral oil is used as the collectionfluid(Lin et al.1999).Both the SKC BioSamplerÒand AGI-30operate under an airflow rate of12.5l min-1through the use of a vacuum pump. During operation of the impinger,the microorganisms are suspended in the collectionfluid,but the high airflow velocity required for efficient particle collection also cau-ses re-aerosolization of the biological particles(Grinsphun et al.1997;Lin et al.1997)and stress that can lead to viability loss(Lin et al.1999,2000).One of the advantages of impingement samplers is the ability to utilize a variety of analytical methods.In addition to culture techniques, samples can also be analyzed via microscopy,flow cytometry,biochemical assays,immunoassays,and molecular techniques such as polymerase chain reaction (PCR)providing better detection of airborne microorganisms which may be non-culturable due to sampling stresses.High-volume samplersAnother class of bioaerosol samplers that has recently evolved due to bioterrorism and biological warfare con-cerns is high-volume samplers.Some examples of these units are the SASSÒ2300(Research International,Mon-roe,WA),BioCaptureÒ560(MesoSystems Technology, Inc,Albuquerque,NM),and the SpinconÒ(Sceptor Industries,Inc,Kansas City,MO).These samplers operate atflow rates of200–450l min-1and the bioaerosols are captured in a concentrated liquid sample.While the high-volume samplers are very costly when compared to units such as the AGI-30and SKC BioSamplerÒ,they are generally more amenable to PCR-based analyses.The ASAPÒmodel2800(Thermo Electron Corporation, Greenbush,NY,USA)sampler has an operationalflow rate of200l min-1,but collects aerosol particles by impaction on polyurethane foam.While the ASAP unit does not use a liquid impingement format like the other high-volume samples,it is currently being marketed as PCR-compatible. At this time,however,a search of the literature reveals a scarcity of peer-reviewed studies with respect to these or comparable units and their operating efficiencies(Bergman et al.2005).For a comprehensive list of commercially available bioaerosol samplers see Grinshpun et al.(2007). Sample processingOnce samples have been collected,choosing the appro-priate analytical technique is important in order to best answer the question of interest.One of the most popular methods to assess microbial populations in aerosol samples has been the use of culture-based techniques.Culture-based techniques were employed in89%of the studies reported here(Table1).As mentioned above,culture-based tech-niques can drastically underestimate the microbial popu-lations in environmental samples as less than10%of the populations may be culturable.In order to improve microorganism detection,some studies have combined the use of culture techniques with other methods such as PCR (16%),microscopy(16%),denaturing gradient gel elec-trophoresis(DGGE,5%),and immunoassays(5%).Sample preparation is important for all of these techniques,as microorganism populations in bioaerosol samples tend to be small and,therefore,concentration of samples is essential.The most commonly used sample preparation methods compatible with the molecular characterization of bioaerosols can be found below.1508World J Microbiol Biotechnol(2009)25:1505–1518 123Concentration andfilter elutionAfter bioaerosols are collected in a liquid impingement solution,it is necessary to concentrate the microorganisms before molecular methods,such as PCR,can be performed. This is necessary because the impingement solution usually contains a relatively low microbial concentration,which must be maximized to ensure sensitivity and quantification for PCR are achieved.A variety offilter materials have been tested for their compatibility with PCR(Table2)such as polytetrafluoroethylene(PTFE),polycarbonate,polyvi-nylidene difluoride,nylon,mixed cellulose ester,and nitrocellulose(Bej et al.1991a).Bej et al.(1991a)reported that PCR was not inhibited by the presence of PTFE and polyvinylidene difluoridefilters,with PTFE giving the greatest sensitivity,but was inhibited by polycarbonate, nitrocellulose,and cellulose acetatefilters.Both Nytran (Alvarez et al.1994)and nitrocellulose(Toranzos and Alvarez1992)filters have been successfully used in solid-phase PCR,where cell lysis and PCR amplification are performed on the membrane.Since DNA does bind to somefilters,it is recommended that allfilters be removed before cell lysis and PCR amplification.Filter materials that have been successfully used in PCR-based bioaerosol studies using liquid samples from glass impingers are Nytran(Alvarez et al.1994), polycarbonate(Paez-Rubio et al.2005),nylon(Alvarez et al.1995),and Teflon(Alvarez et al.1995).Aerosol samples can also be directly impinged ontofilters for subsequent PCR analysis;filters used for this purpose are tracked-etched polyester(Wilson et al.2002a),polycar-bonate(Zeng et al.2004),and polyethersulfone(Sta¨rk et al.1998).Thefilters are added to sterile distilled water (Alvarez et al.1995)or buffer solution(Wilson et al. 2002a;Zeng et al.2004;Paez-Rubio et al.2005)and then the microorganisms are eluted via agitation such as vor-texing,shaking,or sonication.Cell lysis and nucleic acid purificationAfter elution,thefilter is removed and the cells are then prepared for lysis,which can be performed either through physical,chemical,or enzymatic methods.Physical meth-ods include bead beating,sonication,microwave heating, and thermal shock(Roose-Amsaleg et al.2001),but bead beating and sonication can cause significant DNA shearing (Picard et al.1992;Miller et al.1999;Bu¨rgmann et al. 2001).Freeze-thaw lysis has been shown to release70–75% of DNA in bacterial cells after one cycle with complete lysis within six cycles(Bej et al.1991b).Chemical lysis,either alone or in combination with enzymatic methods,has been used extensively.The most widely used detergent is sodium dodecyl sulfate(SDS),whose function is to break up and dissolve cell wall lipids.Detergents are used in combination with heat treatments and chelating agents(e.g.,EDTA)and various buffers(Tris and phosphate).In addition to a detergent,many protocols include enzymatic lysis.Lyso-zyme is a commonly used lytic enzyme that breaks the b-1,4-glycosidic bonds between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan,thereby weakening the cell wall.Some proteases,like proteinase K,are also used to remove contaminating proteins(e.g.,nucleases)that might otherwise degrade nucleic acids during purification. The protease,achromopeptidase,has been used withTable2Filters utilized for preparation of bioaerosol samples for molecular methods including thefilter type,type of sample,and the methods used for sample preparation and analysisFilter Sample type Methods ReferencesPolytetrafluoroethylene, Polyvinylidenedifluoride Bacterial cells in watercollected onfiltersFreeze thaw lysis of cells fromfiltered samples,PCR DNA amplification withfilters presentBej et al.1991a,bPolycarbonate Direct impingement ofbioaerosols onfilter Filters washed in buffer to remove bacteria,DNAextraction(chemical/enzymatic),RT-PCRZeng et al.2004Polycarbonate Bioaerosols collected in liquidimpingers andfiltered Impinger solutionfiltered,DNA extraction,PCR,cloning,sequencingPaez-Rubio et al.2005Track etched polyester Direct impingement ofbioaerosols onfilter Filters washed in buffer to remove bacteria,DNAextraction(physical/chemical/enzymatic),microarray analysisWilson et al.2002aMixed cellulose nylon Bioaerosols collected in liquidimpingers andfiltered Cell lysis and DNA extraction(chemical/enzymatic)performed onfilters,solid-phasePCR used for amplificationAlvarez et al.1994Nitrocellulose Filtration of bacterial cells inwater Cell lysis and DNA extraction(chemical/enzymatic)performed onfilters,solid-phasePCR used for amplificationToranzos andAlvarez1992Polyethersulfone Direct impingement ofbioaerosols onfilter Filters were dried and dissolved in chloroform,DNA extraction(chemical),nested PCR assaySta¨rk et al.1998World J Microbiol Biotechnol(2009)25:1505–15181509123lysozyme to increase the lysis of anaerobic Gram-positive cocci(Ezaki and Suzuki1982)and extraction efficiency of nucleic acids from Frankia(Simonet et al.1984).Detailed methods on the extraction and purification of nucleic acids can be found in Sambrook and Russell(2001) and Ausubel et al.(2002).Purification of nucleic acids in bacterial lysates is generally accomplished byfirst mixing with equal volumes of phenol and chloroform.Phenol is used because it removes the proteins from the aqueous phase;chloroform is generally not necessary,but it is used to remove residual phenol from the aqueous phase.The nucleic acids are then precipitated from the aqueous phase by additions of ethanol and collected by centrifugation. The nucleic acids can then be dissolved in buffer(e.g., Tris-EDTA)and stored at-20°C.Alternatively,nucleic acids can be purified using the many commercially avail-able spin column formats that utilize silica-nucleic acid binding(Qiagen,Inc.,Fremont,CA,USA;Mo Bio Labo-ratories,Carlsbad,CA,USA;Promega,Inc.,Madison,WI, USA;MP Biomedicals,Solon,OH,USA;Invitrogen,Inc., Carlsbad,CA,USA).As a result,the spin kits require no phenol or chloroform purification or alcohol precipitation. After the silica-based membrane has been loaded with cell lysate,the DNA or RNA is cleaned by rinsing with an ethanol-containing buffer,and then eluted using a small volume of buffer or water.The characterization of airborne microorganisms Culture versus molecular-based approachesMany of the available bioaerosol sampling methods rely on culture-based techniques for the characterization and quantification of airborne microorganisms.Microorgan-isms(fungi and bacteria)that are collected on a nutrient agar surface by impaction can be cultivated directly. However,only those cells which survive,reproduce,and produce visible colonies under the specified culture con-ditions will be enumerated.The disadvantage of culture-based techniques is that not all microorganisms are cul-turable,while they still may be viable(Heidelberg et al. 1997).This could lead to an underestimation of the total microorganism concentration in the aerosol sample.With culture-based techniques,non-culturable microorganisms and their associated byproducts that may cause health effects will go undetected.While liquid samples from impingers are commonly used for culture-based analyses, they can also be analyzed by microscopy to determine total microorganism concentrations or by biochemical,immu-nological,and molecular assays to detect specific micro-organisms,both culturable and non-culturable(Cruz and Buttner2007).As an alternative to culture-based techniques,the detection of microorganisms in aerosols by PCR has become increasingly popular over the last two decades (Alvarez et al.1994;Wakefield1996;Sta¨rk et al.1998; Olsson et al.1998;Williams et al.2001;Wu et al.2003; Zeng et al.2004;Paez-Rubio et al.2005;An et al.2006) allowing for the detection of target nucleic acid sequences, thereby eliminating the need to cultivate microorganisms for their detection and identification.This is particularly useful for microorganisms that are difficult to culture,slow growing or have never been cultured before,providing increased sensitivity over traditional culture-based methods (Josephson et al.1993;Alvarez et al.1994).A limitation of the PCR assay,however,is the inability to distinguish between non-viable and viable microorganisms.While non-viable pathogenic microorganisms do not present an infectious disease risk,the presence of their DNA in a sample will often produce a positive PCR result.Therefore, one cannot truly determine if the positive result represents a potential disease threat if the viability of the microor-ganisms in the original sample was unknown.A positive detect for targeted microorganisms only means that a sample contains viable or non-viable cells or both.Non-quantitative PCRTraditional PCR involves the separation of DNA(usually a specific gene or portion of a gene)into two strands,the annealing of oligonucleotide primers to the template DNA, and then the primer-template is elongated by use of a DNA polymerase enzyme(e.g.,Taq polymerase).During PCR, each of the steps is accomplished by regulating the tem-perature of the reaction and,as a result,multiple copies of the template are produced.Guidance on the optimization of PCR can be found in several laboratory manuals(Weiss-ensteiner et al.2003;Hughes and Moody2007).By using carefully designed primers,the genetic sequence of a specific microorganism or microbial function can be tar-geted and amplified.If ribonucleic acid(RNA)is targeted, then the RNA must be converted into complementary DNA (cDNA)through a reverse transcription process,after which the resultant cDNA is PCR amplified.One advan-tage of targeting RNA(e.g.,mRNA)is that it has a very short half-life and,therefore,it is a good indicator of viable microorganisms(Bej et al.1991b).The amplified DNA is visualized most often by running the samples in an electrophoresis gel(e.g.,agarose or polyacrylamide),staining the DNA within the gel with ethidium bromide,and viewing the separated DNA under UV light.A standard molecular weight marker is run along side the samples so the size of the DNA can be determined. The amplified DNA can also be processed for genetic fingerprinting,clone library analysis,and microarray1510World J Microbiol Biotechnol(2009)25:1505–1518 123。
我是小导游介绍恐龙的演化过程英语作文
我是小导游介绍恐龙的演化过程英语作文全文共3篇示例，供读者参考篇1Hello everyone! My name is Jamie and I'm going to be your dinosaur tour guide today at the Natural History Museum. I'm really excited to share what I've learned about the amazing evolution of these prehistoric creatures. Let's start at the very beginning!The earliest dinosaurs emerged around 230 million years ago during the late Triassic period. They descended from a reptile lineage, but these first true dinosaurs were quite different from modern-day reptiles. What set them apart were special holes in their skulls called fenestrae, which made their skulls lighter and allowed for larger jaw muscles and more powerful bites.One of the earliest known dinosaur species was the Eoraptor, a small, light-weight predator only about 3 feet long. It walked on two legs and had characteristics we now recognize as distinctly dinosaurian, like hands with claws, a long neck, and a jaw full of serrated teeth perfect for shredding meat.As the dinosaurs continued evolving into the Jurassic period from 200-145 million years ago, they became much more diverse in size and type. There were the heavy plant-eating Sauropods like the massive Diplodocus with its ridiculously long neck and whip-like tail. These gentle giants could grow over 100 feet long but had very small heads and simple teeth for stripping leaves off branches.In contrast, the theropods were the terrifying meat-eaters like the Allosaurus that stalked its prey on powerful hind legs. With razor-sharp teeth and slashing claws on their hands and feet, these fierce predators were not dinosaurs you'd want to run into in a dark alley!The Jurassic was also when some of the first birds started emerging, evolving from small feathered dinosaurs. Recent fossil discoveries have shown plumes and feathers covering the bodies of many theropods, providing evidence that birds are indeed direct descendents of dinosaurs.Moving into the Cretaceous period from 145-66 million years ago, the diversity of dinosaurs exploded even further with weirder and wackier varieties than ever before. Who could imagine a burrowing mammal-like dinosaur covered in armorwith a parrot-like beak (Edmontonia)? Or a crested "dragon" that could blast out super-heated air (Tsintaosaurus)?Of course, this was also the time of the iconic Tyrannosaurus Rex, which lived towards the very end of the Cretaceous as the last of the giant theropods. At over 40 feet long and 10 tons, with an enormous head and teeth like bananas, this apex predator was basically a real-life monster ripped straight from our nightmares!But the Cretaceous is also famous for the duck-billed hadrosaurs with their batteries of tightly-packed teeth, allowing them to cut through the toughest plants. Dinosaurs like Maiasaura even exhibited some of the earliest known parenting behaviors, including herding young into protected nesting areas.So how did this incredible reign of the dinosaurs finally meet its catastrophic end? The most widely-accepted theory is that around 66 million years ago, a gigantic asteroid or comet struck the earth in what is now the Yucatan Peninsula in Mexico. This cataclysmic impact kicked up billions of tons of dust and ash into the atmosphere, blocking out the sun for years and causing a severe global winter.Vegetation died off first, followed by the plant-eaters, and then the large predators that depended on them. Even animalsthat managed to initially survive experienced disruptions to their food chains, making it impossible for most species to endure over time. Within a few million years, dinosaurs had vanished entirely from the face of the planet.It's hard to comprehend just how dominant and magnificent the dinosaurs were for over 160 million years of prehistory. From the earliest proto-dinosaurs, to the gigantic long-necked giants, to the badass theropod carnivores, to the final bizarre varieties right before their extinction, dinosaurs thrived and adapted to pretty much every environment our planet threw at them.And yet they were wiped out almost instantly by a random stroke of bad cosmic luck. In a brutal act of natural selection, these glorious rulers of the earth eventually made way for smaller warm-blooded mammals to take over and radiate into vast new species, including our own human ancestors.While dinosaurs no longer walk among us, fossil evidence proves they were the ancestors to modern birds. So in a way, dinosaurs did manage to survive by evolving into a new form of feathered, ancestors to everything from tiny hummingbirds to towering ostriches. All birds today are literally dinosaurs reborn!Well that's the broad overview of dinosaur evolution, spanning from their misunderstood origins to their suddendemise by doomsday asteroid. I hope this tour helped bring the ancient world of our planet's past to life in a way you'll never forget. Discovering dinosaurs has a way of captivating kids and sparking our imaginations about the wonders of deep time and life's resilient progression through eons of changes.Thanks for joining me today and letting me share my passion for paleontology! If you want to learn more, be sure to check out our museum exhibits on feathered dinosaurs, dinosaur eggs and nests, and of course the ever-popular Tyrannosaurus Rex fossil display. You'll come away with a profound respect for how these magnificent creatures once dominated the earth as the undisputed ruling raptors of the Mesozoic Era. Until next time, happy dino trails!篇2Hi there! My name is Jamie and I'll be your guide today as we explore the amazing evolution of dinosaurs. Get ready to travel back in millions of years and discover how these incredible creatures emerged, diversified, and ultimately met their demise. It's going to be an adventure through time that you won't want to miss!Our journey begins over 230 million years ago during the Triassic period. This was a pivotal time when the very first dinosaurs appeared on Earth. Now, you might be thinking - didn't dinosaurs live with cavemen? Not quite! Dinosaurs actually preceded humans by a staggering 230 million years.The earliest dinosaurs were quite small, only about 1 meter long. They descended from a reptile lineage and are believed to have originated in modern-day South America. One of the earliest known dinosaurs was Eoraptor, which means "dawn raptor." It walked on two legs and had characteristics similar to later theropods like Tyrannosaurus Rex.As the Triassic period progressed, these small dinosaurs began to diversify into two main groups - ornithischians and saurischians. Ornithischians, like Stegosaurus and Triceratops, were herbivores and often displayed unique features like bony plates or horns. The saurischians split into the huge long-necked sauropods like Brachiosaurus, and the mostly carnivorous theropods such as Velociraptor.After the Triassic mass extinction around 201 million years ago, dinosaurs really had their time to thrive in the Jurassic period that followed. This is probably the era you're most familiar with - it was the zahey age when ginormous dinosaurslike Diplodocus and Brachiosaurus munched on vegetation, while fierce predators like Allosaurus hunted them down.One of the most iconic dinosaurs from this period has to be Stegosaurus. Can you imagine seeing these tanks roaming around with their huge dorsal plates and spiked tails? They used these features to defend themselves from fearsome predators. Speaking of which, allow me to introduce Allosaurus - one of the earliest forms of a giant theropod dinosaur. Measuring up to 12 meters long, this beast was essentially a scaly ancestor to Tyrannosaurus Rex!As we transition into the Cretaceous period around 145 million years ago, dinosaurs just kept getting bigger, weirder, and more diverse. This was the era of the duck-billed hadrosaurs like Parasaurolophus with its bizarre hollow cranial crest. Can you imagine hearing highpitched dinosaur calls echoing through the forests? Absolutely mind-blowing!The Cretaceous was also dominated by the iconic tyrant lizard Tyrannosaurus Rex itself - probably the most famous dinosaur of all. Just picture a 6 ton beast over 12 meters long with teeth as big as bananas and jaws that could crush concrete. I don't know about you, but I'm glad we'll never have to encounter one of those in real life!Of course, all good things must come to an end. Around 66 million years ago, a gigantic asteroid slammed into what is now the Yucatan Peninsula in Mexico. This cataclysmic event triggered extreme climate changes that made it impossible for dinosaurs to survive. Most of them perished, although some dinosaur lineages actually survived and evolved into the birds we see today.Can you believe that birds like sparrows and eagles are technically direct descendants of feathered dinosaurs like Velociraptor? It's pretty amazing to think that dinosaurs still live among us in the form of our feathered friends. The next time you see a bird, remember that you're actually looking at a pint-sized dinosaur!So there you have it - the epic journey of the dinosaurs from their humble beginnings over 230 million years ago to their eventual demise after reigning for nearly 180 million years. They dominated the planet for longer than mammals have even existed! Isn't that just mind-boggling?Although dinosaurs may be extinct, they've left behind an incredible fossil legacy that has allowed scientists to reconstruct their evolution in incredible detail. Who knows - maybe we'llrevive them through DNA someday and get the chance to see living, breathing dinosaurs again! One can dream, right?Well, that's all for our dinosaur journey today. I hope you've enjoyed reliving the rise and fall of these magnificent prehistoric beasts as much as I have. Let me know if you have any other questions - I may be just a kid, but I'm basically a dinosaur expert at this point! Thanks for joining me and happy trails!篇3The Evolution of the Dinosaurs: A Student Tour GuideHello there! My name is Jamie and I'll be your student tour guide today at the Museum of Natural History. We're going to be exploring the fascinating world of dinosaurs and learning all about their incredible evolutionary journey over millions of years. Put on your walking shoes and let's dive into the prehistoric past!Our adventure begins in the late Triassic period, around 230 million years ago. This is when we find the earliest dinosaur fossils. These first dinosaurs were relatively small, about the size of a large dog. One of the earliest known species is Eoraptor, which means "dawn raptor." With its long tail, grasping hands, and large claws on its feet, Eoraptor was likely an agile predator that hunted small animals and insects.From these small beginnings, dinosaurs rapidly evolved into a hugely successful group of animals that dominated the land for over 165 million years. They diversified into a mind-boggling array of shapes and sizes, ranging from the gigantic long-necked plant-eaters to fearsome meat-eaters with massive jaws of serrated teeth.In the Jurassic period, from about 200 to 145 million years ago, we see some of the most iconic dinosaurs take the stage. The Jurassic was the golden age of the sauropods - those massive long-necked plant-eaters like Diplodocus and Brachiosaurus. Can you imagine a creature weighing up to 80 tons and stretching as long as three school buses? It's no wonder the sauropods are among the largest land animals ever!While the sauropods munched on leaves high in the trees, other dinosaurs stalked them as prey. The Jurassic marked the rise of the theropods, the group that includes both modern birds and the meat-eating dinosaurs like Allosaurus and Ceratosaurus. These fierce predators were packed with blade-like teeth and walked on two powerful legs, using a stiff tail for balance while hunting. Some theropods like Dilophosaurus even had bizarre head crests which may have been used for display.As we move into the Cretaceous period, from 145 to 66 million years ago, the dinosaurs reached the apex of their diversity. Iconic creatures like Triceratops, with its massive bony frill and horns, roamed the land alongside duck-billed dinosaurs like Edmontosaurus. These were some of the last great herds of dinosaurs. Soaring overhead were primitive feathered dinosaurs, the ancestors of modern birds, like the crow-sized Archaeopteryx.And who could forget the tyrannosaurs - the last and most fearsome predators of the dinosaur realm? The one and only Tyrannosaurus rex was basically a cross between a muscle car and a monster truck. With banana-sized teeth, powerful jaws, and a thickset body built for hunting, T. rex was the undisputed king at the end of the Cretaceous.But then, around 66 million years ago, the reign of the dinosaurs came crashing to an end. We still don't know the exact cause, but many scientists believe an asteroid or comet impact played a major role. This cataclysmic event triggered climate changes, volcanic eruptions, and ecological collapse that proved too much for the dinosaurs to overcome. Within a few million years, these magnificent creatures that had lorded over the land for eons vanished into extinction.All except for one incredible lineage of feathered theropod dinosaurs. As the world around them changed, these small, warm-blooded dinosaurs adapted to survive and radiate into thousands of new species we call birds. From mighty T. rex to the little finch at your feeder, birds are dinosaurs' living legacy!So as we walk through the exhibits, be sure to look for the amazing evolutionary transitions on display. You'll see fossils that capture the shift from the earliest dinosaurs to the giants of the Jurassic and then into the mind-bending variety we had by the end of the Cretaceous. Pay close attention to details in the skeletons - like the different styles of hips, skulls, teeth, and limbs - which reveal how the major dinosaur groups are related through descent from shared ancestors.Dinosaurs dominated because they were super successful at evolving new body types to adapt to changing environments over their incredible reign. Through millions of years of branching evolution driven by natural selection, dinosaurs gave rise to an astonishing diversity unlike anything the world had ever seen.I hope this whirlwind tour has helped you appreciate just how amazing and bizarre the dinosaurs were - from their origins to their impact on modern life. If dinosaurs have taught usanything, it's that life on Earth is an endless product of evolution, always changing, adapting, and pushing the boundaries of what's possible over vast stretches of deep time.Thanks for joining me! Let me know if you have any other questions as we continue to explore the prehistoric world. Who's ready to see a real T. rex skeleton?。
pinesap音译
pinesap音译English Answer:A Quick Introduction to Pinesap.Pinesap is a genus of parasitic plants, and its members are commonly known as pinesaps. They are found in the understory of coniferous or hardwood forests and are often associated with beech trees. Pinesaps are parasitic on mycorrhizal fungi, primarily those associated with beech, oak, and pine trees. As such, they lack chlorophyll and obtain their nutrients from their host fungi. Their stems are typically pale and fleshy, with reddish or purplishtips that bear flowers. Pinesaps have small, scale-like leaves that are often reduced to bracts.Pinesap species are distributed globally, with the majority of species occurring in temperate regions of North America and Asia. There are approximately 15 recognized species of pinesap, with the most common species beingEastern pinesap (Pinesap squarrosa) and Western pinesap (Pinesap uniflora).Fascinating Facts about Pinesap.1. Parasitic Nature: Pinesaps are obligate parasites, meaning they cannot survive without their host fungi. They penetrate the fungal hyphae with specialized structures called haustoria, which allow them to absorb nutrients from the fungi.2. Lack of Chlorophyll: Pinesaps lack chlorophyll, the green pigment that enables photosynthesis in plants. Instead, they rely on their host fungi for nutrition.3. Pale and Fleshy Stems: Pinesaps have pale and fleshy stems that often appear translucent or whitish. The stems are often covered with reddish or purplish tips.4. Small, Scale-like Leaves: Pinesaps have small,scale-like leaves that are often reduced to bracts. These leaves are typically arranged in a spiral pattern along thestem.5. Inconspicuous Flowers: Pinesaps produce small, inconspicuous flowers that are often white or tinged with pink. The flowers are borne on the upper portion of the stem and are often hidden by the surrounding vegetation.Pinesap in the Environment.1. Habitat: Pinesaps are typically found in the understory of coniferous or hardwood forests. They are often associated with beech trees, as they primarily parasitize fungi associated with these trees.2. Ecological Role: Pinesaps play a minor ecological role in the forest ecosystem. They do not contribute significantly to the food web and are not known to have any major interactions with other organisms.Conclusion.Pinesap is a fascinating genus of parasitic plants thatrelies on mycorrhizal fungi for survival. Their lack of chlorophyll and reliance on host fungi for nutrients make them unique and intriguing members of the plant kingdom. While they play a minor ecological role, their presence in forest ecosystems adds to the overall biodiversity and complexity of these habitats.Chinese Answer:松叶兰简介。
Alzheimer’s Disease Peptide Epitope Vaccine Reduces Insoluble But Not soluble-Oligomeric
Neurobiology of DiseaseAlzheimer’s Disease Peptide Epitope Vaccine Reduces Insoluble But Not Soluble/Oligomeric ASpecies in Amyloid Precursor Protein Transgenic MiceIrina Petrushina,1*Anahit Ghochikyan,3*Mikayel Mktrichyan,3Gregory Mamikonyan,3Nina Movsesyan,1Hayk Davtyan,3Archita Patel,1Elizabeth Head,1,2David H.Cribbs,1,2‡and Michael G.Agadjanyan1,3‡1The Institute for Brain Aging and Dementia and2Department of Neurology,University of California,Irvine,Irvine,California92697-4540,and3The Institute for Molecular Medicine,Department of Immunology,Huntington Beach,California92647Active vaccination of elderly Alzheimer’s disease(AD)patients with fibrillar amyloid-peptide(A42),even in the presence of a potent Th1adjuvant,induced generally low titers of antibodies in a small fraction(ϳ20%responders)of those that received the AN-1792 vaccine.To improve the immunogenicity and reduce the likelihood of inducing adverse autoreactive T-cells specific for A42,we previously tested in wild-type mice an alternative approach for active immunization:an epitope vaccine that selectively initiate B cell responses toward an immunogenic self-epitope of Ain the absence of anti-AT cell responses.Here,we describe a second generation epitope vaccine composed of two copies of A1–11fused with the promiscuous nonself T cell epitope,PADRE(pan human leukocyte antigen DR-binding peptide)that completely eliminates the autoreactive T cell responses and induces humoral immune responses in amyloid precursor protein transgenic2576mice with pre-existing AD-like pathology.Based on the titers of anti-A1–11antibody exper-imental mice were divided into low,moderate and high responders,and for the first time we report a positive correlation between the concentration of anti-A1–11antibody and a reduction of insoluble,cerebral Aplaques.The reduction of insoluble Adeposition was not associated with adverse events,such as CNS T cell or macrophage infiltration or microhemorrhages.Surprisingly,vaccination did not alter the levels of soluble A.Alternatively,early protective immunization before substantial neuropathology,neuronal loss and cogni-tive deficits have become firmly established may be more beneficial and safer for potential patients,especially if they can be identified in a preclinical stage by the development of antecedent biomarkers of AD.Key words:immunotherapy;Alzheimer’s disease;epitope vaccine;antibody;PADRE;-amyloidIntroductionThe age-related accumulation of amyloid-(A)in the CNS has been hypothesized to play a central role in a cascade of events that eventually induces neuronal loss in affected brain regions in Alz-heimer’s disease(AD)(Selkoe,1991,1994;Hardy and Higgins, 1992;Price and Sisodia,1994;Esler and Wolfe,2001;Hardy and Selkoe,2002).Previously,major support for the amyloid cascade hypothesis emerged from A-immunotherapy studies demon-strated that anti-Aantibodies were capable of reducing AD-like pathology(Schenk et al.,1999)and improving behavior in amy-loid precursor protein(APP)transgenic(Tg)mice(Chen et al., 2000;Janus et al.,2000;Morgan et al.,2000).First,immunother-apy clinical trial in AD patients using the AN-1792vaccine con-taining B and T cell“self epitopes”of A42was halted during Phase IIa,whenϳ6%of the participants developed aseptic me-ningoencephalitis(Schenk,2002;Weiner and Selkoe,2002;Hock et al.,2003;Nicoll et al.,2003;Orgogozo et al.,2003;Ferrer et al., 2004;Bayer et al.,2005;Fox et al.,2005;Gilman et al.,2005; Masliah et al.,2005;Patton et al.,2006).Importantly,only vacci-nated participants(nϭ18)developed meningoencephalitis, whereas none of the control patients(nϭ72)injected with pla-cebo developed adverse events(Orgogozo et al.,2003).Data from these trials suggest that the aseptic meningoencephalitis may have been caused by a T cell-mediated autoimmune response(Nicoll et al.,2003;Ferrer et al.,2004),and that anti-Aantibodies were not responsible for the observed adverse effects after active vac-cination.In fact,low/moderate titers of anti-Aantibodies gen-erated in a small subset of immunized patients(19.7%)were capable of reducing parenchymal amyloid pathology(Nicoll et al.,2003,2006;Ferrer et al.,2004;Masliah et al.,2005;Patton et al.,2006;Boche et al.,2007;Nitsch and Hock,2007)and dimin-ishing progressive cognitive decline associated with the disease (Hock et al.,2003;Gilman et al.,2005).However,ϳ80%of the immunized subjects failed to develop anti-Aantibody titers (“nonresponders”),indicating that the Aself-antigen in theReceived July13,2007;revised Oct.1,2007;accepted Oct.7,2007.This work was supported by National Institutes of Health Grants AG20241and NS50895(D.H.C.)and Alzheimer’sAssociation Grant IIRG-03-6279(M.G.A.).Additional support for mice and the production of peptides was providedby University of California,Irvive Alzheimer’s Disease Research Center Grant P50AG16573.Dr.Nina Movsesyan wassupported by National Institute on Aging training Grant AG00096.We thank Adrine Karapetyan for technical helpand valuable comments.*I.P.and A.G.contributed equally to this work.‡D.H.C.and M.G.A.contributed equally to this work as senior authors.Correspondence should be addressed to Dr.Michael G.Agadjanyan,The Institute for Molecular Medicine,16371Gothard Street,H,Huntington Beach,CA92647-3652.E-mail:magadjanyan@.DOI:10.1523/JNEUROSCI.3201-07.2007Copyright©2007Society for Neuroscience0270-6474/07/2712721-11$15.00/0The Journal of Neuroscience,November14,2007•27(46):12721–12731•12721AN-1792vaccine was not a strong immunogen,thus suggesting that alternative immunotherapeutic strategies should be pursued.Based on the results generated in mouse models of AD(Bard et al.,2000;DeMattos et al.,2001;Dodart et al.,2002),a new clinical trial,AAB-001(Elan and Wyeth Pharmaceuticals,http:// /investorrelations/events/elanwyethsymposium_adpd. asp),has been initiated by using passive transfer of a humanized monoclonal anti-Aantibody(bapineuzumab)in an attempt to avoid the problems associated with active immunization of el-derly AD patients.However,the design of this new trial is associ-ated with additional challenges such as multiple injections of high concentrations of anti-Aantibody every13weeks,the high cost of this monoclonal humanized antibody as well as possible side effects of passive vaccination,including microhemorrhages ob-served in passively immunized very old APP Tg mice(Pfeifer et al.,2002;Wilcock et al.,2004;Racke et al.,2005).This suggests that development of safe active immunotherapeutic strategies may still be desirable.Previously,we engineered and tested a first generation epitope vaccine in wild-type mice(Agadjanyan et al.,2005),and here we report the development and testing the safety and efficacy of therapeutic vaccination of APP Tg2576mice with pre-existing AD-like pathology with a second generation epitope vaccine composed of two copies of the B cell epitope,A1–11in tandem with pan human leukocyte antigen DR-binding peptide(PA-DRE),a synthetic,foreign promiscuous T cell epitope[pre-existing AD-like pathology implies the accumulation of soluble oligomeric forms of amyloid-beta peptide leading to the impair-ment of cognitive functions inՆ6-month-old APP Tg2576mice (Lesne et al.,2006)].Materials and MethodsMice,epitope vaccine,peptide immunogens,and experimental protocol. Aged(ϳ9.4months old)female APP Tg2576mice were bred and pro-vided by the animal facility associated with the University of California at Irvine(UCI)Alzheimer’s Disease Research Center.All animals were housed in a temperature and light-cycle controlled facility,and their care was under the guidelines of the National Institutes of Health and an approved Institutional Animal Care and Use Committee protocol at University of California at Irvine.To engineer an epitope AD vaccine,we synthesized the N terminus of an immunodominant B cell epitope of A1–11(McLaurin et al.,2002; Bard et al.,2003;Cribbs et al.,2003)in tandem with a promiscuous foreign T cell epitope,so called pan-DR epitope,PADRE(Alexander et al.,1994).The peptide2A1–11-PADRE was synthesized as a multiple antigenic peptide(MAP),containing a core matrix of4branching lysines (Tam,1988;Chai et al.,1992)to generate2A1–11-PADRE-MAP(In-vitrogen,Carlsbad,CA).A42peptide was synthesized at the Peptide Core Facility at the Institute for Brain Aging and Dementia at UCI by solid-phase Fmoc amino acid substitution and purified by reverse-phase high-pressure liquid chromatography.Mice were immunized with2A1–11-PADRE-MAP or fA42as de-scribed previously(Cribbs et al.,2003;Petrushina et al.,2003;Agadjan-yan et al.,2005).Briefly,2A1–11-PADRE-MAP(500g/ml)or the fibril-lar(Schenk et al.,1999;Cribbs et al.,2003)A42peptide(500g/ml) were mixed with Quil A,a Th1-type conventional adjuvant,and mice were injected with50g of antigen subcutaneously.Experimental mice at age9.4Ϯ0.9months old were immunized with2A1–11-PADRE-MAP(nϭ21)or fA42(nϭ12),whereas the control group of APP Tg 2576mice(nϭ11)was injected with an adjuvant only.All mice were boosted at monthly intervals.Cellular immune responses were analyzed in five mice from each group killed at9d after the fourth immunization. We continued to boost the remaining mice at monthly intervals and sera was collected at8–10d after the third,fourth,sixth and10th immuniza-tions and used for detection of anti-Aantibodies.At the end of the study,after10vaccinations when animals were19.4Ϯ0.9months old, neuropathological changes were compared across groups in response to treatment.In these animals,cellular immune responses also were analyzed.T cell proliferation.The analysis of T cell proliferation was performed in splenocyte cultures from individual animals,as described previously (Cribbs et al.,2003;Agadjanyan et al.,2005).In addition,CD4ϩT cell proliferation was assessed using FACS assay according to the manufac-turer’s instructions(BD Biosciences,San Jose,CA).Briefly,to detect antigen-specific proliferation of CD4ϩT cells,we stained splenocyte cul-tures by1M succinimidyl ester of carboxyfluorescein diacetate(CFSE; Invitrogen)for10min at37°C.After washing,the cells were incubated for3d in culture media alone or with PADRE(5M).After incubation, the cultures were stained with phycoerythrin(PE)-labeled rat anti-mouse CD4monoclonal antibodies(BD Biosciences).Because dead cells might fluoresce nonspecifically,these cells were excluded from the assay using a nucleic acid dye(7-amino actinomycin D from BD PharMingen, San Diego,CA),and proliferation of viable cells was analyzed by FACS-can flow cytometer(BD Biosciences)as described by the manufacturer. CD4ϩpopulation was separately analyzed using CellQuest software(BD Biosciences).Production of cytokines by immune splenocytes.The same splenocytes used to assess T cell proliferation were used for detection of Th1(IFN␥) or Th2(IL4)lymphokines by ELISPOT(BD PharMingen)as described previously(Cribbs et al.,2003;Agadjanyan et al.,2005).In addition,the FACS method was used for detection of specific cytokines by CD4ϩT cells(Pala et al.,2000).Briefly,the cultures of splenocytes from experi-mental and control animals were restimulated for3–4d with PADRE peptide(5M),and then for4–6h with PMA(phorbol12-myristate 12-acetate)and ionophore(ionomycin)(both from Sigma,St.Louis, MO).In addition,we used Brefeldin A(BD Biosciences)to block cyto-kine secretion,which increases intracellular accumulation.Surface stain-ing was performed using FITC-labeled anti-mouse CD4monoclonal an-tibodies(MoAb;BD PharMingen).Cells were washed,fixed, permeabilized,and CD4ϩT cell subset producing IL-4or IFN␥was de-tected using the appropriate PE-labeled anti-mouse cytokine antibodies (BD PharMingen).Detection of anti-Aantibodies by ELISA.Total anti-A42antibodies were detected as described previously(Cribbs et al.,2003;Petrushina et al.,2003)with small modifications:to develop the color reaction we used the3,3Ј,5,5Јtetramethylbenzidine(TMB)peroxidase substrate(Pierce, Rockford,IL),and plates were analyzed spectrophotometrically at450 nm.The standard curve for determination of anti-Aantibody concen-trations in the sera was based on different known concentrations of monoclonal antibody20.1kindly provided by Dr.Van Nostrand(Stony Brook University,Stony Brook,NY).The concentrations of antibody were recorded in micrograms per milliliter.To determine the specific isotypes,pooled sera from mice were diluted1:2500and tested in dupli-cate.As we reported previously,mice of H2bxs immune haplotype(APP Tg2576)do not express IgG2a,producing IgG2c anti-Aantibodies instead(Petrushina et al.,2003).Therefore,in our experiments we used anti-IgG2a b-specific antibodies(BD PharMingen)along with anti-IgG1-,IgG2b-and IgM-specific antibodies(Zymed,San Francisco,CA). Detection of Aplaques in human brain tissues.Sera from immunized mice were also screened for the ability to bind to Aplaques in the human brain as we described previously,using immunohistochemistry (Ghochikyan et al.,2003;Agadjanyan et al.,2005).A digital camera (Olympus,Tokyo,Japan)was used to capture images of the plaques at 20ϫmagnification.The binding of antisera(dilution1:1000)to the -amyloid plaques was blocked by preabsorption of the sera with5M A1–15peptide(1h,37°C).Immunohistochemistry.To analyze the effect of active immunization with the epitope vaccine on neuropathological changes in APP Tg2576 mice(19.4Ϯ0.9months old),the brains were processed for immuno-histochemistry and histochemistry by previously published methods (Ghochikyan et al.,2003;Agadjanyan et al.,2005).Animals were killed under deep Nembutal sodium solution(150mg/kg,i.p.)anesthesia.To ensure proper fixation and immunostaining of brain tissues,mice were exsanguinated by transcardial perfusion with normal saline.Then brains12722•J.Neurosci.,November14,2007•27(46):12721–12731Petrushina et al.•Testing the Epitope Vaccine in APP Tg2576Micewere removed and bisected in midsagittal plane.The right hemispherewas snap frozen for biochemical analysis,whereas the left hemispherewas fixed in4%paraformaldehyde for immunohistochemical analysis.Forty-micrometer-thick free-floating coronal sections of fixed hemi-brains were collected using a vibratome.To assess the extent of neuropa-thology and neuroinflammation that occurs in the brains of mice,thefollowing primary antibodies were used.Adeposits were detected with anti-A42(dilution,1:2000;Invitrogen)and anti-A40(1:10000;Invitro-gen).Activated microglia were detected with the anti-I-A/I-E[marker ofmajor histocompatibility complex(MHC)II alloantigens;1:200;BDPharMingen]and anti-CD45(1:300;Serotec,Raleigh,NC)antibodies.Astrocytes were labeled with anti-glial fibrillary acidic protein(GFAP;1:3000)antibodies(Eng et al.,2000).Infiltration of T cells and macro-phages were analyzed using anti-CD3-(1:50;Santa Cruz Biotechnol-ogy,Santa Cruz,CA),anti-CD4,anti-CD8(1:250;Novocastra Laborato-ries,Newcastle,UK),and anti-F4/80(1:50;Serotec)antibodies,respectively.The tissues from all animals within a given experimentalgroup were processed in parallel.Sections to be immunostained withanti-Aantibodies were pretreated in90%formic acid for4min to enhance Astaining(Kitamoto et al.,1987).Sections for the staining with anti-CD45and anti-I-A/I-E(anti-MHC II)were pretreated withproteinase K(0.03mg/ml)for5–7min at room temperature.Hydrogenperoxide-quenched and blocked sections were incubated with primaryantibody overnight at4°C.Sections were then washed and incubatedwith appropriate biotinylated secondary antibodies(1h at room temper-ature).After multiple washes,the tissues were incubated in ABC for1h,and color development was performed using DAB(3,3Ј-diaminobenzidine)substrate kit(Vector Laboratories,Burlingame,CA).Sections were mounted on Vectabond-coated slides(Vector Laborato-ries),dehydrated,and covered using DPX(BDH Laboratory Supplies,Poole,UK).Fibrillar Adeposits were visualized using Thioflavin S (ThS)as described by(Schmidt et al.,1995).Briefly,mouse brain sectionswere washed with Tris buffer and stained for10min with a solution of0.5%ThS in50%ethanol.Finally,sections were washed in50%ethanoland Tris buffer,then dried and covered using Vectashield(VectorLaboratories).Quantitative image analysis.NIH imaging was used to analyze the areaoccupied by-amyloid and glial reactivity as described previously(Head et al.,2001).Immunostaining was captured using a Sony(Tokyo,Japan)high-resolution CCD video camera(XC-77)and NIH image1.59b5soft-ware.For every animal,12images(525ϫ410m each)of the frontal parietal region in the cortex at approximately the same plane(0.74to Ϫ2.9mm with respect to bregma)of two adjacent sections were captured with a20ϫor40ϫobjective.The samples included six images from thesuperficial layer and the remaining six from the deep layer.NIH imagingwas used to analyze the area occupied by-amyloid(Aload)relative to the background,and expressed as the percentage of area occupied.The threshold for detection of immunoreactivity was established and then held constant throughout the image analysis.ThS-positive plaques were counted by visual inspection of cortical region of all stained sections although blind with respect to treatment condition;a mean semiquanti-tative score was independently determined for each slide by two observers.Prussian blue staining for microhemorrhage.Staining for hemosiderindeposits was performed on duplicate adjacent coronal sections of themice brains containing similar regions located at approximatelyϪ1.5bregma point,50m thick,collected from immunized and naive,age-matched APP Tg2576mice.The sections were stained with Prussian blue working solution(equal parts of freshly made5%potassium ferrocianide and5%hydrochloric acid)for30min at room temperature,washed in deionized water,and counterstained with Nuclear fast red.Possible hem-orrhage events in the form of the number of Prussian blue-positive pro-files were counted in the brains of each mouse on all sections by two independent observers,and the average number of hemosiderin deposits was calculated per each brain hemisphere.Biochemical analysis.Biochemical analysis of the brain tissue was pro-cessed as described previously(Kawarabayashi et al.,2001),except thatcortices of right hemispheres of brains were used.Briefly,frozen corticeswere thawed,minced and then homogenized in50m M Tris-HCl buffer containing2%SDS,pH8.0,and a mixture of protease inhibitors(MP Biomedicals,Solon,OH).Homogenates were centrifuged(100,000ϫg, 1h,4°C)and supernatants were stored atϪ70°C for additional analysis of soluble-amyloid.Seventy percent formic acid was added to the pel-lets for extraction of SDS-insoluble-amyloid.After sonication samples were centrifuged(100,000ϫg,1h,4°C)and supernatants were stored for analysis of insoluble-amyloid.After neutralization of formic acid with 1.0M Tris-base/0.5M NaH2P04,concentrations of insoluble and soluble A40and A42were analyzed using-amyloid ELISA kits(Invitrogen) according to the manufacturer’s recommendations.Plates were analyzed spectrophotometrically at450nm via a microplate reader,and the con-centrations of A40and A42were calculated using standard curves for A40and A42peptide by comparing the sample’s absorbance with the absorbance of known concentrations of a ing the wet weight of cortex region in the original homogenate,the final values of Awere expressed as micrograms per gram wet weight of cortex.Dot blot assay and combination of IP and WB.Soluble fractions from cortical homogenates of experimental and control mice used in ELISA were also used for dot blot assay and for both immunoprecipitation(IP) and Western blot(WB).Total protein concentration in the homogenates was determined using bicinchoninic acid assay(Pierce)and adjusted to4 mg/ml with PBS.Dot blot.Two microliters of sequential dilutions of homogenates were applied to nitrocellulose membrane(GE Healthcare,Piscataway,NJ), air-dried,and blocked with5%fat-free dry milk in TBST(10m M Tris-HCl,pH8.0,150m M NaCl,0.05%Tween20).Oligomers were detected by using HRP-conjugated A11polyclonal antibody(kindly provided by Dr.C.Glabe,University of California,Irvine,Irvine,CA).Blots were developed with ECL detection system(Santa Cruz Biotechnology).Au-toradiograms were scanned,and densitometry of Aoligomer spots was performed with NIH Image J software,version1.36b.The relative optical density was calculated and presented as average valueϮSD for each group.IP and WB.Aliquots of homogenates were pooled into four groups (based on low,moderate,high anti-Aantibody responders and control nonimmunized mice,200g of total protein in each pooled aliquot), diluted to500l with PBS and incubated(overnight at4°C)with anti-A20.1monoclonal antibody immobilized on protein G-sepharose.The beads were washed three times in PBS,and proteins were eluted in20l of SDS-PAGE loading buffer by boiling.Samples were subjected to elec-trophoresis on12.5%SDS-Tris polyacrylamide gel and proteins were electrotransferred to polyvinylidene difluoride membrane(GE Health-care).The membrane was boiled for2min and blocked with5%fat-free dry milk followed by detection of Aoligomers using anti-Abiotinyl-ated20.1monoclonal antibody.Three experiments with the same ho-mogenates were performed and Western blots were scanned and con-verted into digital files.Densitometry of Aoligomer bands were performed using NIH Image J software,version1.36b and data(aver-ageϮSD)were presented from three experiments.Statistical analysis.All statistical parameters(mean,SD,significant difference,etc.)used in experiments were calculated using Prism3.03 software(GraphPad Software,San Diego,CA).Statistically significant differences were examined using an ANOVA and post hoc comparisons were done using Tukey’s test(pϽ0.05was considered as statistically different).ResultsThe second generation epitope vaccine stimulatesCD4؉IFN␥؉T cells specific to PADRE without activation of autoreactive anti-AT helper cellsTo circumvent the side effects of the AN-1792vaccine against AD,we engineered a vaccine in which a foreign T helper cell epitope was incorporated with two copies of the B cell epitope of A42.APP Tg2576mice vaccinated with2A1–11-PADRE-MAP induced T cell responses directed against the foreign antigenic determinant,PADRE,but not against self Aantigen.More spe-cifically,splenocytes isolated from vaccinated animals prolifer-ated after re-stimulation with PADRE,but not A40(Fig.1A).InPetrushina et al.•Testing the Epitope Vaccine in APP Tg2576Mice J.Neurosci.,November14,2007•27(46):12721–12731•12723contrast,control APP Tg2576mice vacci-nated with fibrillar A42antigen(fA42) that has self B and T cell antigenic deter-minants induced only autoreactive T cells activated after restimulation with A40 (Fig.1A).Direct measurement of activation of PADRE-specific CD4ϩT helper cells in vaccinated APP Tg2576mice by a FACS assay confirmed these results.CD4ϩT cells from mice vaccinated with the pep-tide epitope vaccine induced strong prolif-eration of this subset of T cells after re-stimulation with nonself PADRE peptide, but not A40(Fig.1B).In contrast,vacci-nation with fA42formulated in QuilA ad-juvant induced proliferation of CD4ϩT cells activated after re-stimulation with self-antigen A40,but not with nonself T cell epitope PADRE.Of note,CD4ϩT cells from mice injected with adjuvant did not proliferate after restimulation either with PADRE or A40peptides(Fig.1B).To further investigate T cell responses to vaccination,we analyzed Th1(IFN␥) and Th2(IL-4)cytokine production by splenocytes and CD4ϩT cells from im-munized and control APP Tg2576miceusing ELISPOT and FACS assays,respec-tively(Fig.1C,D).In these experiments,splenocytes from mice vaccinated with epitope vaccine were restimulated with PADRE, whereas splenocytes isolated from animals immunized with fA42were restimulated with A40.Groups of APP Tg2576mice injected with the epitope vaccine induced a strong IFN␥(Th1) response based on the number of splenocytes producing this lym-phokine,whereas mice immunized with fA42had low re-sponses.Because we used a Th1adjuvant in both the epitope and fA42vaccinations,it was not surprising that both groups of mice generated less IL-4(Th2)than IFN␥(Th1),whereas splenocytes from naive mice did not generate either IL4or IFN␥cytokines (Fig.1C).We confirmed these results by measuring the produc-tion of IL-4or IFN␥cytokines in CD4ϩT helper cells.We found that the number of PADRE-specific CD4ϩIFN␥ϩT helper cells in mice immunized with the epitope vaccine was higher than the number of anti-ACD4ϩIFN␥ϩT cells in mice immunized with fA42(Fig.1D).The same was true for IL-4-producing anti-PADRE and anti-ACD4ϩT cells,although both groups had significantly lower number of CD4ϩT cells producing IL-4than IFN␥(Fig.1D).Thus,CD4ϩT cells and splenocytes isolated from both vaccinated groups predominantly produced a Th1-type cytokine,IFN␥,consistent with antigens being formulated in a Th1-type adjuvant,Quil A.Cellular immune responses ana-lyzed after4(Fig.1)and10(data not shown)immunizations showed the same specificity and profile.Collectively,the analyses of T cells demonstrated that the epitope vaccine did not activate autoreactive T cells,but stimulated nonself PADRE-specific CD4ϩIFN␥ϩT lymphocytes.Anti-PADRE-specific CD4؉IFN␥؉T lymphocytes help B cells to produce therapeutically potent anti-A1–11specific antibody in vaccinated APP Tg2576To determine the ability of activated PADRE-specific T cells to stimulate anti-AB cells,we measured antibody concentrations in the sera pooled from each group of animals after three,four, six,and10immunizations.After three injections of the epitope vaccine the concentrations of anti-Aantibodies in the sera were higher than that in mice immunized with fA42.However,after the sixth immunization this difference in anti-Aantibody titers steadily diminished,becoming equal after the last two boosts in both groups(Fig.2A).Of note,mice immunized with the Quil A adjuvant alone did not induce anti-Aantibodies(data not shown).As mentioned above,these data were generated with sera pooled from each of the group;however,in individual animals we found significant variability in anti-Aantibody responses(Fig. 2B).Concentrations of anti-A1–11antibodies after10immuni-zations were high in four mice immunized with the epitope vac-cine(176.8Ϯ163.56g/ml),whereas an additional eight and four vaccinated animals induced moderate(22.2Ϯ14.16g/ml) and low levels(1.2Ϯ1.5g/ml)of anti-A1–11antibodies,re-spectively(Fig.2C–E).This individual variability in humoral im-mune responses was also detected in the group of mice immu-nized with fA42antigen(Fig.2C–E):four mice generated high (82.65Ϯ22.68g/ml),one mouse had moderate(23.3g/ml), and two animals had low concentrations(0.9Ϯ0.7g/ml)of anti-A42antibodies.Thus,the epitope vaccine was at least as effective as fA42in induction of antibodies after10immuniza-tions,but was significantly more effective at initiating the anti-body immune responses(Fig.2A).To characterize the types of humoral immune responses from each vaccination group,we measured the production of IgG1, IgG2a b,IgG2b,and IgM anti-Aantibodies in the sera collected from individual animals after a total of10injections of the epitope vaccine or fA42.All mice vaccinated with the epitope vaccine generated comparable amounts of IgG1,IgG2a b,and as a result,the average of IgG1/IgG2a b ratio was close to1(Fig.2F). This ratio implied that the epitope vaccine formulated in Th1Figure1.The second generation peptide epitope vaccine induces T cell response specific to promiscuous and nonself epitope, PADRE.Splenocyteswereisolatedfromindividualmice(nϭ5)afterfourthimmunizationandrestimulated invitro withPADREorA40peptides.A,Proliferationofsplenocyteswasdetectedby3[H]thymidineincorporation.B,D,ProliferationofCD4ϩTcells(B)and production of cytokines by this T cell subset(D)were detected by flow cytometry in pooled splenocyte cultures.Production ofIFN␥and IL4cytokines by pooled immune splenocytes was detected by ELISPOT assay(C).The experiment was repeated after the10th immunization with similar results.***pϽ0.001.12724•J.Neurosci.,November14,2007•27(46):12721–12731Petrushina et al.•Testing the Epitope Vaccine in APP Tg2576Mice。
介绍白芍的英语作文
介绍白芍的英语作文The Captivating Charm of White PeonyWhite peony, scientifically known as Paeonia lactiflora, is a captivating and versatile flowering plant that has long been revered in many cultures around the world. This perennial herbaceous plant, native to Asia, has a rich history and a diverse array of uses, making it a truly remarkable botanical specimen.One of the most striking features of the white peony is its stunning blooms. The large, delicate flowers can reach up to 8 inches in diameter, with a soft, creamy white color that radiates a sense of purity and elegance. The petals are arranged in a gentle, symmetrical pattern, creating a visually captivating display that has captivated the hearts of gardeners and nature enthusiasts alike.Beyond its aesthetic appeal, the white peony holds significant cultural and medicinal significance. In traditional Chinese medicine, the roots of the plant, known as Bai Shao, have been used for centuries to treat a variety of ailments. The roots are believed to possess anti-inflammatory, analgesic, and sedative properties, making them a valuable ingredient in various herbal remedies.The use of white peony in traditional Chinese medicine can be traced back to ancient times, with records of its medicinal applications dating back to the Han Dynasty (206 BC – 220 AD). Traditional Chinese practitioners have long recognized the plant's ability to nourish the blood, regulate the flow of Qi (vital energy), and soothe the liver and spleen. These properties have made white peony a staple in traditional Chinese pharmacopoeia, with its roots being used to treat conditions such as menstrual cramps, muscle spasms, and even certain neurological disorders.In addition to its medicinal uses, the white peony has also played a significant role in the cultural and artistic traditions of Asia. The flower has been a recurring motif in Chinese and Japanese art, appearing in paintings, ceramics, and textiles. The delicate beauty and symbolic significance of the white peony have made it a cherished subject for artists and poets alike, who have sought to capture the essence of this remarkable plant.One of the most notable examples of the white peony's cultural significance can be found in the traditional Japanese art of flower arrangement, known as Ikebana. In this ancient practice, the white peony is often used as a centerpiece, its graceful form and serene hues serving as a focal point for the entire arrangement. Ikebana practitioners view the white peony as a symbol of purity, simplicity,and harmony, making it a highly sought-after element in their artistic compositions.Beyond its cultural and medicinal uses, the white peony is also a beloved garden plant, prized for its stunning visual appeal and easy-to-grow nature. The plant thrives in well-drained, nutrient-rich soil and prefers full sun to partial shade, making it a versatile choice for a wide range of garden settings. The white peony's large, lush foliage and impressive blooms make it a standout feature in perennial borders, rock gardens, and even as a standalone specimen plant.One of the most remarkable aspects of the white peony is its ability to adapt to a variety of climates. While the plant is native to Asia, it has been successfully cultivated in many other regions around the world, including North America and Europe. This adaptability has contributed to the plant's widespread popularity and has allowed gardeners and horticulturists to enjoy the beauty of the white peony in diverse geographic locations.In conclusion, the white peony is a truly captivating and multifaceted plant that has captivated the hearts and minds of people across the globe. From its stunning visual appeal to its rich cultural and medicinal history, this remarkable botanical specimen continues to inspire and delight those who encounter it. Whether in the garden, in traditional medicine, or in the realm of art and culture, the whitepeony remains a testament to the enduring beauty and versatility of the natural world.。
预防骨质疏松的英文作文
预防骨质疏松的英文作文Title: Preventing Osteoporosis: A Guide to Staying Strong.Osteoporosis, a silent disease that weakens the bones, is a significant health concern affecting millions worldwide. As the saying goes, prevention is better than cure, and this adage holds true for osteoporosis. This article aims to provide a comprehensive guide to preventing osteoporosis, enabling individuals to take proactive measures and maintain strong, healthy bones.Understanding Osteoporosis.Osteoporosis is a condition characterized by the thinning of bone tissue, leading to bones becoming fragile and susceptible to fractures. This condition is often asymptomatic until a fracture occurs, making it crucial to take preventive measures. The risk of osteoporosis increases with age, but it can also affect youngerindividuals due to certain medical conditions, lifestyle choices, and genetics.Maintaining a Healthy Lifestyle.Maintaining a healthy lifestyle is the foundation of preventing osteoporosis. A balanced diet rich in calcium, vitamin D, and other bone-healthy nutrients is essential. Calcium is a key component of bone structure, while vitamin D helps the body absorb calcium. Incorporate calcium-rich foods like dairy products, dark green leafy vegetables, and tofu into your diet. Vitamin D can be obtained through sunlight exposure and foods like fatty fish and fortified milk.Exercise is another crucial aspect of maintaining bone health. Weight-bearing exercises, such as walking, running, and lifting weights, help strengthen bones by putting pressure on them. These exercises stimulate bone-building cells, leading to denser, stronger bones. It is recommended to perform weight-bearing exercises for at least 30 minutes per day, three to five days a week.Avoiding Harmful Habits.Certain habits can increase the risk of osteoporosis, so it's important to avoid them. Smoking and excessive alcohol consumption can weaken bones, increasing the risk of fractures. Quitting smoking and limiting alcohol intake are crucial for maintaining bone health.Additionally, avoiding falls and accidents is essential for preventing fractures. Taking precautions like using handrails, wearing slip-resistant shoes, and maintaining a well-lit home can help reduce the risk of falls.Screening and Monitoring.Regular screening and monitoring are essential for early detection and prevention of osteoporosis. Bone density tests, such as dual-energy X-ray absorptiometry (DEXA), can measure bone mineral density and identify osteoporosis. These tests are recommended for individuals at high risk, such as those with a family history of thedisease or certain medical conditions that increase therisk of osteoporosis.If osteoporosis is diagnosed, it is important to follow the treatment plan recommended by a healthcare provider. Treatment may include medication, lifestyle changes, and physical therapy to manage the condition and reduce therisk of fractures.In conclusion, preventing osteoporosis requires a comprehensive approach that includes maintaining a healthy lifestyle, avoiding harmful habits, and regular screening and monitoring. By taking proactive measures, individuals can reduce their risk of osteoporosis and maintain strong, healthy bones. Remember, prevention is better than cure, so it's essential to prioritize bone health and stay strong.。
Active peptides from skate (Okamejei kenojei) skin gelatin diminish
Active peptides from skate (Okamejei kenojei )skin gelatin diminish angiotensin-I converting enzyme activity and intracellular free radical-mediatedoxidationDai-Hung Ngo a ,BoMi Ryu b ,Se-Kwon Kim a ,c ,⇑aMarine Bioprocess Research Center,Pukyong National University,Busan 608-737,Republic of Korea bSchool of Pharmacy,The University of Queensland,Brisbane,Qld 4072,Australia cDepartment of Chemistry,Pukyong National University,Busan 608-737,Republic of Koreaa r t i c l e i n f o Article history:Received 18January 2013Received in revised form 27May 2013Accepted 16July 2013Available online 25July 2013Keywords:Bioactive peptides ACE inhibition ROSAntioxidative enzymes Human endothelial Skate skina b s t r a c tSkin gelatin of skate (Okamejei kenojei )was hydrolyzed using Alcalase,flavourzyme,Neutrase and prota-mex.It was found that the Alcalase hydrolysate exhibited the highest angiotensin-I converting enzyme (ACE)inhibitory activity.Then,Alcalase hydrolysate was further hydrolyzed with protease and separated by an ultrafiltration membrane system.Finally,two peptides responsible for ACE inhibitory activity were identified to be MVGSAPGVL (829Da)and LGPLGHQ (720Da),with IC 50values of 3.09and 4.22l M,respectively.Moreover,the free radical-scavenging activity of the purified peptides was determined in human endothelial cells.In addition,the antioxidative mechanism of the purified peptides was evaluated by protein and gene expression levels of antioxidant enzymes.The current study demonstrated that the peptides derived from skate skin gelatin could be used in the food industry as functional ingredients with potent antihypertensive and antioxidant benefits.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionSkate is one of the most important fish-catches of the world and it is used entirely for the skate fillet production.However,the skate skin is largely and discarded as waste,which leads to environmen-tal pollution.Recent studies have shown that fish skin provides the best source of gelatin because it has high availability,reduces pol-lution,has no risk of disease transmission,no religious barriers and has the possibility for higher yields of collagen (Senaratne,Park,&Kim,2006).Therefore,there has been an increased need to inves-tigate an efficient means to utilise this by-product.Elevated blood pressure (BP)is considered a major risk factor for cardiovascular diseases,including arteriosclerosis,stroke,and myocardial infarction,and is responsible for most preventable deaths in the world.Amongst processes related to hypertension,angiotensin-I converting enzyme (ACE,EC 3.4.15.1)plays an important role in the physiological regulation of blood pressure.Therefore,inhibition of ACE activity is considered to be an impor-tant therapeutic approach for controlling hypertension (Kearney et al.,2005).Excessive reactive oxygen species (ROS)have emerged as a cen-tral common pathway by which disparate influences may induce and exacerbate hypertension (Kizhakekuttu &Widlansky,2010).ROS are resultant products of oxygen metabolism,which include a broad range of undesirable radicals,including superoxide radi-cals,hydrogen peroxide,hydroxyl radicals,peroxyl radicals,alk-oxyl radicals,and singlet oxygen species.The cells are protected by number of protective mechanisms,including intracellular anti-oxidant enzymes,such as superoxide dismutase (SOD)and gluta-thione (GSH)(Al Ghouleh et al.,2011).Marine peptides have attracted a great deal of attention due to their potential effects in promoting health and reducing disease risk.On the basis of structural properties,amino acid composition and sequences,marine bioactive peptides have been shown to dis-play a wide range of biological functions including antioxidant,anti-hypertensive,antimicrobial,opioid agonistic,immunomodu-latory,prebiotic,mineral binding,anti-thrombotic and hypocho-lesterolemic effects (Betoret,Betoret,Vidal,&Fito,2011).Many natural ACE inhibitory (Table 1)and antioxidant peptides (Table 2)have been isolated from different marine proteins such as cod,pollack,sea bream,yellowtail,clam,krill,mussel,oyster,and shrimp (Kristinsson &Rasco,2000;Ngo,Vo,Ngo,Wijesekara,&Kim,2012).By modulating and improving physiological functions,marine bioactive peptides may provide new therapeutic applica-tions for the prevention or treatment of chronic diseases.The0308-8146/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.foodchem.2013.07.067⇑Corresponding author at:Marine Bioprocess Research Center,Pukyong National University,Busan 608-737,Republic of Korea.Tel.:+82516297094;fax:+82516297099.E-mail address:sknkim@pknu.ac.kr (S.-K.Kim).purpose of this study was to purify and isolate ACE inhibitory and antioxidant peptides from the skin gelatin of skate(Okamejei kenojei).2.Materials and methods2.1.MaterialsSkate(O.kenojei)skin was obtained from Jakalchifish market, Busan,South Korea.Human endothelial cells(EA.hy926cells)were obtained from the American Type Culture Collection(Manassas, VA,USA).Protease from Aspergillus saitoi type XIII,P0.6unit/mg solid(P2143),a-chymotrypsin,ACE(from rabbit lung),hippuryl-histidyl-leucine(HHL)as a substrate of ACE and20,70-dichlorofluo-rescein diacetate(DCFH-DA)were products of Sigma Chemical Co. (St.Louis,MO,USA).Alcalase,Neutrase,protamex andflavourzyme were provided by Novozymes Co.(Bagsvaerd,Denmark).All other reagents used in this study were of the highest grade available commercially.2.2.Gelatin extraction and hydrolysisThe skate skins were initially washed to remove surface slime, cut into small pieces(1Â2cm),and soaked in1%Ca(OH)2with a skin/solution ratio of1:4(w/v).The solution was changed every 24h for3days to remove noncollagenous proteins.Alkaline trea-ted skins were then washed with tap water until neutral or slightly basic pH(pH7–7.5)wash water was obtained.Thoroughly washed fish skins were soaked in distilled water at pH6with a skin/water ratio of1:4(w/v)at60°C for30h with continuous stirring to ex-tract gelatin from the skin matter.The mixture was then centri-fuged at30,000g for10min,the supernatant was immediately collected and freeze-dried using a freeze dryer.The dry matter was referred to as gelatin powder.Gelatin was separately hydro-lyzed with four different enzymes:Alcalase(pH7,50°C),flavour-zyme(pH7,50°C),protamex(pH6,37°C)and Neutrase(pH8, 50°C)in0.1M sodium phosphate buffer.At an enzyme to sub-strate ratio of1/100(w/w),1%substrate and enzyme were mixed. The mixture was incubated for6h with stirring and then heated in a boiling water bath for10min to inactivate the enzymes.After that,hydrolysate was separately hydrolyzed with two enzymes, protease(pH7,37°C)and a-chymotrypsin(pH7,37°C)in0.1M sodium phosphate buffer.At an enzyme to substrate ratio of1/ 100(w/w),1%substrate and enzyme were then mixed.The mix-ture was incubated for6h with stirring and then heated in a boil-ing water bath for10min to inactivate the enzyme.The resultant hydrolysate was fractionated into two ranges of molecular weight (MW,over1kDa and below1kDa)using an ultrafiltration(UF) membrane(GE Healthcare Bio-Sciences Corp,14Walkup Drive, Westborough,MA,01581,USA).All recovered hydrolysates were lyophilized in a freeze dry system and stored atÀ80°C until use.2.3.Purification of bioactive peptidesThe peptide was purified from enzymatic hydrolysates(20mg/ ml)using fast protein liquid chromatography(FPLC,AKTA, Amersham Bioscience Co.,Uppsala,Sweden)on a HiPrep16/10 diethylaminoethyl fastflow(DEAE FF)anion-exchange column (16Â100mm,Amersham Biosciences,Piscataway,NJ,USA).The hydrolysate showing the highest ACE inhibition was loaded (2ml)onto a HiPrep16/10DEAE FF anion exchange column equil-ibrated with a20mM sodium acetate buffer(pH4.0),and eluted with a linear gradient of NaCl(0–2M)in the same buffer at aflow rate of2ml/min.Each fraction was monitored at280nm,collected at a volume of4ml and concentrated using a rotary evaporator. Each fraction was analysed for ACE inhibitory activity and the strongest ACE inhibitory-active fraction was lyophilized and used in the next step.The lyophilized fraction was further purified on a GE Healthcare Superdex™Peptide10/300GL gelfiltration col-umn(10Â300mm)equilibrated with distilled water.The column was eluted with distilled water,and1ml of the fractions was col-lected at aflow rate of1ml/min.The fractions were detected at 280nm and the ACE inhibitory activity was investigated.A strong ACE inhibitory fraction was lyophilized,and chromatography was used as the next step.2.4.Determination of molecular weight and amino acid sequenceAn accurate molecular mass and amino acid sequence of the purified peptides were determined using quadrupole time-of-fightTable1ACE inhibitory peptides derived from marine organisms.Source Enzyme Amino acid sequence IC50(l M)Refs.Alaska pollock Alcalase+GPL 2.6Byun and Kim(2001)PRONASE+CollagenaseAlaska pollock Pepsin FGASTRGA14.7Je,Park,Kwon,and Kim(2004)Sea bream Protease VIY7.5Fahmi et al.(2004)Shrimp Protease IFVPAF 3.4Lun,Lan,Yun,Zhong,and Cheng(2006)Bonito protein IKW0.4Hasan et al.(2007)Salmon muscle Alcalase+papain IW 1.2Enari,Takahashi,Kawarasaki,Tada,and Tatsuta(2008) Shark meat Protease MF0.92Wu et al.(2008)Hard clam Protamex YN51Tsai,Chen,and Pan(2008)Microalgae Pepsin VECYGPNRPQF29.6Sheih,Fang,and Wu(2009)Sea cucumber Bromelain+MEGAQEAQGD15.9Zhao et al.(2009)Alcalase+ProteaseRotifer Alcalase DDTGHDFEDTGEAM9.64Lee,Hong,Jeon,Kim,and Byun(2009)Tuna frame Pepsin GDLGKTTTVSNWSPPKYKDTP11.28Lee et al.(2010)Bonito bowels Autolysis IRPVQ 1.4Hideaki et al.(1993)Sardine muscle Alcalase KW 1.63Matsufuji et al.(1994)Bonito muscle Thermolysin LKP0.32Fujita and Yoshikawa(1999)Wakame Pepsin YNKL21Suetsuna and Nakano(2000)Protease IW 1.5Sato et al.(2002)Tilapia Alcalase DPALATEPDPMPF Vo,Ngo,Kim,Ryu,and Kim(2011)Salmon Alcalase FNVPLYE7.72Ahn,Jeon,Kim,and Je(2012)Pacific cod Gastrointestinal endopeptidases LLMLDNDLPP35.7Himaya,Ngo,Ryu,and Kim(2012)IC50value is the peptide concentration needed to inhibit50%ACE activity.D.-H.Ngo et al./Food Chemistry143(2014)246–255247mass spectrometry(Q-TOF MS)(Micromass,Altrincham,UK),cou-pled with an electrospray ionisation(ESI)source.The purified pep-tide was separately infused into the electrospray source after being dissolved in methanol/water(1:1,v/v),and its molecular mass was determined by the doubly charged(M+2H)2+state in the mass spectrum.Following molecular mass determination,the peptide was automatically selected for fragmentation,and sequence infor-mation was obtained by tandem mass spectroscopy analysis.2.5.Assay of ACE inhibitory activityACE inhibitory activity was performed using the method of Cushman and Cheung(1971)with slight modifications.Briefly,a sample solution(50l l)with50l l of ACE solution(25munits/ ml)was incubated at37°C for10min,and the mixture was pre-incubated with150l l of substrate(8.3mM HHL in a50mM so-dium borate buffer containing0.5M NaCl at pH8.3)at37°C for 30min.Finally,the reaction was stopped by the addition of1M HCl(250l l)and the resulting hippuric acid was extracted by the addition of500l l ethyl acetate.After centrifugation(3000rpm, 10min),200l l of the upper layer was transferred into a glass tube and dried at60°C for30min.The hippuric acid was redissolved in 1ml of distilled water and the absorbance was measured at 228nm using afluorescence micro-plate reader(Tecan Austria GmbH,Grodig/Salzburg,Austria).The IC50value is defined as the concentration of inhibitor required to inhibit50%of the ACE activity.2.6.Cell culture and cytotoxicity determinationEA.hy926cells were grown in Dulbecco’s Modified Eagle Med-ium(DMEM)medium containing10%(v/v)FBS,100l g/ml penicil-lin-streptomycin and5%CO2at37°C.Cytotoxicity levels of the samples on cells were measured using the MTT method as de-scribed by Vo,Ngo,and Kim(2012).2.7.Determination of ROS production byfluorescence-activated cell sorting(FACS)analysisIntracellular formation of ROS was assessed according to a pre-vious method,by employing oxidation sensitive dye20,70-dichloro-fluorescein diacetate(DCFH-DA)as the substrate(Ngo et al., 2011b).Intracellular oxidative stress was assayed by measuring the intracellular oxidation of DCFH(Rosenkranz et al.,1992).EA. hy926cells were treated with the samples for12h and then incu-bated with20l M DCFH-DA for1h at37°C.Subsequently,the cells were washed,harvested,and re-suspended into PBS buffer con-taining H2O2(500l M)for1h.Finally,the cells were analysed by flow cytometry(BD Diagnostic Systems,Cockeysville,MD,USA). The numbers in histograms indicate the percentage of DCF forma-tion due to oxidation of DCFH in the presence of several ROS. 10,000events/sample were acquired using XL-MCL™flow cytom-eter equipped with EXPO™32software(Beckman Coulter,Inc.,CA, USA).The phase contrast images of stimulated EA.hy926cells were collected using Leica inverted microscopy(DM IRB)with a digital CCD camera(CTR6000,Leica,Wetzlar,Germany)underfluores-cence.Intracellular ROS was determined by DCFH-DAfluorescent staining.EA.hy926cells were treated with different concentrations of the samples for12h and incubated with20l M DCFH-DA for1h in the dark.Subsequently,the cells were washed and stimulated with H2O2(500l M)for1h.Fluorescence images of the specimens were captured with a Leica system.2.8.Reverse transcriptase-polymerase chain reaction(RT-PCR)RT-PCR was performed to detect the mRNA expression levels of SOD and GSH.Total RNA was extracted from EA.hy926cells after treatment with different concentrations of the samples for12h fol-lowed by Ang II(1l M)treatment for24h.The cells were lysed with TrizolÒand centrifuged at12,000rpm for15min at25°C fol-lowing the addition of chloroform(5:1).The supernatant was sep-arated and isopropanol was added at a1:1ratio.By centrifugationTable2Antioxidant peptides derived from marine sources.Source Amino acid sequence Methods Refs.Microalgae MPGPLSPL DPPHÅ,ÅOH and OÅÀ2radical scavenging(ESR)Ryu,Kang,Ngo,Qian,and Kim(2012) Microalgae VECYGPNRPQF DPPHÅ,ÅOH,OÅÀ2,peroxyl and ABTS radicalscavengingSheih,Fang,Wu,and Lin(2010)Blue mussel FGHPY LAPS andÅOH radical scavenging(ESR)Jung,Rajapakse,and Kim(2005)Rotifer LLGPGLTNHA andDLGLGLPGAHDPPHÅradical scavenging Byun,Lee,Park,Jeon,and Kim(2009)Horse mackerel NHRYDR DPPHÅandÅOH radical scavenging(ESR)Sampath Kumar,Nazeer,and Jaiganesh(2012) Jellyfish NLDTPYCFYSGDYGG OÅÀ2radical scavenging Li et al.(2012)Bullfrog skin LEELEEELEGCE DPPHÅ,ÅOH,OÅÀ2and peroxyl radical scavenging(ESR)Qian,Jung,and Kim(2008) Scalloped hammerhead WDR,PYFNK ABTSÅ,DPPHÅ,ÅOH and OÅÀ2radical scavenging Wang,Li,Chi,Zhang,and Luo(2012) Tuna LPTSEAAKY and PMDYMVT DPPHÅand ferric thiocyanate method Hsu(2010)Pacific cod TCSP and TGGGNV DPPHÅandÅOH radical scavenging(ESR)Ngo et al.(2011a)Tilapia DPALATEPDPMPF DPPHÅ,ÅOH and OÅÀ2radical scavenging(ESR)Ngo,Qian,Ryu,Park,and Kim(2010) Marine algae LNGDVW DPPHÅ,ÅOH and peroxyl radical scavenging(ESR)Ko,Kim,and Jeon(2012)Hoki ESTVPERTHPACPDFN DPPHÅ,ÅOH,OÅÀ2and peroxyl radical scavenging(ESR)Kim,Je,and Kim(2007)Croaker muscle KTFCGRH DPPHÅ,ÅOH radical scavenging,Wistar rat Nazeer,Sampath Kumar,and Jai Ganesh(2012) Fish protein Spontaneously hypertensive rats Boukortt et al.(2004)Marine shrimp Rats Fahmy and Hamdi(2011)Herring(Clupea harengus)Human Lindqvist,Langkilde,Undeland,and Sandberg(2009)Pacific whitingfish Human Marchbank et al.(2008) Cod and salmon Human Parra et al.(2007)ABTS(2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).Linoleic acid peroxidation system(LAPS).2,2-Diphenyl-1-picrylhydrazyl(DPPH),superoxide anion(OÅÀ2),hydroxyl(ÅOH)and carbon-centred(RÅ)radicals.Electron spin resonance(ESR).248 D.-H.Ngo et al./Food Chemistry143(2014)246–255for10min at10,000rpm,the RNA pellet was obtained.After wash-ing with ethanol,extracted RNA was solubilized in diethyl pyrocar-bonate(DEPC)-treated RNase-free water and the amount of RNA was quantified by measuring the absorbance at260nm using a GENiosÒmicroplate reader(Tecan Austria GmbH).RNA(2l g) was then reverse transcribed into cDNA using a master-mix con-taining1Âreverse transcriptase(RT)buffer,1mM dNTPs,500ng of oligo(dT)15primers,140U of murine Moloney leukaemia virus (MMLV)reverse transcriptase and40U of RNase inhibitor,for 45min at42°C.Polymerase chain reaction(PCR)was carried out in an automatic Whatman thermocycler(Biometra,Kent,UK)to amplify SOD,GSH and glyceraldehyde-3-phosphate dehydroge-nase(GAPDH)mRNA as the gene control.Each transcript was iden-tified using specific forward and reverse primers as manufacturer’s instructions(Promega,Madison,WI,USA).The conditions for the amplification cycles were95°C for30s,55°C for30s,and72°C for1min for35cycles.After polymerase chain reaction products were electrophoresed on1.5%agarose gels in1ÂTAE buffer for 30min at100V,they were visualised by ethidium bromide stain-ing using AlphaEaseÒgel image-analysis software(Alpha Innotech, San Leandro,CA,USA).Primer sequences used to amplify the de-sired cDNA were as follows:SOD forward and reverse primers: 50-AGG-GCA-TCA-TCA-ATT-TCG-AG-30and50-TGC-CTC-TCT-TCA-TCC-TTT-GG-30;GSH forward and reverse primers:50-AGC-ATT-TGG-CAA-AGG-AGA-AA-30and50-ATC-CGT-GCT-CCG-ACA-AAT-AG-30;GAPDH forward and reverse primers:50-GAG-TCA-ACG-GAT-TTG-GTC-GT-30and50-GAC-AAG-CTT-CCC-GTT-CTC-AG-30. 2.9.Western blot analysisStandard procedures were used for the Western blotting,in brief;EA.hy926cells were lysed in RIPA(Radio-Immunoprecipita-tion Assay,150mM NaCl,1%IGEPALÒCA-630,0.5%sodium deoxy-cholate,0.1%SDS and50mM Tris,pH8.0)lysis buffer(NP-40, Sigma–Aldrich,USA).Remaining cell debris was removed by cen-trifugation.The protein concentrations of the cell lysates were determined with the Bio-Rad protein assay(Hercules,CA,USA), using bovine serum albumin(BSA)as the standard.Cell lysates containing equal amount of proteins(%20l g of total protein)were diluted in5Âsample buffer[10%sodium dodecyl sulfate(SDS)and 100mM each DTT,glycerol,bromophenol blue and Tris–HCl],sep-arated by10%SDS–polyacrylamide gel electrophoresis(SDS–PAGE) and transferred onto a nitrocellulose membrane.The membranes were blocked with5%(w/v)BSA in TBST(25mM Tris–HCl, 137mM NaCl and0.1%Tween20,pH7.4)for at least1h at4°C. Membranes were incubated with primary antibodies(1:1000)for 1h at4°C.After washing with TBST,the blots were incubated with secondary antibodies(1:5000)for1h at4°C in the dark.They were then washed again three times with TBST,and developed with an enhanced electrochemiluminescence(ECL)assay kit(Amersham Pharmacia Biosciences,UK)according to the manufacturer’s instructions.Western blot bands were visualised using LAS3000ÒLuminescent image analyzer and protein expression levels were quantified by Multi Gauge V3.0software(Fujifilm Life Science,To-kyo,Japan).Detection of b-actin was used as a control for the equal loading of protein.2.10.Statistical analysisAll statistical analysis was performed with independent exper-iments and values are expressed as mean±standard deviation of three independent determinations.The significance of differences between two samples was analysed using the Student’s t-test and a p-value of<0.05was considered to be significant.3.Results3.1.Screening of the gelatin hydrolysates for antihypertensive and antioxidative peptidesEnzymatic hydrolysis is widely used in the food industry,and this technique does not adopt any toxic chemicals compared to sol-vent extraction.Therefore,gelatin was separately hydrolyzed using industrial food-grade proteinases such as Alcalase,Neutrase,fla-vourzyme and protamex to obtain gelatin hydrolysates with vari-ous bioactivities.Among the hydrolysates tested,Alcalase hydrolysate showed the highest ACE inhibitory activity,of72.8% at2mg/ml(Fig.1A).Several studies have reported that Alcalase hydrolysates derived from food proteins showed potent bioactivi-ties,such as antioxidant and ACE inhibitory activities.These bioac-tivities are attributed to the ability of Alcalase to produce various bioactive peptides because of its endo-peptidase properties.Alca-lase produces shorter peptide sequences as well as terminal amino acid sequences responsible for various health beneficial bioactivi-ties,including ACE inhibition(Hyun&Shin,2000).Moreover,Alca-lase affords the highest ACE inhibitory activity in the production of antihypertensive peptides when compared with other specific (pepsin and trypsin)and non-specific(pronase E)proteases(Qian, Jung,Lee,Byun,&Kim,2007).Previous studies have reported that Alcalase is capable of producing bioactive peptides when it is used to hydrolyze food proteins(Li,Wan,Le,&Shi,2006).The potent Alcalase hydrolysate was further hydrolysed using protease and a-chymotrypsin,as mentioned earlier.The obtained hydrolysates were tested for their potential to inhibit ACE.At1mg/ml concen-tration,protease-hydrolysate showed the highest ACE inhibition of86%(Fig.1B).The protease-hydrolysate was chosen to purify and isolate ACE inhibitory peptides.To determinate the appropriate time for hydrolysis,Alcalase hydrolysate was hydrolyzed with protease(pH7,37°C)at 30min,1,2,4,6and10h.The results show that protease-hydroly-sate showed the highest ACE inhibitory activity at6h(Fig.1C).The protease hydrolysate treatment resulted in a dose-dependent inhi-bition of ACE activity,accounting for85%inhibition at the highest concentration(Fig.1D).Protease hydrolysate was separated using an UF membrane(1kDa MWCO membrane)according to molecu-lar size.Among hydrolysates,the protease hydrolysate below 1kDa exhibited the highest ACE inhibitory activity compared to other hydrolysates(Fig.1E).3.2.Identification of the amino acid sequence and molecular weight of the peptidesThe lyophilized protease-hydrolysate was dissolved in20mM sodium acetate buffer(pH4),and loaded onto a Hiprep16/10DEAE FF anion exchange column with a linear gradient of NaCl(0–2M). All eluents were monitored at215nm,and then fractionated into one non-adsorptive portion and two adsorptive portions.Each frac-tion was pooled,lyophilized,and measured for ACE inhibitory activity.Fraction1(F-I)was found to possess the highest ACE inhi-bition,about73%at100l g/ml(Fig.2A).This fraction was lyophi-lized and separated by a GE Healthcare Superdex™Peptide10/300 GL gelfiltration column(10Â300mm),and was divided into four further fractions.Fraction3(FI-3)showed the most potent ACE inhibitory activity about85%at100l g/ml(Fig.2B).The highest ac-tive fraction,3,was further purified to obtain the active peptides.The peptides were analysed by Q-TOF ESI mass spectroscopy to elucidate the constituent amino acid sequences.The amino acid se-quences of the peptides were identified as MVGSAPGVL(829Da; SP1)(Fig.2C)and LGPLGHQ(720Da;SP2)(Fig.2D).The observed molecular mass was in agreement with the calculated molecularD.-H.Ngo et al./Food Chemistry143(2014)246–255249mass of the ing these obtained molecular weights (829Da =829g/mol and 720Da =720g/mol),the concentration of the peptides was calculated in micromoles for further experi-ments.SP1and SP2showed the potent ACE inhibition with the IC 50values of 3.09and 4.22l M,respectively.3.3.The effect of the purified peptides on human umbilical vein endothelial cell viabilityThe purified peptides were tested for their effect on the viability of EA.hy926cells by employing the MTT assay.The purified pep-tides did not show any significant toxicity towards either cell line at the tested concentrations (Fig.3).Therefore,it is confirmed that the purified peptides are safe materials in terms of cellular toxicity and thereby could be used as safe inhibitors of ACE with no cyto-toxicity to EA.hy926cells and as effective antioxidants,having no effect on the cell proliferation.Based on these results,subsequent cellular experiments were restricted to the determined nontoxic concentrations.3.4.Intracellular radical-scavenging effects of the purified peptides in human umbilical vein endothelial cellsThe unregulated production of ROS leads to damage of cellular macromolecules.In order to investigate the intracellular radical-scavenging effects of the purified peptides,EA.hy926cells were la-belled with a fluorecence probe (DCFH-DA).The non-fluorescent DCFH-DA freely penetrated into the cells and was hydrolysed by intracellular esterases to DCFH,which gets trapped inside the cells.Intracellular ROS oxidises DCFH into fluorescing DCF.This emitting fluorescence was measured in 30min increments up to 3h.It is clearly seen that SP1(Fig.4A)and SP2(Fig.4B)treatment de-creased the DCF-fluorescence intensity even at the lowest concen-tration tested,compared to the H 2O 2-only treated control group,confirming the intracellular radical-scavenging effect of the puri-fied peptides.This data suggest that the purified peptides scav-enged intracellular ROS and thereby protected the cellular macromolecules from ROS-mediated damage.ROS production was also measured using flow cytometry and light microscope analysis.The results obtained from flowcytome-activity of hydrolysates (Alcalase,protamex,flavourzyme and Neutrase)from skin gelatin of skate (O.kenojei ).(B)ACE inhibitory -chymotrypsin and protease),Alcalase below 1kDa and VY (Valine-Tyrosine)as the positive control.(C)ACE inhibitory activity times.(D)ACE inhibitory activity of protease hydrolysate at different concentrations.(E)ACE inhibitory activity of proteaseliquid chromatography of protease hydrolysate below1kDa loaded(2ml)onto a HiPrep™16/10DEAE FF anion-exchange column activity(upper panel).Adsorption fractions were eluted with a linear gradient of0–2M NaCl and the ACE inhibitory activity of exclusion chromatography of hydrolysate loaded(100l l)onto a GE Healthcare Superdex™Peptide10/300GL column(lower (upper panel).Identification of the molecular mass and amino acid sequence of the purified peptides SP1(C)and SP2(D)using Q-TOFthe active peptide was acquired over the m/z range50–2500and sequenced by using the PepSeq denovo sequencing algorithm.try analysis showed that H 2O 2-stimulated cells exhibited high fluo-rescence intensity.However,the ROS production was strongly scavenged by the purified peptides treatments in a dose-depen-dent manner (Fig.4C).The scavenging activity of SP1was found to be stronger than that of SP2at the same concentration treat-ment.Likewise,Fig.4D showed that the fluorescence intensity was high in H 2O 2-stimulated cells.Conversely,the presence of the purified peptides alleviated fluorescence intensity,indicating the scavenging effect of the purified peptides on ROS formation.The results of this experiment indicated that the purified peptides exhibited an inhibitory activity against the elevation of intracellu-lar ROS in the activated cells.3.5.Effect of the purified peptides on the expression of antioxidant enzymes in human umbilical vein endothelial cellsA large number of studies have focused on the pathogenetic sig-nificance of oxidative stress in osteoarthritis,which has been asso-ciated with increases in signs of oxidative stress and lower levels of the body’s natural antioxidants such as SOD and GSH with many diseases.SOD is one of the major antioxidant enzymes because it reduces the damaging reactions of superoxide.Thus,it protects cells from the toxicity of superoxide.GSH plays an important role in regulating the intracellular redox status.The increase in GSH le-vel protects cells against death either by removing free radicals or by conjugating with toxicants (Hsu,2010).In order to investigate the intracellular radical-scavenging activities of the purified peptides on antioxidant enzymes,the mRNA and protein expression levels of antioxidant enzymes (SOD and GSH)were determined by RT-PCR and Western blot anal-ysis in EA.hy926cells.As shown in Fig.4E,all the mRNA and pro-tein expression levels of antioxidant enzymes were up-regulated with the treatment of purified peptides,in a dose-dependent man-ner.The results indicate that the purified peptides could be an effective scavenger of radicals,by increasing the expression levels of antioxidant enzymes in EA.hy926cells.4.DiscussionBioactive peptides usually contain 3–20amino acid residues and low molecular weight peptides are more potent as bioactive peptides than high molecular weight peptides.In this study,skate (O.kenojei )skin gelatin was hydrolyzed to produce bioactive pep-tides with low molecular weight (SP1,829Da and SP2,720Da).SP1contains 9amino acid residues (MVGSAPGVL)and SP2con-tains 7amino acid residues (LGPLGHQ).Regarding the relationship between structure and activity of ACE inhibitory peptides,those peptides had proline,phenylalanine,or tyrosine at the C-terminus and isoleucine and valine at the N-terminal,and showed highly po-tent ACE inhibitory activity.Most of the reported ACE inhibitory peptides are usually short peptides with proline residue at the C-terminal.Proline is known to be resistant to degradation by diges-tive enzymes and may pass from the small intestines into the blood circulation in the sequence of short peptides (Korhonen &Pihlanto,2006;Pan,Luo,&Tanokura,2005).Although,the structure–activity relationship of ACE inhibitory peptides has not yet been fully established,the purified peptides from skate (O.kenojei )skin gela-tin showed some common features.Many recent studies have been published on anti-hypertensive and antioxidant activities of marine peptides and hydrolysates using cellular models,animal and human clinical trials (Tables 1and 2).Anti-hypertensive peptides derived from sea cucumber have shown potent ACE inhibitory activity in spontaneously hyper-tensive rats (SHR)(Zhao et al.,2009).In general,the reduction in systolic blood pressure (SBP)following oral administration (10mg/kg of body weight)of peptides was on average 25mmHg compared to controls (Lee,Qian,&Kim,2010).This anti-hyperten-sive activity was similar with captopril,a commercial anti-hyper-tensive drug.Protein hydrolysates derived from oyster proteins and sea bream scale collagen have also exhibited anti-hypertensive activity in SHR (Fahmi et al.,2004).However,variations in sample type,the dosage and duration of administration make it difficult to compare these hydrolysates in terms of SBP reduction.Boukortt et al.(2004)measured the effects of fish protein on blood pressure,glycemia and antioxidant status in SHR with strep-tozotocin-induced diabetes.After intake of the fish protein,antiox-idative enzyme activities and antioxidant substances were determined in organs (liver,kidney and heart),and vitamin C in plasma.After two months,the antioxidant status had increased significantly compared to the control group.Fish protein has a ben-eficial effect on blood pressure in SHR,and plays an important role in antioxidative defence.This protein may be useful in future treat-ments of such diseases as diabetes and hypertension.Human clinical trials are conducted to confirm both bioavail-ability and function of antioxidants in the body.Results from hu-man in vivo studies are essential before giving dietary recommendations or gaining approval to use health claims in func-tional foods or nutraceutical formulations.Marchbank,Limdi,Mahmood,Elia,and Playford (2008)examined the protective effect of a commercial protein hydrolysate from pacific whiting (Merluc-cius productus )on small intestinal damaging side effects of the non-steroidal anti-inflammatory drug,indomethacin in a pilot clinical trial.The fish protein hydrolysate reduced the degree of small intestinal injury and glutamine constituents in the fish pro-tein hydrolysate may have contributed to antioxidant activity via stimulation of glutathione production.Parra,Bandarra,Kiely,Thorsdottir,and Martinez (2007)investigated the oxidative stress level in individuals subjected to energy-restricted diets that were isocaloric and with the same dietary macronutrient distribution,but with different protein and fat sources.The four diets were fish oil supplemented,salmon-based,cod-based and fish restricted (control).Lean meat was the main protein source in the control and fish oil supplemented diets.In contrast,the cod-based diet sig-nificantly increased plasma total antioxidant status and decreased oxidative stress,measured as plasma malondialdehyde.Many studies reported that oligopeptides (2–10amino acids)are more potent ACE inhibitors and antioxidants than their parent native proteins or polypeptides (10–50amino acids or more)(Erd-mann,Cheung,&Schroder,2008).Peptides derived fromdifferent3.Cell viability determined by the MTT assay.Cytotoxic effects of the purified peptides derived from skin gelatin of skate (O.kenojei )on EA.hy926cells were compared with the peptides of the non-treated group (blank).Values are expressed the means ±SD of three replicate experiments.143(2014)246–255。
生化英汉互译-名词解释
肽peptide 糖异生gluconeogenesis甘油三酯triglyceride 信号肽signal peptide磷脂phospholipid 翻译translation胆固醇cholesterol 遗传密码genetic codon脂蛋白lipoprotein 生物转化biotransformation脂肪酸fatty acid 胆汁酸bile acid乳糜微粒chylomicra CM 蛋白激酶protein kinase PK酮体ketone bodies 结构域domain极低密度脂蛋白very low density lipoprotein 变性denaturation低密度脂蛋白low density lipoprotein 碱基base高密度脂蛋白high density lipoprotein 双螺旋double helix转氨基作用transamination 酶enzyme关键酶key enzyme 核酶ribozyme半保留复制semiconservative replication 酶原zymogen端粒telomere 同工酶isoenzyme逆转录酶reverse transcriptase 变构调节allosteric编码链coding strand 共价修饰调节covalent modification 外显子exon 呼吸链respiratory chain内含子intron 氧化磷酸化oxidative phosphorylation 剪接cleavage splicing 葡萄糖glucose启动子promoter 反密码子anticodon有氧氧化aerobic oxidation 脂类lipids糖酵解glycolysis 柠檬酸循环citrate cycle三羧酸循环tricarboxylic acid cycle 糖原glycogen磷酸戊糖途径pentose phosphate pathway 血糖blood sugar生化名词解释1、结构域：指一些较大的蛋白质分子，其三级结构中具有两个或多个在空间上可明显区别的局部区域。
apoptosis词源
apoptosis词源Apoptosis词源Apoptosis一词源自希腊单词“apo”和“ptosis”，其中“apo”意为“离开”或“分离”，“ptosis”意为“落下”或“掉落”。
因此，apoptosis可以被理解为“离开或掉落”。
在生物学中，apoptosis指的是一种程序性细胞死亡的过程，也被称为细胞凋亡。
细胞凋亡是一种高度有序的细胞死亡方式，它在维持正常生物体内细胞数量和组织结构中起着重要作用。
这种细胞死亡方式是通过一系列复杂的信号传导和分子调控来实现的。
它与细胞生长和发育密切相关，对于维持生物体内细胞的稳态和功能的平衡至关重要。
细胞凋亡的过程包括凋亡信号的传递、凋亡程序的启动和执行以及凋亡细胞的清除。
在凋亡信号传递过程中，细胞内外的多种信号分子参与其中，如细胞因子、生长因子、DNA损伤等。
这些信号可以通过细胞膜上的受体、细胞内的信号转导通路和调节因子来传递和调控。
凋亡程序的启动和执行是通过一系列蛋白质分子的激活和调控来实现的。
其中，凋亡信号经过细胞内的凋亡信号转导通路，最终激活一类特殊的蛋白酶，称为半胱天冬酶家族(caspase)。
这些酶可以分解细胞内的关键蛋白质，如细胞骨架蛋白和DNA修复酶，从而导致细胞的瓦解和死亡。
凋亡细胞的清除是通过其他细胞类型来完成的，如巨噬细胞和其他被称为“吞噬细胞”的细胞。
这些细胞通过识别和吞噬凋亡细胞，将其分解和清除，以维持组织的稳定和功能。
在生物体内，细胞凋亡发挥着多种重要的生理和病理功能。
在发育过程中，细胞凋亡参与形成器官和组织的精细调控。
在免疫系统中，细胞凋亡起着清除异常和受损细胞的作用，以维持免疫系统的正常功能。
在肿瘤发生和发展过程中，细胞凋亡的紊乱往往导致肿瘤细胞的无限增殖和存活。
细胞凋亡还与多种疾病的发生和发展密切相关，如心血管疾病、神经系统疾病和自身免疫性疾病等。
因此，对细胞凋亡的研究不仅可以增加对生命基本过程的理解，还可以为疾病的诊断和治疗提供新的思路和方法。
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Apoptogenic peptides from Tityus discrepans scorpion venom acting against the SKBR3breast cancer cell lineGina D ’Suze a ,*,Arnaldo Rosales a ,Víctor Salazar b ,Carlos Sevcik aa Laboratory on Cellular Neuropharmacology,Biophysics and Biochemistry Center,Instituto Venezolano de Investigaciones Cienti ficas (IVIC)Caracas,Venezuela bHistology Service,Biophysics and Biochemistry Center,Instituto Venezolano de Investigaciones Cienti ficas (IVIC)Caracas,Venezuelaa r t i c l e i n f oArticle history:Received 27May 2010Received in revised form 14September 2010Accepted 22September 2010Available online 1October 2010Keywords:Scorpion venom SKBR3FasLAntineoplastic natural compounds Apoptosis BcL-2a b s t r a c tTwo novel peptides named neopladine 1and neopladine 2were puri fied from Tityus discrepans scorpion venom and found to be active on human breast carcinoma SKBR3cells.Mass spectrometry molecular masses of neopladine 1and 2were 29918and 30388Da,respectively.Their N -terminal sequences were determined by Edman degradation.The peptides induced apoptosis of SKBR3cells but had a negligible effect on non-malignant MA104monkey kidney cells.Neopladine 1and 2induced 6.3and 4.1%of SKBR3apoptosis,respectively,in 5h of exposure;the effect was larger with more prolonged exposures.Inmunohistochemistry showed that neopladines bind to SKBR3cell surface inducing FasL and BcL-2expression.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionSome scorpion venoms inhibit the growth of various types of cancers.Only a few toxins have been found to be the responsible for these anticancer effects and exert their action by three different mechanism:1)blocking a speci fic ion channel (Jäger et al.,2004);2)inhibiting cancer cells invasion binding to a speci fic site (different from an ion channel)on the plasmatic membrane (Deshane et al.,2003);3)activating intracellular pathways inducing apoptosis (Gupta et al.,2010).Apoptosis is a type of pro-grammed cell death,initiated by several factors involving a cascade of intracellular events,leading to the termination of cell survival (Green and Reed,1998).There are two major pathways initiating apoptosis,the extrinsic way mediatedby a death receptor and the intrinsic pathway mediated by mitochondrial damage (Khan et al.,2007).Apoptosis is the underlying mechanism accounting for the action of clini-cally useful antineoplastic drugs such as vinblastine,cisplatin and paclitaxel (Harmon et al.,1994;Villunger et al.,1997;Reshkin et al.,2003).Recent evidence indi-cates that antineoplastic drugs induce a chain of events where MAP kinases,FasL,matrix metalloproteinases,P38,P53or BcL-2are involved in tumor cell death (Müller et al.,1997;Villunger et al.,1997;Deshane et al.,2003;Reshkin et al.,2003;Vousden,2005).Fas ligand (FasL)expression accompanies tumor cell death;the activation of Fas signaling by induction of FasL expression has been proposed as an extrinsic mechanism of apoptosis (Friesen et al.,1996;Müller et al.,1997;Villunger et al.,1997;Khan et al.,2007;Reshkin et al.,2003).Breast cancer is one of the most common malignancies worldwide.Although several novel therapeutic methods have been utilized,curative effects in advanced breast cancer remains poor.Developing new drugs against breast cancer will be essentials for improving therapeutic*Corresponding author.IVIC CBB,Apartado 20632,Caracas 1020A,Venezuela.Tel.:þ582125041225,þ584129217482(Mobile);fax:þ582125041093.E-mail addresses:gina.dsuze@ ,gdsuze@ivic.gob.ve (G.D ’Suze).Contents lists available at ScienceDirectToxiconjournal hom epage:/locate/toxicon0041-0101/$–see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.toxicon.2010.09.008Toxicon 56(2010)1497–1505intervention and prognosis for this malignancy.Scorpion venoms are complex mixtures which may contain over two hundred different peptidic compounds(Batista et al., 2006).These compounds act directly on a broad spectrum of cell membrane ion channels(Possani et al.,1999),and indirectly by activating cellular metabolic pathways,prob-ably,via secondary messengers(López,1999)or by elec-trostatic interaction with the cell membrane(Corzo et al., 2001;Boman,2003).In recent years there have been many studies(Liu et al.,2002;Fu et al.,2007;Mamelak and Jacoby,2007;Gao et al.,2008;Gupta et al.,2010;Lee et al., 2010)aimed at identifying scorpion peptides with anti-neoplastic activity.Scorpion venoms and toxins isolated from Leiurus quinquiestratus,Buthus martensii and Hetero-metrus bengalensis have been found to kill specifically human glioma and leukemic cells(Soroceanu et al.,1998; Wang and Ji,2005;Gupta et al.,2010).Chlorotoxin, a peptide isolated from L.quinquiestratus,specifically binds to glioma cells and prevents their proliferation(Deshane et al.,2003).Recent advances in nanotechnology have led to the development of biocompatible nanoparticles for in vivo molecular imaging and for targeted therapy using chlorotoxin(Lee et al.,2010).The existence of a chlor-otoxin-like peptide has been reported in the venom of B.martensii(Wang and Ji,2005).Also,bengalin a peptide of 72kDa isolated from the venom of H.bengalensis,has antiproliferative and apoptogenic activities against human leukemic cells(Gupta et al.,2010).In this work,we studied the action of Tityus discrepans scorpion venom and two purified peptidic components on SKBR3human breast cancer cells and on non-malignant MA104cell line.Two purified peptides,called here neo-pladine1and2,were found to be active on SKBR3human breast carcinoma cells and had negligible effects on MA104 cells.This work demonstrated that neopladine1and2, induce apoptosis on SKBR3cells via the activation of Fas signaling by induction of FasL expression.2.Material and methods2.1.Venom source and extractionAdult T.discrepans scorpions were collected around Caracas(Venezuela)and kept in plastic boxes with water and food ad libitum.Adult specimens(z200)were anes-thetized once a month with CO2and milked for venom by electrical stimulation.Venom was dissolved in double distilled water and centrifuged at15,000g for15min at 5 C.Protein content was estimated based on absorbance at 280nm,assuming that1mg/ml protein produces1unit of absorbance per unit path length.The supernatant was freeze-dried and stored atÀ80 C until use.2.2.Peptides isolation proceduresVenom fractionation was accomplished using a SephadexÒG-50column(0.9Â200cm)equilibrated and eluted at25 C with20mM ammonium acetate buffer,pH 4.7(19ml/h/cm2).Fractions were collected,lyophilized and stored atÀ80 C until used.Fraction I from SephadexÒG-50 was further fractionated through a semipreparative C4reversed phase column(250Â10mm,Vydac214TP1010, Hesperia,CA,USA),at aflow-rate of1mL/min using a linear gradient from solution A[water with0.12%trifluoroacetic acid(TFA)]to80%solution B(acetonitrile with0.10%TFA)in 80min.Fractions were collected manually and dried using a Savant Speed-Vac dryer.Active fractions were further purified by analytical reverse phase chromatography on a C4column(250Â4.6mm,Vydac214TP54,Hesperia,CA, USA)using a linear gradient from38to53%solution B in 30min(0.5%solution B/min).2.3.Mass and N-terminal sequence determinationMolecular weights of neopladine1and2were deter-mined at the Harvard University FAS Center for Systems Biology Mass Spectrometry and Proteomics Resource Laboratory(MSPRL),by matrix-assisted laser desorption time-of-flight mass spectrometry(MALDI-TOF)performed on a Waters MALDI Micro MX(Waters Corp.,Milford MA) using a standard steel well plate and sinapic acid matrix in linear positive ion mode.N-terminal amino acid sequences of neopladines were determined by automated Edman degradation on an Applied Biosystems Procise494-HT Protein Sequencer(Life Technologies,Carlsbad CA)using manufacturer recommended chemistry cycles and reagents.2.4.Cell lines and cultureHuman breast carcinoma cell line SKBR3(ATCCÒNumber:HTB-30Ô)and normal monkey kidney cell line MA104(ATCC Number:CRL-2378.1Ô)were obtained from the American Type Culture Collection(Rockville,MD).Cells were grown in Dubelco/Vogt modified Eagle’s minimal essential medium(DMEM)supplemented with10%heat-inactivated fetal bovine serum(FBS)(Sigma Chemical Co., St.Louis,MO),penicillin(100U/mL),gentamicin(48m g/mL) and amphotericin B(3m g/mL)under an humidified atmo-sphere of5%CO2and95%air at37 C.2.5.Viability assaysSKBR3or MA104cell lines were cultured to confluence in96-well plates in DMEM and FBS culture medium.Before exposure to venom,culture medium was changed to FBS free DMEM.Both cell lines were incubated with SephadexÒG-50fractions FI to FV(0.15m g/m L),cell viability was determined after48h using thiazolyl blue tetrazolium bromide(Sigma Chemical Co.,St.Louis,MO)(Mosmann, 1983).Cell death was determined by reading absorbance at570nm.2.6.Morphological assessment and apoptosis–necrosisquantificationMA104and SKBR3cell lines were cultured to confluence on22Â22mm2cover slides kept in culture plates with DMEM-FBS.Cell death was determined as apoptosis or necrosis by epifluorescence microscopy using the DNA binding dyes ethidium bromide(100m g/mL)and acridine orange(100m g/mL)(EB/AO)in PBS(McGahon et al.,1995;G.D’Suze et al./Toxicon56(2010)1497–1505 1498Jolly et al.,1997).This method measures differential dye uptake in viable and nonviable cells.In this assay,the apoptotic index and cell membrane integrity are assessed simultaneously and there is no cellfixation step(Renvoize et al.,1998;Mironova et al.,2007).Venoms’(1,15and30m g/m L)and neopladines’(1m g/m L or z33m M)capacity to induce apoptosis or necrosis were measured on cell lines after6and5h exposition respec-tively.The apoptotic or necrotic effects as function of exposure time to whole venom(5m g/m L)was determined in both cell lines after1,2,3,4,5and6h.Percentages of apoptosis or necrosis were determined on at least811cells counted from randomly selected microscopefields.Controls were exposed to FBS free DMEM.Positive necrosis controls were exposed for1h to1mM H2O2to induce oxidative stress,followed by4h recovery in fresh medium(Dini,2005).Positive apoptosis controls were exposed to6nM paclitaxel(ClitaxelÒ,Nolver C.A., Venezuela)or vinblastine(Vinblastina TN,Elmor S.A.,Cara-cas,Venezuela)during5h;both drugs are known to induce apoptosis in human breast cancer(Reshkin et al.,2003).2.7.ImmunohistochemistrySKBR3monolayers incubated with whole venom,neo-pladines or fresh medium were washed with PBS,scraped and resuspended in FBS free DMEM,centrifuged3times and smeared on subbed chrome-gelatin slides,fixed with Bouins’fluid for5min,washed with PBS,discolored with 0.5%w/v ammonium acetate to eliminated picric acid,and washed with bidistillated water.Slides were dehydrated for 1h in70%ethanol supplemented with0.25%NH4OH,then rehydrated with50%ethanol for10min and transferred to PBS(Baschong et al.,2001).Antigen was retrieved by boiling slides in0.01M citrate buffer(pH6.0)in a500W microwave oven twice for5min, and extensively washed with PBS(Farioli-Vecchioli et al., 2001).Unspecific binding sites were blocked with PBS containing5%skim milk,0.2%triton X-100for1h at25 C. Four slides of experimental and control groups were incu-bated overnight with the primary antibodies at4 C with 0.2mg/mL rabbit polyclonal anti-Tityus venom IgG (1:1000),or with polyclonal sheep anti-recombinant full-length BcL-2oncoprotein IgG(ab16120,Abcam),or with rabbit polyclonal antibody against FasL synthetic peptide (human)IgG(ab15285,Abcam Inc.)all diluted1:1000in PBS containing2.5%skim milk and0.1%triton X-100.All incubation steps were followed by extensive washing in PBS.Then,slides were incubated at25 C for1h with the secondary antibodies(1:5000in PBS):goat polyclonal anti-rabbit IgG coupled with FITC(ab6717Abcam Inc.)for Tityus–venom and FasL,and donkey polyclonal anti-sheep IgG coupled with rhodamine(ab6897Abcam Inc.)for BcL-2.Nuclei were stained with DAPI(1m g/mL)for5min and washed with PBS.Finally slides were mounted with50%v/v glycerol in PBS.Control slides were incubated only with antibodies.Slides were observed using phase contrast illumination and epifluorescence on a Nikon E600light microscope (Nippon Kogahu KK,Tokyo,Japan)with40Âand100Âplan-apochromatic objectives.For epifluorescence a100W mercury lamp light source and cubes UV-2A,B-2A and G-2A(Nikon)were used to visualizes DAPI,FITC and rhoda-mine respectively.Slides were digitally photographed with a Nikon Coolpix8700digital camera.The2560Â1920pixel images were saved as24-bit color TIFFfiles.The ImageJ (version 1.34w)public domain Java image processing package(National Institute of Health,Bethesda,MA,USA) was used to merge images.2.8.Statistical proceduresSome data are presented as medians and ranges(n¼2). Large samples of viable,apoptotic or necrotic cells (n!811),guarantee that the binomial distribution of the proportions is asymptotically Gaussian.Consequently proportions(p)are presented as percentages with bars indicating standard deviation,s(p),calculated as indicated in Fleiss(1973)as follows:sðpÞ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffip$ð1ÀpÞnr:Statistical significance of the differences between proportions was calculated also with the Gaussian asymptote of the binomial distribution as follows:z¼j p1Àp2jffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffis2ðp1Þþs2ðp2Þp¼j DðpÞjffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffis2ðp1Þþs2ðp2Þp;where z is a normalized Gaussian variable;DðpÞwas considered significant if z!1:9601ÀPðzÞ0:025which corresponds to a confidence level of P0.05for a two tailed test(Fleiss,1973).3.Results3.1.Active peptides isolationVenom fractionation on SephadexÒG50columns yiel-ded5fractions called FI to FV(Fig.1A,right inset).Further fractionation of FI by HPLC rendered26fractions,out of which components labeled N1and N2with retention times of57.6and58.3min respectively were preferentially toxic on SKBR3cell line and were called neopladines(Fig.1A). The left inset in Fig.1A,shows sharp HPLC peaks which confirm the homogeneity of N1and N2,this was confirmed by mass spectrometry data where only one molecular mass of29918Da for N1and one of30388Da for N2were obtained;also,in the amino terminal sequencing only one residue per cycle was observed.The N-terminal amino acid sequence of N1was SMG RQD KILV and is different from the N2first15amino terminal residues which were AENLFSMG RQD KCLV(Fig.1B).3.2.Toxicity assaysThe differential EB/AO uptake stains nuclei of living cell in green,nuclei of early apoptotic cells with condensed or fragmented chromatin in bright green,and nuclei of late apoptotic cells with condensed and fragmented chromatin in orange;necrotic cells had orange nuclei.Fig.2A showsG.D’Suze et al./Toxicon56(2010)1497–15051499Fig.1.Venom fractionation and neopladines puri fication A)Right inset :Chromatographic pro file of T.discrepans venom fractionated on a Sephadex ÒG-50column (0.9Â200cm)equilibrated and eluted at 25 C with 20mM ammonium acetate buffer,pH 4.7(19ml/h/cm 2).I ¼FI,II ¼FII,III ¼FIII,IV ¼FIV,V ¼FV,an asterisk identi fies the active fraction.NEV ¼Normalized elution volume.A)Chromatographic pro file of FI eluted through a C4semipreparative column,at 1mL/min using a linear gradient from solution A [water with 0.12%tri fluoroacetic acid (TFA)]to 80%solution B (acetonitrile with 0.10%TFA)in 80min.A)Left insets :N1and N2Active components puri fication by elution through a C4analytical column using a linear gradient from 38to 53%solution B in 30min.B)neopladine 1and 2molecular masses determined by MALDI-TOF.Neopladine 1and 2N -terminal amino acid sequences determined by automated Edman degradation.Other details in Material and Methodssection.Fig.2.Apoptosis and necrosis percentages as function of time and venom concentration .A)Apoptosis -and necrosis A percentages of normal kidney cell (MA104)and human cancer breast cell (SKBR3)lines after 6h incubation with 1,15and 30m g/m L whole venom.B)Apoptosis -or necrosis A percent of MA104and SKBR3cell lines induced by 5m g/m L venom as function of time;insets are controls cell lines exposed to FBS free DMEM.Percentages were determined on >811cells counted from randomly selected microscope fields.Bars indicate the standard deviation of the percentages.Other details in Material and Methods section .G.D ’Suze et al./Toxicon 56(2010)1497–15051500that incubation of SKBR3cells with whole venom(1and15, 30m g/m L)for6h induced a z10fold larger effect than on the non-malignant cell line MA104incubated under similar conditions.The lowest venom concentration studied(1m g/ m L)induced more apoptosis than necrosis on the SKBR3cellline.Fig.2B shows that the SKBR3cell line suffered4.6%of apoptosis and1.4%necrosis after treatment with venom (5m g/m L)after a6h exposure.The same conditions induced 1%of apoptosis and0.1%of necrosis in MA104cells.The SKBR3cell death was time and venom concentration dependent.SKBR3cells incubated with FI,FII,FIII,FIV or FV(0.15m g/ m L)for48h,were killed in proportions of70,7,10,0and 26%,respectively(Fig.3A).After treatment with FI,SKBR3 cell were round,shrunk,had protruding elongated spikes at the cell surface,and died.Cell detachment from the substrate was also observed(Fig.3B right).These changes were not observed in the MA104cell line exposed to FI under same conditions where only16%of the cells were killed.Control SKBR3cells exposed to FBS free DMEM for 48h maintained its morphology and remained organized as a monolayer(Fig.3B left).3.3.Apoptotic and necrotic effectsNeopladine1(1m g/m L,z33m M,5h exposure)induced 2.1Æ0.28%(meanÆsd)apoptosis and0.31Æ0.11%of necrosis in MA104cells(n¼2620).Neopladine2(1m g/m L, z33m M,5h exposure)induced2.02Æ0.26%apoptosis and 0.28Æ0.10%of necrosis in MA104cells(n¼2868).Neo-pladine1(1m g/m L,z33m M,5h exposure)induced 6.31Æ0.48%apoptosis and0.57Æ0.15%of necrosis in SKBR3(n¼2613)cells.Neopladine2(1m g/m L,z33m M,5h exposure)induced4.04Æ0.29%apoptosis and0.24Æ0.07% of necrosis in SKBR3cells(n¼4649).The neopladines were also applied in combination(z33m M of each one,5h exposure);this treatment produced 5.06Æ0.56%of apoptosis and 2.92Æ0.43%of necrosis in SKBR3cells (n¼4649)(Fig.4A).We used two positive control drugs:vinblastine and paclitaxel(both6nM with a5h exposure).Paclitaxel induced0.42Æ0.21%apoptosis and no necrosis in MA104 cells(n¼952);the same drug induced 3.21Æ0.43% apoptosis and0.47Æ0.17%of necrosis in SKBR3cells (n¼1714).Vinblastine induced3.2Æ0.56%apoptosis and 0.2Æ0.14%of necrosis in MA104cells(n¼999);the same drug induced4.69Æ0.50%apoptosis and1.87Æ0.32%of necrosis in SKBR3cells(n¼1814)(Fig.4A).In the micrographies in Fig.4B it is shown that expo-sures to neopladine1or2produced nuclear fragmentation (F),cytoplasm condensation(C),membrane protrusions (P),induction of vesicles(V)and loss of cellular confluence (L)of SKBR3cells.3.4.Immunohistochemical venom localizationFig.5B–D show that whole venom and neopladines bind to SKBR3cell membranes.This is observed as anintenseFig.3.SephadexÒG50fractions effect on malignant cell line Viability.A)Cell death percentages induced on SKBR3confluent layers after48h exposure to SephadexÒG-50fractions(0.15m g/m L).Cell death was determined by absorbance at570nm.These percentages were deduced from the absorbance at570nm of cells in96-well plates after treatment with MTT.The bars are means and ranges(brackets on top of the bars)of two replicates.B)Morphology changes of SKBR3 confluent layers after48h incubation with FBS free DMEM culture medium(control,CNT)and SephadexÒG-50fraction I(0.15m g/m L).Bars¼25m m.Other details in Material and Methods section.G.D’Suze et al./Toxicon56(2010)1497–15051501green fluorescence surrounding the cells,due to FITC-labeled anti-rabbit IgG which recognizes anti-T.discrepans rabbit IgG,coupled to either venom (Fig.5B),neopladine 1(Fig.5C)or neopladine 2(Fig.5D)bound to the cell membrane.The FITC-IgG was absent from control SKBR3cells exposed only to fresh medium (Fig.5A).3.5.Venom enhanced FasL and BcL-2expressionFig.5F through Fig.5H show that SKBR3cells treated with either venom (Fig.5F),neopladine 1(Fig.5G)or neopladine 2(Fig.5H)expressed Fas ligands.This appears as an intense green fluorescence at the cell membrane,produced by FITC-labeled anti-rabbit IgG which recognizes rabbit polyclonal anti-FasL IgG coupled to FasL expressed by the cells.This fluorescence was absent in control SKBR3cells (Fig.5E).Fig.5J through Fig.5L show that BcL-2is expressed by SKBR3cells treated with venom (Fig.5J),neopladine 1(Fig.5K)or neopladine 2(Fig.5L).This is seenas an intense red fluorescence inside the cells,due to rhodamine-labeled anti-sheep IgG recognizing sheep anti-BcL-2IgG which bind to the expressed BcL-2.SKBR3control group did not exhibit fluorescence (Fig.5I).4.DiscussionScorpion venom may contain hundreds of compounds with different pharmacological activities.Venoms from scorpions from the Buthidae family can induce human death.However,not all venom compounds are toxic to humans,specially the ones with high molecular masses.T.discrepans venom induced apoptosis in a concentra-tion similar or lower than other scorpion venoms (Wang and Ji,2005;Gupta et al.,2010);four of the five venom fractions isolated on Sephadex G50were able to kill SKBR3cells.The most potent fraction,FI,contains the highest molecular weight components and is harmless to mammalians (D 0Suze et al.,1995).Neopladines 1and2Fig.4.Human cancer breast cell death induced by puri fied neopladines.A)Apoptosis (crisscrossed bars)and necrosis (open bars)percentages of SKBR3cell lines after 5h treatment with puri fied neopladines 1(N1)and 2(N2)(z 33m M)alone and in combination (N1þN2,z 33m M of each one).Positive apoptosis controls were exposed for 5h to 6nM paclitaxel (PCT)(Clitaxel ÒNolver C.A,Venezuela)or 6nM vinblastine (VBL)(Vinblastina TN ,Elmor S.A.,Venezuela).Control (CNT)group were incubated 5h with FBS free DMEM culture medium.Brackets on top of the bars indicate its standard deviation calculated as indicated in Section 2.8.B)In as little as 5h,neopladine 1and 2induced nuclear fragmentation (arrow labeled F),cytoplasm condensation (arrow labeled C),membrane protrusions (arrow labeled P),induction of vesicles (arrow labeled V)and loss of cellular con fluence (arrow labeled L)on con fluent layers of SKBR3cell lines (1m g/m L,33m M).Control (CNT)group were incubated 5h with FBS free DMEM culture medium.Bars ¼25m m.G.D ’Suze et al./Toxicon 56(2010)1497–15051502z 30KDa eluted with fraction FI.The N -terminal amino acid sequence of neopladine 2was similar to one toxin reported in the proteomic study of T.discrepans venom (Batista et al.,2006),with retention time of 50.88min (z 30kDa).Both neopladines induced nuclear fragmenta-tion,loss of cellular con fluence and apoptosis in SKBR3cell cultures.The apoptotic effects resemble those caused by B.martensii (Karsch)venom on human glioma,and by bengalin,from H.bengalensis venom,on leukemic cells (Wang and Ji,2005;Gupta et al.,2010).Chlorotoxin,iso-lated from L.quinquiestratus venom,inhibits glioma growth by interacting with the plasmalemma matrix metal-loproteinase-2(Deshane et al.,2003).Chlorotoxin effect occurs at concentrations ranging between 10and 20m M,close to the neopladines ’z 33m M.Neopladines act on SKBR3cells,if this interaction occurs by binding to membrane receptors (Ehrenpreis et al.,1969)or if they directly activate second messengers which trigger the expression of FasL,caspases or BcL-2(Villunger et al.,1997;Reshkin et al.,2003),is still unknown.A group of snake venom toxins block malignant cell migration and invasion via an RGD-amino acid sequence dependent interaction (Ritter et al.,2000;Schmitmeier et al.,2000;Sheu et al.,1997).We found that both neo-pladines ’N -terminal have a RQD-amino acid sequence perhaps determining a similar toxin –cell interaction.Neopladine 1and 2bind to SKBR3cell membrane,induce FasL expression and trigger programmed cell death;similar apoptotic mechanisms are known for other drugs (Villunger et al.,1997;Reshkin et al.,2003).FasL expression occurs during methotrexate-or doxorubicin-induced apoptosis in CEM T-ALL cells (Friesen et al.,1996)and bleomycin-and cisplatin-induced apoptosis in HepG2hepatoma cells (Müller et al.,1997).BcL-2,an integral outer mitochondrial membrane protein,suppresses apoptosis in a variety of cell systems;yet,malignant cells under the action of apoptotic drugs,produce BcL-2(Chao and Korsmeyer,1998).Thus,in ef fi-cient apoptotic drugs,the cytotoxic effect prevails over the antiapoptotic BcL-2effect.Neopladines were abletoFig.5.Immunohistochemical localization of venom,neopladine 1,neopladine 2,FasL and BcL-2on malignant treated cell.SKBR3con fluent layers after exposure to fresh medium,whole venom or neopladine 1or 2.Whole venom (B),neopladine 1(C)and neopladine 2(D)was immunohistochemical localized as a fluorescent green color at the malignant cell surface membrane.Similar immunohistochemical localization of FasL is shown as an intense green color at SKBR3cell membrane as response to venom (F),neopladine 1(G)and neopladine 2(H).Immunohistochemical localization of BcL-2is shown as an intense red color inside SKBR3cells as response to venom (J),neopladine 1(K)and neopladine 2(L)treatment.Control SKBR3cells were exposed to either culture medium and to primary and secondary antibodies anti-Tityus (A),anti-FasL (E)and anti-BcL-2(I).Sections were observed by epi fluorescence microscopy.Bars ¼2m m.Other details in Material and Methods section.G.D ’Suze et al./Toxicon 56(2010)1497–15051503promote BcL-2in SKBR3cells,and still were active proa-poptotic compounds.We observed3populations of cells in cultures exposed to antineoplastics:viable cells,apoptotic cells and necrotic cells.With the doses and the observation periods used,only vinblastine(P¼2Â10À7)and neopladine1(P¼0.009), produced significant necrosis on SKBR3cells when compared with the controls.Neither paclitaxel(P¼0.054) nor neopladine2(P¼0.266)necrotic SKBR3cell frequen-cies were statistically different from the controls.Neo-pladine1induced larger percentages of SKBR3apoptosis than paclitaxel(P¼6Â10À7)and vinblastine(P¼0.009). Neopladine2was as apoptogenic for SKBR3as paclitaxel (P¼0.053)and as vinblastine(P¼0.129).The SKBR3 percent apoptosis when the two neopladines were applied together,was weakly significantly lower than the SKBR3 apoptotic effect of neopladine1(P¼0.044)and not different from neopladine2(P¼0.052);still,the SKBR3 necrotic cell percentage of the two neopladines combined was the largest one observed(P¼0.025versus vinblastine, and P<10À7versus any of the other conditions tested).Vinblastine(P¼0.006)increased apoptosis slightly above the control in MA104cells;neopladine1(P¼0.115) and neopladine2(P¼0.152)were not apoptotic in these cells.Neither the neopladines nor vinblastine had necroticeffects above the control values for MA104culturesðP! 0:069Þ,paclitaxel produced no necrosis at all.Taken together with the results discussed in the preceding paragraph the neopladines seem to be apoptotic peptides with selectivity for SKBR3breast cancer cells rather than for non-malignant MA104monkey kidney cells. Promoting apoptosis is the mechanism underlying the effect of efficient chemotherapeutic drugs such as vinblastine(Harmon et al.,1994)cisplatin and paclitaxel (Villunger et al.,1997;Reshkin et al.,2003).We must, however,deal with the bigger necrotic pool observed with an apparently unchanged apoptotic pool,when the neo-pladines were applied together.To explain this apparent paradox,we will consider3possible kinetic relationships between the cell pools under consideration.Two of these models are shown in Fig.6.At the top of thefigure we present what we call a sequential model where the drug induces apoptosis at a rate k A,and the apoptotic cells die, becoming necrotic,at a rate k N.The bottom of Fig.6pres-ents an alternative model(parallel model)where a direct pathway between the viable and necrotic pools exists (dotted line)in parallel with the sequential model.A third alternative could be similar to the parallel model,but with no connection between the apoptotic and necrotic pools; since this implies that the apoptotic cells are immortal,we will disregard this third possibility.From the sequential model it is very easy to explain that a drug,such as the neopladines,at short times and/or at low concentrations,may have a significant apoptotic effect without apparent necrosis.It is also intuitively clear that as the apoptotic pool increases with time,a necrotic pool must become evident.The time and concentrations of drug required depend on f¼k A=k N,the ratio between the rates of apoptosis and necrosis.When the viable pool is very large the model could reach a state where the apoptotic pool seems constant while the necrotic pool increases;implicit in the model is that an apoptotic drug will somehow increase k A and perhaps also k N,thus the sequential model is quite compatible with our results with neopladines1and2,applied alone or in combination.To explain our results with the neopladines1or2not in combination,with the parallel model,the branch repre-sented by the dotted line must correspond to either a very low k N’or to a cooperative process which starts with a delay.Otherwise,some increase in the necrotic pool size should occur in parallel with an increase in the apoptotic pool.We will accept the sequential model as valid,for a reason of simplicity for the time being,until we are able to do a full synergism study of the neopladines.The statistical analysis clearly shows that neopladines are apoptotic peptides on SKBR3cell and have little or no effect on MA104cells.Furthermore,the apoptogenic effects of the neopladines were statistically significantly larger than the apoptogenic effect of paclitaxel and vinblastine. The neopladines also had negligible necrotic effects on SKBR3cells when applied separately.Also the percent apoptosis induced by the neopladines may seem small due to the short observation period,but is completely equiva-lent to the effects of two classical antineoplastic drugs (paclitaxel and vinblastine)studied under equivalent conditions.Regarding the comparison of the pure peptides with the whole venom,please notice that the venom is a cocktail of bioactive peptides with a wide spectrum of toxicities;it is not thus surprising tofind that whole venom would be more toxic to anything than its individual components. Neopladines are a new kind of antineoplastic peptides, which are interesting per se,as tools to understand the physiopathology of cancer,or as templates for novel anti-neoplastic drugs of clinical interest.Given the chlorotoxin success at labeling malignant cells to better remove tumors (Veiseh et al.,2007),peptides such as neopladines,which Fig.6.Two possible models to produce apoptosis and necrosis.We observed3 populations of cells in cultures exposed to antineoplastics:viable cells, apoptotic cells and necrotic cells.At the top of thefigure we present what we call a sequential model where the drug induces apoptosis at a rate k A,and the apoptotic cells die,becoming necrotic,at a rate k N.The bottom offigure presents an alternative model(parallel model)where a direct pathway (dotted line)between the viable and necrotic pools exists in parallel with the sequential model with a rate constant k N0.G.D’Suze et al./Toxicon56(2010)1497–1505 1504。