Changes in membrane-associated H+-ATPase activities and
Chaperone-mediated autophagy, machinery, regulation and biological consequences
REVIEWChaperone-mediated autophagy:machinery,regulation and biological consequencesWenming Li •Qian Yang •Zixu MaoReceived:12July 2010/Revised:7October 2010/Accepted:8October 2010/Published online:26October 2010ÓSpringer Basel AG 2010Abstract Degradation of dysfunctional intracellular components in the lysosome system can occur through three different pathways,i.e.,macroautophagy,micro-autophagy and chaperone-mediated autophagy (CMA).In this review,we focus on CMA,a type of autophagy distinct from the other two autophagic pathways owing to its selectivity,saturability and competitivity by which a subset of long-lived cytosolic soluble proteins are directly deliv-ered into the lysosomal lumen via specific receptors.CMA participates in quality control to maintain normal cell functions by clearing ‘‘old’’proteins and provides energy to cells under nutritional stress.Deregulation of CMA has recently been shown to underlie some diseases,especially neurodegenerative disorders for which the decline with age in the activity of CMA may become a major aggravating factor.Therefore,targeting aberrant alteration in CMA under pathological conditions could serve as a potential therapeutic strategy for treating related diseases.Keywords CMA ÁLysosome ÁHsc70ÁLamp2a ÁAutophagy ÁUnibiquitinIntroductionMaintaining a balance between protein synthesis and deg-radation is absolutely essential for proper cellular function,homeostasis and survival in a changing extracellularenvironment.The two major protein degradation mecha-nisms in eukaryotes are the ubiquitin-proteasome system (UPS)and the autophagy-lysosome system [1,2].Collec-tively,they (1)exert quality control by clearing damaged or incorrectly synthesized proteins,(2)recycle proteins that are no longer needed into constitutive amino acid compo-nents,(3)mount the cellular defense by breaking down components of various invading pathogens,and (4)regu-late cellular response to stress or the alteration in extracellular micro-environments,assisting cells in adapt-ing to new changes [3,4].The UPS is a multi-subunit protease complex in the cytosol that permits entry and subsequent degradation of proteins tagged with one or more covalently bound ubiquitin molecules [5].Ubiquitin is a small protein highly conserved from yeast to man.It was first described in the context of protein degradation,but later shown to participate in regulation of other cellular processes,such as endocytosis,signal transduction and DNA repair.Conjugation of ubiquitin is a complex reaction that requires E1,E2and E3enzymes,and leads to the for-mation of an isopeptide bond between the C-terminal glycine of ubiquitin and the 3-amino group of a lysine residue of the substrate protein [6,7].Subunits of the regulatory complex of the proteasome recognize the ubiquitin tag,remove it and mediate the unfolding of the substrates required to gain access to the catalytic region of the proteasome barrel.With certain exceptions,most proteasome substrates have short half-lives [6,8].Inter-estingly,recent studies suggest that ubiquitin and lysosomal pathways are linked.For example,new evi-dence suggests that attachment of ubiquitin to various cellular cargos not only constitutes a degradation signal for proteasome,but also serves to signal for cargo removal by lysosomal system via autophagy [2,9].W.Li ÁQ.Yang ÁZ.Mao (&)Departments of Pharmacology and Neurology,Emory University School of Medicine,Atlanta,GA 30322,USAe-mail:zmao@Cell.Mol.Life Sci.(2011)68:749–763DOI 10.1007/s00018-010-0565-6Cellular and Molecular Life SciencesAutophagy is a conserved cellular ‘‘self-eating’’process that involves sequestration and delivery of cytosolic components to the lysosome for degradation and recycling [10].This evolutionarily conserved process can be cate-gorized into three classes depending on their respective sequestration and delivery mechanism (Fig.1)[11].In macroautophagy,a double-membrane vesicle termed the autophagosome is formed to engulf long-lived proteins and organelles.The autophagosome is subsequently fused with a lysosome,releasing its cargo for degradation by lyso-somal hydrolases.The resultant nucleotides,amino acids and fatty acids are eventually recycled back into the cytosol for reuse.In microautophagy,the sequestration of cytosolic content is facilitated by direct invagination or exvagination of lysosomal membrane,and subsequent budding of the invaginated vesicles into the lysosomal lumen releases the sequestered cytosolic material.In con-trast to the vesicle-mediated substrate delivery of macro and microautophagy,chaperone-mediated autophagy (CMA)targets and delivers substrate proteins directly across the lysosomal membrane via the specific receptor [9,12].Only proteins containing a consensus peptide sequence are recognized by a chaperone complex.This CMA sub-strate-chaperone complex locates to the lysosome through interaction with the lysosome receptor and translocates the substrate across the lysosomal membrane with the assis-tance of lysosomal chaperones on the lumenal side [12,13].In this review,we first describe the machinery of CMA,summarize the regulation mechanisms involved in activation of CMA,and finally discuss the biological consequences of CMA under various physiological and pathological conditions,and relevant therapeutic strategies via regulating CMA.History of CMAIn the early 1980s,Professor Dice’s group first reported that radiolabeled RNase A introduced into the cytoplasm of human fibroblasts by using erythrocyte-mediated microin-jection or osmotic lysis of pinosomes was degraded with a half-life of approximately 90h in the presence of serum,whereas in response to serum deprivation its rate of deg-radation was enhanced 1.6-fold [14].This enhanced breakdown following serum withdrawal was highly selec-tive and based on a feature present within the N-terminal 20amino acids of RNase A [15].During subsequent years,the essential motifs related to KFERQ were identified in proteins serving as substrates of this selective degradation pathway,which is referred to as the selective pathway for degradation of cytosolic proteins by lysosomes [16,17].In 1989,a 73-kDa heat shock cognate protein (Hsc73,now commonly referred toas Hsc70)was found to bind to KFERQ-like regions in intracellular proteins that are tar-geted for lysosomal degradation in response to serum deprivation [18].In 1994,isolated rat liver lysosomes were used to probe the selective binding and uptake of RNase A and glyceraldehyde-3-phosphate dehydrogenase.Their uptake and degradation by lysosomes were progressively activated in rat liver by starvation [19].In 1996,Lamp2a,also named Lgp96(lysosomal glycoprotein of 96kDa),was identified as a receptor for the selective import and degradation of proteins within lysosomes [20].In 1997,an intralysosomal Hsc73was determined to be required for the selective pathway of lysosome-mediated protein deg-radation [21].Since 2000,this pathway has been formally named chaperone-mediated autophagy (CMA)[22,23].Throughout the subsequent decade,new substrates,moreFig.1Autophagy refers to the conserved degradation of intracellular components by lysosomes.In mammals,three types of autophagy have been described:macroautophagy,microautophagy and CMA.Adapted from [11]with permission750W.Li et al.detailed machinery,new components,physiological roles and associated diseases for CMA have been extensively investigated and elucidated [3].Machinery of CMACMA uses the unique and distinctive machinery from the other two autophagy pathways to carry out this process [3,24].The basic machinery consists of at least three types of proteins,including (1)chaperone proteins,which are responsible for recognizing substrates based on their spe-cific motifs and delivering them to lysosomes,(2)receptor proteins,which bind and transport/pull substrates into lysosome lumens,and (3)substrate proteins,which are a subset of soluble cytosolic proteins containing specific motifs related to KFERQ.Following activation of CMA,these three subsets of proteins collaborate and complete the process (Fig.2)[3,25].Workshop—lysosomeLysosomes are the primary catabolic compartment of eukaryotic cells.The name lysosome derives from the Greek words lysis,which means dissolution or destruction,and soma,which means body [24].Lysosomes were dis-covered by the Belgian cytologist Christian de Duve in 1949[26].They are created by the addition of hydrolyticenzymes to early endosomes from the Golgi apparatus [24].The membrane around a lysosome allows the digestive enzymes to work at pH 5.1–5.5,which is optimal to these acidic hydrolases.Lysosomes fuse with vacuoles and dis-pense their enzymes within digesting the contents [27].A healthy cell is dependent on the proper targeting of newly synthesized lysosomal proteins.Two classes of proteins are essential for the function of lysosomes:soluble lysosomal hydrolases (i.e.,acid hydrolases)and integral lysosomal membrane proteins (LMPs)[24].Lysosomal acidic pH and the large variety of hydrolases present in the lysosomal lumen (including proteases,lipases,glycosi-dases and nucleases)confer upon this organelle its high capacity of degradation and mediate complete breakdown of all types of molecules.In addition to bulk degradation,lysosomal hydrolases are involved in antigen processing,degradation of the extracellular matrix and initiation of apoptosis [24,28].LMPs reside mainly in the lysosomal limiting membrane and have diverse functions,including acidification of the lysosomal lumen,protein import from the cytosol,membrane fusion and transport of degradation products to the cytoplasm [29].The main LMPs are lyso-some-associated membrane protein 1and 2(Lamp1and 2),lysosomal integral membrane protein 2and tetraspanin CD63[24].Substrates can reach lysosomes via heteroph-agy,in which the cargo to be degraded originates at the plasma membrane or extracellularly,or via autophagy,for cargo located in the cytosol [3,24,30].Fig.2The diagram ofproposed mechanisms of CMA:a Hsc70with co-chaperones recognizes a KFERQ-related peptide in cytosolic substrate proteins;b the complex binds to the Lamp2a receptor on the lysosomal membrane;c the substrate protein is unfolded before traversing the lysosomal membrane;d lys-Hsc70pulls the substrate into the lysosome matrix;e the substrate protein is degraded by lysosomal proteases;f the Hsc70-cochaperone complex is released from the lysosomal membrane;g Hsc70is available to bind to another CMA substrate.Adapted from [25]with permissionBasics of chaperone-mediated autophagy 751Chaperones—Hsc70and partnersHsc70,a73-kDa protein,is the constitutive member of the heat shock protein70family of chaperone[19,25].Hsc70s have been found to be involved in many cellular processes including dissociation of clathrin and assembly proteins [25,31].Binding of Hsc70to substrate proteins is regu-lated by ATP binding and hydrolysis,and the ADP-bound form of Hsc70has the highest affinity for protein substrates for CMA[19,32].Hsc70is located in the cytosol or in the lumen of lysosomes.Cytosolic Hsc70(cyt-Hsc70)can recognize a peptide sequence including the KFERQ motif in CMA substrate proteins and aid in their transport to the lysosomal receptor[3,19].Cyt-Hsc70docked to the lysosomal membrane helps to unfold substrate proteins,a necessary step for their entry into lysosomes[33].Other co-chaperones interact with Hsc70and regulate its activities.Hsp40may activate the ATPase activity of Hsc70to facilitate substrate binding;and the Hsp70-interacting protein(Hip)stimulates the assembly of Hsc70 with Hsp40and the protein substrate[25].Cell division cycle48(Cdc48)can also enhance the activity of Hsc70-Hsp40complexes[32,34].Hsc70-Hsp90organizing pro-tein acts as an adapter between Hsc70and Hsp90,which recognize unfolded regions within proteins and prevent substrate protein aggregation[25,32].There are co-chap-erones of Hsp90that may be in the molecular chaperone complex.Activator of Hsp90ATPase is a family of heat shock proteins that activates the ATPase activity of Hsp90 and thereby stimulates both protein binding and release [32,35].The Bcl2-associated athanogene1protein(Bag-1) was initially described as a co-chaperone of Hsc70that uncouples the ATPase cycle from substrate binding[36]. But several subsequent studies showed that it functions as a nucleotide exchange factor that stimulates substrate release [37].The carboxyl terminus of Hsc70-interacting protein (Chip),which can regulate protein refolding[38],acts as a chaperone-associated ubiquitin ligase to stim-ulate the degradation of Hsc70client proteins[39].The chaperone complex present on the cytosolic face of lyso-somal membranes is linked to Lamp2a by the substrate protein via a site that is different from sites of interaction for the molecular chaperone complex.Each of the com-ponents in the molecular chaperone complex is required for transport of substrates into the lysosome lumen[25,32].Both Hsc70and Hsp90have been found to be also present in the lysosomal lumen.Lysosomal-Hsc70(lys-Hsc70)is required for the CMA pathway[21].The more active population of lysosomes contains abundant lys-Hsc70,whereas the less active ones contain little Hsc70. The latter group of lysosomes can be made more active for CMA if they are allowed to take up Hsc70.A role for lys-Hsc70in the uptake of substrate proteins has been demonstrated in cultured confluent humanfibroblasts.It has been speculated that lys-Hsc70may be required to pull proteins into the lysosomal lumen because of analogous roles of Hsp70s in the translocation of proteins into the endoplasmic reticulum,mitochondria and chloroplasts [21,25].Nearly half of the lysosomal Hsp90s associate with the lumenal side of the lysosomal membrane where this chaperone may contribute to the stabilization of essential components of the translocation complex when it is organized into a multimeric structure[3,40].Receptor—Lamp2aIn1996,a lysosomal membrane glycoprotein Lgp96 (called Lamp2a in human)was identified as a receptor for binding and uptake of lysosome substrates[20].Lamp2a is one of the three splice variants of the Lamp2gene that gives rise to three single-span membrane proteins, Lamp2a,b and c.These variants all have a common highly N-glycosylated lumenal region,but possess different transmembrane and C-terminal cytosolic tail regions. Lamp2a has a short cytosolic tail(GLKRHHTGYEQF)to which CMA substrate proteins bind[25].The positively charged residues in the Lamp2a cytosolic tail are important for binding of substrate proteins.However,the specific amino acids on the CMA substrate proteins required for receptor binding remain elusive[41].Binding of substrate proteins to Lamp2a is a rate-limiting step for the CMA process,and overexpression of Lamp2a in Chinese hamster ovary cells can increase CMA mp2a is not limited to acting as a classic receptor.Increasing evidence shows that Lamp2a is involved in many other aspects of the CMA process such as substrate translocation[3,25,40]. Lamp2deficiency in mice causes extensive accumulation of autophagic vacuoles in many tissues[42].Contrary to the proposed receptor function of Lamp2a in CMA,con-fluent mouse embryonicfibroblasts deficient in lamp2 appear to have normal levels of CMA-associated lysosomal proteolysis after prolonged serum withdrawal,suggesting that Lamp2a may not be the only receptor for CMA [29,43].Substrates—KFERQ motifSubstrates for CMA are defined by an amino acid sequence motif related to KFERQ[3,16].This pentapeptide target-ing motif wasfirst identified in microinjected RNase A[44].It consists of a glutamine(Q)preceded or followed bya combination of four amino acids that are basic(R,K), acidic(D,E),or bulky and hydrophobic(F,I,L,V)resi-dues.In some cases,Q may be substituted by the related N [45].Antibodies raised against KFERQ can immunopre-cipitate30%of cytosolic proteins in mammalian cells[46].752W.Li et al.Without denaturation,more than80%of cytosolic pro-teins that contain KFERQ motifs can be recognized by the antibody to this motif,indicating that most KFERQ motifs in these proteins are exposed.However,certain proteins such as aldolase B have hidden KFERQ motifs due to multimeric formation.The may dissociate the aldolase tetramer and result in the exposure of KFERQ motif[25,47].In addition,certain monomers may have hidden KFERQ sequences that may be exposed following partial unfolding.Therefore,the presence of a KFERQ sequence alone in the primary structure of a protein is not sufficient for determining them as substrates of CMA[48]. Such identification requires rigorous experimental proof. Recently,we have identified MEF2D,a protein known to promote neuronal survival as a CMA substrate.Interest-ingly,MEF2D uses a set of over-lapping imperfect KFERQ motifs to mediate its interaction with Hsc70[49]. Characteristics of CMACompared to macro-and microautophagy,which occurs in a wide range of eukaryotes including mammals,plants and fungi,CMA has only been described in mammals.This process has some distinctive features.SelectivityLysosome-mediated degradation had traditionally been perceived as a process performed in bulk with poor selectivity.The initial idea of selective autophagy origi-nated from the observation that starvation in animals or serum removal in cultured cells accelerates the degradation of particular cytosolic proteins in lysosomes but not others[50].Selectivity thus became one of the hallmarks of CMA[51].CMA mediates selective targeting of non-essential proteins for degradation to obtain the amino acids required for the synthesis of essential proteins.The intrinsic selec-tivity of CMA is also well suited for the removal of specific proteins damaged during stress without interfering with nearby normally functioning forms of the same protein[51, 52].This selectivity is achieved by making the KFERQ motifs in the altered protein accessible to the chaperone but inaccessible when it is properly folded or pare to CMA,macroautophagy has traditionally been consid-ered as a nonselective degradation process.However, recent evidence suggests that this view needs modification. New experimental data demonstrate that some macro-autophagic processes,termed chaperone-assisted selective autophagy,are assisted by chaperone proteins and can be selective in targeting protein complexes,organelles and microbes[2].In this process,macroautophagy has been proposed to be initiated by a selective ubiquitylation of cellular targets and followed by recognition via autophagic ubiquitin adaptors such as p62,NBR1and HDAC6.These molecules can mediate docking of ubiquitinated proteins or damaged organelles to autophagosomes and lysosomes, thereby ensuring their selective degradation[2,53,54].SaturabilityThe unusual characteristics of CMA are not limited to its selectivity.As the mechanism for cargo delivery was revealed,it became evident that,in contrast to the other forms of autophagy,vesicle formation was not required in CMA.Instead,the substrate proteins were translocated across the lysosomal membrane[55].The receptor Lamp2a is mainly responsible for this translocation.Because of the requirement for receptor binding before translocation can occur,this delivery process becomes saturable.In contrast to Hsc70,which is often in excess in the cytosol,levels of Lamp2a are limiting for CMA and hence subjected to tight regulation[23].CompetitivityThe selectivity and saturability of CMA directly lead to the competitive binding of CMA substrates.During the process of CMA,different substrates compete for binding Hsc70 and limited pool of Lamp2a[23,56].Several studies have shown that degradation of some substrates is slowed down by overexpression of other substrates.This has been proposed to underlie the pathogenesis of some diseases [49,57].For example,both the wild-type a-synuclein and neuronal transcription factor MEF2D are substrates of CMA.Elevated levels of wild-type a-synuclein may reduce the degradation of MEF2D via CMA[49].Regulation of CMAThe signal transduction pathways involved in the regula-tion of CMA from cell membranes to cytosolic chaperone proteins and lysosomes remain largely elusive[3].The p38 MAPK inhibitor can partly prevent the activation of CMA, implicating this pathway in CMA[45].On the other hand, the extensively investigated local regulation of CMA activity in the lysosomes has precise,fine-tuned mecha-nisms[3,25].Regulation of CMA via Lamp2aThe level of Lamp2a at the lysosomal membrane is pro-portional to the activity of CMA.Therefore,changes in the Lamp2a level at the lysosomal membrane can quickly regulate the activity of CMA.The level of Lamp2a at theBasics of chaperone-mediated autophagy753lysosomal membrane may be changed by synthesis,deg-radation and redistribution[41,58].De novo synthesis of Lamp2a or its by protein synthesis inhibitors can directly change the level of Lamp2a in cells,but the physiological or pathological relevance remains unclear [3,59].The distribution of Lamp2a at the lysosomal membrane is regulated by its dynamic association with discrete membrane lipid microdomains[60].Under basal conditions,sequestration of Lamp2a in cholesterol-enri-ched regions favors its cleavage by two proteases, including an unidentified metalloprotease at the membrane and a serine protease—cathepsin A,which associates dynamically with the lumenal side of the lysosomal mem-brane[60,61].Exclusion of Lamp2a from these regions allows its multimerization(see below),a step required for the uptake of CMA substrate by lysosomes.Interestingly, the degradation of Lamp2a can be reduced by the presence of substrates or stimuli that induce the activation of CMA. This blockage in degradation,rather than the increase of synthesis,to increase Lamp2a at the lysosomal membrane is particularly advantageous to cells with limited access to amino acids such as during nutrient deficit[3,62].The levels of Lamp2a can be further increased at the lysosomal membrane through the mobilization of the pool normally resident in the lysosomal lumen.The exact nature of this luminal pool of Lamp2a remains unclear,but intact molecules of this protein exist inside lysosomes,and a gradual decrease in the percentage of Lamp2in this com-partment occurs as activation of CMA persists beyond1 day[3,60].Membrane chaperones and an intact membrane potential are needed for the mobilization of Lamp2a from the lumen to membrane[3].Fractionation studies have shown that luminal Lamp2a associates with lipid,indicat-ing the possible existence of luminal Lamp2a-containing micelles.It is thought that these micelles fuse or integrate into the lysosomal membrane under specific stress,result-ing in the incorporation of Lamp2a in the membrane and exposure of its C terminus to the cytosol[60,63].Lamp2a can undergo cycles of rapid assembly into a 700-kDa protein complex at the lysosomal membrane. Monomers of Lamp2a at the lysosomal membrane can accept substrate proteins,and this interaction drives the organization of Lamp2a into multimeric complexes needed for substrate translocation into lysosomes.Once the sub-strate protein reaches the lysosomal lumen,Lamp2a will disassemble from the multimeric complex to enable sub-sequent rounds of substrate binding[3,40].This continuous assembly and disassembly of Lamp2a from the multimeric translocation complex highlights the impor-tance of the lateral mobility of this protein in the lysosomal membrane[40].Chaperone proteins located at both sides of the lysosomal membrane may regulate the lateral mobility of Lamp2a.Lys-Hsc70induces disassembly of Lamp2a from the700-kDa complex once the substrate has crossed the membrane.A lysosome-associated form of the glial fibrillary acidic protein(GFAP),a component of the intermediatefilament network,associates to Lamp2a once it is organized into multimers and contributes to stabilizing the CMA translocation complex against the disassembling activity of Hsc70,whereas GTP-mediated release of elongation factor-1a from the lysosomal membrane pro-motes self-association of GFAP,disassembly of the CMA translocation complex and the consequent decrease in CMA[12,64].In addition,Hsp90at the lumenal side is also required to preserve the stability of Lamp2a during the transition[40].Regulation of CMA via Hsc70The other limiting lysosomal component is lys-Hsc70.As indicated in the previous section,the presence of this chaperone at the luminal side of the membrane is necessary for substrate translocation[18].Levels of lys-Hsc70 increase gradually with increasing CMA activity,although the mechanisms modulating this increase are still poorly understood.Although Hsc70contains two KFERQ sequences and is a putative substrate of CMA[21,25],it appears that neither CMA nor macroautophagy is involved in delivering lys-Hsc70to lysosomes[3,65].It is possible that this chaperone reaches lysosomes through maturation of late endosomes,a compartment in which high levels of luminal Hsc70have been detected,thus highlighting a possible relationship between CMA and endocytosis [3,65,66].Regulation of CMA via macroautophagyThe activity of CMA is also directly modulated by changes in other autophagic and proteolytic systems inside the cell. Cells in culture respond to CMA blockage by upregulating macroautophagy.Similarly,blockage of macroautophagy results in constitutive activation of CMA[65].These pathways are clearly not redundant,as CMA is,for example,unable to degrade organelles normally turned over by macroautophagy,whereas macroautophagy lacks the selectivity of CMA in the degradation of individual soluble cytosolic proteins[67].Nevertheless,the compen-satory activation of one form of autophagy when the other is compromised allows cells to preserve homeostasis,at least under basal conditions.Additionally,blockage of either form of autophagy also has a direct impact on pro-teasomal activity[3].The molecular mechanisms that regulate crosstalk between these two different pathways are currently under investigation.In the case of the interrela-tionship between macroautophagy and CMA,continuous fusion of autophagosomes to lysosomes when754W.Li et al.macroautophagy is upregulated results in transient dissi-pation of the lumenal lysosomal pH,which negatively affects lys-Hsc70stability.In fact,although lys-Hsc70is normally stable at pH ranges of5.2–5.4,changes in pH values above5.6in the lysosomal lumen result in its rapid degradation in this compartment.The reduced levels of lys-Hsc70in those lysosomes decrease their capability to perform CMA[68].Regulation of CMA via UPSUPS and autophagy were long viewed as independent, parallel degradation systems with no point of intersection. Increasing evidence shows that the UPS and autophagy are functionally interrelated catabolic processes.Specifi-cally,these degradation systems share certain substrates and regulatory molecules,and show coordinated and,in some contexts,compensatory function.For example,the neuronal protein a-synuclein can be degraded by the UPS,macroautophagy and CMA.Under conditions in which the UPS is compromised,enhanced degradation by CMA and macroautophagy may become critical to maintaining pools of amino acids for protein synthesis and may protect against the accumulation of a toxic species[1,3].On the other hand,during the acute stages of CMA blockage,there is an accumulation of poly-ubiquitinated proteins,often in the form of protein aggregates,attributable to the observed reduction in their removal through the proteasome system,and the under-lying mechanisms remain to be clarified[69]. Interestingly,recent evidence suggests that ubiquitin may play a key role in the crosstalk between proteasome-mediated degradation and selective autophagy[2,70]. Furthermore,ubiquilin,a ubiquitin-like protein,functions to regulate macroautophagy by facilitating maturation of LC3protein,and is also a substrate of CMA,indicating that ubiquilin may also be at a crossroad between protein degradation pathways[71,72].However,detailed mechanisms are under investigation.Physiological relevanceSelective degradation of cytosolic proteins via CMA con-tributes to both quality control(housekeeping)and response to stress[2,3].CMA was initially identified as an inducible pathway in response to stress.However, increasing evidence shows that there is a certain level of CMA activity under basal conditions.Most cell types analyzed to date display some level of continuous CMA activity detectable in the absence of typical CMA-inducing conditions.Basal CMA requires participation of the same effectors at the lysosomal membrane—the membrane chaperones and the protein translocation complex.It has been speculated that there is a difference between basal and inducible CMA.Whether the regulation of basal and inducible CMA occurs through different signaling mecha-nisms is under investigation[65].However,both basal and inducible CMA may have physiological relevance.Recycling and quality controlThe delivery of intracellular substrates such as misfolded proteins and damaged organelles from the cytosol to the lysosomes for degradation is crucial for cell survival. Under physiological conditions,renewal of cytosolic pro-teins is needed to maintain their normal function through recycling their old versions[4].Besides bulk autophagy (microautophagy and macroautophagy),CMA efficiently facilitates the transit of specific proteins from the cyto-plasm to lysosomes and is responsible for their recycling.If this process is inhibited,excessive old and dysfunctional proteins will accumulate in the cytosol and disturb the physiological functions of cells[3,49].Immune responseDurable adaptive immunity is dependent on CD4?T cell recognition of the major histocompatibility complex (MHC)class II molecules that display peptides from exogenous and endogenous antigens.Specialized antigen-presenting cells use the endosomal/lysosomal systems to internalize exogenous antigens,which can then be pre-sented on MHC to CD4?T cells.Beside the proteasome and macroautophagy in processing and MHC loading with endogenous and exogenous antigens,CMA has also been recently investigated in antigen processing/presentation. Cells with reduced levels of Lamp2a or Hsc70exhibit decreased presentation of cytoplasmic epitopes on class II molecules[73].Conversely,an increase in cytoplasmic autoantigen presentation is observed upon overexpression of either Lamp2a or Hsc70.Furthermore,there is cross-talk between autophagy pathways in the expression of MHC class II molecules.Macroautophagy can deliver antigens into autophagosomes for processing by acidic proteases before MHC class II presentation.However,other endog-enous antigens are processed by cytoplasmic proteases, yielding fragments that translocate via CMA into the endosomal network to intersect MHC class II.This cross-talk,particularly in response to stress,appears to balance the relative efficiency of each pathway,limits redundancy and gives MHC class II broader access to antigens within different intracellular compartments.Whether alteration in CMA activity such as its reduction with age could con-tribute to the altered immune response requires further investigation[74].Basics of chaperone-mediated autophagy755。
纳米颗粒与细胞相互作用的研究进展
P.G. Kremsner, J.F.J. Kun, Recognition of Plasmodium falciparum proteins by mannan-binding lectin, a component of the human innate immune system, Parasitol. Res.2002,88 :113~117.13 G raudal N, Madsen H0, Tarp U, et al. The association of variant mannose-binding lectin genotypes with radiographic outcome in rheumatoid arthritis.Arthritis Rheum, 2000,43(3):515~521.14 T urner MW. Mannose-binding lectin:the pluripotent molecule of the innate immune system.Immunol Today,1996,17(11):532-540.15 T re'goat V, Montagne P, Be'ne'M.C, and Faure G. Changes in the Mannan Binding Lectin (MBL) Concentration in Human Milk During Lactation.Journal of Clinical Laboratory analysis,2002,16:304~307.16 R antala A,Lajunen T,Juvonen R et al.Low mannose-binding lectin levels and MBL2 gene polymorphisms associate with Chlamydia pneumoniae antibodies.Innate Immun,2011,17(1):35~40.17 F idler K.J,Wilson P,Davies J.C,et al.Increased incidence andseverity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin.Intensive Care Med,2004,30:1438~1445.18 Y tting H,Christensen I J,Christian J.et al.Preoperative mannose-lectin pathway and prognosis in colorectal cancer.Cancer Immunol Immunother,2005,54:265~272.19 B onioto M, Braida L, Spano A, et al. Variant mannose-binding lectin aleles are associated with celiac disease .Immunogenetics, 2002, 54(8):596~598.20 M atsushita M,Hijikata M,Ohta Y. et al.Hepatitis C virus infection and mutations of mannose-binding lectin gene MBL.Arch Virol,1998,143:645~651.21 H alla MC,do Carmo RF,Silva Vasconcelos LR et al.Association of hepatitis C virus infection and liver fibrosis severity with the variants alleles of MBL2 gene in a Brazilian population.Hum Immunol, 2010,71(9):883~887.作者单位： 510282 南方医科大学珠江医院2009级本科（刘印） 510282 南方医科大学珠江医院 （田京） *通讯作者 纳米技术是当前生物医学研究的热点。
基于膜片钳放大器传递特性的细胞膜电容测量方法道
基于膜片钳放大器传递特性的细胞膜电容测量方法道摘要膜片钳细胞膜电容测量技术是检测细胞分泌活动的重要手段之一。
由于生物膜的电容率近似为常数，细胞膜电容及其变化是细胞膜表面积及其变化量的重要量化指标。
细胞胞吐过程中，囊泡膜与质膜融合将其内容物释放到胞外空间，此时将导致细胞膜表面积增加;而胞吞过程中囊泡膜回收则使细胞膜面积减小。
因此，研究和改进膜电容测量方法和技术对于更加准确地检测细胞膜电容及其变化量，推动对细胞胞吞和胞吐机制的研究具有重要意义。
为了明确研究膜片钳细胞膜电容测量方法的方向，在对基于阻抗分析的膜电容测量方法进行深入分析的基础上，将膜电容测量方法的关键技术概括为映射机制和校正机制。
映射机制是将单一频率下测量的细胞复导纳和钳位电压下的直流电导变换为细胞的三个等效电路参数;校正机制是为了消除膜片钳放大器(PCA)内部个功能模块电路对细胞响应电流产生的影响，从而获得真正的细胞复阻抗。
针对由Gillis提出并被广泛使用的利用软件锁定放大器SLIA进行细胞膜电容测量的方法在校正机制方面的不足进行了扩展改进，提出了基于膜片钳放大器传递特性的细胞膜电容测量方法(Z-H方法)，即利用PCA探头反传递函数Hsys估算细胞等效电路参数的值。
Z-H方法沿用馈电阻复阻抗Zf和通道SLIA中的sine dc方法进行参数映射，但通过划分校正类别，即硬件校正和幅度/相位校正，提出了全新的膜电容测量的校正方法。
Z-H 方法中硬件校正的目的是准确确定 PCA 中与细胞膜电容测量相关的硬件参数的实际值，是基于 PCA 传递特性的膜电容测量的预校正环节。
硬件相关参数包括PCA 探头反馈电阻的阻值和杂散电容值，PCA 输入电容、注入电容和 BNC 连接器的分布电容等。
这些参数对Z-H 方法准确确定膜电容的绝对值起至关重要的作用。
本研究提出频域方法，即f-方法，以精确确定上述各硬件参数。
f-方法利用正弦信号而非传统的方波信号，通过进行幅度/相位测量确定 PCA 的上述硬件参数。
BmNPV_侵染后不同抗性家蚕品系中肠组织转录组学分析
引用格式：刘 勇，龚椿营，艾均文，等. BmNPV侵染后不同抗性家蚕品系中肠组织转录组学分析[J]. 湖南农业科学，2023（11）：1-9，13. DOI:DOI:10.16498/ki.hnnykx.2023.011.001蚕桑文化是中国文明的起点，至少已有4 000 a 以上的历史。
家蚕作为重要的经济昆虫对人类文化、经济发展贡献巨大。
种桑养蚕至今仍是部分地区农民的重要收入来源。
然而生产上，家蚕始终受到细菌病、病毒病等的侵害，给农户造成一定的经济损失[1]。
其中，以家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus，BmNPV）引起的血液型脓病对蚕桑产业的威胁最大，其传染性极强，一旦发病就难以控制[2]。
目前，环境消毒仍是预防蚕桑病害的主要措施，但该防治方法费时费力，严重制约了蚕桑业的发展。
在此背景下，学者们通过传统杂交育种、分子标记育种和转基因育种等方式选育抗性品种来预防家蚕频发性血液型脓病，虽然这种方式前期投入大、开发周期长，但是一旦成功选育出抗性品种，其经济效益、社会效益和生态效益都是不可估量的[3]。
因此，基于材料间所存在的抗性差异而进行家蚕抗病基因筛选、抗病机制解析成为近年来蚕桑领域的热门课题。
BmNPV是一种环状双链DNA的核型多角体病毒，具有包涵体衍生病毒（occlusion-derived virus，ODV）和出芽型病毒（budded virus，BV）2种不同形式，侵染家蚕的方式包括ODV引起的食下感染和BV引起的创伤感染[3]。
BmNPV在家蚕体内复制增BmNPV侵染后不同抗性家蚕品系中肠组织转录组学分析刘 勇，龚椿营，艾均文，薛 宏，何行健，贾超华，陈卓华，任立志（湖南省蚕桑科学研究所，湖南长沙 410127）摘 要：家蚕核型多角体病毒（BmNPV）是造成蚕业严重经济损失的主要病原体之一，主要通过食下感染引发家蚕血液型脓病，中肠是免疫病原体的重要组织器官。
Part 3 membrane
细胞生物学The World of the Cell生命科学学院生物科学系细胞生物学教研室Shaojun Li1The World of the CellMembrane: their structure, function Membrane:their structure functionand chemestry生物膜2014曹建军2014An essential feature of every cell is the presence of membranes that An essential feature of every cell is the presence of membranes thatdefine the boundaries of the cell and its various internal compartments.•Plasma (or cell)Membrane（质膜、细胞膜），Intracellular membranes（细胞内y的膜）and The Endomembrane System（细胞内膜系统）FIGURE 7-1 The Prominence of Membranes Around and Within Eukaryotic Cells. Among the structures of eukaryotic cells that involve membranes are the plasma membrane, nucleus, chloroplasts, mitochondria, endoplasmic reticulum (ER),Functions of Membranes（生物膜的功能）FIGURE 7-2 Functions ofMembranes. Membranes notonly define the cell and itsorganelles but also have anumber of important functions,number of important functionsincluding transport, signaling,and adhesion.Models of membrane structureModels of membrane structure（生物膜结构模型）•Fluid mosaic model(流动镶嵌模型)：envisions amembrane as two quite fluid layers of lipids, withmembrane as two quite fluid layers of lipids,withproteins localized within and on the lipid layers andoriented in a specific manner with respect to theinner and outer membrane surfaces.inner and outer membrane surfaces•Integral membrane proteins (Bacteriorhodopsin)•Peripheral proteins•Lipid-anchored proteins•Lipid raft (脂筏)FIGURE 7-3 Timeline for Development of the Fluid Mosaic Model. The fluidmosaic model of membrane structure that Singer and Nicolson proposed in1972 was the culmination of studies dating back to the 1890s (a)–(e). Thismodel (f) has been significantly refined by subsequent studies (g and h).Northwest A&F UniversityThe FF luid 流动Mosa镶嵌模aic Mo型odelFIGURE 7-5 The Fluid Mosaic Model of Membrane Structure. These drawings show(a) representative phospholipids and proteins in a typical plasmamembrane, with closeups of (b) an integral membrane protein and (c) one of its transmembrane segments.The chemistry of Th h i fmembrane:membrane lipidsb li idand proteins•Membrane lipids: the“fluid” part of the“fl id”t f thfluid mosaic model.•Phospholipids（磷脂）•Glycolipids 糖脂）Gl li id甾醇•Sterols（甾醇）•Phospholipids•PhosphoglyceridesPh h l id•sphingolipids•Glycolipids•Tay-Sachs disease(台萨氏病): absent ofβ-N-acetylhexosaminidase A in l ysosomes•ABO blood groups•Chloroplasts: Monogalactosyldiacylglycerol (单半乳糖二脂酰甘油，MGDG) and digalactosyldiacylglycerol (DGDG)d di l t ldi l l l(DGDG)Sterols：a fluidity buffer.•a fluidity buffer.•Cholesterol，phytosterols、ergosterol(麦角甾醇)。
小檗碱抑制类风湿关节炎患者的成纤维样滑膜细胞的自噬并促进其凋亡：基于下调ROS
类风湿关节炎（RA ）是一种全身性自身免疫性疾病，主要临床表现为关节滑膜炎症、滑膜异常增生、血管翳形成以及骨和软骨破坏［1］。
成纤维样滑膜细胞（FLSs ）［2］约占关节滑膜细胞总量的70%［3］，是RA 主要的效应细胞［4］，在疾病进展过程中呈现“类肿瘤样增殖”，同时分泌多种基质金属蛋白酶（MMPs ），包括MMP2、MMP9等，以及大量促炎症细胞因子包括肿瘤坏死因子-α（TNF-α）、白细胞介素-1β、白细胞介素-6等，进而导Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathwayZONG Shiye 1,ZHOU Jing 2,CAI Weiwei 1,YU Yun 1,WANG Ying 1,SONG Yining 1,CHENG Jingwen 1,LI Yuhui 1,GAO Yi 1,WU Baihai 1,XIAN Hao 1,WEI Fang 11School of Pharmacy,Bengbu Medical College,Bengbu 233030,China;2Department of Pharmacy,Hangzhou Hospital of Traditional Chinese Medicine,Hangzhou 310007,China摘要：目的探究小檗碱（BBR ）对类风湿关节炎（RA ）成纤维样滑膜细胞（FLSs ）凋亡/自噬失衡的调控作用及机制。
方法CCK-8法检测BBR 对RA-FLSs 的增殖抑制作用，实验设空白对照组、TNF-α（25ng/mL ）组、TNF-α+BBR （10、20、30、40、50、60、70、80µmol/L ）组，Annexin V/PI 双染流式法和JC-1免疫荧光染色检测BBR 对RA-FLSs 凋亡的影响，Western blot 检测BBR 对RA-FLSs 自噬和凋亡相关蛋白表达水平的影响。
信号分子和细胞信号转导
• Reversion
配体-受体结合曲线
2. Receptors distribution
in either cell suface membrane or cytosol
Classification ：
Cell surface membrane receptors
For ligands: Water soluble, can’t enter cell Growth factor, Cytokine, Water-soluble hormone, Cell adhesion molecule Diffuse or compartmentation
Primary way to exchange of information or materials Effective in cell proliferation, differentiation even in mammalian cells
Hormone regulation:
Conmunication between cells, Long distant away
B. Transform the ligand to down-steam signal to which the cell respond.
The characters of receptor-ligand binding
• Strong specificity • High affinity • Saturation
Contact communication of cell surface molecules 膜表面分子接触通讯
T淋巴细胞
T cell: FasL (one kind of cytokine)
氧化稳定性
1.氧化稳定性测试总结：氧化稳定性是指物质抵抗氧气或其他氧化性试剂的作用而保持其性质不发生永久变化的能力。
测定方法：夏老师：分别将不同比例的复合膜放在80℃含有2mg/LFeSO4，3％wt.H2O2溶液中(此种溶液称为Fenton溶液)，通过记录膜开始溶解的时间作为评价其氧化稳定性的依据．（也有文献采用如下测定配比，高分子电解质膜的氧化稳定性的测定方法是将大小为(1厘米×1厘米)的膜浸泡在Fenton’s试剂(30 ppm浓度的FeSO4溶于30%过氧化氢的溶液，其中ppm指的是mg/L)中，温度为80o C。
该膜的氧化稳定性用膜在试剂中完全分解所用的时间表征。
）依据夏学长的测定方法，实验方法如下：1.配制1L的Fention试剂：量取30%的双氧水90ml，转移至1000ml容量瓶中，再称取七水合硫酸亚铁0.0037g在烧杯中用适量蒸馏水混合，转移至容量瓶中，加水至刻线；（30%的双氧水密度为11.1g/ml，3%的双氧水约为1.0，算得欲配制1000ml 的3%双氧水溶液，需要30%的双氧水90ml）；2.取八个烧杯，分别加入100ml的Fetion试剂，在80℃甘油浴中预热5min；3.裁取1*1cm2大小的膜，将膜置于Fenten试剂中，用保鲜膜封住烧杯口，并开始计时；3.观察，记录下膜开始溶解和彻底溶解所用的时间。
小结：一定要用保鲜膜盖住烧杯口，不然会大量蒸发；恒温不是很稳定，显示温度与温度计测量温度相差四五度；刚放进去的膜会卷曲，浮在表面，用夹子挤出其中空气使之沉下去，随着时间的推移，膜会变薄，容易浮起来挂在烧杯壁上，此时要注意观察，可以倾斜烧杯使膜泡在溶液中。
膜的变化过程大致为，先变薄，再浮到表面，然后破裂成碎片，下沉到杯底，最后渐渐消失。
溶胀:聚合物因吸收液体或气体而发生体积膨胀的现象。
离子交换树脂是亲水性高分子化合物，当将干的离子交换树脂浸入水中时，其体积常常要变大，这种现象称为溶胀。
氧化稳定性
1.氧化稳定性测试总结：氧化稳定性是指物质抵抗氧气或其他氧化性试剂的作用而保持其性质不发生永久变化的能力。
测定方法：夏老师：分别将不同比例的复合膜放在80℃含有2mg/LFeSO4，3％wt.H2O2溶液中(此种溶液称为Fenton溶液)，通过记录膜开始溶解的时间作为评价其氧化稳定性的依据．（也有文献采用如下测定配比，高分子电解质膜的氧化稳定性的测定方法是将大小为(1厘米×1厘米)的膜浸泡在Fenton’s试剂(30 ppm浓度的FeSO4溶于30%过氧化氢的溶液，其中ppm指的是mg/L)中，温度为80o C。
该膜的氧化稳定性用膜在试剂中完全分解所用的时间表征。
）依据夏学长的测定方法，实验方法如下：1.配制1L的Fention试剂：量取30%的双氧水90ml，转移至1000ml容量瓶中，再称取七水合硫酸亚铁0.0037g在烧杯中用适量蒸馏水混合，转移至容量瓶中，加水至刻线；（30%的双氧水密度为11.1g/ml，3%的双氧水约为1.0，算得欲配制1000ml 的3%双氧水溶液，需要30%的双氧水90ml）；2.取八个烧杯，分别加入100ml的Fetion试剂，在80℃甘油浴中预热5min；3.裁取1*1cm2大小的膜，将膜置于Fenten试剂中，用保鲜膜封住烧杯口，并开始计时；3.观察，记录下膜开始溶解和彻底溶解所用的时间。
小结：一定要用保鲜膜盖住烧杯口，不然会大量蒸发；恒温不是很稳定，显示温度与温度计测量温度相差四五度；刚放进去的膜会卷曲，浮在表面，用夹子挤出其中空气使之沉下去，随着时间的推移，膜会变薄，容易浮起来挂在烧杯壁上，此时要注意观察，可以倾斜烧杯使膜泡在溶液中。
膜的变化过程大致为，先变薄，再浮到表面，然后破裂成碎片，下沉到杯底，最后渐渐消失。
溶胀:聚合物因吸收液体或气体而发生体积膨胀的现象。
离子交换树脂是亲水性高分子化合物，当将干的离子交换树脂浸入水中时，其体积常常要变大，这种现象称为溶胀。
细胞凋亡及细胞程序性坏死和细胞焦亡的研究进展
中华实用诊断与治疗杂志2021年3 J!第犯卷第3期J Chin P h u m Dia仰Ther/Mar. 2〇21. V〇l. 3：,. \〇. 3• 321••综述•细胞凋亡及细胞程序性坏死和细胞焦亡的研究进展郭双.邢栋.吕勃哈尔滨医科大学附属第二K院心内科.黑龙江哈尔滨150081摘要：细胞死亡方式包括细胞凋亡、细胞程序性坏死和细胞焦亡。
细胞凋亡丨(4于非炎症性细胞死f方式.形态学不涉及 细胞质、细胞膜的破裂。
细胞程序性坏死和细胞焦亡在形态‘7:丨M]•友现为细胞质、细胞膜破裂.释放一系列炎性w f-.弓丨起炎性反应•属于炎症性细胞死亡方式。
三#在形态学及发卞机制丨:有所K別•但/(•:某邱方面乂存f t符联系。
在某邱疾 病的发卞过程中.并非仅出现一种细胞死亡方式。
水文就3种细胞死亡方式的形态学改变、发生机制及x者间关系的研 究进展作一综述。
关键词：细胞凋亡；细胞程序性坏死；细胞焦1_’：Advances in apoptosis, necroptosis and pyroptosisGUO Shuang. XIN(i Dong, LYU BoDepart/mjnt o f Curdiolof^y *the Second A J f i l i a t e d Hos/^ilal o f Harbin M edical U niversity •H a rb in，H ei/ongjiu ng l j〇081 . ChinaCorresponding author：LYU Bo, E-mail：2855928554@Abstract：C'ell death includes apoptosis, necroptosis and pyroptosis. Apoptosis is the first clise«avered mode of cell death and l)〇longs to non inflfimmntory cell death. and its morphology involves no rupture of cytoplasm or membrane. But nccroj)tosis and pyroptosis are demonstrated by rupture of cytoplasm and membrane in morphology, releasing a scries of inflammatory factors, and causing inflammatory response, therefore, necroptosis and pyroptosis belong to inflammatory cell death. Apoptosis, necroptosis and pyr〇i)tosis are different in morphology Jind have different mechanisms. l>ut they arc related with each other in some respects, and exist in some diseases at the same time. This paper reviews the morphological changes and mechanisms of these three types of cell death and the relationships among them.Keywords：ap〇i)tosis：necroptosis；pyroptosis细胞死亡是正常细胞的然终点，是不可逆的细 胞功能的终结。
IB数学SL真题
2210-600721 pagesM10/4/BIOLO/HPM/ENG/TZ1/XX+Monday 17 May 2010 (afternoon)BiologyHigHer level PaPer 1INSTRUCTIONS TO CANDIDATES• Do not open this examination paper until instructed to do so.• Answer all the questions.• For each question, choose the answer you consider to be the best and indicate your choice onthe answer sheet provided.1 hour© International Baccalaureate Organization 2010221060071. The lengths of a sample of tiger canines were measured. 68 % of the lengths fell within a rangebetween 15 mm and 45 mm. The mean was 30 mm. What is the standard deviation of this sample?A. 5 mmB. 15 mmC. 7.5 mmD. 30 mm2. Which of the following are features of prokaryotes and eukaryotes?70S ribosomes80S ribosomes Naked DNA DNA associatedwith proteinsA.prokaryote eukaryote prokaryote eukaryoteB.eukaryote prokaryote eukaryote prokaryoteC.eukaryote prokaryote prokaryote eukaryoteD.prokaryote eukaryote eukaryote prokaryote3. Which statement is part of the cell theory?A. Cells are composed of organic molecules.B. Cells have DNA as their genetic material.C. Cells have cytoplasm surrounded by a membrane.D. Cells come from pre-existing cells.2210-60074. What route is used to export proteins from the cell?A. Golgi apparatus → rough endoplasmic reticulum → plasma membraneB. Rough endoplasmic reticulum → Golgi apparatus → plasma membraneC. Golgi apparatus → lysosome → rough endoplasmic reticulumD. Rough endoplasmic reticulum → lysosome → Golgi apparatus5. Which of the following take(s) place during either interphase or mitosis in animal cells?I. Re-formation of nuclear membranesII. Pairing of homologous chromosomesIII. DNA replicationA. I onlyB. I and II onlyC. II and III onlyD. I and III only6. Which substance in prokaryotes contains sulfur?A. DNAB. PhospholipidsC. ProteinsD. Antibiotics2210-6007Turn over2210-60077.Which describes these molecules correctly?I.II.III A.ribose amino acid B.glucose amino acid C.ribose fatty acid D.glucosefatty acidC OCH 2OHH C C C C OH HH OHH OHH O HOC OH(C H 2)nCH 38. What sequence of processes is carried out by the structure labelled X during translation?XU A CA U G C C G U A C G A U C5′3′A. Combining with an amino acid and then binding to an anticodonB. Binding to an anticodon and then combining with an amino acidC. Binding to a codon and then combining with an amino acidD. Combining with an amino acid and then binding to a codon2210-6007Turn over2210-60079.The diagram below shows a biochemical pathway in a yeast cell. Which of the following correctlyidentifies a compound in the diagram?A. I is fat.B. II is pyruvate.C. III is lactate.D.IV is carbon dioxide.10. Which of the following factors influence(s) the rate of oxygen production in photosynthesis?I. TemperatureII. Wavelength of lightIII. Number of mitochondriaA. I onlyB. I and II onlyC. II and III onlyD. I, II and III11. In some people, hemoglobin always contains the amino acid valine in place of a glutamic acid at oneposition in the protein. What is the cause of this?A. An error in transcription of the hemoglobin geneB. An error in translation of the mRNAC. Lack of glutamic acid in the dietD. A base substitution in the hemoglobin gene12. What is a suspected heterozygous individual crossed with in a test cross?A. Homozygous dominantB. Homozygous recessiveC. Heterozygous dominantD. Heterozygous recessive2210-6007Turn over2210-600713. Which of the following genotypes is possible in the offspring of a homozygous male with bloodgroup A and a female with blood group B? A. I A I A B. I A i C. iiD.I B i14. What type of inheritance is shown in this pedigree chart?A. X-linked dominantB. Y-linked dominantC. X-linked recessiveD.Y-linked recessive2210-6007Turn over15. What is a community? A. A group of organisms living and interacting in the same trophic level B. A group of populations living and interacting in a food chainC. A group of organisms of the same species living and interacting in an ecosystemD.A group of populations living and interacting in an area16. What are the units of a pyramid of energy? A. kJ m yrB. kJ m yrC. J m sD.J m s17. Population size is influenced by certain factors.If I = immigration, E = emigration, N = natality, M = mortality and (N + M) = (I + E) = 0, whichphase is this population in? A. Exponential phase B. Transitional phase C. Plateau phaseD.Growth phase18. An animal has radial symmetry, a sac-like body with only one opening and tentacles with stingingstructures. To which phylum does this animal belong? A. Annelida B. Cnidaria C. MolluscaD.Porifera1− 2− 1− 1− 1− 3− 2 1−19. Which of the following are used as evidence for evolution?I. Homologous structuresII. Selective breeding of domesticated animalsIII. Overproduction of offspringA. I and II onlyB. I and III onlyC. II and III onlyD. I, II and III20. Which of the following is correct for lipase?Substrate Source pH optimumA.triglycerides pancreas pH = 8B.fatty acids small intestine pH = 7C.triglycerides small intestine pH = 9D.fatty acids pancreas pH = 921. What prevents antibiotics from being effective against viruses?A. Viruses have a high rate of mutation.B. Viruses have no RNA.C. Viruses have no metabolism.D. Viruses have a protein shell that protects them.2210-600722. The diagram below shows the human heart. What structures are indicated by the labels X, Y and Z?XZY[Source: adapted from /102spareparts/images/heart2.gif]X Y ZA.left atrium aorta semi-lunar valveB.left atrium aorta atrio-ventricular valveC.right atrium pulmonary artery atrio-ventricular valveD.right atrium pulmonary artery semi-lunar valve23. Which muscles contract to cause air to pass into the lungs through the trachea?A. Internal intercostal muscles and diaphragmB. Internal intercostal muscles and abdomen wall musclesC. External intercostal muscles and diaphragmD. External intercostal muscles and abdomen wall muscles1224. The diagram below shows the changes in membrane potential during an action potential. What occurs at the stages labelled 1 and 2?M e m b r a n e p o t e n t i a l / m V+ 60+ 40– 40– 60–100Action potential Threshold potential Resting potential0 1 2 34 5 6 7 8Time / msec12A.Na + ions diffuse in;inside becomes more positive K + ions diffuse out;inside becomes more negative B.K + ions diffuse out;inside becomes more negative Na + ions diffuse in;inside becomes more positive C.Na + ions diffuse out;inside becomes more negative K + ions diffuse out;inside becomes more positive D.Na + ions diffuse in;inside becomes more positiveK + ions diffuse in;inside becomes more negative25. What are the roles of testosterone in males? A. Stimulation of FSH production and growth in pubertyB. Pre-natal development of genitalia and development of secondary sexual characteristicsC. Development of genitalia and pre-natal secondary sexual characteristicsD.Stimulation of FSH production and pre-natal development of secondary sexual characteristics26. What is the reason for Okazaki fragments being formed during DNA replication?A. To enable replication of the 3′ → 5′ (lagging) strandB. To form the template for the RNA primersC. To initiate replication on the 5′ → 3′ (leading) strandD. To help the DNA helicase unwinding the DNA helix27. Which is correct for the non-competitive inhibition of enzymes?Inhibitor resemblessubstrate Inhibitor binds to active siteA.yes yesB.yes noC.no yesD.no no28. What is removed during the formation of mature RNA in eukaryotes?A. ExonsB. IntronsC. CodonsD. Nucleosomes29. During glycolysis a hexose sugar is broken down to two pyruvate molecules. What is the correctsequence of stages?A. Phosphorylation → oxidation → lysisB. Oxidation → phosphorylation → lysisC. Phosphorylation → lysis → oxidationD. Lysis → oxidation → phosphorylation30. Where are the light-dependent and light-independent reactions taking place in the diagram below?I IIIV IIILight-dependent Light-independentA.I IVB.II IIIC.III IID.IV I31. What are differences between monocotyledonous and dicotyledonous plants?Monocotyledonous DicotyledonousA.parallel venation;floral organs in multiples of 4 or 5net-like venation;floral organs in multiples of 3-like venation;floral organs in multiples of 3parallel venation;floral organs in multiples of 4 or 5-like venation;floral organs in multiples of 4 or 5parallel venation;floral organs in multiples of 3D.parallel venation;floral organs in multiples of 3net-like venation;floral organs in multiples of 4 or 532. The diagram below shows part of the vascular system of a dicotyledonous plant. Which process isindicated by the arrows?[FREEMAN, SCOTT, BIOLOGICAL SCIENCE, 3rd Edition, copyright 2008, p.828. Reprinted by permission of Pearson Education, Inc.,Upper Saddle River, NJ]A. Passive translocation in the phloem of sucrose from the sink to the sourceB. Active translocation in the phloem of sucrose from the source to the sinkC. Passive translocation in the xylem of sucrose from the sink to the sourceD.Active translocation in the xylem of sucrose from the source to the sink33. What controls the flowering process in long-day plants? A. P fr is converted by red light to P r which acts as a promoter of flowering. B. P r is converted by red light to P fr which acts as an inhibitor of flowering. C. P r is converted by red light to P fr which acts as a promoter of flowering.D.P fr is converted by red light to P r which acts as an inhibitor of flowering.34. A test cross resulted in these recombinants:tB Tbtb tbWhich of the following was the parental test cross?A.TB tb tb×tbB.TB tb tB×TbC.Tb tb tB×tbD.TB TB tb×tb35. What occurs during the blood clotting process?A. Prothrombin is converted into thrombin which acts on fibrinogen.B. Prothrombin is converted into thrombin which acts on fibrin.C. Fibrinogen is converted into fibrin which acts on prothrombin.D. Fibrinogen is converted into fibrin which acts on thrombin.36. What is indicated by the letters X, Y and Z?X Y ZA.sarcomere myosin filaments actin filamentsB.sarcomere actin filaments myosin filamentsC.dark band myosin filaments actin filamentsD.dark band actin filaments myosin filaments37. The diagram below shows some stages in the production of monoclonal antibodies. What are stagesX, Y and Z?[Used with the permission of Access Excellence @ the National Health Museum]XYZA.injection of antibody isolation of B-cell fusion between B-cell and tumour cell resulting in plasma cellB.injection of antibody isolation of T-cell fusion between T-cell and tumour cell resulting in plasma cellC.injection of antigen isolation of T-cell fusion between T-cell and tumour cell resulting in hybridoma cellD.injection of antigenisolation of B-cellfusion between B-cell and tumour cell resulting in hybridoma cellXYZTumour cellsTissue cultureMonoclonal antibodies isolated for cultivation38.In the diagram of the nephron below, what structures are indicated by the letters Y and Z?[Source: adapted from /sjones/SGHL12007 files/image005.jpg]YZA.glomerulus collecting ductB.Bowman’s capsule collecting ductC.Bowman’s capsule distal convoluted tubuleD.glomerulusdistal convoluted tubuleYZ– 21 –M10/4/BIOLO/HPM/ENG/TZ1/XX+ 39. What are the roles of the following structures in the production of semen?Epididymis Seminal vesicle Prostate glandA.production of a fluidproduction of fructose maturation of spermcontaining alkalinemineralsB.maturation of sperm production of a fluidproduction of fructosecontaining citric acidC.maturation of sperm production of fructose production of a fluidcontaining alkalinemineralsD.production of a fluidmaturation of sperm production of fructosecontaining alkalineminerals40. What is the role of HCG in early pregnancy?A. It prevents the degeneration of the corpus luteum.B. It initiates the development of the uterus lining.C. It inhibits the production of estrogen.D. It stimulates the degeneration of the corpus luteum.2210-6007。
抗癫痫药物的分类、作用机制与不良反应
抗癫痫药物的分类、作用机制与不良反应吴冬燕;朱国行【摘要】抗癫痫药物按作用机制可分为膜稳定剂、减少神经递质释放的药物、提高γ-氨基丁酸能的药物和其他4类。
许多抗癫痫药物的作用机制复杂，致使临床中治疗药物的选择及其不良反应均存在不确定性。
有些抗癫痫药物有肝酶诱导或抑制作用，故在多药联合治疗或与其他药物合并使用时的代谢速率发生改变。
临床上应注意抗癫痫药物的非特异性不良反应，如困倦、头晕等；也应注意各种抗癫痫药物治疗的比较严重的特异性不良反应，如拉莫三嗪可引起严重皮疹、卡马西平会引起白细胞计数降低等。
%According to drug mechanism, antiepileptic drugs can be divided into four groups: membrane stabilization, reducing neurotransmitters release, increasing γ-aminobutyric acid mediated inhibition, and the others. Many antiepileptic drugs have more than one mechanism, leading to the uncertainty of drug selection and adverse effects. Some antiepileptic drugs are liver enzyme inducers or inhibitors, resulting in changes in the metabolism, when used in multiple treatment or in combination with other drugs. Antiepileptic drugs have not only non-speciifc side effects such as drowsiness and dizziness, but also a variety of speciifc serious adverse effects such as severe rashes caused by lamotrigine and leukopenia caused by carbamazepine.【期刊名称】《上海医药》【年(卷),期】2015(000)009【总页数】5页(P3-7)【关键词】癫痫;抗癫痫药物;作用机制;不良反应【作者】吴冬燕;朱国行【作者单位】复旦大学附属华山医院神经内科上海 200040;复旦大学附属华山医院神经内科上海 200040【正文语种】中文【中图分类】R971.6吴冬燕*朱国行**（复旦大学附属华山医院神经内科上海 200040）Classification, mechanism and adverse effects of antiepileptic drugsWU Dongyan*, ZHU Guoxing**(Department of Neurology, Huashan Hospital, Fudan University, Shanghai 200040, China)抗癫痫药物的化学结构和作用机制各异，且人们对其临床效用和作用机制之间的相互关系还不完全了解，有些作用机制相似的抗癫痫药物的临床效用却存在着较大的差异。
抗生素耐药的机理(英文)
• Trigger membrane associated autolytic enzymes that destroy cell wall
• Inhibit bacterial endopeptidase and glycosidase enzymes which are involved in cell wall growth
Clin. Microbiol. Rev.10:781-791, J.Infect.Dis.162:705-710
Result
• All PBPs in S.aureus become redundant –MRSA is resistant to all ß-lactams
Mutation by Recombination with Foreign DNA
– Reduced affinity to beta lactams
• Seen as penicillin resistant Pneumococci
Beta Lactam Activity Against 100 Penicillin Resistant Pneumococci from Spain
CHз
HO
o
Carbapenems 1976-
SR N
COOH
HO
o
N
o
Clavulanic acid 1976 COOH
Mobactams
R
R-CONH
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Monobactam 1981-
N
R
Mechanisms of Action
畜牧兽医专业英语十篇课文翻译
畜牧兽医专业英语十篇课文翻译部门： xxx时间： xxx整理范文，仅供参考，可下载自行编辑畜牧兽医专业英语十篇课文翻译Lesson 1 skeletonThe skeleton is the basic framework of the animal. It giv es it shapeandsize.The skeleton carries and supports the weight of the body . It protects some organs from external damage ,e.g.ribs ribs protect the heart and lungs and skull protect s the allow the animal to move. The bones contain reserves o f some elements which the animal can mobilize when they are needed by the body(1>b5E2RGbCAP动物骨架的基本框架。
它给出了它的形状和尺寸。
骨架进行和支持身体的重量。
它可以保护从外部损伤某些器官，egribs肋骨保护心脏和肺部，骷髅保护动物移动。
骨骼含有的一些元素储备的动物时，可以调动他们所需要的身体<1）p1EanqFDPwThe skeleton develops in the unborn animal from cartilage which hardens as chemical salts are deposited in it.DXDiTa9E3d 骨架在未出生的动物从化学盐沉积在软骨硬化的发展。
In the aged animal the amount of mineral of in the bone increases. This makes the bones brittle. Bones grow in both length and thickness. The bones of the skeleton do not deve lop at the same rate in the growing animal (2> . The skull and hind limbs of a new-born lamb form a high percentage of the skeleton but this decreases as the lamb grows. Hind limbs develop faster than for e limbs.RTCrpUDGiT在老年动物矿物在骨的增加的量。
英文名词解释
Introduction of Pathophysiology1.PathophysiologyPathophysiology is a science to study on the mechanisms and laws of occurrence and development of diseases.Conspectus of Disease1.DiseaseDisease is referred as an aberrant manifestation of homeostatic disturbances caused by harmful agents.2.Causative factor / pathogenic causeCausative factor or pathogenic cause is referred as the factor that can cause a disease and determine its specificity.3.Alternation of the cause and effectAlternation of the cause and effect means that during the development of a disease, the original cause can result in certain changes of the body and these changes can in turn lead to another alternations under some conditions. That is to say, the original pathogenic factor of a disease leads to a result and the latter can be transformed into the cause of further alternations under certain conditions. In this way, the successive alternation and transformation of cause and effect promote the progression of the disease.4.Vicious cycleVicious cycle is a circular process of the alternate cause-effect, in which every alternation of cause and effect will bring about more serious injuries to the body and further spoil the health condition. The final result of this kind of alternative circulation is to cause the patient to die.5.Brain deathBrain death is a state of permanent irreversible cessation of whole brain activity. At that time, the function of the patient's body as a whole body has stopped forever.Water and Sodium Balance and Imbalance1.Hypovolemic hyponatremiaHypovolemic hyponatremia is hyponatremia with decreased extracellular fluid volume. In this situation, sodium loss is more than water loss, and serum sodium concentration falls below 130mmol/L and plasma osmotic pressure is less than 280mmol/L. It is also termed hypotonic dehydration or hypo-osmotic dehydration.2.Hypovolemic hypernatremiaHypovolemic hypernatremia is hypernatremia with decreased extracellular fluid volume. In this situation, water loss is more than sodium loss, serum sodium concentration is more than 150mmol/L, and plasma osmotic pressure is more than 310mmol/L. It is also termed hypertonic dehydration or hyperosmotic dehydration.3.Isotonic dehydrationIsotonic dehydration means that water loss is proportional to salt loss, so both the serum sodium concentration and plasma osmotic pressure are normal, but blood volume is decreased. It is also termed iso-osmotic dehydration. 4.EdemaEdema means that excessive fluid accumulates in interstitial compartment and some cavities in the body.5.HypokalemiaHypokalemia is defined as a decrease in serum K+ concentration less than 3.5 mmol/L.6.HyperkalemiaHyperkalemia is defined as an increase in serum K+ concentration more than 5.5 mmol/L.7.Hypervolemic hyponatremiaHypervolemic hyponatremia is hyponatremia with increased extracellular fluid volume resulting from excessivewater intake, meanwhile accompanied by decreased excretory function of kidney that leads to the accumulation of hypotonic fluid in exterior and interior of cells. In this situation, serum sodium concentration and plasma osmotic pressure are less than 130mmol/L and 280mmol/L, respectively, but the total amount of body sodium is normal or increased. It is also called water intoxication or hypo-osmotic overdehydration.8.PseudohyperkalemiaIn some conditions, the increase of serum K+concentration results from an artifact of laboratory measurement (due to release of potassium from blood cells during or after drawing of the blood specimen), but in fact the K+ level of plasma or serum within the body is not increased. This hyperkalemia is referred to as pseudohyperkalemia.9.Hyperpolarization blockingIn acute hypokalemia, the ICF/ECF ratio of K+ concentration increases, and the K+ efflux out of cells increases, so the voltage of the membrane potential becomes more negative than the normal resting membrane potential. At this time, the membrane becomes hyperpolarized; the difference between the resting membrane potential and the threshold potential increases with the hyperpolarization of the cell membrane; the cell membrane becomes less reactive to any stimulus that would initiate an action potential under normal circumstances. This state is referred to as hyperpolarization blocking.10.Depolarization blockingIn severe acute hyperkalemia, the resting membrane potential decreases and may fall close to the threshold potential. At this time, the cell membrane is too depolarized, which causes many voltage-gated Na+ channels to inactivate, and the action potential will not be initiated by any normal stimulus. That is, the irritability of the nerve and muscle cells is decreased. This phenomenon is termed depolarization blocking.Acid-base Balance and Imbalance1.Metabolic acidosisIt is a kind of simple acid-base disorder, which results from multiple causes and is characterized by primary reduction of [HCO3-] in plasma.2.Respiratory acidosisIt is a kind of simple acid-base disturbance，which results from the exhaust deficiency or abnormal inhale of CO2 and is characterized by primary increase of [H2CO3] in plasma or PaCO2.3.Paradoxical aciduriaIt refers to the situation that the patient with alkalemia exhibits abnormally acidic urine. Generally, the urine is basic when the patient has an alkalosis. But in the body of patient with hypokalemia, potassium ions would shift out of the cells because of low [K+] in extracellular fluid. Meanwhile, hydrogen ions outside the cells move into the cells, as a result, alkalosis in extracellular fluid ensues. At this time, because of the increase of H+and decrease of K+ in the cells, the epithelium of renal tubules secretes more H+ and less K+ into the urine to make it become abnormally more acidic.4.Paradoxical alkaluria (alkaline urine)It refers to the situation that the patient with acidemia exhibits abnormally alkaline urine. Generally, the urine is asidic when the patient has an acidosis. But in the body of patient with hyperkalemia, potassium ions would move into the cells because of high [K+] in extracellular fluid. Meanwhile, hydrogen ions in the cells move out, as a result, acidosis in extracellular fluid ensues. At this time, because of the increase of K+ and decrease of H+ in the cells, the epithelium of renal tubules secretes more K+ and less H+ into the urine to make it become abnormally more alkaline or exhibit neutral. This situation can be also found in those patients with renal tubular acidosis (RTA).5.PaCO2It refers to the partial pressure exerted by CO2 gas molecules dissolved in arterial plasma, which pressure range is normally 33～46mmHg (4.39～6.25kPa), and its average is normally 40mmHg (5.32kPa).6.Standard bicarbonate (SB)It refers to the concentration of bicarbonate in arterial plasma, which should be measured under the standard conditions. The standard conditions include a temperature of 38℃, the hemoglobin oxygen saturation of 100% and the balanced CO2 gas partial pressure of 40mmHg(5.32kPa). The normal range of this parameter is 22～27 mmol/L with an average of 24 mmol/L.7.Actual bicarbonate (AB)Under the conditions of actual hemoglobin oxygen saturation and PaCO2, the bicarbonate concentration of arterial plasma in airtight blood sample is measured. This measured parameter is termed as actual bicarbonate and reflects the actual status of an individual. Its normal value should be consistent with that of the standard bicarbonate.8.Buffer base (BB)It is the summation of all alkaline buffer substances with negative charges in blood, mainly including HCO3-, HPO42-, Pr-, Hb--, HbO2-, etc. The normal value range is 45～52mmol/L with an average of 48mmol/L9.Base excess (BE)It is a parameter measured under the standard conditions of the temperature of 38℃, the hemoglobin oxygen saturation of 100%, and PCO2 of 40mmHg. Under these conditions, the milligram molecular weight of acid or base consumed in titrating 1 litre blood or plasma sample to make its pH to 7.4. The value of base excess is positive if acid is needed, and negative if base is needed. The normal range of base excess is 0±3 mmol/L.10.Anion gap (AG)It is the deference between the concentrations of unmeasured anion (UA) and unmeasured cation (UC) in plasma (AG=UA-UC). The AG value can be obtained by calculating the difference between plasma concentration of major measured cation (Na+) and the sum of the plasma concentrations of major measured anions (Cl-and HCO3-), that is, AG = [Na+] - ([Cl-] + [HCO3-]). Its normal range is 12±2 mmol/L.11.Fixed acidsFixed acids, which are also called nonvolatile acids, are referred to as the substances which cannot be converted into gases to be removed from the body by the lungs but can be eliminated in urine by the kidneys. For example, the catabolism of some bodily substances such as certain amino acids，phospholipid and nuclear acids that contain nitrogen, sulphur and phosphorus can produce sulphuric acid, phosphoric acid and uric acid; The incomplete oxidation of carbohydrates and fats can yield some glyceric acid, pyruvic acid, lactic acid, ß-hydroxybutyric acid and acetoacetate, etc.12.Metabolic alkalosisIt is a kind of simple acid-base disorder，which is caused by multiple causes and characterized by primary increase of [HCO3-] in plasma.13.Respiratory alkalosisIt is a kind of simple acid-base disorder, which results from pulmonary hyperventilation and is characterized by primary decrease of [H2CO3] in plasma or PaCO214.Mixed acid-base disorderIt means the concurrence of two or more kinds of simple acid-base disorders in the same patient. But it is impossible that both respiratory acidosis and respiratory alkalosis occur simultaneously in the same body15.Concentrated alkalosisIt is a kind of alkalosis in which the total amount of HCO3- in the extracellular comparment does not change but its concentration rises with the decrease (or contraction) of extracellular fluid volume because of the loss of NaCl solution (e.g. administration of diuretics except the carbonic anhydrase inhibitors).16.CO2 narcosisIt refers to the situation that when PaCO2rises over 80mmHg (10.7KPa) because of the retention of CO2, the excessive high PaCO2 exerts a narcotic effect on the central nervous system.The patient with CO2 narcosis would produce those symptoms and signs including headache, fainting, dysphoria, alalia, asterixis, delirium, somnolence, twitch, even coma and respiratory inhibition, etc.17.Acid-base disturbanceAcid-base disturbance is a common basic pathological process in which, because of the actions of certain causative factors, the quantitative abnormal changes of acidic or alkaline substance within the body (overload or obvious shortage) are beyond the regulative abilities of the body, or the regulative mechanisms themselves are disrupted, or the coexistence of these two situations occurs, the normal acid-base homeostasis is damaged.ctic acidosisWhen oxygen's deficiency occurs due to various causes, glucose can be converted into lactic acid by the way of anaerobic glycolysis. If the increase of lactic acid exceeds the utilization ability of the liver, or if the lactic acid cannot be utilized sufficiently by the severely damaged liver, the concentration of lactic acid in blood will rise remarkably and a metabolic acidosis follows. At this time, the sodium bicarbonate in blood is consumed in the process of neutralizing increased hydrogen ions dissociated from lactic acid. Meanwhile, the latter's remainder, the radical of lactic acid, joins the AG. Therefore lactic acidosis is a kind of metabolic acidosis with a high AG. 19.Keto-acidosisUnder the conditions of diabetes, hunger or alcohol intoxication, etc., because of the shortage or metabolic obstruction of glucose, the catabolism of fats is accelerated so as to produce large amount of fatty acid. The latter is brought by the blood flow to the liver where it is split into ketone body. The ketone body possesses strong acidity. When the amount of ketone body exceeds the oxidative ability of peripheral tissue as well as the removal ability of the kidneys, it would be accumulated in the blood and this situation is then called keto-acidosis, which belongs to the kind of metabolic acidosis with a high AG.20.Renal tubular acidosis (RTA)It is a kind of disorder that mainly results from a defect in renal tubular excretion of hydrogen ion or in reabsorption of bicarbonate, or both. The main causes of this disorder include chronic renal failure, insufficient aldosterone secretion (Addison's disease), and some hereditary or acquired disorders leading to decreased renal tubular function, such as Fanconi's syndrome etc. Nevertheless the glomerular function maintains normal. Under this situation, serious acidemia can occur while the urine is paradoxically alkaline or neutral.Hypoxia1.HypoxiaHypoxia can be defined as a deficiency in either oxygen delivery or its utilization at the tissue level or the deficiency of both, which can lead to changes in function, metabolism and even structure of the body.2.CyanosisCyanosis refers to the violaceous color of skin and mucous membranes which occurs as the deoxyhemoglobin concentration of the blood in capillaries becomes greater than 5g/dl.3.Hemic hypoxiaHemic hypoxia refers to hypoxia resulting from a low carrying capacity of oxygen in the blood caused by an altered affinity of Hb for oxygen or a decrease in the amount of Hb in the blood.4.Enterogenous cyanosisWhen pickled vegetables containing nitrate are consumed in large amounts, the reabsorbed nitrate reacts with HbFe2+to form HbFe3+OH. The color of skin becomes coffee color. This phenomenon is called enterogenous cyanosis.5.Partial pressure of oxygen (PO2)PO2 is the tension produced by oxygen molecules physically dissolved in plasma.6.Oxygen binding capacity of hemoglobin (CO2max)CO2max is the maximal amount of O2 that can be potentially combined to hemoglobin in 100ml blood.7.Oxygen content in blood (CO2)CO2is the actual oxygen content in 100ml blood, including oxygen combined to Hb and oxygen physically dissolved in plasma (only 0.3ml/dl).8.Oxygen saturation of hemoglobin (SO2)SO2 is the percentage of haemoglobin present as oxyhaemoglobin, normally 97～99% in arterial blood and about 75% in venous blood.9.Ischemic hypoxiaThe deficiency of blood perfusion to tissues caused by decreased arterial pressure or obstruction of arteries is called ischemic hypoxia.Fever1.FeverFever is a complicated pathological process characterized by a regulated elevation of core body temperature, in which the hypothalamic set point is temporarily reset at an elevated temperature in response to pyretic substances.2.Pyrogenic activatorsFever can be caused by a number of microorganisms and non-microbial pyretic substances, which are collectively called pyrogenic activators.3.Endogenous pyrogensEndogenous pyrogens are described as cytokines inducing fever, which are produced and released by EP cells, such as interleukin-1, tumor necrosis factor, etc.4.HyperthermiaHyperthermia is described as the elevation of body temperature that occurs without changes of the set point in the hypothalamic thermoregulatory center. It occurs when the thermoregulatory mechanisms are overwhelmed.5.Exogenous pyrogensMicroorganisms and their products are also called exogenous pyrogens.Stress1.StressIt is defined as a systemic nonspecific response of the body to environmental demands or pressures made upon it.2.General adaptation syndromeIt refers to a series of physiological, psychological and behavioral adaptive responses of the body after its homeostasis is threatened and disturbed. When the stressor continues its effect on the body, general adaptation syndrome manifest a dynamic, continuous process, and can finally lead to collapse of adaptive mechanism, occurrence of diseases and even death. General adaptation syndrome is a collective term for various damages and injuries of the body caused by stress response.3.Heat shock proteinsHeat shock proteins are a family of stress proteins which synthesis is elicited or up-regulated in response to a variety of stimuli such as “heat stress”. They are considered intracellular, and non-secreted proteins which functions include helping the proper folding of newly synthesized proteins and guiding their movement, helpingthe repair, removal, and proteolysis of damaged proteins.4.StressorThe stimulus that provokes a stress response is referred to as stressor.5.Acute phase proteinIn response to stressors, such as infection, inflammation, or tissue injuries, the body is elicited to evoke rapid-mobilized defensive and non-specific responses, such as rise of body temperature, raised level of blood glucose, increase of white blood cell amounts, nuclear left shift, and increase of some plasma protein concentrations. The responses listed above are called “acute phase responses”. And the plasma proteins which level in plasma increase quickly is called 'acute phase proteins' and are secreted proteins.6.Molecular chaperoneIt is also termed as “heat shock protein”. It can participate in the proper folding, movements and maintenance of newly synthesized proteins. It also can recognize and bind to the exposed hydrophobic domain of denatured proteins to prevent them from aggregating. Then it will help the protease system produce proteolystic effects on those proteins or refold them into nature conformation.Shock1.ShockShock is a pathological process caused by various drastic etiological factors, which is characterized by microcirculation failure resulting from decreased effective circulatory blood volume and inadequate tissue perfusion with the results of cellular metabolism impediment and dysfunction of multiple vital organ.2.Auto blood transfusionAt the early stage of shock, vessel constriction because of release of a large amount of vasoconstrictors may mobilize the stored blood to participate in the circulation, which is considered as compensation of venous return.3.Auto fluid transfusionAt the early stage of shock, significant decrease of hydrostatic pressure in capillary may drive fluid to shift from interstitial space to the vascular compartment, and as a result, the plasma volume can be partly restored as a compensatory responseCoagulation-Anticoagulation Balance and Imbalance1.3P test3P test is a test for detecting fragment X of FDP. Normal serum contains no detectable level of FDP. In patient with DIC, the activated fibrinolysis can result in an increase in fragment X of FDP which usually combines with fibrinmonomer to form soluble fibrinmonomer complex. Fragment X may dissociate from the soluble complex and then fibrinmonomers are polymerized to form gelatinous fibrin precipitate in the presence of protamine. The positive result of 3P test indicates the activation of fibrinolysis and the presence of DIC.2.Disseminated intravascular coagulation (DIC)DIC is a pathological process caused by disturbance of the kinetic balance between coagulation and anticoagulation systems (including fibrinolytic system). Etiologic factors activate extensive intravascular coagulation and secondary fibrinolysis. The clinical features of DIC are bleeding, shock, organ dysfunction and microangiopathic hemolytic anemia.3.Fibrin degradation products (FDP)Fibrinolysis is initiated by fibrin clot and plasminogen activator in DIC. Plasmin can degrade fibrin to form a series of protein fragments, called fibrin degradation products (FDP). FDP may act as antithrombin to inhibit coagulation process.Heart failure1.Heart failureHeart failure is a pathological process in which the systolic or/ and diastolic function of the heart is impaired, and as a result, cardiac output decreases and is unable to meet the metabolic demands of the body.2.High-output failureHigh-output failure indicates that the cardiac output may be supra- normal but inadequate owing to excessive metabolic needs. The causes of high-output heart failure include severe anemia, fever, hyperthyroidism and pregnancy, etc.3.Myogenic dilationThe heart is overfilled to such an extent that the contractility produced by the slide of actin and myosin filaments decreases, with the result, the further increase of ventricular filling may produce a decrease in cardiac output. This kind of cardiac dilation is called myogenic dilation.4.Concentric hypertrophyIt is a type of myocardial hypertrophy. It, as a myocardial response to pressure overload, is associated with increased numbers of sarcomeres arranged in parallel (parallel hyperplasia of myocardial fibers) which lead to an increase in cardiac wall thickness without the increase of internal chamber size.5.Eccentric hypertrophyIt is a type of myocardial hypertrophy and it, as a myocardial response to volume overload, results from increased numbers of sarcomeres arranged in series (series lengthening of myocardial fibers) which produce an decrease in cardiac wall thickness with increase of internal chamber size.6.OrthopneaOrthopnea refers to shortness of breath that occurs when the patient with heart failure and pulmonary circular congestion is lying down in a horizontal position. Because of that, the patient has to sit up in a forward-leaning posture or by supporting the back of his/her body with several pillows so as to lessen dyspnea. The horizontal position redistributes body fluid, increases blood return from the extremities, leads to abdominal contents to exert pressure on the diaphragm, or reduces the efficiency of the respiratory muscles.7.Paroxysmal nocturnal dyspneaParoxysmal nocturnal dyspnea is a sudden attack of dyspnea that occurs during sleep in a recumbent posture. The patients with left heart failure wake up at night gasping for air and have to sit up or stand to relieve the dyspnea. It is precipitated by the development of interstitial pulmonary edema because of the redistribution of body fluid.8.Myocardial remodelingMyocardial remodeling is a process based on the alteration of gene expression involving myocardial cells, non-myocardial cells and extracellular matrix (ECM). It includes myocardial hypertrophy and phenotype alteration, non-myocardial cellular proliferation and ECM rebuilding.Respiratory Failure1.Dead space like ventilationIt indicates that the blood flow in the ventilated alveoli is reduced because of diseases. So the air in these alveoli cannot be exploited sufficiently. This situation is just like dead space ventilation.2.Functional shuntBecause of obstructive or restrictive ventilation dysfunction, the involved alveoli have hypoventilation but their blood flow does not reduce, thus the ratio of V A/Q is lowered, leading to the venous blood passing through the alveolar capillaries to flow into pulmonary vein without enough oxygenation. This condition is called venousadmixture, just like artery-vein shunt.3.Pulmonary encephalopathyIt means the central nervous system dysfunction resulting from chronic respiratory failure. The reason why it occurs is that hypoxia, hypercapnia and acidosis produce the damage effects on the cerebral vessels and cerebral cells, resulting in cerebral vasodilation, the increases in cerebral blood flow and cerebral vascular permeability, the edema and electric activity dysfunction of brain cells and intracellular lysosomal enzyme release.4.Acute respiratory distress syndrome (ARDS)It is a pathological process caused by severe shock, infection and intoxication, et al. Its basic pathological change is the acute injuries of alveolar- capillary membrane. ARDS induces acute respiratory failure, characterized by progressive dyspnea and refractory hypoxemia.5.Respiratory failureRespiratory failure is a syndrome, in which the severe external respiratory dysfunction leads to a PaO2 below 60 mmHg (8.0kPa) with or without a PaCO2 above 50 mmHg (6.67kPa) in a resting subject breathing air at sea level.6.Restrictive hypoventilationIt means hypoventilation caused by the restriction of alveolar dilation or inflation during inspiration.7.Obstructive hypoventilationIt means hypoventilation that caused by the stenosis or obstruction of airway and the increased airway resistance.Hepatic Failure1.Hepatic insufficiencyHepatic insufficiency is referred to a syndrome, in which severe liver damage results in severe dysfunctions, including jaundice, bleeding, infection, renal dysfunction or encephalopathy.2.Hepatic failureHepatic failure is a terminal stage of hepatic insufficiency. Hepatic encephalopathy and hepatorenal syndrome are the primary clinical manifestations.3.Hepatic encephalopathyHepatic encephalopathy is a complex, potentially reversible disturbance in central nervous system that occurs as a consequence of severe liver diseases. It is characterized by neuropsychical manifestations ranging from a slightly altered mental status to coma.4.Gamma-aminobutyric acid (GABA) hypothesisGamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. Increased GABA is observed in patients with cirrhosis, perhaps because of decreased hepatic metabolism of GABA When GABA crosses the blood-brain barrier of patients with cirrhosis, it interacts with supersensitive postsynaptic GABA receptors. Activation of the GABA receptor increases neuronal membrane permeability to Cl-by opening the Cl- ionophore. When the Cl- resting potential of the neurons is more negative than the neuronal resting membrane potential, Cl- will enter the neurons causing membrane hyperpolarization, thus leading to the dysfunction of CNS.5.Ammonia intoxication hypothesisAmmonia intoxication hypothesis holds that the patient with hepatic cirrhosis has hyperammonemia and the occurrence of hepatic encephalopathy is due to entering of ammonia into the brain. All of the neuropsychiatric symptoms are due to the poisonous action of ammonia to central nervous system.6.False neurotransmitter hypothesisIn the patient with hepatic failure, false neurotransmitters are accumulated in the synapse of the reticular structure in the brain stem. The false neurotransmitters can compete with true neurotransmitters because their chemical structure is similar to the true neurotransmitters. When false neurotransmitters replaces true neurotransmitter inthe reticular structure, the disorders of CNS occur.Renal Failure1.Renal osteodystrophyRenal osteodystrophy is a serious complication of CRF (especially, of uremia), which includes renal rickets (for children), adult osteomalacia, osteitis fibrosa, osteoporosis, osteosclerosis, and etc.2.Trade-off hypothesisTrade-off hypothesis indicates that as the nephrons are progressively destroyed, increased blood concentration of some solutes stimulate over-secretion of some related regulatory factors (such as hormones) in order to increase the excretion function. At the same time, however, high blood levels of the regulatory factors will result in some other metabolic disorders.3.UremiaIt comes from the Greek 'urine in the blood'. Uremia is a clinical and biochemical syndrome that occurs either abruptly or gradually as renal function declines acutely or chronically. Uremia is the end-stage of renal failure, with which the patients have to receive treatment in the form of dialysis or renal transplantation.4.Acute renal failure (ARF)Acute renal failure is a pathological progress, which is characterized by a deterioration of renal function over a period of hours to days, resulting in failure of kidney to excrete nitrogenous waste products and to maintain fluid, electrolyte homeostasis and acid-base balance. The patients often present azotemia, water intoxication, hyperkalemia and metabolic acidosis.5.Chronic renal failure (CRF)Chronic renal failure is a syndrome of impaired homeostasis owing to structural damage (reduced functional nephrons) of the kidneys. The disturbances are characterized by metabolic acidosis, hypocalcemia, hyperphosphatemia, alteration in vitamin D metabolism and the presence of certain toxic materials in body fluid.Ischemia- reperfusion injury1.Ischemia- reperfusion injuryThe restoration of blood flow after transient ischemia may be associated with further reversible or irreversible cell damage, which is called ischemia-reperfusion injury or reperfusion injury.2.Calcium overloadCalcium overload refers to that intracellular content of calcium is increased abnormally during ischemia and reperfusion, which results in the disorder of cellular structure and function.3.No-reflow phenomenonNo-reflow phenomenon refers to a paradoxical phenomenon that the relief of blood vessel occlusion (or other causes responsible for ischemia) cannot make the ischemic area obtain sufficient blood perfusion. The significant impairment of flow may occur at microvascular level. The main determinant of no-reflow is the activation of neutrophils in microvesseles.4.Respiratory burstNeutrophils (NADPH oxidase etc) activated by ischemia may obviously increase the production of oxygen free radicals (e.g., O2－·, H2O2) during reperfusion regaining O2 supplement, the phenomenon is called respiratory burst.5.Oxygen paradoxOxygen paradox refers to that the restoration of the oxygen partial pressure in perfusion solution after transient hypoxia may be associated with increase in membrane permeability and cell death.。
陈景元,男,1962年6月出生,辽宁宽甸人,医学博士
陈景元简历陈景元，男，1962年6月出生，辽宁宽甸人，医学博士。
第四军医大学军事预防医学系主任、长江学者特聘教授、博士生导师，国家重点学科(军事预防医学)、教育部“211工程”重点建设学科（军事特殊环境医学）带头人。
1985年7月毕业于第二军医大学临床医学专业，获医学学士学位； 1991年7月毕业于第四军医大学军队卫生学专业，获医学硕士学位；1996年7月毕业于第四军医大学生理学专业，获医学博士学位；2000－2002，美国哥伦比亚大学公共卫生学院从事博士后研究。
主要学术兼职有国际神经毒理学会（INA）常务理事，中国毒理学会理事，中华预防医学会理事，中华预防医学会环境卫生分会常委，全军医学计量科学技术委员会常委，全军军队卫生学专业委员会副主任委员，陕西省环境卫生专业委员会主任委员等，国内外多家学术期刊编委和审稿人。
学科专业：劳动卫生与环境卫生学；科研方向：环境医学和环境神经毒理学。
近年来主要从事环境神经毒理学研究和高原高寒危险因素评估及防治措施研究。
近5年共发表SCI收录论文20余篇, 5年内共主持国家“十一五”科技支撑计划重点课题、国家自然科学基金重点项目、国家863、国家重大新药创制计划项目、全军“十五” 和“十一五”专项课题13项，第一完成人获省部级（军队）科技进步二等奖3项（2005，2007，1998）。
主编（副主编）专著、教材4部。
培养博士后3名，博士研究生15名，硕士研究生18名。
总后科技银星，总后优秀教师，2008年获中华公共卫生与预防医学发展贡献奖。
近年发表科研论文目录：1.Du K, Chen Y, Dai Z, Bi Y, Cai T, Hou L, Chai Y, Song Q, Chen S, Luo W, Chen J*.Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing. Biochem Biophys Res Commu.2010, 391: 267–271.2.Luo W, Li J, Zhan D, Cai T, Song L, Yin X.-M, Desai D, Amin S, Chen J*. Bid MediatesAnti-Apoptotic COX-2 Induction Through the IKK/NF B Pathway Due to 5-MCDE Exposure. Curr Cancer Drug Tar, 2010, [Epub ahead of print].3.Wang Q, Luo W, Zhang W, Liu M, Song H, Chen J*. Involvement of DMT1 +IRE in thetransport of lead in an in vitro BBB model. Toxicol in Vitro. 2009, [Epub ahead of print].4.Liu M, Cai T, Zhao F, Zheng G, Wang Q, Chen Y, Huang C, Luo W, Chen J*. Effect ofMicroglia Activation on Dopaminergic Neuronal Injury Induced by Manganese, and Its Possible Mechanism. Neurotox Res. 2009, 16(1):42-9.5.Zheng G, Zhang W, Zhang Y, Chen Y, Liu M, Yao T, Yang Y, Zhao F, Li J, Huang C, LuoW, Chen J*. γ-aminobutyric acid A (GABA A) receptor regulates ERK1/2 phosphorylation in rat hippocampus in high doses of Methyl Tert-Butyl Ether (MTBE)-induced impairment of spatial memory. Toxicol and Appl Pharmacol. 2009; 236( 2): 239-245.6.Yang RH, Wang WT, Chen JY, Xie RG, Hu SJ. Gabapentin selectively reduces persistentsodium current in injured type-A dorsal root ganglion neurons. Pain. 2009; 143(1-2):48-55.7.Zhao F, Cai T, Liu M, Zheng G, Luo W, Chen J*. Manganese induces dopaminergicneurodegeneration via microglial activation in a rat model of manganism. Toxicol Sci. 2009;107(1):156–164.8.Luo W, Chen Y, Liu M, Chen J*. EB1089 induces Skp2-dependent p27 accumulation,leading to cell growth inhibition and cell cycle G1 phase arrest in human hepatoma cells.Cancer Invest. 2009; 27(1):29-37.9.Jin C, Bai L, Wu H, Teng Z, Guo G, Chen J*. Cellular uptake and radiosensitization ofSR-2508 loaded PLGA nanoparticles. J Nanopart Res. 2008; 10 (6):1045-105210.Zheng G, Chen Y, Zhang X, Cai T, Liu M, Zhao F, Luo W, Chen J*. Acute cold exposureand rewarming enhanced spatial memory and activated the MAPK cascades in the rat brain.Brain Res. 2008;1239:171-180.11.Yang RH, Hu SJ, Wang Y, Zhang WB, Luo WJ, Chen JY*. Paradoxical sleep deprivationimpairs spatial learning and affects membrane excitability and mitochondrial protein in the hippocampus. Brain Res. 2008;1230:224-32.12.Jin C, Bai L, Wu H, Liu J, Guo G, Chen J*. Paclitaxel-loaded poly(D,L-lactide-co- glycolide)nanoparticles for radiotherapy in hypoxic human tumor cells in vitro. Cancer Biol Ther.2008;7(6):911-6.13.Luo W, Liu J, Li J, Zhang D, Liu M, Addo JA, Patil S, Zhang L, Yu J, Buolamwini JK, ChenJ*, Huang C. Anti-cancer Effects of JKA97 are Associated with its Induction of Cell Apoptosis via a Bax-dependent, and p53-independent Pathway. J Biol Chem. 2008, 283(13):8624-33.14.Ouyang W, Luo W, Zhang D, Jian J, Ma Q, Li J, Shi X, Chen J, Gao J, Huang C. PI-3K/AktPathway-Dependent Cyclin D1 Expression Is Responsible for Arsenite-Induced Human Keratinocyte Transformation. Environ Health Persp. 2008, 116(1): 1-6.15.Du K, Chai Y, Hou L, Chang W, Chen S, Luo W, Cai T, Zhang X, Chen N, Chen Y, Chen J*.Over-expression and siRNA of a novel Environmental lipopolysaccharide responding gene on the cell cycle of the human hepatoma derived cell line HepG2. Toxicology, 2008, 243:303-310.16.Zhang XP, Zheng G, Zou L, Liu HL, Hou LH, Zhou P, Yin DD, Zheng QJ, Liang L, ZhangSZ, Feng L, Yao LB, Yang AG, Han H, Chen JY*. Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells. Mol Cell Biochem. 2008, 307(1-2):101-8.17.Cai T, Yao T, Li Y, Chen Y, Du K, Chen J*, Luo W. Proteasome inhibition is associatedwith manganese-induced oxidative injury in PC12 cells. Brain Res. 2007, 1185: 359-65.18.Zheng G, Zhang X, Chen Y, Zhang Y, Luo W, Chen J*. Evidence for a role of GABAAreceptor in the acute restraint stress-induced enhancement of spatial memory. Brain Res.2007, 1181:61-73.19.Wang Q, Luo W, Zheng W, Liu Y, Xu H, Zheng G, Dai Z, Zhang W, Chen Y, Chen J*. Ironsupplement prevents lead-induced disruption of the blood-brain barrier during rat development. Toxicol and Appl Pharmacol. 2007, 219(1):33-41.20.Wang Q, Luo W, Zhang W, Dai Z, Chen Y, Chen J*. Iron supplementation protects againstlead-induced apoptosis through MAPK pathway in weanling rat cortex. NeuroToxicology.2007, 28(4):850-859.21.Liu YL, Bi H, Chi SM, Fan R, Wang YM, Ma XL, Chen YM, Luo WJ, Pei JM, Chen JY*.The Effect of Compound Nutrients on Stress-induced Changes in Serum IL-2, IL-6 and TNF-a Levels in Rats. Cytokine. 2007, 37:14-21.22.Ding J, Li J, Chen J, Chen H, Ouyang W, Zhang R, Xue C, Zhang D, Amin S, Desai D,Huang C.. Effects of Polycyclic Aromatic Hydrocarbons (PAHs) on Vascular Endothelial Growth Factor Induction through Phosphatidylinositol 3-Kinase/AP-1-dependent, HIF-1{alpha}-independent Pathway. J Biol Chem. 2006, 281(14):9093-100. （Co-first author）23.Chen J, Yan Y, Li J, Ma Q, Stoner GD, Ye J, Huang C. Differential requirement of signalpathways for benzo[a]pyrene (B[a]P)-induced nitricoxide synthase (iNOS) in rat esophageal epithelial cells. Carcinogenesis 2005, 26(6):1035-104324.Meller E, Shen C, Nikolao TA, Jensen C, Tsimberg Y, Chen J, Gruen RJ. Region-specificeffects of acute and repeated restraint stress on the phosphorylation of mitogen-activated protein kinases．Brain Res. 2003, 979: 57-64.25.Chen J, Shen C, Meller E. 5-HT1A receptor-mediated regulation of mitogen- activated proteinkinase phosphorylation in rat brain．Eur J Pharmacol 2002, 452；155-162.26.Louis ED, Zheng W, Jurewicz EC, Watner D, Chen J, Factor-Litvak P, Parides M. Elevationof blood carboline alkaloids in essential tremor. Neurology. 2002, 59:1940–1944.27.Chen JY, Tsao GC, Zhao Q, Zheng W. Differential Cytotoxicity of Mn(II) and Mn(III):Special Reference to Mitochondrial [Fe-S] Containing Enzymes．Toxicol and Appl Pharmacol. 2001, 175, 160–168.。
内质网-线粒体结构偶联及运动应激研究进展
内质网-线粒体结构偶联及运动应激研究进展孙易;丁树哲【摘要】诸多生命活动依赖于线粒体和内质网的协作.MAMs是存在于线粒体和内质网间,由蛋白质复合体组成的特殊结构,其结构的完整性和生理功能的正常运行是保证线粒体动态变化、细胞凋亡和内质网应激等生命过程有序进行的前提.对MAMs的最新研究动态进行归纳,提出Ca2+在MAMs调控中的决定性作用,MAMs在ROS、内质网应激、细胞凋亡、细胞自噬、线粒体动态变化及流动性和炎症等过程中扮演的重要角色,探讨运动应激对MAMs的可能调节机制及MAMs相关分子介导运动适应的途径,从而为未来运动适应机制的探索提供新的研究方向.【期刊名称】《体育科学》【年(卷),期】2017(037)008【总页数】8页(P50-57)【关键词】线粒体;内质网;MAMs;运动应激;Ca2+【作者】孙易;丁树哲【作者单位】华东师范大学青少年健康评价与运动干预教育部重点实验室,上海200241;华东师范大学体育与健康学院,上海 200241;华东师范大学青少年健康评价与运动干预教育部重点实验室,上海 200241;华东师范大学体育与健康学院,上海200241【正文语种】中文【中图分类】G804.6膜包裹细胞器的出现标志着物种进化进入新的时代，不同属性的生化反应得以在相对隔离的微环境中进行。
与此同时，为了保障生命活动和分子反应的正常运行，牟定在细胞器间的固态连接网络（physical contact）衍生而来，以利于代谢底物和生物信号在细胞器间半自由传递。
固态连接网络同时肩负着细胞凋亡、免疫调节和调控细胞器动态变化等重任。
存在于相邻细胞器间的并由两部分细胞器膜组成的特殊结构称作膜连接部位（MCSs，membrane contact sites）［14］。
MCSs具有以下4个特点：1）两种细胞器膜间距一般小于30 nm；2）膜互不融合：3）某些蛋白和脂质在MCSs处格外丰富汇聚，充当系带（tether）的作用；4）MCSs的特有结构影响两细胞器中至少1种的结构或功能［42］。
线粒体E3泛素连接酶MARCH5表达上调促进肝癌生长
•424 •现代生物医学进展 Progress in Modern Biomedicine VoL21 NO*3 FEB»2021doi: 10.13241/ki.pmb.2021.03.005线粒体E3泛素连接酶MARCH5表达上调促进肝癌生长*耿西林1张静1李晖1杨宇宇2张煜1常虎林1A(1陕西省人民医院肝胆外科陕西西安7丨〇〇68;2通用环球西安西航医院外一科陕西西安710021)摘要目的：膜相关辞指蛋白MARCH5(membrane-associatedRING-C H5)是定位于线粒体外膜的E3泛素连接酶，在调控线粒体分裂融合相关蛋白的表达中发挥重要作用。
以往研究在多种肿瘤中证实了线粒体分裂融合的异常，但目前MARCH5在肝癌中的表达与生物学作用均不清楚。
本研究旨在探讨MARCH5在肝癌组织与细胞系中的表达及其在肿瘤生长中的调控作用。
方法：1).利用免疫组化实验检测62对肝癌癌与癌旁组织中MARCH5表达，以明确MARCH5在肝癌中的表达是否发生了异常改变。
2)_ 利用qRT-PCR与Western b lo t实验检测4株肝癌细胞（SNU-354、SNU-368、H LE与HLF)与1株正常肝细胞HL7702中MARCH5表达，进一步分析MARCH5在肝癌细胞系中的表达改变。
3).下调肝癌细胞中MARCH5表达后，利用E D U实验与克隆形成实验分析对肝癌细胞增殖与克隆形成能力的影响。
结果：1).MARCH5在肝癌组织中表达显著高于癌旁组织。
2).MARCH5 在4株肝癌细胞中的表达均显著高于正常肝细胞。
3).下调MARCH5表达可显著抑制肝癌细胞的增殖与克隆形成。
结论：MARCH5在肝癌中表达显著上调并通过诱导增殖与克隆形成而促进肝癌的生长。
关键词:MARCH5;线粒体；增殖；克隆形成；肝癌中图分类号:R-33;Q244;R735.7 文献标识码:A文章编号：1673-6273(2021 )03-424-05Over-expression of MARCH5 Promotes Tumor Growth in Liver Cancer* GENGXi-Iin', ZHANG Jing1, LIH ui1, YANG Yu-yii, ZHANG Yu', CHANG Hu-lin'A(1 Department o f H epatobiliary Surgery, Shaanxi Provincial People's Hospital, Xi'an, Shaanxi, 710068, China;2 Department o f g eneral surgery, Xi'an Xihang hospital, Xi'an, Shaanxi, 710021, China)ABSTRACT Objective:MARCH5 (membrane-associated RING-CH 5) is an E3 ubiqutin-protein that localized in mitochondrial membrane and functions as a regulator of mitochondrial morphology.Previous studies have indicated the close link between mitochondrial dynamic dysfunction and cancer.However,the expression and biological functions of MARCH5 in liver cancer remains unclear. The present study aims to explore the expression and tumor growth promotive role of MARCH5 in liver cancer.Methods: 1.Immunohis-tochemistry analysis was applied to evaluate the expression of MARCH5 in 62-paired tumor and peritumor tissues of liver cancer. 2. MARCH5 expression was further evaluated in four human liver cancer cell lines(SNU-354, SNU-368, HLE and HLF)and one normal liver cell line HL-7702. 3.The effects of MARCH5 knockdown on the proliferation and colony formation were determined by EDU (5-Ethynyl-2'-deoxyuridine)and colony formation assays in liver cancer SNU-368 cells.Results: 1.MARCH5 expression was significantly increased in tumor tissues of liver cancer when compared with peritumor tissues(P<0.001). 2.MARCH5 expression was significantly higher in four liver cancer cell lines than in normal liver cancer cell. 3.MARCH5 knockdown suppressed both proliferation and colony formation in liver cancer cells.Conclusion:MARCH5 over-expression promotes liver cancer growth through increasing cell proliferation and colony formation.Key words:MARCH5; Mitochondrial;Proliferation;Colony formation;Liver cancerChinese Library:R-33; Q244; R735.7 Document code:AArticle ID: 1673-6273(2021)03-424-05刖目线粒体在细胞能量代谢、凋亡、钙离子与氧化还原稳态调 控中发挥重要作用M。
植物光信号转导
植物光信号转导LI Xiukun;XU Dongqing【摘要】植物的生长发育受到多种环境因素的影响,而光信号是其中最重要的环境信号之一.从植物的种子萌发开始到完成整个生命周期,光参与调控其中几乎每一个生长和生理发育过程.植物自身进化了一整套复杂而精细的光信号转导系统,以应对自然界中时刻变化的光环境.经过30多年的研究,植物光生物学家确定了光受体-E3泛素化连接酶复合体-转录因子为主要调控途径的光信号转导体系.这一信号转导网络控制了植物体内将近1/3基因的表达,从而在分子层面上确保植物的正常生长发育.【期刊名称】《自然杂志》【年(卷),期】2019(041)003【总页数】5页(P183-187)【关键词】光信号;光受体;COP/DET/FUS【作者】LI Xiukun;XU Dongqing【作者单位】;【正文语种】中文太阳光是植物能量的主要来源。
除此之外，光还作为一个重要的环境信号因子影响和调控植物的诸多生长发育和生理过程。
自20世纪90年代以来，植物光生物学家借助分子生物学和分子遗传学的发展和各种新的技术方法，对植物光信号转导进行研究并有突破性的进展。
植物接受光信号并传递至下游，进而作出适时的应答反应，以适应时刻变化的光环境。
植物是固生生物，从种子落地发芽开始，就会在一个固定的地点完成整个生命周期。
但是，植物并不像我们看到的那样静止不动，在微观层面上可谓是瞬息万变。
不同光环境下，植物启动精密的光信号转导系统以应对不同的光质、光强、光照时间和方向，确保其自身的健康生长。
1 植物识别光信号的物质——光受体阳光普照一切生物，为大自然带来生机。
所谓大海航行靠舵手，万物生长靠太阳。
自1906年美国科学家Garner和Allard发现光对植物生长发育的影响开始，全世界的科学家就从未停止过光对植物作用的研究。
在数万年的进化过程中，植物为了感知和识别光信号已经进化出几类不同的光受体。
它们是识别280～315 nm 的 UV-B 信号的紫外光受体 UVR8[1-2] 、吸收波段为315～500 nm 的 UV-A和蓝光受体隐花色素 1 和 2(CRY1和CRY2)以及向光素 1 和 2(Phot1和Phot2)[3-5] ，而光敏色素(PHYA～PHYE)主要吸收600～750 nm 的红光和远红光[6] (图1)。
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Changes in membrane-associated H+-ATPase activities andamounts in young grape plants during the cross adaptationto temperature stressesJun-Huan Zhang a,b,Yue-Ping Liu a,c,Qiu-Hong Pan a,Ji-Cheng Zhan a,Xiu-Qin Wang a,Wei-Dong Huang a,*a College of Food Science and Nutritional Engineering,China Agricultural University,Beijing100083,Chinab Institute of Forestry and Pomology,Beijing Academy of Agriculture and Forestry Science,Beijing100093,Chinac Department of Biology Technology,Beijing Agriculture College,Beijing102206,ChinaReceived14July2005;received in revised form17November2005;accepted18November2005Available online9December2005AbstractProton pumps make a critical contribution to the physiology of plants,although it remains unclear whether or not membrane-associated H+-ATPase is involved in the cross adaptation to different temperature stresses.This experiment investigated the changes in membrane-associated H+-ATPase activities that associated with chilling-treated plants after heat acclimation(HA,388C/10h)and with heat-treated plants after cold acclimation(CA,88C/2.5days)in annual young grape plants(Vitis vinifera L.cv.Jingxiu)using biochemical and electron microscopic cytochemical assay methods in which cerium trichloride(CeCl3)precipitation was adopted.The results indicated that plasma membrane H+-ATPase activity increased as a result of both pretreatments,while V-type and F-type H+-ATPase activity hardly changed.Under subsequent cross temperature stresses,however,the three H+-ATPase types did maintain higher activity levels than that of the control.Thisfinding suggests that either a HA or CA pretreatment may promote stability in membrane-associated H+-ATPase.A western-blotting assay of the plasma membrane H+-ATPase(P-H+-ATPase)indicated that the immuno-signal intensity of a100kDa peptide was visibly stronger in the HA and CA pretreated plants than in the control both before and after stress.This suggests that the HA-or CA-induced P-type H+-ATPase activation can be partly attributed to a new synthesis of the enzyme protein.Further,the results also suggested that membrane-associated H+-ATPase was involved in the HA-induced chilling resistance and the CA-induced thermo-tolerance in grape plants and that they had a similar regulating mechanism.#2005Elsevier Ireland Ltd.All rights reserved.Keywords:Cold acclimation;Cross adaptation;H+-ATPase;Heat acclimation;Young grape plants1.IntroductionTemperature induced stress is an important environmental factor that influences the growth and development of plants. Previous studies on a number of plant species have shown that thermo tolerance can be enhanced by heat acclimation[1,2],while chilling injury can reduce using cold acclimation techniques[3,4].In addition,an increasing number of studies have shown the existence of cross adaptation in plants where exposure of a plant to a moderate stress not only induces resistance to a specific stress,but can also improve the plant’s tolerance to other stresses as well[5,6].For example,Fu et al.[7]found that CA puma rye seedlings had a higher heat tolerance when exposed to cold than seedlings not exposed to cold or only partially cold hardened.Ma et al.[8]reported that HA and CA induced thermotolerance to cucumber seedlings by increasing both salicylic acid and soluble protein content,as well as various enzyme activities(peroxidase,superoxide dismutas and catalase)during heat stress.Structural and functional stability of membrane is crucial in plant adaptation to temperature stresses[9,10],and damage to/locate/plantsciPlant Science170(2006)768–777Abbreviations:ATP,adenosine triphosphate;BCIP/NBT,5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium;BSA,bovine serum albu-min;CA,cold acclimation;DTT,dithiothreitol;EDTA,ethylenediamine terra-acetic acid;HA,heat acclimation;Mes,2-(N-morpholino)ethane sulfonic acid;PM,plasma membrane;PMSF,phenylmethylsulfonylfluoride;PVPP,poly-vinylpolypyrrolidone;Tris,tris(hydroxymenthyl)-amino methane*Corresponding author.Tel.:+861062737024;fax:+861062737553.E-mail address:huanggwd@(W.-D.Huang).0168-9452/$–see front matter#2005Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.plantsci.2005.11.009membrane structure can led to changes in membrane semi-permeability as well as changes in some membrane-localized enzymes[11,12].H+-ATPase is a membrane-localized protein its plasma membrane form is often referred to as the‘‘master enzyme’’of plant cells which plays important role in plant physiology and biochemistry[13].Both the plasma membrane H+-ATPase and tonoplast H+-ATPase act as primary transpor-ters by pumping protons out of the cell or into the lumen of the vacuole,and thus creating pH and electrical potential differences across the plasma membrane or tonoplast.This electrochemical gradient is subsequently used as the driving force for the secondary transport of ions and nutrients into and out of the cell[14,15].Because of its importance,H+-ATPase activity is expected to be modulated to cope with environmental and metabolic changes[16–18].Recent studies have shown H+-ATPase to be very sensitive to abiotic stresses,such as salinity, drought and low temperature[19–21].At the same time, impairment of tonoplast H+-ATPase is considered to be the initial physiological response of cells to chilling in mung bean [22],while H+-ATPase activity in leaf cells subject to heat shock was enhanced by moderate heat pretreatment in maize seedlings[23].Zhang et al.[24]found that the stability of the cell membrane system in young grape plants under chilling stress can be increased by a HA pretreatment,and that a CA pretreatment can protect the membrane structure against subsequent heat stress.The purpose of the current study is to examine the contribution of H+-ATPase to the mechanism of plant cross adaptation to temperature stresses in young grape plants. Furthermore,the changes in amounts of plasma membrane H+-ATPase were also analyzed by Western blot.2.Materials and methods2.1.Plant materialAnnual young grape plants(Vitis vinifera L.cv.Jingxiu) were planted in pots containing a mixture of soil,vermiculite and humus(1:1:1,v/v,ratio).The young plants were grown in a greenhouse under controlled conditions(278C day/188C night cycle,200m mol photons mÀ2sÀ1light intensity and relative humidity of75–80%).Plants with10functional leaves were chosen for experimentation.2.2.Heat acclimation pretreatment and chilling stress treatmentYoung plants were transferred to a chamber set at 38.0Æ0.58C for10h under200m mol/m2per s light intensity with75–80%relative humidity,and then allowed to recover at 25.0Æ0.58C for2h before being subjected to chilling stress. Control plants were pretreated in another chamber set at 25.0Æ0.58C under the same conditions.Both the recovered plants and the control plants were transferred to0.0Æ0.58C chamber with a darkness regimen of0,1,3,6and12h.Samples were collected and immediately used or frozen in liquid nitrogen atÀ808C before storage.The samples used for cytochemical analysis were taken from the leaf blades of the inferior fourth leaf of HA plants,non-acclimated(NA)plants, HA and chilled plants(HAC),and NA and chilled plants(NAC) after cold treatment for4or10h,respectively.Each treatment had at least three independent replicates,each replicate consisting of nine plants.2.3.Cold acclimation pretreatment and then heat stress treatmentYoung plants were transferred to a chamber set at 8.0Æ0.58C(the temperature used was confirmed by preliminary experiments)under the same photoperiod and the same relative humidity as described above for2.5days. Control plants were simultaneously transferred to another chamber set at25.0Æ0.58C under the same conditions.Then, the CA plants were allowed to recover at25.0Æ0.58C for2h. Afterwards,for the high-temperature stress,both the recovered plants and the control plants were transferred to45.0Æ0.58C for0,0.5,1,3and6h.Samples were taken at different times and immediately used or frozen in liquid nitrogen atÀ808C before storage.The samples used for cytochemical analysis were taken from the leaf blades of the inferior fourth leaf of CA plants,non-acclimated plants(NA),CA and heat-stressed plants(CAH),and non-acclimated and heat-stressed plants (NAH)under heat stress for2or4h,respectively.Each treatment had at least three independent replicates and each replicate contained nine plants.2.4.Determination of membrane permeabilityMembrane permeability was determined by conductivity (L t/L0)[25].Leaves from treated plants were sliced into small discs(1.0cm diameter,0.5g)and placed inflasks with30mL deionized water.Theflasks were slightly shaken on a rotary shaker at258C for30min and the electrical conductivity,L t,of the solution measured using a conductivity meter.Flasks with solution were then heated at1008C for15min,cooled quickly, and the electrical conductivity of the solution(L0)measured. Each experiment was repeated three times.2.5.Cytochemical localization and quantification of of H+-ATPase activityATPase activity was cytochemically analyzed according to methods described in Jian et al.[26]and Peng et al.[27]with some modification.Rectangular segments(1mmÂ2mm) were cut from the leaf between the second and third principal vein,5mm from the midvein.The shorter axis of the segment was always situated along the vein.Segments were prefixed at room temperature for2h with a mixture of4%(w/v) paraformaldehyde and1%(v/v)glutaraldehyde(buffered to pH7.2with50mM sodium cacodylate).The penetration of the mixture was improved by vacuum pumping.The leaf samples were then washed twice(0.5h each time)with50mM sodium cacodylate(pH7.2)and with50mM Tris–maleate buffer(pH 7.2).The samples were incubated at378C for2h in a reactionJ.-H.Zhang et al./Plant Science170(2006)768–777769medium consisting of50mM Tris–maleate buffer(pH7.0), 2mM ATP-Na2,5mM MgSO4and3mM CeCl3.Two control reactions were conducted to demonstrate the specificity and reliability of the assay:(1)ATP(substrate)was omitted from the reaction medium;(2)10mM sodiumfluoride(NaF)was added to the incubation medium.After incubation,the samples were rinsed with50mM Tris–maleate buffer several times and twice with50mM sodium cacodylate(each for0.5h).After rinsing,the samples werefixed in a cacodylate buffer(pH7.2) containing3%(v/v)glutaraldehyde for3h at48C,and then rinsed with50mM sodium cacodylate three times(each for 0.5h).The samples were then post-fixed overnight in a cacodylate buffer(pH7.2)containing1.5%osmium tetroxide (OsO4)at48C.Following another extensive rinsing with a cacodylate buffer and double-distilled water,the samples were dehydrated in a graded ethanol series(30–100%)(v/v)and 100%(v/v)acetone.The leaf samples were infiltrated with Spurr resin(Dow Chemical Co.,New York,USA)for24h at room temperature.Polymerization was conducted at608C for 24h.Ultrathin sections(60–80nm)were cut with a diamond knife on an LKB-8800ultramicrotome,mounted on copper thin-bar grids(100mesh)coated with0.3%Formvarfilm, stained with uranyl acetate/lead citrate,and photographed under the JEM-100S transmission election microscope(JEM Technologies,Kyoto,Japan).Three leaves were collected from different plants subjected to the specified temperature treat-ments.Five segments were cut from each leaf and several cells in each segment were analyzed.Quantification of labeling H+-ATPase activity:Sampling was carried out over a number of micrographs taken randomly from all the cells of interest on each grid.The number of the density of the electron grains representing H+-ATPase activity was defined as the number of grains per area unit(m m2).This number of grains was determined by hand counting particles over the plasma membrane.For each stage labeling density was expressed as mean labeling density of all micrographsÆS.D.2.6.Microsome preparationMicrosome from leaves was prepared according to methods described in Ferrol[28]with minor modification.Leaves from treated plants were frozen with liquid nitrogen and then crushed.The crushed leaves were homogenized in a1:3(w/v) cold medium(leaves:medium)containing100mM Tris–HCl (pH7.5),250mM sucrose,1mM MgCl2,2mM EDTA,10% (v/v)glycerol,5mM ascorbic acid,1mM PMSF,5mM dithiothreitol(DTT),5m g/mL Leupeptin,5m g/mL Pepstatin, 5m g/mL Aprotinin and2%(w/v)PVPP.The homogenate was filtered through four layers of600m m nylon cloth,and the filtrate centrifuged at12,000Âg for20min to yield a supernatant.The supernatant was centrifuged again at 100,000Âg for1h and the resulting microsomal pellet re-suspended in a suspension buffer containing5mM Tris–Mes (pH7.0),1mM DTT,0.5mM PMSF and10%(v/v)glycerol. The suspended microsome was either kept at08C for immediate use or frozen with liquid nitrogen and stored at À808C.All steps were performed between0and48C.2.7.H+-ATPase activity assayATPase hydrolysis assays were performed according to Peng et al.[27].Microsome proteins(10m g)were added to0.5mL of reaction medium(30mM Tris–Mes[pH6.5for P-type H+-ATPase(=P-ATPase),pH8.0for V-type H+-ATPase(=V-ATPase),F-type H+-ATPase(=F-ATPase)],3mM MgSO4, 0.5mM NaMoO4,0.01%(v/v)Triton X-100and corresponding inhibitors(50mM KNO3and1mM NaN3for P-ATPase assay, 0.1mM NaVO4and1mM NaN3for V-ATPase assay,and 0.1mM NaVO4and50mM KNO3for F-ATPase assay))using 30mM of ATP to start the reaction.After incubation for30min at378C,the reaction was stopped by adding250m L10%(w/v) sodium dodecyl sulphate(SDS).Total inorganic phosphate(Pi) produced from the ATP hydrolysis was determined according to methods described in Ames[29].Phosphatase activity was estimated at both pH6.5and pH8.0as the difference between the value measured in the ATPase assay medium in the absence of0.5mM NaMoO4and that in the presence of0.5mM NaMoO4.2.8.Protein determinationProtein concentration was determined according to Bradford [30]using a bovine albumin(BSA)as standard.2.9.Western blot analysis and quantitative estimation of P-H+-ATPaseThe microsome protein was separated by SDS-PAGE according to Laemmli[31]and electro-transferred to nitro-cellulose(0.45m m,Amersham LIFE SCIENCE)using a transfer apparatus(Bio-Rad)described in Zhang and Wang [32].After rinsing in TBS buffer(10mM Tris–HCl,pH 7.5,150mM NaCl),the membrane was blocked for2h at room temperature with3%(w/v)BSA in0.05%(v/v)Tween20and TBS,then incubated with gentle shaking for3h at room temperature in a1000-fold diluted primary antibody against plasma membrane H+-ATPase(a kind gift from Dr.R.Serrano). Following extensive washes with TBST buffer[TBS,0.05%(v/ v)Tween20],the membrane was incubated with goat anti-rabbit IgG-alkaline phosphatase conjugate(Sigma,St.Louis; 1:1000diluted in TBST)at room temperature for1h and then washed with TBST and TBS.The membrane was stained with a 10mL solution containing nitro blue tetrazolium(NBT)and5-bromo-4-chloro-3-indolyl phosphate(BCIP)in the dark,and the reaction terminated by adding double distilled water.The amount of P-H+-ATPase protein was quantified by scanning the NC membranes after immunoblotting with densitometer using ImageQuant software.2.10.StatisticsAll treatments were repeated at least three times and all samples were analyzed three times.Means and standard errors were calculated from pooled data.In thefigures,the vertical line associated with each point represents the standard error.J.-H.Zhang et al./Plant Science170(2006)768–777 7703.Results3.1.Effects of HA or CA pretreatment on the membrane permeability under chilling or heat stress in young grape plantsPrice and Hendry have suggested that the physical state of cell membranes is highly sensitive to temperature stress and may be the first part of the plant damaged when subjected to chilling injury [33].Both solute and electrolyte leakage are generally used as direct parameters for describing membrane injury.As shown in Fig.1a,the relative conductivity of control leaves (quantified by L t /L 0)increased to a much higher level than that of HA leaves during chilling treatment,especially after 3h,suggesting that HA pretreatment could effectively alleviate the damage caused by chilling stress in grape plants.No significant difference was found between the relative conductivity of the CA and control plants before heat treatment.During the subsequent heating treatment,however,the relative conductivity in the CA plants was always much lower than thatJ.-H.Zhang et al./Plant Science 170(2006)768–777771Fig.1.Effects of heat acclimation (HA,388C/10h)and cold acclimation (CA,88C/2.5days)on the relative conductivity in the leaves of young grape plants under cross temperature stresses.Following that the plants were treated as described in Section 2,the leaves were collected form each treated plants and the relative conductivity was measured.(a)Changes in relative conductivity in HA-pretreated leaves during cold (08C)stress.(b)Changes in relative conductivity in CA-pretreated leaves during heat (458C)stress.Values were means ÆS.D.(n =3).Fig.2.Effects of heat acclimation (HA,388C/10h)on the membrane-associated P-,V-and F-type H +-ATPase and phosphatase (PPase)activities in the leaves of young grape plants under cold (08C)stress.The plants were treated as described in Section 2.The microsomes for H +-ATPase assay were prepared from HA-pretreated leaves and control leaves under cold stress for 0,0.5,1,3,6and 12h,respectively.P-type H +-ATPase was assayed at pH 6.5and V-,F-type H +-ATPase at pH 8.0.(a)The changes in P-type H +-ATPase activities.(b)V-type H +-ATPase activities.(c)F-type H +-ATPase activities.(d)Phosphatase activities were measured at pH 6.5and 8.0and they were undetectable at pH 8.0;so only those measured at pH 6.5,which were very low,are presented in figure.Values were means ÆS.D.(n =5).of the control plants (Fig.1b),indicating that damage caused by heat stress might be reduced when CA is used as a pretreatment.3.2.The activities of microsomal membranes H +-ATPase during cross adaptationAs shown in Fig.2,HA pretreatment activated both P-and V-type ATPase,while F-ATPase activity decreased after HA.During the subsequent chilling stress,the activities of P-and F-type ATPase were sharply reduced in both the control and HA pretreated leaves,with the greatest reduction in activity occurring in the control leaves (Fig.2a and c).For V-ATPase,overall activity remained elevated during the chilling stress (0.5–6h)and reached a peak value (11.38m mol Pi mg À1protein)at 6h before decreasing thereafter (but still higher than that in normal leaves).When compared with HA plants,V-ATPase activity in the control plants was significantly reduced throughout the chilling stress,and always much lower than that in HA plants (Fig.2b).Phosphatase-like activity at pH 8.0(at which V-,F-ATPase was assayed)was undetectable and possibly inhibited by molybdate in the grape leaves (the data was not shown).As shown in Fig.2d,phosphatase-like activity at pH 6.5(at which P-ATPase was assayed)was very low and apparently not induced by HA pretreatment in spite of the chilling stress (with or without).This suggests that the divergence in the ATPase assay from the phosphatase activity is negligible in plants.The results also suggest that HApretreatment induced the stability of H +-ATPase activity under chilling stress.The CA pretreatment did not lead to any significant decrease in either V-H +-ATPase or F-H +-ATPase activity,and only a very slight increase in P-H +-ATPase activity (Fig.3).However,during subsequent heat stress conditions,the activity of all three types of H +-ATPase in the CA-pretreated leaves was significantly higher than that in the corresponding controls,although H +-ATPase activity decreased continuously (Fig.3a–c).As with the HA pretreatment (Fig.2d),membrane-associated phosphatase activity for the CA pretreatment was very low and could not effectively activate phosphatase,and thus reduce damage to the enzymes during subsequent heat stress (Fig.3d).This supports the belief that the CA pretreatment can protect H +-ATPase activity from inactivation due to heat stress in grape plants.3.3.Subcellular observation of H +-ATPase during cross adaptationWhen sample sections were incubated in a H +-ATPase complete reaction medium,ATP was hydrolyzed by the H +-ATPase to ADP and inorganic phosphate (Pi).The Pi was then precipitated out by cerium to fine-grained products of cerium phosphate (CePO 4),which could be seen under an electron microscope (EM).Membrane-associated phosphatase may also hydrolyze ATP and interfere with this technique,but theJ.-H.Zhang et al./Plant Science 170(2006)768–777772Fig.3.Effects of cold acclimation (CA,88C/2.5days)on membrane-associated P-,V-and F-type H +-ATPase and phosphatase (PPase)activities in the leaves of young grape plants under heat (458C)stress.The plants were treated as described in Section 2.The microsomes for H +-ATPase assay were prepared from CA-pretreated leaves and control leaves under heat stress for 0,0.5,1,3and 6h,respectively.P-type H +-ATPase was assayed at pH 6.5and V-,F-type H +-ATPase at pH 8.0.(a)P-type H +-ATPase activities.(b)V-type H +-ATPase activities.(c)F-type H +-ATPase activities.(d)Phosphatase activities were measured at pH 6.5and 8.0and they were undetectable at pH 8.0;so only those measured at pH 6.5,which were very low,are presented in figure.Values were means ÆS.D.(n =5).J.-H.Zhang et al./Plant Science170(2006)768–777773Fig.4.Effects of heat acclimation(HA,388C/10h)on the cytochemistrical localization and activity of plasma membrane H+-ATPase in the mesophyll cell of young grape plants under cold(08C)stress.(A)Image of electron microscopy and(B)quantitative analysis of H+-ATPase activity labeling in plasma membrane.(a)Cytochemical localization of H+-ATPase on the plasma membrane in leaf cells of control grape plants under optimum temperature.(b)Heat acclimated plants cells,showing the higher plasma membrane H+-ATPase activity(P-H+-ATPase)than those in control plants.(c)Control plants treated at08C for4h,showing the decrease of P-H+-ATPase activity.(d)HA plants treated at08C for4h,there was no obvious change in P-H+-A TPase activity,as compared with the HA plants without cold stress. The less V-H+-ATPase activity was also observed.(e)There was a further decrease of ATPase activity in control plants treated at08C for10h,and the plasma membrane was detached from the cell wall.(f)HA plants treated at08C for10h,still remaining the higher P-H+-ATPase activity.(g)There was no P-H+-ATPase activity without ATP in the incubating substrate.(h)P-H+-ATPase activity was abolished after NaF(0.01mol/L)was added into the incubating substrate.Bar=1m m.phosphatase activities at pH 8.0were undetectable and those at pH 6.0were very low suggesting that the influence of phosphatases on subcellular labeling of ATPase activity was negligible in this study.The higher the density (or the larger thesize)of the electron grains,the higher the H +-ATPase activity.When tissue samples were incubated in the 2control reaction media without ATP or with NaF in ATPase activity labeling assay,a few grains could be seen on the plasma membraneJ.-H.Zhang et al./Plant Science 170(2006)768–777774Fig.5.Effects of cold acclimation (CA,88C/2.5days)on the cytochemical localization and activity of membrane-associated H +-ATPase in the mesophyll cell of young grape plants under heat (458C)stress.(A)Image of electron microscopy and (B)quantitative analysis of H +-ATPase activity labeling in plasma membrane.(a)Cytochemical localization of H +-ATPase on the plasma membrane in leaf cells of control grape plants under optimum temperature.(b)Cold acclimated plants cells,showing the higher plasma membrane H +-ATPase activity (P-H +-ATPase)than those in control plants.(c)Control plants treated at 458C for 2h,showing the decrease of P-H +-ATPase activity.(d)CA plants treated at 458C for 2h,showing a slightly decrease of the P-H +-ATPase activity,but still higher than that in control plants (Fig.5c).(e)There was almost no ATPase activity in control plants treated at 458C for 4h,and the plasma membrane was severely detached from the cell wall.(f)HA plants treated at 458C for 4h,showing a further decrease but still remaining some P-H +-ATPase activity.Bar =1m m.(Fig.4g and h)suggesting the results were specific and reliable and that the localization and the H+-ATPase activity could be visualized by the location and density of the grains.Observations made using an electron microscopic revealed that the cerium phosphate grains were localized mainly on the plasma membrane in the leaf cells of young grape plants under optimum temperature conditions(Fig.4a).The number of cerium phosphate grains in the plasma membrane increased after the HA pretreatment(Fig.4b),and no obvious changes were found during a subsequent chilling stress at0.0Æ0.58C for4h(Fig.4c and d).After10h of chilling stress,the level and localization of plasma membrane H+-ATPase was reduced in both the HA and control grape plants,although the number of grains in the HA pretreated leaves was much greater than that in control(Fig.4e and f).These results were consistent with the previous reporting about P-ATPase activity presented in Fig.2a.Similarly,the number of cerium phosphate grains in the plasma membrane also increased after CA pretreatment(Fig.5a and b).When the control plants were heated at458C for2h or longer,the density of grains became less and less(Fig.5c and e).In contrast,a large number of reaction products still remained on the plasma membrane in the CA pretreated plants during the same prolonged heat stress(Fig.5d and f), confirming the results of the biochemical analysis of P-ATPaseactivity presented in Fig.3a.3.4.Immunoblotting of P-H+-ATPase during cross adaptationA100kDa peptide was detected on plasma membrane H+-ATPase using SDS-PAGE gels of the leaves for microsomal proteins(Fig.6)indicating that P-H+-ATPase existed on both the HA,CA and control grape plants.However,the immune signal in the HA pretreated leaves was noticeably stronger than that in the control leaves,and there were no significant difference between CA and control leaves.During chilling stress,the intensity of the immune signal gradually weakened in both the HA pretreated and control plants.However,the signal in the HA pretreated leaves was always much stronger than that in control leaves(Fig.7).J.-H.Zhang et al./Plant Science170(2006)768–777775Fig.6.Western blot analysis of plasma membrane H+-ATPase in mesophyll cell of heat acclimation(HA),cold acclimation(CA)and control young grape plants. The protein samples in Lanes HA,CA and control were extracted from CA,HA and control leaves,respectively.Equal amounts of protein(12m g)were subjected to SDS-PAGE and transferred to a nitrocellulose membrane.Thereafter,the P-H+-ATPase contents were immunodetected with the specific antibody.(a)Image of immunoblotting and(b)quantitative analysis of immunoblottingbands.Fig.7.Western blot analysis of plasma membrane H+-ATPase in mesophyll cell of heat acclimation(HA)and control young grape plants under cold stress. The protein samples in Lanes1,3,5,7and9were extracted from HA-pretreated leaves under cold stress for0.5,1,3,6and12h,respectively.Correspondingly, the protein samples in Lanes2,4,6,8and10were extracted from control leaves under cold stress for0.5,1,3,6and12h,respectively.Equal amounts of protein (12m g)were subjected to SDS-PAGE and transferred to a nitrocellulose membrane.Thereafter,the P-H+-ATPase contents were immunodetected with the specific antibody.(a)Image of immunoblotting and(b)quantitative analysis of immunoblottingbands.Fig.8.Western blot analysis of plasma membrane H+-ATPase in mesophyll cell of cold acclimation(CA)and control young grape plants under heat stress. The protein samples in Lanes1,3,5and7were extracted from CA-pretreated leaves under heat stress for0.5,1,3and6h,respectively.Correspondingly,the protein samples in Lanes2,4,6and8were extracted from control leaves under heat stress for0.5,1,3and6h,respectively.Equal amounts of protein(12m g) were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Thereafter,the P-H+-ATPase contents were immunodetected with the specific antibody.(a)Image of immunoblotting and(b)quantitative analysis of immu-noblotting bands.。