Molecular Basis for Interactions of G Protein Subunits with Effectors
含金属离子的蛋白靶点与小分子对接
含金属离子的蛋白靶点与小分子对接Metal ions play crucial roles in biological processes and are often involved in various enzymatic reactions. Certain proteins are specifically designed to bind these metal ions, known as metalloproteins. Understanding the interaction between metal ion-containing protein targets and small molecules is of great importance in drug discovery and pharmacological research.金属离子在生物过程中起着至关重要的作用，并经常参与各种酶反应。
某些蛋白质专门设计用来结合这些金属离子，被称为金属蛋白质。
了解含有金属离子的蛋白靶点与小分子之间的相互作用对于药物研发和药理学研究非常重要。
One commonly studied example of a metalloprotein is cytochrome P450, which contains a heme group coordinatedwith an iron ion. These proteins are involved in drug metabolism and catalyze many important reactions in the body. Interactions between small molecule drugs and cytochrome P450 can have significant effects on drugefficacy and metabolism.一个常被研究的金属蛋白质的例子是细胞色素P450，其中包含一个与铁离子配位的血红素组分。
胶原蛋白的细胞粘附基序
胶原蛋白的细胞粘附基序英文回答：Collagen, a major component of the extracellular matrix, provides a structural scaffold for cells and regulates various cellular processes. Its interactions with cell surface receptors are crucial for cell adhesion, migration, and differentiation. The specific amino acid sequenceswithin collagen that mediate cell adhesion are known ascell adhesion motifs or recognition sites.One of the well-characterized cell adhesion motifs in collagen is the RGD (Arg-Gly-Asp) sequence. This motif is recognized by integrins, a family of transmembranereceptors that play a key role in cell-matrix interactions. Integrins bind to the RGD motif through their extracellular ligand-binding domains, leading to conformational changes that transmit signals into the cell, triggeringintracellular signaling pathways and regulating cell behavior.In addition to the RGD motif, other cell adhesion motifs have been identified in collagen, including the GFOGER (Gly-Phe-Orn-Gly-Glu-Arg) sequence and the DGEA (Asp-Gly-Glu-Ala) sequence. These motifs are recognized by other types of cell surface receptors, such as syndecans and discoidin domain receptors, respectively.The interactions between collagen and its cell adhesion motifs are essential for tissue development, wound healing, and immune responses. Dysregulation of these interactions can lead to various pathological conditions, including cancer, fibrosis, and autoimmune diseases. Understanding the molecular basis of collagen-mediated cell adhesion is therefore of great importance for developing therapeutic strategies to modulate these processes.中文回答：胶原蛋白是细胞外基质的主要成分，为细胞提供结构性支架并调节各种细胞过程。
分子生药学英文
分子生药学英文Molecular Pharmacology: A Profound Exploration of the Microscopic MarvelsIn the intricate tapestry of the human body, a symphony of molecular interactions orchestrates the delicate balance that sustains our well-being. Molecular pharmacology, a discipline at the forefront of modern medicine, delves into the hidden world of these microscopic marvels, unraveling the complex mechanisms that govern the ways in which drugs and other therapeutic agents interact with the body. This captivating field offers a glimpse into the inner workings of the human organism, paving the way for more targeted and effective treatments.At the heart of molecular pharmacology lies the study of the intricate dance between drugs and their target biomolecules. These biomolecules, often proteins or nucleic acids, play crucial roles in the body's physiological processes. When a drug binds to its target, it can either enhance or inhibit the biomolecule's function, leading to the desired therapeutic effect. By understanding the precise nature of these drug-target interactions, researchers can design more potent and selective drugs, minimizing side effects and maximizingthe therapeutic benefits.One of the cornerstones of molecular pharmacology is the investigation of drug receptor interactions. Receptors are specialized proteins found on the surface of cells or within their interiors, which serve as the primary targets for many drugs. When a drug binds to its receptor, it can trigger a cascade of cellular responses, ultimately leading to the desired therapeutic outcome. Researchers in this field employ advanced techniques, such as X-ray crystallography and molecular modeling, to study the intricate three-dimensional structures of these receptors and the way they interact with different drug molecules.Another crucial aspect of molecular pharmacology is the exploration of drug metabolism and pharmacokinetics. This area examines how the body absorbs, distributes, metabolizes, and eliminates drugs, providing valuable insights into the drug's fate within the human system. By understanding these processes, researchers can optimize drug dosing, improve bioavailability, and minimize the risk of adverse effects. Techniques like mass spectrometry and enzyme kinetics are used to analyze the complex metabolic pathways that drugs undergo, enabling the development of more effective and safer therapeutic interventions.Advances in molecular biology and genetics have further expandedthe horizons of molecular pharmacology. The field now encompasses the study of how genetic variations can influence an individual's response to drugs, a concept known as pharmacogenomics. By identifying genetic markers associated with drug sensitivity or resistance, researchers can tailor treatments to individual patients, ushering in an era of personalized medicine. This approach holds immense promise in improving therapeutic outcomes and reducing the risk of adverse drug reactions.Moreover, molecular pharmacology plays a pivotal role in the development of novel therapeutic agents. From small-molecule drugs to biopharmaceuticals, such as monoclonal antibodies and gene therapies, this discipline provides the foundational knowledge and tools necessary for the rational design and optimization of these cutting-edge treatments. By understanding the intricate mechanisms underlying disease pathogenesis, researchers can target specific molecular pathways and develop more effective and targeted therapies.The impact of molecular pharmacology extends beyond the realm of drug development. This field also contributes to the understanding of the fundamental biological processes that underlie human health and disease. By elucidating the molecular mechanisms involved in the pathogenesis of various disorders, researchers can identify new therapeutic targets and develop innovative approaches to diseasemanagement.As the field of molecular pharmacology continues to evolve, it holds the promise of revolutionizing the way we approach healthcare. By harnessing the power of these microscopic marvels, researchers and clinicians can design more personalized and effective treatments, ultimately improving the quality of life for patients around the world. The journey of molecular pharmacology is one of unraveling the complexities of the human body, unlocking the secrets of disease, and paving the way for a future where targeted, individualized care becomes the norm.。
发育生物学研究英语作文
发育生物学研究英语作文Developmental Biology: Unraveling the Mysteries of Life.Developmental biology delves into the intricate processes that guide the transformation of a single-celled zygote into a fully formed organism. This multifaceted discipline encompasses a wide array of scientific approaches, ranging from molecular genetics to embryology,to elucidate the fundamental principles governing the development and growth of organisms.During early embryonic development, a remarkable cascade of events unfolds, orchestrated by a complex interplay of genetic and environmental factors. Thefertilized egg undergoes a series of rapid cell divisions, forming a blastula and subsequently a gastrula, which establishes the primary germ layers—the building blocks from which all tissues and organs will arise.Underlying these developmental processes is a symphonyof molecular events. Gene expression, controlled by a myriad of regulatory elements, dictates the fate of differentiating cells, specifying their identity and function. Morphogens, signaling molecules that diffuse through tissues, create concentration gradients that guide the organization and patterning of the embryo.As development progresses, tissues begin to specialize and organize into organs, a process known as organogenesis. The heart, brain, and lungs are just a few examples of the intricate structures that emerge during this critical period. The formation of these organs involves a concerted interplay of cell migration, cell adhesion, and tissue remodeling.Modern developmental biology has witnessed significant advancements, particularly in the realm of molecular genetics. The advent of techniques such as gene editing and genome sequencing has empowered researchers to identify and manipulate genes involved in developmental processes. This has led to a deeper understanding of the molecular basis of congenital malformations and diseases.Moreover, the integration of computational modeling and bioinformatics has facilitated the creation of sophisticated simulations that can predict how developmental processes will unfold. Such models have proven invaluable for exploring the complex interactions between different molecular pathways and for elucidating the genetic basis of human traits.Furthermore, developmental biology has far-reaching implications for understanding evolution. By studying how genes regulate the development of organisms, scientists can gain insights into the evolutionary forces that have shaped the diversity of life on Earth. Comparative developmental biology, which explores similarities and differences in developmental processes across species, provides a unique perspective on evolutionary relationships.The knowledge gleaned from developmental biology has profound implications for human health. Developmental disorders, such as neural tube defects and limb malformations, can arise from disruptions in the intricatedevelopmental processes that guide fetal development. Understanding the molecular and genetic basis of these disorders holds the potential to improve diagnosis, treatment, and prevention strategies.Additionally, regenerative medicine, which aims to repair or replace damaged tissues, draws heavily upon the principles of developmental biology. By manipulating developmental pathways, researchers seek to stimulate the regeneration of tissues that have been lost or damaged due to disease or injury.In conclusion, developmental biology stands at the forefront of scientific discovery, bridging the gap between the microscopic and macroscopic worlds. By unraveling the intricate mechanisms that govern the development and growth of organisms, researchers are gaining a deeper understanding of life's origins, evolution, and the human condition. As the field continues to advance, it promises to provide transformative insights into human health and disease, and to illuminate the boundless possibilities of life itself.。
核磁共振英文
核磁共振英文Nuclear Magnetic Resonance (NMR) is a powerful analytical technique that is commonly used in chemistry, biochemistry, and materials science. NMR can provide detailed information about the molecular structure and dynamics of a wide range of compounds, including small molecules, proteins, and polymers. In this review, we will provide an overview of the principles and applications of NMR spectroscopy.Principles of NMR SpectroscopyNMR works by exploiting the magnetic properties of certain atomic nuclei, most commonly the hydrogen nuclei (protons) in organic molecules. When a sample is placed in a strong magnetic field, the protons align themselves with the applied field. The energy required to flip the proton spin from one direction to the other can be measured by applying a radiofrequency pulse at the resonant frequency of the proton. This frequency is determined by the magnetic field strength and the local electronic environment of the proton.The NMR spectrum is recorded by measuring the absorbed and emitted frequencies of the protons as they relax back to their equilibrium state. The frequency difference between the applied pulse and the emitted signal is called the chemical shift, which is measured in parts per million (ppm) relative to a standard reference such as tetramethylsilane (TMS). The chemical shift indicates the electron density around the proton, which is influenced by nearby atoms and functional groups. The number of peaks in the spectrum corresponds to the number of unique environments in the molecule, while the relative intensities of the peaks reflect the number of protons in each environment.In addition to chemical shift, NMR provides information about spin-spin coupling, which arises from the magnetic interactions between pairs of protons that are close together in the molecule. The coupling can give rise to splitting of the NMR peaks, which allows the identification of neighboring protons and the determination of their relative positions in the molecule.Applications of NMR SpectroscopyNMR is widely used for the structural and functional characterization of organic molecules. It can be used to identify unknown compounds or confirm the identity of synthesized compounds. By measuring the chemical shift and coupling patterns, NMR can provide information about the functional groups, stereochemistry, and conformation of the molecule.NMR is also used in biochemistry and biophysics to study the structures and interactions of proteins and nucleic acids. In these applications, NMR provides information about the three-dimensional structure of the molecule, as well as the dynamics of the molecule on various timescales. For example, NMR can be used to study the folding of proteins, the binding of ligands to proteins, and the interactions between nucleic acids and proteins.NMR is also an important tool in materials science, where it is used to study the properties and structures of polymers, nanoparticles, and materials. In these applications, NMR can provide information about the composition, chain length, branching, and end groups of polymers, as well as the size, shape, and surface properties of nanoparticles.ConclusionNMR spectroscopy is a powerful and versatile analytical technique that has broad applications in chemistry, biochemistry, and materials science. NMR provides detailed information about the molecular structure, dynamics, andinteractions of compounds, making it essential for a wide range of research and industrial applications. With ongoing advancements in NMR instrumentation and methodology, the range of applications and the level of detail that can be obtained are expanding rapidly, making NMR an increasingly important tool for chemical and biological research.。
Interactions Between the Microbiotaand the Immune System
The Gut MicrobiotaREVIEWInteractions Between the Microbiota and the Immune SystemLora V.Hooper,1*Dan R.Littman,2Andrew J.Macpherson 3The large numbers of microorganisms that inhabit mammalian body surfaces have a highly coevolved relationship with the immune system.Although many of these microbes carry out functions that are critical for host physiology,they nevertheless pose the threat of breach with ensuing pathologies.The mammalian immune system plays an essential role in maintaining homeostasis with resident microbial communities,thus ensuring that the mutualistic nature of the host-microbial relationship is maintained.At the same time,resident bacteria profoundly shape mammalian immunity.Here,we review advances in our understanding of the interactions between resident microbes and the immune system and the implications of these findings for human health.Complex communities of microorganisms,termed the “microbiota,”inhabit the body surfaces of virtually all vertebrates.In the lower intestine,these organisms reach extraordi-nary densities and have evolved to degrade a variety of plant polysaccharides and other dietary substances (1).This simultaneously enhances host digestive efficiency and ensures a steady nutrient supply for the microbes.Metabolic efficiency was likely a potent selective force that shaped the evolution of both sides of the host-microbiota lions of years of coevolution,however,have forged pervasive interconnections between the physiologies of microbial commu-nities and their hosts that extend beyond metabolic functions.These interconnections are particularly apparent in the relationship between the microbiota and the immune system.Despite the symbiotic nature of the intestinal host-microbial relationship,the close association of an abundant bacterial community with intesti-nal tissues poses immense health challenges.The dense communities of bacteria in the lower intes-tine (≥1012/cm 3intestinal contents)are separated from body tissues by the epithelial layer (10m m)over a large intestinal surface area (~200m 2in humans).Opportunistic invasion of host tissue by resident bacteria has serious health consequences,including inflammation and sepsis.The immune system has thus evolved adaptations that work to-gether to contain the microbiota and preserve the symbiotic relationship between host and microbiota.The evolution of the vertebrate immune system has therefore been driven by the need to protect thehost from pathogens and to foster complex micro-bial communities for their metabolic benefits (2).In this Review,we survey the state of our understanding of microbiota-immune system in-teractions.We also highlight key experimental challenges that must be confronted to advance our understanding in this area and consider how our knowledge of these interactions might be harnessed to improve public health.Tools for Analyzing the Microbiota –Immune System RelationshipMuch of our current understanding of microbiota –immune system interactions has been acquired from studies of germ-free animals.Such animals are reared in sterile isolators to control their exposure to microorganisms,including viruses,bacteria,and eukaryotic parasites.Germ-free animals can be studied in their microbiologically sterile state or can serve as living test tubes for the establishment of simplified microbial ecosystems composed of a single microbial species or defined species mixtures.The technology has thus come to be known as “gnotobiotics,”a term derived from Greek meaning “known life.”Gnotobiotic ani-mals,particularly rodents,have become critical experimental tools for determining which host immune functions are genetically encoded and which require interactions with microbes.The current impetus for gnotobiotic exper-imentation has been driven by several impor-tant technical advances.First,because any mouse strain can be derived to germ-free status (3),large numbers of genetically targeted and wild-type inbred isogenic mouse strains have become avail-able in the germ-free state.The contribution of different immune system constituents to host-microbial mutualism can thus be determined by comparing the effects of microbial colonization in genetically altered and wild-type mice (4,5).Second,next-generation sequencing tech-nologies have opened the black box of micro-biota complexity.Although advances in ex vivo culturability are still needed,the composition ofhuman and animal microbiotas can be opera-tionally defined from polymorphisms of bacterial genes,especially those encoding the 16S ribo-somal RNA sequences.Such analyses have made possible the construction of defined microbiotas,whose distinct effects on host immunity can now be examined (6).Moreover,these advances allow the study of experimental animals that are both isobiotic and,in a defined inbred host,isogenic.A dominant goal of these efforts is to benefit hu-man health [see Blumberg and Powie (7)].With the developing technology,the species differ-ences can be closed using mice with a defined humanized microbiota (8).On the horizon,there is even the prospect of humanized isobiotic mice that also have a humanized immune system (9).A third advance has been the development of experimental systems that allow the uncoupling of commensal effects on the immune system from microbial colonization.This cannot be achieved by antibiotic treatment alone because a small pro-portion of the targeted microbes will persist.Deletion strains of bacteria lacking the ability to synthesize prokaryotic-specific amino acids have been developed that can be grown in culture but do not persist in vivo,so the animals become germ-free again.This allows issues of mucosal immune induction,memory,and functional protection to be explored without permanent colonization (10).Finally,important insights about the impact of resident microbial communities on mammalian host biology have been acquired by using high-throughput transcriptomic and metabolomic tools to compare germ-free and colonized mice (11,12).These tools include DNA microarrays,which have led to a detailed understanding of how microbiota shape many aspects of host physiology,includ-ing immunity (13,14)and development (15),as well as mass spectrometry and nuclear magnetic resonance spectroscopy,which have provided im-portant insights into how microbiota influence metabolic signaling in mammalian hosts (12).The application of these new approaches to the older technology of gnotobiotics has revolutionized the study of interactions between the microbiota and the immune system.Looking Inside-Out:Immune System Control of the MicrobiotaA major driving force in the evolution of the mammalian immune system has been the need to maintain homeostatic relationships with the microbiota.This encompasses control of micro-bial interactions with host tissues as well as the composition of microbial consortia.Here,we dis-cuss recent insights into how the immune system exerts “inside-out ”control over microbiota local-ization and community composition (see Fig.1).Stratification and compartmentalization of the microbiota.The intestinal immune system faces unique challenges relative to other organs,as it must continuously confront an enormous micro-bial load.At the same time,it is necessary to avoid1The Howard Hughes Medical Institute and Department of Im-munology,The University of Texas Southwestern Medical Center at Dallas,Dallas,TX 75390,USA.2Howard Hughes Medical Institute and Molecular Pathogenesis Program,The Kimmel Center for Biology and Medicine of the Skirball Institute,New York University School of Medicine,New York,NY 10016,USA.3Maurice Müller Laboratories,University Clinic for Visceral Sur-gery and Medicine,University of Bern,Bern,Switzerland.*To whom correspondence should be addressed.E-mail:lora.hooper@8JUNE 2012VOL 336SCIENCE1268 o n M a y 20, 2015w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mpathologies arising from innate immune signaling or from microbiota alterations that disturb essential metabolic functions.An important function of the intestinal immune system is to control the expo-sure of bacteria to host tissues,thereby lessening the potential for pathologic outcomes.This oc-curs at two distinct levels:first,by minimizing direct contact between intestinal bacteria and the epithelial cell surface(stratification)and,second, by confining penetrant bacteria to intestinal sites and limiting their exposure to the systemic im-mune compartment(compartmentalization).Several immune effectors function together to stratify luminal microbes and to minimize bacterial-epithelial contact.Intestinal goblet cells secrete mucin glycoproteins that assemble into a~150-m m-thick viscous coating at the intestinal epithelial cell surface.In the colon,there are two structurally distinct mucus layers.Although the outer mucus layer contains large numbers of bacteria,the inner mucus layer is resistant to bacterial penetration (16).In contrast,the small intestine lacks clearly distinct inner and outer mucus layers(17).Here, compartmentalization depends in part on antibac-terial proteins that are secreted by the intestinal epithelium.RegIII g is an antibacterial lectin that is expressed in epithelial cells under the control of Toll-like receptors(TLRs)(18–20).RegIII g limits bacterial penetration of the small intestinal mucus layer,thus restricting the number of bacteria that contact the epithelial surface(5).Stratification of intestinal bacteria on the luminal side of the epithelial barrier also depends on secreted immunoglobulin A(IgA).IgA spe-cific for intestinal bacteria is produced with the help of intestinal dendritic cells that sample the small numbers of bacteria that penetrate the over-lying epithelium.These bacteria-laden dendritic cells interact with B and T cells in the Peyer’s patches,inducing B cells to produce IgA directed against intestinal bacteria(21).IgA+B cells home to the intestinal lamina propria and secrete IgA that is transcytosed across the epithelium and deposited on the apical surface.The transcytosed IgAs bind to luminal bacteria,preventing micro-bial translocation across the epithelial barrier(22).Mucosal compartmentalization functions to minimize exposure of resident bacteria to the sys-temic immune system(Fig.1B).Although bacteria are largely confined to the luminal side of the epithelial barrier,the sheer number of intestinal bacteria makes an occasional breach inevita-ble.Typically,commensal microorganisms that penetrate the intestinal epithelial cell barrier are phagocytosed and eliminated by lamina propria macrophages(23).However,the intestinal im-mune system samples some of the penetrant bac-teria,engendering specific immune responses that are distributed along the length of the intes-tine(21).Bacteria that penetrate the intestinal barrier are engulfed by dendritic cells(DCs)re-siding in the lamina propria and are carried alive to the mesenteric lymph nodes.However,these bacteria do not penetrate to systemic secondarylymphoid tissues.Rather,the commensal-bearingDCs induce protective secretory IgAs(21),whichare distributed throughout all mucosal surfacesby recirculation of activated B and T cells.Thus,distinctive anatomical adaptations in the mucosalimmune system allow immune responses directedagainst commensals to be distributed widely whilestill being confined to mucosal tissues.Other immune cell populations also promotethe containment of commensal bacteria to in-testinal sites.Innate lymphoid cells reside in thelamina propria and have effector cytokine pro-files resembling those of T helper(T H)cells(24).Innate lymphoid cells that produce interleukin(IL)–22are essential for containment of lymphoid-resident bacteria to the intestine,thus preventingtheir spread to systemic sites(25).The compartmentalization of mucosal andsystemic immune priming can be severely per-turbed in immune-deficient mice.For example,mice engineered to lack IgA show priming ofserum IgG responses against commensals,indi-cating that these bacteria have been exposed tothe systemic immune system(22).A similar out-come is observed when innate immune sensingisFig.1.Looking inside-out:immune system control of the microbiota.Several immune effectors function together to stratify luminal microbes and to minimize bacterial-epithelial contact.This includes the mucus layer,epithelial antibacterial proteins,and IgA secreted by lamina propria plasma partmen-talization is accomplished by unique anatomic adaptations that limit commensal bacterial exposure to the immune system.Some microbes are sampled by intestinal DCs.The loaded DCs traffic to the mesenteric lymph nodes through the intestinal lymphatics but do not penetrate further into the body.This compartmentalizes live bacteria and induction of immune responses to the mucosal immune system. There is recirculation of induced B cells and some T cell subsets through the lymphatics and the bloodstream to home back to mucosal sites,where B cells differentiate into IgA-secreting plasma cells. SCIENCE VOL3368JUNE20121269SPECIAL SECTIONThe Gut Microbiotadefective.Mice lacking MyD88or TRIF signal-ing adaptors for TLR-mediated sensing of bacteria also produce serum IgG responses against com-mensals(26).This probably results from the fact that in these settings,large numbers of commensals cross the epithelial barrier and phagocytic cells are less able to eliminate the penetrant organisms.Immune system control of microbiota com-position.The development of high-throughput sequencing technologies for microbiota analysis has provided insight into the many factors that determine microbiota composition.For example nutrients,whether derived from the host diet (27)or from endogenous host sources(28),are critically important in shaping the structure of host-associated microbial communities.Recent evidence suggests that the immune system is also likely to be an important contributor to“inside-out”host control over microbiota composition.Certain secreted antibacterial proteins produced by epithelial cells can shape the composition of in-testinal microbial communities.a-defensins are small(2to3kD)antibacterial peptides secreted by Paneth cells of the small intestinal epithelium.Anal-ysis of the microbiota in mice that were either de-ficient in functional a-defensins or that overexpressed human a-defensin-5showed that although there was no impact on total numbers of colonizing bacte-ria,there were substantial a-defensin–dependent changes in community composition,with reciprocal differences observed in the two mouse strains(29).An interesting question is how far secreted in-nate immune effectors“reach”into the luminal microbial consortia.For example,the impact of hu-man a-defensin-5on luminal community composi-tion contrasts with the antibacterial lectin RegIII g, which limits penetration of bacteria to the epithelial surface but does not alter luminal communities(5). This suggests that some antimicrobial proteins,such as a-defensins,reach into the lumen to shape overall community composition,whereas others,such as RegIII g,have restricted effects on surface-associated bacteria and thus control microbiota location relative to host surface tissues.Questions remain as to ex-actly how a-defensin-5controls luminal community composition,however.In one scenario,these small antimicrobial peptides diffuse through the mucus layer and directly act on bacteria that inhabit the lu-men.Another possibility is that a-defensin-5exerts its antibacterial activity on bacteria that are trapped in the outer reaches of the mucus layer,with those bac-teria acting as reservoirs that seed luminal commu-nities and thus dictate their composition.Answering these questions will require improved tools for fine-mapping microbiota composition and consortia from the surface of the intestine to the interior of the lumen.The impact of the immune system on micro-biota composition is also suggested by several im-mune deficiencies that alter microbial communities in ways that predispose to disease.For example, Garrett et al.studied mice that lack the transcription factor T-bet(encoded by Tbx21),which governs inflammatory responses in cells of both the innate and the adaptive immune system(30).WhenTbx21–/–mice were crossed onto Rag2–/–mice,which lack adaptive immunity,the Tbx21–/–/Rag2–/–progeny developed ulcerative colitis in a microbiota-dependent manner(30).Remarkably,this colitisphenotype was transmissible to wild-type mice byadoptive transfer of the Tbx21–/–/Rag2–/–micro-biota.This demonstrated that altered microbiotawere sufficient to induce disease and could thus beconsidered“dysbiotic.”Similarly,mice lacking thebacterial flagellin receptor TLR5exhibit a syn-drome encompassing insulin resistance,hyper-lipidemia,and increased fat deposition associatedwith alterations in microbiota composition(31).These metabolic changes are transferable to wild-type mice that acquire the Tlr5–/–gut microbiota.A third example of immune-driven dysbiosis isseen in mice deficient for epithelial cell expres-sion of the inflammasome component NLRP6.These mice develop an altered microbiota withincreased abundance of members of the Bacte-roidetes phylum associated with increased intes-tinal inflammatory cell recruitment and susceptibilityto chemically induced colitis.Again,there is evi-dence that dysbiosis alone is sufficient to drive theintestinal inflammation,because conventionallyraised wild-type mice that acquire the dysbioticmicrobiota show similar immunopathology(32).Together,these findings suggest that the im-mune system affords mammalian hosts some con-trol over the composition of their resident microbialcommunities.It is also clear that these commu-nities can be perturbed by defects in the host im-mune system.This leads to the idea of the immunesystem as a form of ecosystem management thatexerts critical control over microbiota compo-sition,diversity,and location[see Costello et al.(33)].However,a number of questions remain.First,although it is apparent that the immune sys-tem shapes community composition at the specieslevel,it is not yet clear whether the immune sys-tem shapes the genetics and physiology of indi-vidual microbial species.Second,how much doesthe immune system combine with gastric acid andintestinal motility to control the longitudinal dis-tribution of microbial species in the gastrointes-tinal tract?Finally,it will be important to determinethe extent to which the immune system also con-trols microbial community composition and loca-tion in other organ systems,such as the respiratorytract,urogenital tract,and skin.Looking Outside-In:How MicrobiotaShape ImmunityThe earliest comparisons of germ-free and colonizedmice revealed a profound effect of microbial colo-nization on the formation of lymphoid tissues andsubsequent immune system development.It wasthus quickly apparent that the microbiota influ-ence the immune system from“outside-in.”Recentstudies have greatly amplified this understandingand have revealed some of the cellular and mo-lecular mediators of these interactions(see Fig.2).The impact of the microbiota on lymphoidstructure development and epithelial function.The tissues of the gastrointestinal tract are rich inmyeloid and lymphoid cells,many of whichreside in organized lymphoid tissues.It has longbeen appreciated that the gut microbiota have acritical role in the development of organized lym-phoid structures and in the function of immunesystem cells.For example,isolated lymphoid fol-licles in the small intestine do not develop ingerm-free mice,and such mice are also deficientin secretory IgA and CD8ab intraepithelial lym-phocytes.The specific microbial molecules en-dowed with this inductive function have not yetbeen described,however.Sensing of commensal microbiota through theTLR-MyD88signaling pathway triggers severalresponses that are critical for maintaining host-microbial homeostasis.The microbiota inducerepair of damaged intestinal epithelium through aMyD88-dependent process that can be rescued inmicrobe-depleted animals by gavage with bacteriallipopolysaccharide(LPS).The innate signals,con-veyed largely through myeloid cells,are required toenhance epithelial cell proliferation(34,35).Asdiscussed above,MyD88-dependent bacterial sig-nals are also required for the induction of epithelialantimicrobial proteins such as RegIII g(5,19).Thisexpression can be induced by LPS(19,20)or flagel-lin(36).The flagellin signals are relayed throughTLR5expressed by CD103+CD11b+dendritic cellsin the lamina propria,stimulating production of IL-23that,in turn,promotes the expression of IL-22by innate lymphoid cells(37).IL-22then stimu-lates production of RegIII g,which is also secretedupon direct activation of MyD88in epithelialcells(5,20).This is one clear example of theimportance of commensals in the induction of hostinnate responses,but it likely represents a tinyfraction of the multitude of effects of microbiota onthe host immune system.Microbiota shaping of T cell subsets.It hasrecently become evident that individual commensalspecies influence the makeup of lamina propria Tlymphocyte subsets that have distinct effector func-tions.Homeostasis in the gut mucosa is maintainedby a system of checks and balances between poten-tially proinflammatory cells,which include T H1cellsthat produce interferon-g;T H17cells that produceIL-17a,IL-17f,and IL-22;diverse innate lymphoidcells with cytokine effector features resemblingT H2and T H17cells;and anti-inflammatory Foxp3+regulatory T cells(T regs).Colonization of mice withsegmented filamentous bacteria(SFB)results inaccumulation of T H17cells and,to a lesser extent,inan increase in T H1cells(38,39).SFB appear able topenetrate the mucus layer overlying the intestinalepithelial cells in the terminal ileum,and they in-teract closely with the epithelial cells,inducing hostcell actin polymerization at the site of interactionand,presumably,signaling events that result in aT H17polarizing environment within the laminapropria.There is little known about host cell8JUNE2012VOL336SCIENCE 1270signaling pathways initiated by SFB.It is possible that SFB influence epithelial gene expression,re-sulting,for example,in expression of antimicro-bial proteins such as RegIII g and of molecules that participate in T H 17cell polarization.SFB may also act directly on cells of the immune sys-tem,either through interactions with myeloid cells that extend processes through the epithelium to the mucus layer or by production of metabolites that act on various receptors expressed by host cells.Other bacteria have been shown to enhance the anti-inflammatory branches of the adaptive immune system by directing the differentiation of T regs or by inducing IL-10expression.For example,coloniza-tion of gnotobiotic mice with a complex cocktail of 46mouse Clostridial strains,originally isolated from mouse feces and belonging mainly to cluster IVand XIV a of the Clostridium genus,results in the expansion of lamina propria and systemic T regs .These have a phenotype characteristic of T regs in-duced in the periphery in response to transforming growth factor (TGF)–b and retinoic acid [in contrast to thymic-derived natural (n)T regs (40)],and manyof these inducible T regs (iT regs )express IL-10.The exact Clostridial strains within the complex exper-imental mixture that drive this regulatory response remain to be defined.Furthermore,polysaccharide A (PSA)of Bacteroides fragilis induces an IL-10response in intestinal T cells,which prevents the expansion of T H 17cells and potential damage to the mucosal barrier (41).In contrast,mutant B.fragilis lacking PSA has a proinflammatory profile and fails to induce IL-10.Production of PSA by B.fragilis has been proposed to be instrumental for the bac-terium ’s success as a commensal.Within the intestine,the balance of effector lym-phoid cells and T reg cells can have a profound in-fluence on how the mucosa responds to stresses that elicit damage.The relative roles of commensal-regulated Tcells differ according to the models used to study inflammation.For example,in mice sub-jected to chemical or pathogen-induced damage to the mucosa,T H 17cells have a beneficial effect that promotes healing.In contrast,T H 1and T H 17cells,as well as IL-23–dependent innate lymphoid cells,promote colitis in models in which T reg cells aredepleted.It is likely that inflammatory bowel dis-eases in humans can be similarly triggered by commensal-influenced imbalance of lymphoid cell subsets.This is supported by numerous observations,including the strong linkage of IL23R polymor-phisms with Crohn ’s disease,a serious condition with relapsing intestinal inflammation and a risk of malignancy,and the severe enterocolitis associated with IL10and IL10R mutations (42,43).Microbiota effects on systemic immunity.The influence of commensal bacteria on the balance of T cell subsets is now known to extend well beyond the intestinal lamina propria.Homeostatic T cell proliferation itself is driven by the microbiota or their penetrant molecules (44).Systemic auto-immune diseases have long been suggested to have links to infections,but firm evidence for causality has been lacking.Recent studies in animal models,however,have reinforced the notion that commen-sal microbiota contribute to systemic autoimmune and allergic diseases at sites distal to the intestinal mucosa.Several mouse models for autoimmunity are dependent on colonization status.Thus,germ-free mice have marked attenuation of disease in models of arthritis and experimental autoimmune encephalomyelitis (EAE),as well as in various colitis models.In models of T H 17cell –dependent arthritis and EAE,monoassociation with SFB is sufficient to induce disease (42,45,46).In all of these models,induction of T H 17cells in the in-testine has a profound influence on systemic dis-ease.Exacerbation of arthritis and EAE is likely the consequence of an increase in the number of arthritogenic or encephalitogenic T H 17cells that traffic out of the lamina propria.The antigen spec-ificity of such cells remains to be examined.Induction of iT regs by the cluster IV and XIV a Clostridia also has a systemic effect on inflamma-tory processes.Colonization of germ-free mice with these bacteria not only results in attenuated disease after chemical damage of the gut epithelium but also reduces the serum IgE response after immuni-zation with antigen under conditions that favor a T H 2response (40).As with pathogenic T H 17cells,the antigen specificity of the commensal-induced iT regs that execute systemic anti-inflammatory func-tions is not yet known,although at least some of the T regs in the gut have Tcell receptors with specificity for distinct commensal bacteria (47).Finally,B.fragilis PSA affects the develop-ment of systemic T cell responses.Colonization of germ-free mice with PSA-producing B.fragilis results in higher numbers of circulating CD4+T cells compared to mice colonized with B.fragilis lacking PSA.PSA-producing B.fragilis also elicits higher T H 1cell frequencies in the circulation (48).Together,these findings show that commen-sal bacteria have a general impact on immunity that reaches well beyond mucosal tissues.Microbiota influences on invariant Tcells and innate lymphoid cells.A recent study extends the role of microbiota to the control of the function invariant natural killer T cells (iNKT cells),whichFig.2.Looking outside-in:how microbiota shape host immunity.Some of the many ways that intestinal microbiota shape host immunity are depicted.These include microbiota effects on mucosal as well as systemic immunity.ILFs,isolated lymphoid follicles.SCIENCEVOL 3368JUNE 20121271SPECIAL SECTION。
生物化学笔记英文
生物化学笔记英文Biochemistry NotesBiochemistry is a fascinating discipline that explores the chemical processes and substances within living organisms It plays a crucial role in understanding life at the molecular level and has applications in various fields such as medicine, agriculture, and biotechnology In this article, I'll share some important points and concepts from my biochemistry studiesOne of the fundamental concepts in biochemistry is the structure and function of biomolecules Proteins, for example, are essential molecules that perform a wide range of functions in the body They are composed of amino acids linked together in a specific sequence, and their threedimensional structure determines their activity Enzymes, which are a type of protein, act as catalysts to speed up biochemical reactions Understanding the structure and function of enzymes is crucial for explaining metabolic processesAnother important aspect is metabolism Metabolism encompasses all the chemical reactions that occur within an organism to maintain life There are two main types: anabolism, which involves the synthesis of complex molecules from simpler ones, and catabolism, which is the breakdown of complex molecules to release energy The energy currency of cells, ATP （adenosine triphosphate)， is constantly generated and consumed during metabolic processesCarbohydrates also play significant roles Glucose is a primary source of energy for cells Glycolysis is the first step in the breakdown of glucose toproduce ATP Polysaccharides like starch and glycogen are storage forms of carbohydrates in plants and animals, respectivelyLipids are another class of biomolecules Fats and oils are important for energy storage and insulation Phospholipids form the basis of cell membranes, which separate the internal environment of the cell from the outsideNucleic acids, including DNA and RNA, are responsible for storing and transmitting genetic information DNA contains the instructions for building and maintaining an organism, while RNA is involved in protein synthesisBiochemical pathways are tightly regulated to ensure the proper functioning of cells and organisms Feedback inhibition is a common regulatory mechanism where the end product of a pathway inhibits an earlier step to prevent overproduction Hormones also play a role in regulating metabolism and other biochemical processesBiochemistry is closely linked to medicine Disorders such as diabetes, where insulin regulation of glucose metabolism is disrupted, are studied and treated based on biochemical principles Understanding the biochemistry of drugs and their interactions with biological molecules is essential for developing effective therapeuticsIn agriculture, biochemistry is applied in areas such as pest control and crop improvement Understanding the biochemical processes of plants and pests helps in developing targeted strategies for enhancing crop yields and protecting them from damageIn biotechnology, recombinant DNA technology and genetic engineering rely on our knowledge of biochemistry to manipulate genes and produce desired proteins or organismsIn conclusion, biochemistry provides a deep understanding of the molecular basis of life and has wideranging applications that impact various aspects of our lives It continues to be a field of active research, uncovering new insights and providing solutions to complex problems in health, agriculture, and beyond。
光谱法研究药物小分子与蛋白质大分子的相互作用的英文
Spectroscopic Study of the Interaction between Small Molecules and Large Proteins1. IntroductionThe study of drug-protein interactions is of great importance in drug discovery and development. Understanding how small molecules interact with proteins at the molecular level is crucial for the design of new and more effective drugs. Spectroscopic techniques have proven to be valuable tools in the investigation of these interactions, providing det本人led information about the binding affinity, mode of binding, and structural changes that occur upon binding.2. Spectroscopic Techniques2.1. Fluorescence SpectroscopyFluorescence spectroscopy is widely used in the study of drug-protein interactions due to its high sensitivity and selectivity. By monitoring the changes in the fluorescence emission of either the drug or the protein upon binding, valuable information about the binding affinity and the binding site can be obt本人ned. Additionally, fluorescence quenching studies can provide insights into the proximity and accessibility of specific amino acid residues in the protein's binding site.2.2. UV-Visible SpectroscopyUV-Visible spectroscopy is another powerful tool for the investigation of drug-protein interactions. This technique can be used to monitor changes in the absorption spectra of either the drug or the protein upon binding, providing information about the binding affinity and the stoichiometry of the interaction. Moreover, UV-Visible spectroscopy can be used to study the conformational changes that occur in the protein upon binding to the drug.2.3. Circular Dichroism SpectroscopyCircular dichroism spectroscopy is widely used to investigate the secondary structure of proteins and to monitor conformational changes upon ligand binding. By analyzing the changes in the CD spectra of the protein in the presence of the drug, valuable information about the structural changes induced by the binding can be obt本人ned.2.4. Nuclear Magnetic Resonance SpectroscopyNMR spectroscopy is a powerful technique for the investigation of drug-protein interactions at the atomic level. By analyzing the chemical shifts and the NOE signals of the protein in thepresence of the drug, det本人led information about the binding site and the mode of binding can be obt本人ned. Additionally, NMR can provide insights into the dynamics of the protein upon binding to the drug.3. Applications3.1. Drug DiscoverySpectroscopic studies of drug-protein interactions play a crucial role in drug discovery, providing valuable information about the binding affinity, selectivity, and mode of action of potential drug candidates. By understanding how small molecules interact with their target proteins, researchers can design more potent and specific drugs with fewer side effects.3.2. Protein EngineeringSpectroscopic techniques can also be used to study the effects of mutations and modifications on the binding affinity and specificity of proteins. By analyzing the binding of small molecules to wild-type and mutant proteins, valuable insights into the structure-function relationship of proteins can be obt本人ned.3.3. Biophysical StudiesSpectroscopic studies of drug-protein interactions are also valuable for the characterization of protein-ligandplexes, providing insights into the thermodynamics and kinetics of the binding process. Additionally, these studies can be used to investigate the effects of environmental factors, such as pH, temperature, and ionic strength, on the stability and binding affinity of theplexes.4. Challenges and Future DirectionsWhile spectroscopic techniques have greatly contributed to our understanding of drug-protein interactions, there are still challenges that need to be addressed. For instance, the study of membrane proteins and protein-protein interactions using spectroscopic techniques rem本人ns challenging due to theplexity and heterogeneity of these systems. Additionally, the development of new spectroscopic methods and the integration of spectroscopy with other biophysical andputational approaches will further advance our understanding of drug-protein interactions.In conclusion, spectroscopic studies of drug-protein interactions have greatly contributed to our understanding of how small molecules interact with proteins at the molecular level. Byproviding det本人led information about the binding affinity, mode of binding, and structural changes that occur upon binding, spectroscopic techniques have be valuable tools in drug discovery, protein engineering, and biophysical studies. As technology continues to advance, spectroscopy will play an increasingly important role in the study of drug-protein interactions, leading to the development of more effective and targeted therapeutics.。
分子堆积——影响固态有机小分子力响应发光行为的关键因素
第42卷第3期2021年3月发光学报CHINESE JOURNAL OF LUMINESCENCEVol.42No.3Mar.,2021文章编号：1000-7032(2021)03(283-13分子堆积——影响固态有机小分子力响应发光行为的关键因素李爱森1,王金凤2,李振(1.天津大学-新加坡国立大学福州联合学院天津大学福州国际校区，福建福州350207；2.天津大学分子聚集态科学研究院，天津300072；3.武汉大学化学与分子科学学院，湖北武汉430072)扌摘要：力刺激响应型有机发光小分子因其独特的发光性质在信息加密、光学存储、压力传感器等领域具有潜在应用，引起了广泛关注。
其相关发光特性并不只来源于单分子结构，更与分子聚集态不同的堆积模式密切相关，因此，如何调控分子堆积模式是获得功能化发光材料的关键。
本文主要从力致变色和力致发光材料体系岀发，概述了几种调控分子间相互作用的策略，突岀强调了分子堆积与发光性能之间的关系，为研究具有新颖发光特性的发光材料提供了研究思路，希望促进有机发光材料的进一步发展。
关键词：外力；力致变色；力致发光；分子堆积；分子间相互作用中图分类号：O482.31文献标识码：A DOI；10.37188/CJL.20200354Molecular Stacking一Key Factor in Mechanical-responsive Luminescent Behaviors of Solid Organic Small MoleculesLI Ai-sen1,WANG Jin-feng2,LI Zhen1,2,3*(1.Joint School of National University of Singapore and Tianjin University,International Campus qf Tianjin University,Fuzhou350207,China；2.Institute of Molecular Aggregation Science,Tianjin University,Tianjin300072,China；3.College of Chemistry and Molecular Sciences,Wuhan University,Wuhan430072,China)*Corresponding Author,E-mail:lizhentju@Abstract:Mechanical-responsive organic luminogens have potential applications in the fields of information encryption,optical storage,pressure sensors,etc.due to their unique luminescent properties,and have attracted widespread attention.Their luminescent properties are not only derived from the single-molecule structure,but also closely related to the different stacking modes of molecular aggregates.Therefore,how to control the molecular stacking mode becomes a key issue for obtaining functional luminescent materials.This review mainly focuses on the material system of mechano-chromism and mechanoluminescence,outlines several strategies to regulate the intermolecular interactions,and highlights the relationship between molecular stacking and luminescent performance, which paves a new way to design novel luminescence materials and further promotes the development of organic luminescent materials.Key words:external force；mechanochromism；mechanoluminescence；molecular stacking；intermolecular interactions收稿日期；2020-11-22；修订日期；2021-01-24基金项目：天津大学启动经费资助项目Supported by Start-up Funds for Tianjin University284发光学报第42卷1引言有机发光小分子可通过分子间非共价键相互作用形成分子聚集体。
【word】植物与内生真菌互作的生理与分子机制研究进展
植物与内生真菌互作的生理与分子机制研究进展第28卷第9期2008年9月生态ACTAEC0L0GICASINICAV o1.28.No.9Sep.,2008植物与内生真菌互作的生理与分子机制研究进展袁志林,章初龙‟,林福呈,(1.浙江大学生物技术研究所,杭州310029;2.中国林业科学研究院亚热带林业研究所,富阳311400)摘要:在自然生态系统中,植物组织可作为许多微生物定居的生态位.内生真菌普遍存在于植物组织内,与宿主建立复杂的相互作用(互惠,拈抗和中性之间的相互转化),并且存在不同的传播方式(垂直和水平传播).内生真菌通过多样化途径来增强植物体的营养生理和抗性机能.但这种生理功能的实现有赖于双方精细的调控机制,表明宿主和真菌双方都进化形成特有的分子调控机制来维持这种互惠共生关系.环境因子(如气候,土壤性质等),宿主种类和生理状态,真菌基因型的变化都将改变互作结果.此外,菌根真菌和真菌病毒等也可能普遍参与植物一内生真菌共生体,形成三重互作体系,最终影响宿主的表型.研究试图从形态,生理和分子水平阐述内生真菌与植物互作的基础.关键词:内生真菌;植物;互作机制文章编号:10000933(2008)09.4430.10中图分类号:Q143,Q948文献标识码:A Recentadvancesonphysiologicalandmolecularbasisoffungaiendophyte- plantinteractionsYUANZhi—Lin‟一.ZHANGChu.Long一,UNFu—Cheng‟1InstituteofBiotechnology,ZhejiangUniversity,Hangzhou,ZhejiangProvi nce,310008,China2Instituteofsubtropicalforesto”,ChineseAcademyofForestry,Fuyang,Zhe jiangProvince310029,ChinaActaEcologicaSinica.2008.28(9):4430～4439.Abstract:Innaturalecosystems,plantsconstitutesuitablehabitatsandniches forthecolonizationofadiversityofmicroorganisms.Endophyticfungi,livingintheinnerofhealthyplanttissues ubiquitously,exhibitcomplexinteractionswiththeirhosts(acontinuumofmutualism,antagonismandneutralism)anddi fferenttransmissionmodes(horizontalorvertica1).Plantnutrientacquisitionandstresstolerancemaybestrengthenedbythefungalsymbiont.However,a successfulplant—fungalendophyteinteractionrequiresthefinetuningofant agonisticinteractions,whichimpliesthe evolutionofuniqueself—regulatingmechanismsonbothsidestomaintaina mutualisticinteraction.Hostspecies,fungal geuotypeandenvironmentalfactorssuchasclimateandsoilpropertiesaswell asthenutritionalstatusoftheplantwill ultimatelydeterminetheoutcomeoftheinteraction.Additionally,ternaryinte ractionswithmycorrhizalfungiand mycovirusesmayalsobeimportantcomponentsparticipatingintheplant?en dophyteassociationand,thus,influencethehostphenotype,Inthisreview,weattempttooutlinetheinteractivemechanismsof plant—fungalendophyteassociationsonamorphological,physiologicalandmolecularleve1.KeyWords:endophyticfungi;hostplant;interactivemechanisms基金项目:国家自然科学基金资助项目(30600002)收稿日期:2008—0227;修订日期:2008—05—23作者简介:袁志林(1979～),男,苏州人,博士生,从事植物?共生真菌基础理论与应用研究.E-mail:zhi—*********************}通讯作者Correspondingauthor.E—mail:******************.cn;***************.e nFoundationitem:TheprojeetwasfinanciallysupportedbytheNationalNatur alScienceFoundationofChina(No.30600002)Receiveddate:2008—02-27;Accepteddate:2008—05—23 Biography:YUANZhi—Lin,Ph.D.cadidate,mainlyengagedinplant—fun galendophytesinteractions.E-mail:zhi一/in—*****************http://www.ecologica.ca9期袁志林等:植物与内生真菌互作的生理与分子机制研究进展植物与周围环境生物的互作是一种普遍现象,其中植物-微生物的相互作用是重要形式之一.在叶围(phyllosphere)和根围(rhizosphere)区域,植物体时刻与众多的有害,有益和中性微生物同生存,并产生直接或间接的接触.在长期的协同进化过程中,植物对微生物的侵染已经形成一种适应性的机制,既能够识别来自微生物的信号分子并作出相应的生理反应,包括亲和性的互作(compatibleinteractions)和非亲和性的互作(incompatibleinteracti0ns).植物为了适应复杂的生态环境,进化成很多形式的植物?微生物共生体系统.C同位素标记技术显示根系组织中存在相当复杂的植物一真菌,植物一细菌共生体,对提高植物抗性和促进养分吸收起重要作用].现有的一些结果表明,植物与真菌的相互关系可能更为古老,化石证据证实了早在4亿多年前就已经存在植物与真菌的共生体,而且这种共生关系在植物由水生演化到陆生过程中扮演了重要角色口].在自然界中植物与真菌的非致病性的共生关系(nonpathogenicsymbiosis)具有普遍性,而致病性的亲和互作只是个例].因此研究植物.共生真菌的相互关系有助于更好阐明真菌在自然界的生态功能.有两类共生真菌能够侵染并定植在植物组织中,其中菌根真菌(mycorrhizalfungi)只与植物根系建立互惠关系,在根部组织中能形成特定的功能性结构,如AM菌根中存在丛枝和泡囊,外生菌根中可形成菌套(mantle)和哈帝氏网(Hartignet)等,涉及土壤一真菌.植物根系3个界面;而内生真菌(endophyticfungi)普遍存在于植物的地上和地下部分,且只存在植物组织一真菌的互作界面,侵染根系的内生真菌也不形成明显的结构特征.从已积累的研究结果看,内生真菌一植物共生体可能远比菌根共生体复杂.植物一内生真菌共生体是继豆科植物.根瘤共生体,菌根共生体后发现的植物与微生物共生关系的又一种表现形式,业已成为国际研究热点.但从研究历史,深度看,与病原真菌,菌根真菌比较,内生真菌的工作才处于基础探索阶段.在近10a中,在植物内生真菌的研究领域已经取得了很大的进步,从传统的分离培养,类群分析和分类鉴定逐渐过渡到从形态,生理和分子水平阐明互作机理.本文针对这几个方面的问题加以详细叙述和评论.1内生真菌定义及不同类型的生物学特性比较有关内生真菌的定义,国际上一直存有争议,现在普遍接受的是Petrini提出的概念J,即生活史的一部分能侵染并定殖在植物组织器官中,宿主无明显感染症状的一类真菌.绝大部分属于子囊菌和半知菌,担子菌和卵菌作为植物的内生菌也有报道..‟.一般将内生真菌分为两个类群,即禾草内生真菌(grassendophytes orbalansiaceousendophytes)和非禾草内生真菌(non—grassendophytes).它们之间的区别见表1ts,38].表1禾草内生真菌和非禾草内生真菌生物学特性比较[8,38]Table1Comparisonofcharacteristicsoftwogroupsofendophyficfungiinpla nk[8,38]禾草内生真菌Endophytesofgrasshosts非禾草内生真菌Endophytesofnongrasshosts种类较少,主要是麦角菌科的子囊真菌Fewfungalspecies,Clavicipitaceae侵染宿主组织面积广,胞问定殖Extensivecolonizationofgrassleavesandsheath,intercellular与宿主是”组成性互惠关系”,对宿主的增益作用明显“Constitutivemutualism”系统性种子垂直传播,但ichlo#也可水平传播V erticallytransmittedviaseeds绝大多数子囊菌,半知菌和和少数的卵菌,担子菌Manyspecies,taxonomicallydiverse主要以局部组织的定殖侵染为主,胞内或胞问生存Restrictedcolonizationofplanttissues,intracellularorintercellular与宿主是”诱导性互惠关系”,多因素影响互作结果“Inducedmutualism‟‟or”conditionalmutualism”.manyfactors determiningtheoutcomeoftheassociation非系统性孢子水平转播,极少数可垂直传播Horizontallytransmittedviasporesfromplantstoplants2内生真菌侵染宿主的形态学,生理学机制及双方各自的生理反应与菌根真菌和病原真菌相似,内生真菌的侵染过程也经历一系列复杂过程,包括孢子与亲和性宿主的识别,活化,黏附表面基质,萌发直到侵染组织内部_9.在侵染早期,双方各自均释放信息素类似物并被彼此识别,由此在代谢水平上做出相应的调整.在丛枝菌根和外生菌根研究中发现,气生菌丝具有对宿主植物根系定位的能力,在真菌和根系非直接物理接触前就开始了信号的交流”].已经证明根系分泌物中的倍半萜类,黄酮类物质充当了信号分子,促进孢子萌发.在内生真菌与宿主互作中也发现有类似现象,通过建立体外共培养技术(invitrodualculturesystem),观察内生真菌与无菌苗(或愈伤组织)共培养时各自的生理反生态28卷应,研究表明内生真菌菌丝对宿主的某些化学物质具有趋化性(chemotaxis),且生长良好;而在非宿主组织中并无此现象,表明如果在内生真菌一宿主互作中存在明显的趋化信号,该内生真菌并非仅仅是随机性侵染体(incidentalopportunists),而是在进化过程中已经形成对宿主的适应机制|I引.另一方面内生真菌的成功侵染也必须穿透宿主的机械屏障并克服其防御反应.现已发现内生真菌产生的胞外酶系统也相当丰富,如纤维素酶(cellulases),漆酶(1accase),木聚糖酶(xylanase),蛋白质酶(protease)等,在侵染初期破坏植物角质层和皮层细胞的细胞壁,利于菌丝进入组织内部.与菌根真菌相似,植物对于这类共生真菌的侵染所引发的防御反应程度较弱_1,推测一方面内生真菌携带的低毒力因子,菌丝分泌某些糖蛋白或细胞壁组成的改变,从而削弱宿主对其强烈的识别能力8j.最近发现在拟南芥根部细胞内质网中存在一种p一葡萄糖苷酶(PYK10),能限制内生真菌印度梨形孢(Piriformosporaindica)的侵入,从而削弱宿主产生的防御反应.植物组成型次生代谢产物如皂角苷(saponin),精油(essentialoils)等是一种进化形成的抵抗外界病原物的机制.某些病原真菌为了成功侵染产生相应的策略,分泌解毒酶(detoxificationenzyme),能分解利用次生代谢产物,克服宿主的防御系统¨博.内生真菌也有类似的机制,而且内生真菌对次生代谢产物的生物转化能力在一定程度也定了决定其宿主的范围.侵染组织后,为了行使特定的生物学功能,互作双方在形态,生理和分子水平均发生着深刻的变化,从而建立稳定有效的共生体.由于绝大多数内生真菌在细胞间隙繁殖生存的,因此质外体空间(apoplasticspace)是两者信号物质,营养交换的主要场所.对P.indica.大麦共生体系的生理与分子机制研究发现:菌株侵染根系后能削弱根部细胞HvBI一1基因的表达,HvB1—1基因的过表达反能限制菌丝的侵染强度.HvBI一1基因在真核生物中很保守,能抑制细胞程序性死亡,这表明菌丝在宿主体内的生长和繁殖需要植物组织细胞一定程度的死亡,最终两者达到平衡状态.但在侵染过程中,真菌在识别新的环境(如在宿主胞外体空间存在的碳水化合物等)中所发生的一系列生物学行为和反应机制,到目前为止涉及很少.有研究者利用激光共聚焦技术观察了GFP标记的哈茨木霉(Trichodermaharzianum)菌株侵染番茄根系时菌丝形态水平的变化,结果发现共培养2d后菌丝顶端呈现酵母状的乳突型细胞,推测这种特异性的形态变化有助于双方营养的交换.在植物.病原真菌互作研究中,已经发现菌丝的程序性死亡(programmedcelldeath)或自噬(autophagy)对于其成功侵染组织是必需的‟引.研究内生真菌不同侵染时期的基因差异表达,有助于了解其互作的关键调控因子以及分析比较不同植物一真菌相互关系的异同.3内生真菌改变植物生理代谢并增强宿主抗逆性的现象和机制分析过去的研究往往只关注菌根真菌对植物个体,种群和群落结构的影响,而忽视了内生真菌的生理生态功能.自发现禾草地上部分内生真菌Epichlo~/Neotyphodium在增强宿主生物量,抗逆方面具有独特作用,科学家普遍认为植物体进化形成不同类型的真菌共生体对其生存起着关键作用.随着研究的深入,对于水平传播内生真菌的生物学特性也逐渐涉及,尤其在探讨根系内生真菌增强宿主生理功能的机制方面尤为活跃.研究内生真菌自身的生物学特性及其对植物初级代谢和次级代谢的调节有助于我们更好地阐明植物受益的机理.内生真菌赋予植物优良生长性状的特点与菌根真菌类似,如促进植物营养生长,光合作用增强,增加生物量(产量)并提高在逆境中的生存能力.担子菌P.indica能分泌生长素如吲哚乙酸促进植物生长…,内生镰刀菌通过抑制植物体内的乙烯信号途径来提高植物生长活力¨.内生真菌还能通过活化硝酸还原酶,分泌铁载体和磷酸酶等形式促进植物养分吸收,从而更利于植物生长(图1)拍.有些真菌还能分泌多糖类黏液物质,并在根表面形成菌膜(biofilm),协同植物抗旱.Carroll 归纳总结了内生真菌互利共生的5种特性:(1)该内生真菌在特定的宿主植物中普遍存在,地理分布较广,植物不表现任何明显的病症;(2)该真菌能垂直传播或水平传播的效率很高;(3)内生真菌在整株植物组织均能生长定殖,如果只在某一器官中生长,则该组织器官感染内生真菌的强度比较高;(4)内生真菌能分泌毒性或抗生物质;(5)该内生真菌在分类单位上与病原物拮抗菌很接近.因此通过常规的菌株分离,鉴定工作,并结合上述原则,可以筛选出具有9期袁志林等:植物与内生真菌互作的生理与分子机制研究进展特定生物学功能的内生真菌.从热带兰科植物根系中分离出内生真菌,其中有两个菌株Trichodermachlorosporum和Clonostachysrosea,在分类单位上均属于生防菌株,接种试验表明这些菌株能显着提高无菌苗移栽成活率和生物量.植物育种专家通过现代分子生物学技术已经初步阐明植物体存在一些抗逆基因来参与逆境调节,但至少有很多的工作表明植物体的某些抗性特征与内生真菌的存在有关.甚至有专家预测这种内生真菌生物技术可能与传统的抗逆育种和转基因培育技术并驾齐驱.无论是垂直传播内生真菌还是水平传播内生真菌都能增强在生物(病原物)和非生物胁迫(热,盐胁迫等)中的生存能力.内生真菌协同植物适应极端环境有3种假说:①适应性生态位共生(habitat—adapted symbiosis),既一种胁迫环境下植物的内生真菌不能使另一种逆境条件下的植物受益,只能增强原胁迫环境下植物的生理功能;如耐盐植物内生真菌对热胁迫植物无生物学效应;农作物抗病内生真菌对植物耐盐无贡献等.这种植物通过共生真菌的功能来实现抗逆是一种基于基因组间的表观遗传学作用机制(intergenomic epigenetics).②共生体调节(symbioticmodulation),植物,内生真菌双方由于受到周围微环境变化的选择压力,具有选择性地改变另一方的能力.这可能是一种跳一分布于叶片组织间隙的内生真菌菌丝————————卜一EndophyticroyceIiumoccur叶围PhyllospheretInIecellul|dr1yinleavestissues①次生代谢产物②诱导子③铁载体④植物激素⑤侵染定殖…一一一………根-R.ph.陀,根系内生真菌Rootfungalendophytes菌根真菌Mycorrhizalfungi图1植物地上和地下部分组织真菌共生体及可能的生理功能【～]Fig.1Fungalsymbiontsoccurringinabovegroundandbelowground planttissuesandpossiblefunctionstheyplayed①Secondarymetabolites;②Elicitors;③Siderophores;④Phytohormones;⑤Infectionandcolonization;⑥Promotinghost growthandyield;⑦Induceddiseaseresistance;⑧ConferringhostI℃sistancet0abi0ticstress【-36]跃式的协同进化方式,使共生体快速适应环境胁迫.③共生体生活方式的转换(symbioticlifestyleswitching),某些所谓的”病原真菌”,如一些炭疽菌属真菌(Colletotrichumspp.),在一种植物中表现致病,而在另一宿主中却表现互惠共生;病原真菌的单基因突变引起其生活方式的转变,由致病性向互利共生性转化引.但这些假说几乎都基于对现象或通过接种实验来证明,深层次的机理挖掘却很少.在非生物逆境中(如盐胁迫),受内生真菌侵染的植物能更快识别周围的逆境,而且可能通过增强植物组织脯氨酸(proline)的生物合成来消除活性氧的积累_3J.最近有研究者发现一种根际真菌Paraphaeosphaeriaquadriseptata能分泌一种能抑制植物热激蛋白HSP90活力的活性物质,从而提高植物的耐热程度,是否在内生真菌一植物互作体系中也存在类似的作用机制值得深入探讨帅J.目前对于内生真菌的抗病机制的认识还处于起步阶段,但不外乎以下几种途径:(1)分泌抗生物质;(2)生态位竞争;(3)重寄生作用;(4)诱导植物抗性47I引.如禾草内生真菌在离体(invitro)或活体(inplanta,invivo)条件下均能产生一系列生物碱类物质,能有效抗虫;非禾草内生真菌增强植物抗虫能力的发现是源于对虫生真菌的(entom叩athogenicfungi)生态功能的重新认识,某些感染病原虫体的真菌在植物组织中具有内生性特点,对虫体具有很强的抗性.研究最透彻的虫生真菌是Beauveriabassiana,能够与玉米,棕榈,咖啡树和罂粟等共生.B.bassiana能分泌多种代谢产物如bassianin,beauvericin,bassianolide,beauveriolide等,对病原虫体均产生毒害作用.植物体对病原物的抗性有两种不同的机制,即系统获得抗性(systemicacquiredresistance,SAR)和诱导型系统抗病性(inducedsystemicresistance,ISR).SAR反应主要受到病原菌,生防真菌或一些化合物诱导产生,显着特征是水杨酸,茉莉酸和乙烯等物质参与,并伴有病程相关蛋白(pathogenesis—relatedproteins,PR)如几生态28卷丁质酶,葡聚糖酶和多酚氧化酶等的积累,而ISR反应并没有PR蛋白的积累.早前人们对于植物根圈促生细菌(plantgrowthpromotingrhizobacteria,PGPR)引起植物诱导型系统抗病性有深入的认识.近期的研究也表明内生真菌也能诱导植物体产生相似的抗性反应_5卜j,如菌丝分泌的某些小分子蛋白或代谢产物能够作为诱导子引发植物抗性;植物组织内的谷胱甘肽?抗坏血酸代谢途径(glutathione—ascorbatepathway)被激活,从而增强了细胞抗氧化能力引.但内生真菌诱导植物抗性可能有其独特性和复杂性的一面,P.indica和Sebacinavermifel‟a接种烟草的试验表明,这两种内生真菌均能有效促进植物生长,但同时伴随着对烟草天蛾(Manducasexta)抗性能力的下降,结果证明了内生真菌的侵染导致植物体内一种防御蛋白活力的降低一胰蛋白酶抑制剂(trypsinproteinaseinhibitors,TPI)[57j.在镰刀属内生真菌(Fusariumsolani)也发现类似的现象,F.solani接种番茄对病原尖孢镰刀菌(F.oxysporum)的抗性是依赖乙烯合成途径的,但S.vermifera促进烟草生长却是通过抑制乙烯信号转导来实现的.有趣的是,将P.indica 接种大麦后却显现出多重有益效应,能同时抗病,耐盐和提高产量,这项研究结果对传统的观点:”植物抗病,增产不可兼得”提出了挑战.这些有趣的现象提示:在不同基因型的植物一内生真菌互作中所产生的表型可能会发生变化,而且不同的功能性内生真菌进化程度可能存在差别,有些是潜在的病原菌,而有些则是”真正内生菌”(trueendophytes)Lsj.很多报道表明PGPR微生物如假单孢菌属(Pseudomonasspp.)细菌和菌根真菌对根部土传病害的病原菌都具有良好的防治效果,但对植物地上部分病原物的防治能力有一定的局限性引,除非这些微生物能诱导植物系统获得抗性;而内生真菌能稳定存在于植物的整个组织器官中,受到植物体本身机械组织的保护,因此对士传,气传病原物的抗性持久有效.虽然大多数内生真菌增强植物抗病性的报道还多局限于室内盆栽试验,是否在田问试验中也有类似的功效是值得关心的问题.鉴于木本植物内生真菌种群多样性,植物体的抗性反应可能得益于诸多内生真菌发挥生理功能的总和,因此考虑优势内生真菌之问的协同作用可能使植物受益更明显].从大量的研究结果看,植物不同组织器官富含的内生真菌对植物的贡献能力也有差异,这对筛选功能性菌株具有一定的指导意义.根系和叶片组织是植物体受外界生物和非生物因子胁迫最大的部位,因此根系和叶片内生真菌对于保护植物组织免受伤害起着尤为重要的作用.归纳目前的研究结果并加以分析可以看出,内生真菌对植物各组织器官生理效应的影响能力有以下趋势:根系>叶片>茎.植物体根围区域是一个能量和物质交换异常活跃的区域,植物和土壤微生物相互影响,而且根系被认为是一个动态的”碳库(carbonsink)”,营养物质充足,是众多微生物相互竞争的部位j.因此在这种复杂的环境中植物的生存策略之一就是容纳了与之互惠共生的内生真菌;而且根系内生真菌的侵染程度较地上部分要强(extensiveorsystemiccolonization),而叶片内生真菌多以局部侵染为主(1ocallylimitedcolonization).红色不育真菌(SRF,sterileredfungus)和暗色有隔内生菌(DSEs,darkseptateendophytes)是根系非菌根共生真菌的典型代表鲫‟.,某些DSEs甚至能形成类似菌根的侵染结构,国外已经有很多综述文章对这两类真菌的生理生态功能加以评论,虽然存在相矛盾的观点,但不容置疑的是这种在植物根系普遍存在的真菌群体必然扮演着特殊的角色,如Phialocephalasp.能降解根部老化和木栓化的细胞,从而有利于减少植物体能量的耗损.需要指出的是,某些土壤习居菌如一些木霉属(Trichodermaspp.)真菌,镰刀属真菌(Fusariumspp.),也能侵染根系皮层细胞成为无毒”机会性”内生真菌,发挥着与其作为生防菌株类似的功能.可见要严格区分内生真菌和土壤真菌是有一定难度的,长期的选择进化改变了真菌的生活方式.4内生真菌-植物互作体系中的重要分子元件及调控机制当前关于豆科植物菌根共生体和根瘤共生体建立过程中的信号转导研究比较透彻,揭示出植物与微生物形成共生关系具有很大的保守性.研究证实至少有3个植物信号元件参与其中,分别是受体样激酶(DMI2),离子通道(DMI1),依赖钙调蛋白的激酶(DMI3).此外还发现其他重要功能蛋白,如植物质体蛋白和某些核孔蛋白对于真菌和细菌进人根系形成共生关系也至关重要.研究发现日本百脉根(Lotusjaponicus)中存在两种同源的质体蛋白基因(CASTOR和POLLUX)和编码一种核孑L 蛋白的基因NUP85,主要调节质体与胞液9期袁志林等:植物与内生真菌互作的生理与分子机制研究进展之间的离子流量并激活ca信号,被认为是植物与微生物形成内共生体所必需的保守元件..但至今还很少报道有关内生真菌共生体中信号分子的化学本质和维持这种互惠关系的必备元件.在拟南芥突变体Pii.2根部细胞质膜中分离到两个富含亮氨酸重复体(LRR)的蛋白质:At1g13230和A~g16590.其中Atlg13230含有一个内质网滞留信号,A~g16590是一种非依赖磷酸化的信号转导中的受体蛋白,参与识别P.indica的侵染.这两种蛋白对于P.indica发挥生理功能至关重要;但拟南芥的DMI-1突变体却未影响与P.indica的互惠关系,这可能说明植物体已经进化形成多种重要蛋白分子以此来响应不同的微生物类群.与此对应的是,在内生真菌中也存在一些关键的酶基因来执行特定的功能,这是共生体双方相互适应的结果.BarryScott研究小组长期致力于禾草内生菌与宿主的相互关系,阐述了维持这种动态平衡的分子机制,内生真菌Nox基因可以通过调控胞间合成ROS(活性氧)来控制菌丝在宿主中的生物量,从而建立互惠共生关系;Nox的突变体菌株侵染牧草能导致植物严重感病坏死,而且菌丝在组织中生长杂乱无序J.进一步的深入研究发现在E.festucae中的SakA基因编码一种MAP激酶,能调控Nox复合体的活性,一突变体导致ROS增加加;此外在禾草内生真菌中还克隆到一个编码非核糖体多肽合成酶(nonribosomalpeptidesymthetase,NPS)的基因,该基因参与合成铁载体(siderophores),同样NPS基因的突变体导致不能正常合成铁载体,也导致植物的病变坏死,表明内生真菌分泌铁载体能力的缺失改变了共生体铁离子的动态平衡(ironhomeostasis),引起转录水平的重调,最终导致其互惠性向拮抗性转化”J.5内生真菌对宿主表型的可塑性绝大多数内生真菌发挥有益生理功能是在一定条件下实现的,与宿主是诱导型互惠关系.根据内生真菌起源于病原真菌这一观点,Schulz和Boyle等提出了维持植物一内生真菌和谐共生的动态拮抗平衡假说(balanceofantagonisms),既只有内生真菌携带的毒性因子和植物的防御反应处于相平衡状态,才能建立稳定有效的共生体,任何一方的失调就能打破这种平衡关系,导致内生真菌不能成功侵染宿主或使植物感病.诸多因素能够影响内生真菌一植物互作结果,其中宿主基因型和生理状态,真菌基因型,环境因子和土壤的营养水平是最主要的因素.如上所述,Freeman等在1993就发现致病刺盘孢菌(Collectotrichumspp.)的一个单基因突变就能使其生活方式由致病性向互惠内共生性转变.后来证明这个单基因位点编码一种胞外丝氨酸蛋白酶.后来他们又发现,同一种病原菌,接种在不同植物产生的表型却不一样,有的致病,有的却表现互惠共生.特别是在水平传播内生真菌中,表现出了灵活的生活方式,在植物正常生理状态下呈现互惠共生(mutualism),而在宿主遭受逆境胁迫下有表现寄生性(parasitism);在当植物组织衰老死亡时,腐生(saprophytism)生活成为其主要方式.也许正是内生真菌所显现的表型可塑性(phenotypicplasticity)给研究带来了一定难度.6菌根真菌,内生真菌和植物三者之间的关联以上讲述的均是单一的植物一内生真菌互作,但在自然界中,植物体却能够容纳丰富的微生物类群,因此在植物个体组织中也必然存在复杂的植物一微生物,微生物一微生物之间的相互作用.利用免培(cultureindependent)环境PCR方法,提取植株健康根系总基因组DNA,通过真菌特异性引物扩增发现,根系中存在丰。
细胞生物学(第4版)翟中和 名词解释
癌基因（oncogene）：通常表示原癌基因（proto oncogene）的突变体，这些基因编码的蛋白使细胞的生长失去控制，并转变成癌细胞，故称癌基因。
氨酰-tRNA合成酶（aminoacyl tRNA synthetase）：将氨基酸和对应的tRNA的3′端进行共价连接形成氨酰-tRNA的酶。
不同的氨基酸被不同的氨酰－tRNA合成酶所识别。
暗反应（light independent reaction）：光合作用中的另外一种反应，又称碳同化反应（carbon assimilation reaction)。
该反应利用光反应生成的ATP和NADPH中的能量，固定CO2生成糖类。
白介素-1β转换酶（interleukin-1β converting enzyme，ICE）：Caspase-1，Caspase家族成员之一，线虫Ced3在哺乳动物细胞中的同源蛋白，催化白介素-1β前体的剪切成熟过程。
半桥粒（hemidesmosome）：位于上皮细胞基底面的一种特化的黏着结构，将细胞黏附到基膜上。
胞间连丝（plasmodesma plasmodesma）：相邻植物细胞之间的联系通道，直接穿过两相邻细胞的细胞壁。
胞内体（endosome）：动物细胞内由膜包围的细胞器，其作用是转运由胞吞作用新摄取的物质到溶酶体被降解。
胞内体被认为是胞吞物质的主要分选站。
胞吐作用（exocytosis）：携带有内容物的膜泡与质膜融合，将内容物释放到胞外的过程。
胞吞作用（endocytosis）：通过质膜内陷形成膜泡，将细胞外或细胞质膜表面的物质包裹到膜泡内并转运到细胞内（胞饮和吞噬作用）。
胞外基质（extracellular matrix）：分布于细胞外空间、由细胞分泌的蛋白质和多糖所构成的网络结构，如胶原和蛋白聚糖等，在决定细胞形状和活性的过程中起着一种整合作用。
胞质动力蛋白（cytoplasmic dynein）：由多条肽链组成的巨型马达蛋白，利用ATP水解释放的能量将膜泡或膜性细胞器等沿微管朝负极转运。
中药网络药理学_理论_方法与应用_英文_李梢
110 Chin J NatMed Mar. 2013 V ol. 11 No.22013年3月 第11卷 第2期Chinese Journal of Natural Medicines 2013, 11(2): 0110-0120doi: 10.3724/SP.J.1009.2013.00110Chinese Journal of Natural MedicinesTraditional Chinese medicine network pharmacology:theory, methodology and applicationLI Shao *, ZHANG BoBioinformatics Division and Center for Synthetic and Systems Biology, TNLIST/Department of Automation, Tsinghua University, Beijing 100084, ChinaAvailable online 20 Mar. 2013[ABSTRACT] Traditional Chinese medicine (TCM) has a long history of viewing an individual or patient as a system with different statuses, and has accumulated numerous herbal formulae. The holistic philosophy of TCM shares much with the key ideas of emerging network pharmacology and network biology, and meets the requirements of overcoming complex diseases, such as cancer, in a system-atic manner. To discover TCM from a systems perspective and at the molecular level, a novel TCM network pharmacology approach was established by updating the research paradigm from the current “one target, one drug” mode to a new “network target, multi-components” mode. Subsequently, a set of TCM network pharmacology methods were created to prioritize disease-associated genes, to predict the target profiles and pharmacological actions of herbal compounds, to reveal drug-gene-disease co-module associa-tions, to screen synergistic multi-compounds from herbal formulae in a high-throughput manner, and to interpret the combinatorial rules and network regulation effects of herbal formulae. The effectiveness of the network-based methods was demonstrated for the discovery of bioactive compounds and for the elucidation of the mechanisms of action of herbal formulae, such as Qing-Luo-Yin and the Liu-Wei-Di-Huang pill. The studies suggest that the TCM network pharmacology approach provides a new research paradigm for translating TCM from an experience-based medicine to an evidence-based medicine system, which will accelerate TCM drug discov-ery, and also improve current drug discovery strategies.[KEY WORDS] Traditional Chinese medicine; Network pharmacology; Network target; Theory; Methodology; Application[CLC Number] R96 [Document code] A [Article ID] 1672-3651(2013)02-0110-111 TCM Network Pharmacology: An Emerging Subject Traditional Chinese medicine (TCM) has developed overthousands of years and has accumulated abundant clinicalexperience, forming a comprehensive and unique medicalsystem. The administration of TCM herbal formulae is a re-markable feature of treatment based on Syndrome (ZHENG inChinese) differentiation, as well as holistic thinking in TCMtheory. Recently, TCM has excited worldwide interest [1-4].However, understanding the scientific basis of TCM herbalformulae at the molecular level and from a systems perspective[Received on] 12-Feb.-2013[Research funding] This project was supported by the National Natural Sciences Foundation of China (Nos. 81225025, 60934004, and 91229201). [*Corresponding author] LI Shao: Prof., Email: shaoli@mail.tsinghua. These authors have no conflict of interest to declare.is still one of great challenges for evidence-based TCM [3-4]. The recent application of cutting-edge technologies in ana-lytical chemistry and chemical biology to characterize com-monly used herbs or herbal formulae has provided the means to identify the active ingredients in TCMs and their biologi-cal targets [5-11]. Indeed, it is likely that the investigation of the molecular basis of herbal formulae will increase the ac-ceptance of TCM worldwide [12]. Such efforts have facilitated the identification of the main active ingredients and synergis-tic ingredient pairs and, in some cases, have led to drug dis-coveries based on TCM. However, as a large number of in-gredients are included in TCM herbs or herbal formulae, and many molecular changes are involved in diseases and TCM Syndromes, the combinatorial rules and roles of most herbal formulae in complex diseases remain to be elucidated. Al-though some studies have successfully reported the extraction of a single active ingredient from an herb or herbal formula, and the identification of its biological activities and targets, a better understanding of how the multiple ingredients in anherbal formula act in synergy, and what effect they can have on2013年3月 第11卷 第2期Chin J NatMed Mar. 2013 V ol.11 No. 2 111multiple targets of a disease, is both biologically and clinically important for modern studies of TCM [12].With the rapid progress of bioinformatics, systems biol-ogy and polypharmacology, network-based drug discovery is considered a promising approach toward more cost-effective drug development [13-16]. In TCM, the perspective of holism has long been central to herbal treatments for various dis-eases. Characterized by holistic theory, and a rich experience in multicomponent therapeutics, TCM herbal formulae offer bright perspectives for treating complex diseases in a system-atic manner. Thus, bridging the emerging network science and ancient TCM will provide novel methodologies and op-portunities for discovering bioactive ingredients and bio-markers, potentially revealing mechanisms of action, and exploring the scientific evidence of herbal formulae on the basis of complex biological systems. As a beginning of the “TCM network pharmacology”, Li proposed that there was a possible relationship between TCM Syndrome and molecularnetworks in 1999 [17], and then in 2007 he established a net-work-based TCM research strategy [18] and conducted a net-work study for Cold/Hot Syndromes and Hot/Cold herbal formulae [19]. Subsequently, Li updated the TCM research framework as an “Herb network-Biological network-Pheno-type network” [20] and proposed a new concept of “Network target” [21]. A series of methods were also created by Li’s laboratory to provide methodological support for TCM net-work pharmacology [22-44] (Table 1). At nearly the same time, the subject of “Network Pharmacology” was proposed in 2007 and 2008 [45-46], and it is rapidly becoming a cut-ting-edge research field in current drug studies and the next-generation mode of drug research.Here, recent progress in this laboratory on the theory, methodology and application of TCM network pharmacol-ogy is reviewed to provide a reference for the moderniza-tion of TCM by combining computational and experimental efforts.Table 1 Concepts, methods and databases created by LI Shao’s laboratory in TCM network pharmacologyCategory TermDescriptionYearRef.and molecular networks1999 [17] Proposed a network-based TCM research framework related to TCM network pharmacology2007 [18] A network-based case study on Cold/Hot herbal formu-lae and Hot/Cold Syndromes2007 [19] Proposed the “Herb network-Biological net-work-Phenotype network”2009 [20] ConceptsTCM network pharmacologyProposed the new concept of “Network target”2011 [21, 22] CIPHER Network-based prediction for disease genes2008[24]drugCIPHERNetwork-based prediction for drug (herbal ingredient)targets and functions2010 [23, 35] comCIPHER Drug–gene–disease co-module analysis 2012 [34] CIPHER-HIT Modularity-based disease gene prediction2011[25]DMIMHerb network construction and co-module analysis for herbal formulae2010 [31] NADA Network-based assessment for drug (herbal ingredient) action2010 [32] NIMSNetwork-based identification of multi- component synergy and drug (herbal ingredient) combinations2011 [22, 36, 37] Drug combinationmodelA formal model for analyzing drug combination effects2010 [33] LMMA Disease-specific biomolecular network construction 2006 [26] CSPN Disease-specific pathway network construction 2010 [27] MethodsClustEx Disease-specific responsive gene module identification2010[28] HerbBioMapA molecular data source for herbs and TCM phenotypes 2010[40] DatabasesdbNEIA database for neuro-endocrine-immune interactions anddrug-NEI-disease network2006, 2008[38, 39]2 Network Target: A Key Concept of TCMNetwork Pharmacology2.1 Network: a computable representation of complex bio-logical systemsIn TCM network pharmacology, a “network” is a mathematical and computable representation of various con-nections between herbal formulae and diseases, particularly in complex biological systems. Fig. 1 shows the basic network topological measures that allow for the characterization of different drug treatments from a network perspective. For instance, in the “Herb network-Biological network-Phenotype network” [20], a “node” denotes the following: (i) a gene, gene product or any biological entity in the biomolecular networkincluding the protein-protein interaction network, gene regu-112 Chin J NatMed Mar. 2013 V ol. 11 No.22013年3月 第11卷 第2期latory network, genetic interaction network, metabolic net-work, and signaling network; (ii) an herb, herb ingredient or drug in the herb network; (iii) a clinical phenotype of a dis-ease in the phenotype network. An “edge” is an association, interaction, or any other well-defined relationship. The “de-gree” of a node is the number of edges connected to it. The “betweenness” of a node is the number of shortest paths that go through a given node. The nodes with high centrality (e.g., network degree, modular structure, and betweenness) can beviewed as key nodes in a network. Network parameters such as degree, betweenness, shortest path and modules can be used to measure directly the targeted key druggable proteins or protein interactions, and indirectly the targeted key un-druggable proteins by network propagation. The introduction of a “n etwork” in drug discovery incorporates the assessment of network topology, as well as dynamics, and thus offers a quantifiable description of the complex biological system and its response to various drug/herbal treatments.Fig. 1 Some basic measurements of network topological properties2.2 Paradigm shift from the “one target, one drug” to the “network target, multi-components”TCM network pharmacology highlights a paradigm shift from the current “one target, one drug” strategy to a novel version of the “network target, multi-components” strategy. As shown in Fig. 2, the new concept of “network target” was proposed by this laboratory as a core in the “Herb net-work-Biological network-Phenotype network”. The “network target” is an attempt to treat a disease-specific biomolecular network as a therapeutic target to help design appropriate treatments [18-22]. Researchers have realized that the disrup-tion of biomolecular networks can act as sensors and drivers of common human diseases [47-48]. Therefore, it was consid-ered that the mechanism of action of a herbal formula is to adjust, not for single molecules, but for imbalances in the status of disease-specific networks, which refers to the net-work interaction and node activity or expression in a given disease context. Moreover, an herbal formula is a compli-cated chemical system involving a mixture of many types of chemical compounds, and the “Multiple targets” model is not sufficient to account for the combinatorial principle of Sov-ereign-Minister-Assistant-Envoy (Jun-Chen-Zuo-Shi in Chi-nese) of herbal formulae. Based on these concerns and related studies [18-44], it was proposed that the principles of herbal formulae are considered to act on the “Network target” of specific diseases. This concept attempts to comprehensively describe all of the possible vulnerable targets for clarifying the efficiency and toxicity of drug treatments, such as herbal formulae. It represents an evolutionary approach to the prob-lems of the design and optimization of network-based multi-component therapeutics [21-22].In the “Network target” theory, the establishment of mo-lecular connections between drug/herbal formulae and dis-eases/TCM Syndromes is crucial. These molecular connec-tions are derived from a disease-specific network, which can be formed from the interactions of genes or gene products, signaling pathways and the co-functions of biological proc-esses. The network targets are those key components with important topological, dynamic and functional properties in a disease-specific molecular network. Moreover, as the effi-ciency and toxicity of drug treatments can be predicted by network-based approaches [21-23, 49], the network targets pro-vide the means to decipher the mechanisms of the therapeutic effects of drugs, or TCM herbal formulae, as well as the means to understand their possible toxicity and unknown pharmacological activities. As such, network targets have the potential to determine drug or TCM herbal formulae on-target and off-target effects that may serve as the basis for the rational design of drug combinations. 2.3 Network target vs Multiple targetsIn comparison with the former concept of “multiple tar-gets”, several unique characteristics of network targets are important to bear in mind. First, a network target is defined in relation to a particular disease and, accordingly, each disease has its own unique network target. Second, the mechanismsof drugs/herbal formulae and diseases/TCM Syndromes2013年3月 第11卷 第2期Chin J NatMed Mar. 2013 V ol.11 No. 2 113Fig. 2 The framework of TCM network pharmacology and the network target conceptshould be simultaneously considered in the network target theory. A disease-specific network target may not be identical to other network targets, but it is likely to overlap with these other network targets, which means that a particular drug or herbal formula can be used to treat different diseases. Several drug-specific network targets can be implicated in a disease network, indicating that these drugs or herbal formulae can treat the same disease. Third, from a network perspective, the systematic modulation of diseases or TCM Syndromes is achieved by targeting specific network targets due to network propagation, although some components of such network targets are not druggable. Lastly, a network target can provide predictive and quantitative measures to the mechanistic role of drugs or herbal formulae in the treatment of diseases. These characteristics aid in the network target identification process, an important step in TCM network pharmacology.3 Methodologies of TCM Network Pharmacol-ogyMethodology is the mainstay of any new subject. Net-work pharmacology is a multidisciplinary research field that integrates a large amount of information to make new dis-coveries by combining both computational and experimental approaches. The computational approaches mainly include graph theory, statistical methods, data mining, modeling, and information visualization methods. The experimental ap-proaches include various high-throughput omics technologies and biological and pharmacological experiments. As listed in Table 1 and illustrated in Fig. 3, a series of TCM network pharmacology methods were created, including the net-work-based prediction of disease genes [24-25], drug targets [23] and drug functions [22,23], the construction of disease-specific networks [26-29], the construction of herb networks [31], and a drug-gene-disease co-module quantitative analysis [31-34]. These methods and patented key procedures [35-37] and data-bases [38-40] have provided a solid platform for performing network target studies. Indeed, the promise of the networktarget approaches in drug discovery is best illustrated in the area of TCM. These approaches have continuously led to (i) the identification of active ingredients and synergistic ingre-dient pairs in TCM herbal formulae [22, 31-32, 41-42] and (ii) ex-ploration of the network characteristics of the classic theory of TCM herbal formulae, such as Cold or Hot herb properties [19], and the combinatorial rules of ‘Jun-Chen-Zuo-Shi ’ [31]. Furthermore, these network characteristics can be exploited to predict clinical biomarkers of TCM herbal formulae and rationally design multi-component therapeutics.3.1 Network-based global prediction of disease genes and drug targetsThe realization of the full potential of network target ap-proaches is dependent on identifying the genes and proteins related to diseases and TCM Syndromes, and the target pro-files of the drugs and herbal ingredients. This will require the use of new technologies, including systems or network biol-ogy with associated computational approaches, because of the high cost of experimental biology methods, such as func-tional genomics and chemoproteomics, and their inability to perform whole-genome screening for diseases or drugs. Based on these concerns, the TCM holistic analogism has been employed to develop the comparative analysis methods of complex network systems. This strategy has allowed the successful prediction of disease-related genes and drug target profiles. Two methods were developed in our laboratories, CIPHER (Correlating protein Interaction network and PHE-notype network to pRedict disease genes) [24] and drugCI-PHER (Correlating protein Interaction network and chemi-cal/PHEnotype network to pRedict drug targets) [23]. They have demonstrated highly accurate and genome-wide infer-ence capabilities for disease-related genes and drug targets, and were used for building disease-biomolecular networks and drug-biomolecular networks, respectively. To predict drug target profiles, drugCIPHER can be used to infer drug-target interactions on a genome-wide scale. Three linearregression models were proposed that relate drug therapeutic114 Chin J NatMed Mar. 2013 V ol. 11 No.22013年3月 第11卷 第2期Fig. 3 A general schematic diagram of TCM network pharmacology in the discovery of an herbal formulasimilarity, chemical similarity and their combination, respec-tively, to the relevance of the targets on the basis of a pro-tein-protein interaction network. Based on drugCIPHER, a genome-wide map of drug biological fingerprints can be constructed with high accuracy. This work demonstrated that the integration of phenotypic and chemical indexes in phar-macological space and protein-protein interactions in ge-nomic space can speed the genome-wide identification of drug targets, and also find new applications or side effects for existing drugs or herbal ingredients [23]. Another scoring sys-tem of CIPHER for disease-related gene prediction is based on a similar integrative model. The CIPHER method calcu-lates the correlation between the closeness profile of the can-didate genes in the PPI network and the similarity profile of the query phenotypes, and assigns a score to the candidate gene [24]. Recently, a modularity measurement was introduced into the algorithm framework of CIPHER to improve the prediction accuracy [25].CIPHER and drugCIPHER are in accordance with the rationale of “like attracts like”. The interactome and func-tional relationship networks can be integrated to reveal genes or proteins potentially involved in disease pathogenesis or drug mechanisms of action. Several characteristics are impli-cated in these methods. First, the relationships among nu-merous diseases or drugs are included instead of the investi-gation of only a single disease or drug. Second, the informa-tion of the interactome, and the knowledge of all the avail-able known disease-gene or drug-target relationships can be integrated to determine the molecular basis of a given disease or drug (herbal compound). Lastly, such systematic ap-proaches can be used for the large-scale prediction of dis-eases or drugs on a whole-genome level to obtain a compre-hensive molecular understanding of diseases or drugs. Thesetwo methods provide the possible means to map diseases, TCM Syndromes, the drugs and the herbal formulae, into the biomolecular network.3.2 Construction of a disease- or TCM syndrome-specific biomolecular networkAs the functional interdependencies between the mo-lecular components in a human cell are ubiquitous, a disease or TCM Syndrome is rarely a consequence of an abnormality in a single gene. Rather, it may reflect an imbalance of com-plex intracellular and intercellular network states that link tissue and organ systems [18, 20]. The inherent mechanisms of a disease or a TCM Syndrome can be characterized with the biomolecular network model. Based on the network resulting from CIPHER and drugCIPHER, the biomolecular network specific for a disease or TCM Syndrome can be further con-structed by combining various types of datasets, including multi-level high-throughput omics data, biomedical literature, and the human interactome maps and databases. For example, through integrating these datasets, a construction strategy for a network of a particular disease or TCM Syndrome was proposed [26]. Various datasets have also been integrated to analyze and evaluate the network balance at the molecular and signaling pathway levels [27-30]. Integrative network con-struction and analysis strategies are applicable to can-cer-related inflammation and angiogenesis [26-30], as well as Cold Syndrome and Hot Syndrome [19, 43-44].3.3 Drug-gene-disease co-module analysis based on a net-work targetThe drug-gene-disease co-module analysis based on a network target [31, 34, 39] is primarily focused on mapping dis-ease phenotypes and herbal compounds into biomolecular2013年3月 第11卷 第2期Chin J NatMed Mar. 2013 V ol.11 No. 2 115networks and then conducting qualitative and quantitative analyses of their molecular interactions. Such an analysis can help discover active ingredients and their synergistic combi-nations, elucidate the mechanisms of action of herbal formu-lae, and develop modern rational drug design strategies for TCM. The basic principle of the drug-gene-disease co-module analysis is that the interactions between diseases and drugs can be viewed as the interaction between dis-ease-specific molecules and drug-targeting proteins in the network target. By calculating the node importance and the distance from the network target, one can gain a comprehen-sive insight into drug efficiency and toxicity. Meanwhile, this computational model also can identify the synergistic drug combinations of numerous therapeutic agents and allow the quantitative evaluation of synergistic effects and the better understanding of the molecular mechanisms of the observed combinatorial effects based on the network target. This novel method (network target-based identification of multicompo-nent synergy, or “NIMS”) is a first-step computational ap-proach toward the identification of synergistic drug combina-tions at the molecular level, and it has been used to select synergistic agent pairs from TCM herbal formulae for a pathological process demonstrated by angiogenesis [22]. Clearly, such an approach would reduce the search range and experimental cost for multicomponent therapeutics.To understand the synergistic effects of drugs in a dose-response manner, a formal model for quantitatively analyzing drug combination effects was also proposed by simulating the kinetics of the key elements (e.g., a biochemi-cal pathway) in the network target [33]. As an example in that study, a model of the TNF-NF κB pathway, using dose-response data for therapeutic agents targeting proteins in the pathway, identified synergistic combinations between certain agents, such as the IKK inhibitor PS-1145 and the HSP90 inhibitor geldanamycin [33]. This suggests that this approach can help identify reasonable targets for creating effective ingredient combinations from herbal formulae.4 Applications of TCM Network PharmacologyPowered by the above methodologies of network-target- based TCM network pharmacology [18-44], a novel strategy can be established to elaborate the combinatorial rules of TCM herbal formulae and discover active ingredients and synergistic ingredient pairs for TCM drug development, as illustrated in Fig. 3.4.1 Identification of active herbal ingredients and synergis-tic combinationsOne of the great challenges in the modernization of TCM is to identify the active herbal ingredients and ingredient pairs that produce the therapeutic effects or the adverse ef-fects. The “prediction and discovery” of active ingredients or synergistic ingredient pairs in herbal formulae is recognized as a major goal of TCM network pharmacology. We selected a Xin-An medical family’s anti-rheumatoid arthritis (RA)herbal formula “Qing-Luo-Yin” (QLY) [50] as an example. This formula consists of four herbs, Ku-Shen (Sophora fla-vescens ), Qing-Feng-Teng (Sinomenium acutum ), Huang-Bai (Phellodendron chinensis ) and Bi-Xie (Dioscorea collettii ). Following the steps shown in Fig. 3, some anti-angiogenic and anti-inflammatory active ingredients, such as kurarinone, matrine, sinomenine, berberine, and diosgenin, can be identi-fied among the 235 ingredients of QLY by predicting each ingredient’s target profile using drugCIPHER and performing a network target analysis [42]. Moreover, the synergistic ef-fects of major ingredients, such as matrine and sinomenine, in QLY can be identified, and may be derived from the feed-back loop and compensatory mechanisms by targeting the TNF- and VEGF-induced signaling pathways involved in rheumatoid arthritis [22, 32, 42]. Several ingredient groups such as saponins and alkaloids that act as active components in QLY were also identified [42].To identify more herbs and their ingredients that are ac-tive in angiogenesis, the herbs in QLY were treated as seeds, and a method was established called the Distance-based Mu-tual Information Model (DMIM) to extend the herb pairs from 3685 collateral-related herbal formulae [31]. The DMIM approach, combining mutual information entropy and the ‘Jun-Chen-Zuo-Shi ’ relationship, offers a new approach for identifying herb networks, which in turn, can recommend candidate effective herb pairs with synergistic and antagonis-tic relationships [31]. From such an herb network, some novel angiogenesis inhibitors were discovered, such as vitexicarpin from Man-Jing-Zi (Fructus viticis ) in an herbal co-module with Huang-bai in QLY [31]. Using target prediction and ex-perimental validation, vitexicarpin was found to target key molecules (AKT and SRC) in the VEGF pathway to exert anti-angiogenic effects [41]. These efforts demonstrate the effectiveness of the network target approach in identifying active ingredients (as well as ingredients that caused side effects) and synergistic ingredient pairs from numerous compounds in a given formula.4.2 Understanding the combinatorial rules of TCM herbal formulaeBecause many pathological processes are involved in a complex disease, and because they can be organized into different functional modules in a disease-specific biomolecu-lar network, it is reasonable to assume that the “Jun-Chen-Zuo-Shi ” principle of herbal formulae can be explained by the actions of herbs on network-based func-tional modules. Given the ability to predict the target profiles of all available ingredients in an herbal formulae, it is possi-ble to reveal the combinatorial rule of “Jun-Chen-Zuo-Shi ’” based on the target interactions on the disease-specific mo-lecular network. Moreover, the interactions of the target proteins of herbal ingredients may contribute to the “Emer-gence” of the comprehensive effects of TCM herbal formu-lae [21-22]. Therefore, it is promising to interpret the scientificbasis and combinatorial rules of herbal formulae by the net-116 Chin J NatMed Mar. 2013 V ol. 11 No.22013年3月 第11卷 第2期work target analysis of herbal ingredients, as indicated by the example of QLY . According to the procedures in Fig. 3, the network target analysis for all available ingredients in QLY further suggests that Ku-Shen (Sophora flavescens ; Jun herbs) acts on the targets enriched in RA-related pathological proc-esses, such as inflammation, the immune response and an-giogenesis. Qing-Feng-Teng (Sinomenium acutum ; Chen herb), and Huang-Bai (Phellodendron chinensis ) and Bi-Xie (Dioscoreae collettii ; Zuo-Shi herbs) can augment or modulate the therapeutic effects of the Jun herb by targeting RA-related pathological processes, particularly, by syner-gistically acting on the compensatory pathway and feedback loop in the TNF/IL1B/VEGF-induced NF κB pathway [42]. These results address the possible molecular basis underly-ing the combinatorial rules and pharmacological activities of QLY .4.3 Elucidation of the Formula-Syndrome relationshipA characteristic signature that distinguishes herbal for-mulae and multicomponent therapeutics is the clinical guid-ance of TCM Syndrome theory. The Formula-Syndrome rela-tionship can be reflected by the rules of “the same treatment for different diseases” and “the same disease with different treatments” in TCM. Network target approaches have been applied to explore the mechanisms as well as biomarkers of the Formula-Syndrome relationship, and they greatly facili-tate the mechanistic interpretation of herbal formulae and TCM Syndrome. For instance, the co-module analysis pre-sented by the Liu-Wei-Di-Huang (LWDH) formula nicely illustrates “the same treatment for different diseases” [31]. This is demonstrated by a study that showed that multiple LWDH formula-treated diseases share a common network target associated with the neuro-endocrine-immune (NEI) pathways, as well as the imbalance of the human body [31]. Further investigation suggested that the key genes regulated by the LWDH formula are enriched in NEI pathways, and are also significantly close to the genes associated with cancer, diabetes and hypertension in the network target (P < 0.000 1). These LWDH-treated diseases share an overlapping molecu-lar basis and show high phenotypic similarity (P = 0.025) [31]. To understand the interplay between networks and TCM Syndromes, and to determine the mechanism of “the same disease with different treatments”, molecular networks asso-ciated with Cold Syndrome or Hot Syndrome were identified, providing useful indicators for predicting Cold or Hot Syn-drome diseases [19]. Indeed, evidence from experiments using an collagen induced arthritis rat model indicates that the Hot formula (Wen-Luo-Yin ) acts on the hub nodes of the Cold Syndrome network, whereas the Cold formula (Qing-Luo-Yin ) tends to target the hub nodes of the Hot Syndrome network, which is in agreement with the TCM therapeutic principles of “Warming the Cold and Cooling the Hot”. The fact that the network can lead to the differentiation of the effects of Cold and Hot formulae suggests that the network target will be-come an essential component of TCM Syndrome research strategies. In addition, the networks of Cold and Hot Syn-dromes have been applied to clinical research for predictionof network biomarkers, suggesting that the Cold and Hot networks can give rise to different clinical phenotypes [43-44]. Therefore, increasing efforts are being made in TCM Syn-drome studies based on the theory of network targets, which may be promising for TCM modernization and, in the future of personalized medicine [51].4.4 Rational design and optimization of drug discovery from herbal formulaeTCM is a rich source of therapeutic leads for the phar-maceutical industry, and also has a well-developed theory of prescriptions. TCM network pharmacology, as an integrative approach based on the network target theory, can provide some feasible strategies and recommendations to increase the success rate of modern drug discovery. For example, vitexi-carpin and several ingredient pairs in QLY (discussed above) have been demonstrated as possible anti-inflammatory and anti-angiogenesis treatments using network target ap-proaches [22, 41-42]. Additionally, TCM network pharmacology can also be used to refine the experience by identifying and optimizing the synergistic and antagonistic herb combina-tions in an herbal formula, which in turn benefits combinato-rial drug development. Therefore, the goal of rational design and optimization based on TCM network pharmacology is determining the best method of designing an optimal pre-scription of multiple components with a clear understanding of its pharmacology and potential drug-related adverse effects. In the future, this question will be further explored as follows: (1) recognizing the optimal ways that natural products work against a disease-specific network target; (2) quantifying the biological impact of network perturbations caused by natural products and their combinations with high efficacy and low side effects; and (3) detecting and making use of the charac-teristics of the combinatorial rules of TCM herbal formulae from a network perspective.5 TCM Network Pharmacology: a New Avenuefor Discovering Traditional Chinese Medi-cinesMore than 10 000 herbs used in more than 100 000 herbal formulae have been recorded in traditional Chinese medicine [3], posing a huge challenge to the pursuit of a better understanding of the molecular mechanisms of TCM herbal formulae. Our recent studies have demonstrated that the TCM network pharmacology strategy has a wide variety of practi-cal applications for understanding TCM herbal formulae (Fig. 4). Meanwhile, with the availability of various databases and data resources related to TCM and biological systems [52-64] (Table 2), many researchers have continued to modernize TCM in the context of network pharmacology. As listed in Table 3, researchers have conducted many network-based computational and experimental studies to detect effectivesubstances and determine the mechanisms of action of herbal。
分子动力学的英文
分子动力学的英文English: Molecular dynamics is a computer simulation method used to study the movement and interactions of atoms and molecules in a system. It is based on the principles of classical mechanics and uses potential energy functions to calculate the forces acting on the particles. By integrating the equations of motion over small time increments, molecular dynamics can simulate the behavior of a system over time, providing insights into its structure, dynamics, and thermodynamic properties. This method is widely used in various fields such as material science, chemistry, and biology, and has contributed significantly to our understanding of complex systems at the molecular level.中文翻译: 分子动力学是一种计算机模拟方法，用于研究系统中原子和分子的运动和相互作用。
它基于古典力学原理，利用势能函数计算作用在粒子上的力。
通过在小时间增量上积分运动方程，分子动力学可以模拟系统随时间的行为，提供对其结构、动力学和热力学性质的洞察。
蛋白质和聚合物之间作用力
Intermolecular Forces between Proteins and PolymerFilms with Relevance to FiltrationJeffrey A.Koehler,Mathias Ulbricht,†and Georges Belfort* Rensselaer Polytechnic Institute,Howard P.Isermann Department of Chemical Engineering,Troy,New York12180-3590Received January3,1997XIn order to understand the effects of protein fouling during ultrafiltration of biological fluids,we have investigated the molecular interactions between a thin polysulfone film and hen egg lysozyme with the surface forces apparatus(SFA).The normalized forces between the adsorbed protein layers and polymer films were measured below,at and above the p I of lysozyme,and compared with four different permeation fluxes obtained from ultrafiltration experiments.The intermolecular forces between two protein layers were also measured at the different pH values.Adsorption kinetics of lysozyme onto mica were also obtained.Buffer and lysozyme solutions at similar pH values and concentrations were filtered with6kD polysulfone membranes to obtain flux decline and hence fouling measurements.Hydrophobic membranes, such as polysulfone,exhibit extremely long-range attractive interactions(on the order of1500-2000Å) with proteins such as lysozyme.Even in the presence of electrostatic repulsion at pH values above the isoelectric point of lysozyme(when both lysozyme and polysulfone were negatively charged),a long-range attractive interaction of around210µN/m was observed.Such interactions were absent with measurements between adsorbed lysozyme-lysozyme layers.From these measurements,simple linear correlations were found relating the normalized forces to the fluxes from the ultrafiltration experiments.With respect to fouling,protein-protein and protein-polymer interactions are about equally important during ultrafil-tration.This suggests that both the surface chemistry of the membrane and the solution conditions could be chosen to minimize fouling for specific protein solutions.Hence,as a result of this study,fouling of polysulfone membranes with lysozyme solutions can be reduced if(i)filtration is conducted at pH values above the pI of lysozyme(approximately10.8)and(ii)the membranes are modified such that the long-range attractive interactions are reduced.These results support those from previous phenomenological studies on membrane filtration of protein solutions and are the first evidence relating intermolecular force interactions with macroscopic events in membrane fouling.IntroductionMembrane fouling is composed of pore plugging,pore narrowing,and cake deposition.All of these phenomena share two important factors in the filtration of biological fluids:the positive interactions between dissolved protein and itself(aggregation)and between dissolved protein and the membrane surface(adsorption).The mechanisms that underlie these attractive forces at the molecular level are unknown.This is significant because,in principle, membranes could be produced that exhibit smaller at-tractive forces between the membrane surface and the protein.This,in turn,should yield membranes that have a longer operational life and exhibit higher performance characteristics(i.e.retention and flux).To date,a great deal of work has been performed on membrane filtration using various macromolecules in-cluding proteins(such as bovine serum albumin,BSA), dextrans,polyethylene glycol,and others.Different flux-decline rates occurred for all the various cases,showing a solute dependent process.Thus,each new membrane or macromolecule system must be individually analyzed. Among the many reasons that have been proposed for this result are the following:(1)protein adsorption (specifically protein-membrane and protein-protein interactions and their dependence on pH and ionic strength,1-6(2)reduced driving force due to an osmotic back-pressure from solute buildup at the membrane surface,7-12(3)increased resistance due to protein deposi-tion and cake formation along with the increased viscosity of the fluid near the membrane surface.13-17†Current Address:Fachbereich Chemie,Institut fu¨r Organische und Bioorganische Chemie,Humboldt-Universita¨t zu Berlin,Berlin, Germany.*Corresponding author.Telephone:(518)276-6948.Fax:(518) 276-4030.E-mail:belfog@.X Abstract published in Advance ACS Abstracts,July1,1997.(1)Fane,A.G.;Fell,C.J.D.;Suki,A.The effect of pH and ionic environment on the ultrafiltration of protein solutions with retentive membranes.J.Membr.Sci.1983,16,195.(2)Mattiasson,E.The role of macromolecular adsorption in fouling of ultrafiltration membranes.J.Membr.Sci.1983,16,23.(3)Nystrom,M.;Laatikainen,M.;Turku,M.;Jarvinen,P.Resistance to fouling accomplished by modification of ultrafiltration membranes. Prog.Colloid Polym.Sci.1990,82,321.(4)McDonogh,R.;Bauser,H.;Stroh,N.;Chmiel,H.Concentration polarization and adsorption effects in cross-flow ultrafiltration of proteins.Desalination1990,79,217.(5)Robertson,B.C.;Zydney,A.L.Protein adsorption in asymmetric ultrafiltration membranes with highly constricted pores.J.Colloid Interface Sci.1990,134(2),563.(6)Meireles,M.;Aimer,P.;Sanchez.Albumin denaturation during ultrafiltration:effects of operating conditions and consequences on membrane fouling.V.Biotechnol.Bioeng.1991,38,528.(7)Nabetani,H.;Nakajima,M.;Watanabe,A.Effects of osmotic pressure and adsorption on ultrafiltration of ovalbumin.AICHE J.1990, 36(6),907.(8)Goldsmith,R.L.Macromolecular ultrafiltration with microporous membranes.Ind.Eng.Chem.Fundam.1971,10,113.(9)Leung,W.F.;Probstein,R.F.Low polarization in laminar ultrafiltration of macromolecular solutions.Ind.Eng.Chem.Fundam. 1979,18(3),274.(10)Vilker,V.L.;Colton,C.K.;Smith,K.The osmotic pressure of concentrated protein solutions.Effect of concentration and ph in saline solutions of bovine serum albumin.J.Colloid Interface Sci.1981,79 (2),548.(11)Jonsson,G.Boundary Layer Phenomena during the Ultrafil-tration of Dextran and Whey Protein Solutions.Desalination1984,51, 61.(12)Wijmans,J.G.;Nakao,S.;Smolders,C.A.Flux limitation in ultrafiltration:osmotic pressure and gel layer model.J.Membr.Sci. 1984,20,115.(13)Blatt,W.F.;Dravid,A.;Michaels,A.S.;Nelson,L.Solute polarization and cake formation in membrane ultrafiltration.Causes, consequences and control techniques.In Membrane Science and Technology;Flinn,J.E.,Ed.;Plenum Press:New York,New York, 1970;pp47-97.4162Langmuir1997,13,4162-4171S0743-7463(97)00010-3CCC:$14.00©1997American Chemical SocietyWith BSA as a model protein,experiments have shown that fluxes are higher when the pH of the solution is not equal to the p I of the protein,1,3,4,14,17when the ionic strength of the solution is low,14and when the surface of the membrane is hydrophilic.18In several recent papers, Zydney and co-workers19,20and Tracey and Davis21have proposed a two-step mechanism to describe BSA fouling and flux decline in stirred cell rge BSA aggregates are convectively dragged toward the membrane surface constricting and blocking the pores,with these aggregates serving as nucleation or attachment sites for further BSA deposition.By filtering(i)prefiltered BSA solutions or(ii)nonaggregating BSA(cysteinyl-and carboxymethyl-mediated cap of the free sulfhydryl group on BSA),Kelly and Zydney20have shown that minimal flux decline occurs.When they used a prefiltered BSA solution,they were able to show that,in the absence of aggregates,there was little,if any,fouling.Also,the rate of mixing and hence back-migration had no effect on the results.With mixtures of unfiltered and prefiltered BSA (through a100kDa molecular weight cutoff ultrafiltration membrane),they showed that“flux-decline was deter-mined entirely by the deposition of aggregates and was unaffected by the concentration of native(monomeric) BSA”.20Kim and Fane22have compared the performance of four commercial ultrafiltration(UF)membranes with the same nominal molecular weight cutoff but with different hy-drophilicities using a0.1wt%BSA solution in a crossflow test cell.They obtained“enhanced UF fluxes with slower flux loss and lower solute resistance”for the hydrophilic membranes as compared to the unmodified hydrophobic membranes(as measured by contact angle).They also report that the“hydrophilic membranes were not neces-sarily easier to clean”.In addition,they observed,as others have before,that UF fluxes were always greater when the solution pH was away from the p I.Maa and Hsu23provide convincing evidence that the presence of aggregates in solution is not the only potential cause of fouling with recombinant human growth hormone (rhGH).They suggest that“aggregation/adsorption in the filter pores during filtration is a better explanation for membrane fouling”.High pH,low salt,and the presence of a nonionic detergent all resulted in improved flux. These phenomenological studies are extremely useful in suggesting the causes of fouling,since they provide indirect evidence of the effects of fouling.To understand the mechanisms and to provide direct evidence of fouling, intermolecular force measurements were performed be-tween a model protein(lysozyme)and a thin,hydrophobic polymer film(polysulfone).Polysulfone was chosen as the polymer because it is a commonly used membrane material in industry due to its chemical and structural stability.The forces were compared with four permeation flux criteria from membrane filtration results in an attempt to relate the molecular scale measurements with the macroscopic observations.A correlation is presented that shows a simple relation between the forces and flux decline.Materials and MethodsProtein.Lysozyme,Lz(Pharmacia Biotech(Lot#4100267011,Piscataway,NJ),was chosen for this experiment because it isnot readily denatured and has an intermediate adiabaticcompressibility value.The adiabatic compressibility, s,oflysozyme is4.67×10-12cm2/dyn.24This is a median valuebetween very rigid proteins such as cytochrome c( s)0.066×10-12cm2/dyn)and very flexible proteins such as BSA( s)10.5×10-12cm2/dyn).24Lysozyme has a prolate spheroid shape with the dimensions30×30×45Å3.It has a molecular mass of14400Da with129amino acids and4disulfide bonds,and theisoelectric point is10.8-11.4with a mean about11.1.From thespace-filled model of the Lz crystal structure(not shown),it canreadily be seen that Lz has both hydrophobic and hydrophilicamino acids exposed to the exterior.25Film and Membrane.Both the films(Udel3500,UnionCarbide,Danbury,CT)and the membranes were made ofpolysulfone,the chemical structure of which is given byThe polysulfone ultrafiltration membranes were of the typegr81pp from Dow Danske(Nakskov,Denmark)and have anominal molecular weight cutoff(MWCO)of6kDa.The films were prepared by spin-coating a drop of a3%(w/v)solution of polysulfone dissolved in1,2-dichlorobenzene onto micawhich had been glued onto the SFA lens(see below).It was spunat a rate of500rpm for5s on a spin-coating apparatus(Photoresist spinner,Headway Co.,Garland,TX)followedimmediately at a rate of5000rpm for40s to smooth out the film.Drying was done in a convection oven for2h at80°C.Solventselection was crucial to achieve both continuous homogeneousfilms and films strongly adhering to the mica surface.Filmsformed under these spin-coating conditions had a reproduciblethickness of430(15Å,measured via ellipsometry.For theellipsometry measurements(Model II-004,Rudolph,Fairfield,NJ),the polysulfone was spun onto a silicon wafer for easydetermination of the film thickness.Surface Characterization.The films and membranes werestudied by attenuated total reflectance-Fourier transforminfrared spectroscopy(ATR-FTIR)and atomic force microscopy(AFM)to determine the chemical nature and morphology of thesurfaces.ATR-FTIR was used to confirm that both surfaces werechemically similar.AFM was used to ascertain if the film surfacewas smooth enough to be used in the SFA;i.e.,the surfaceroughness should not be much greater than about10Å.ATR-FTIR spectra were taken using a45°germanium crystal on aNicolet Magna-IR550Spectrometer Series II with AuxiliaryExperiment Module(Madison,WI).The AFM scans wereperformed in noncontact mode using a0.2µm ultralever on anAutoprobe CP(Park Scientific Instruments,Sunnyvale,CA).Theprobe tip was Si3N4and had a tip diameter of approximately100(14)Fane,A.G.;Fell,C.J.D.;Waters,A.G.Ultrafiltration of protein solutions through partially permeable membranes.The effect of adsorption and solution environment.J.Membr.Sci.1983,16,211.(15)Nakao,S.-I.;Nomura,T.;Kimura,S.Characteristics of mac-romolecular gel layer formed on ultrafiltration tubular membrane. AICHE J.1979,25,615.(16)Nakao,S.-I.;Kimura,S.Analysis of solute rejection in ultra-filtration.J.Chem.Eng.Jpn.1981,14(1),32.(17)Tirmizi,N.P.Study of ultrafiltration of macromolecular solutions.PhD Thesis,Department of Chemical Engineering,Rennselaer Polytechnic Institute,Troy,NY,1990.(18)Hannemaaijer,J.H.;Robbertsen,T.;van den Boomgaard,Th.; Gunnink,J.W.Characterization of clean and fouled ultrafiltration membranes.Desalination1988,68,93.(19)Kelly,S.T.;Opong,W.S.;Zydney,A.L.The influence of protein aggregates on the fouling of microfiltration membranes during stirred cell filtration.J.Membr.Sci.1995,80,175.(20)Kelly,S.T.;Zydney,A.L.Mechanisms for BSA fouling during microfiltration.J.Membr.Sci.1995,107,115.(21)Tracey, E.M.;Davis,R.H.BSA fouling of track-etched polycarbonate microfiltration membranes.J.Colloid Intferface Sci.1994, 167,104.(22)Kim,K.-J.;Fane, A.G.Performance evaluation of surface hydrophilized novel ultrafiltration membranes using aqueous proteins. J.Membr.Sci.1995,99,149.(23)Maa,Y.-F.;Hsu,C.C.Membrane fouling in sterile filtration of recombinant human growth hormone.Biotechnol.Bioeng.1996,50, 319.(24)Kharakoz,D.P.;Sarvazyan,A.P.Hydrational and intrinsic compressibilities of globular proteins.Biopolymer1993,33,11.(25)Wilson,K.P.;Malcolm,B.A.;Matthews,B.W.Structural and thermodynamic analysis of compensating mutations within the core of chicken egg white lysozyme.J.Biol.Chem.1992,267,10842.Forces between Proteins and Polymer Films Langmuir,Vol.13,No.15,19974163Å.For the captive bubble contact angle measurements,small diameter air bubbles(approximately2-3mm)were injected from a syringe into a glass chamber containing deionized water.26The bubble was released from the tip of a needle and floated to the surface of the inverted immersed membranes and films.The contact angles were then measured using a SIT camera(SIT66, Dage-MTI Inc.,Michigan City,IN)with a lens and observed on a video screen.The values for the contact angles were averaged over four to six different air bubbles.In order to measure advancing and receding angles,the air bubbles were inflated and deflated with air and the respective contact angles were measured.The glass chamber was washed with DI water while the syringe and the needle were washed with acetone and then rinsed carefully with water before use.Surface Forces Apparatus(SFA).The SFA was used to measure intermolecular forces between the two layers;one,an adsorbed layer of lysozyme,and the other,a thin film of polysulfone or another adsorbed layer of lysozyme.The layers were adsorbed onto mica that had been glued to half-cylindrical silica lenses.The distance between the two layers was deter-mined by interferometry.This separation was used with a known spring force constant to give the forces.The method has been described previously.27It has been used to measure adhesion and forces between inorganic surfaces,28,29proteins,30-39sur-factants,40-43polymers,33,44-46glycolipids,47biological ligands,48 and thin hydrophobic surfaces.24,49-53In our experiment,the surfaces were submerged in a10-2M KOH/HNO3solution in a Teflon bath.The ionic strength of the solution remained the same while the pH of the solution was changed from experiment to experiment by mixing proper amounts of10-2M KOH or10-2 M HNO3.The room temperature was controlled at21(1°C. Details of the SFA used in this study have been given previ-ously.31,32The hard wall interaction was chosen as the zero distance reference point for all of the SFA measurements so that all of the results could be easily compared.Adsorption Kinetics.The adsorption kinetics of lysozyme onto mica were performed using ATR-FTIR.The amplitude of the amide II peak(1550cm-1)was related to the amplitude of the mica peak at950cm-1to determine the lysozyme con-centration.54-56A calibration curve was measured by placing known quantities of lysozyme solution on a specific size of mica and allowing all of the water to evaporate forcing all of the lysozyme to remain on the surface.The adsorption kinetics were measured at the four different pH values with respect to time.Filtration Measurements.The filtration experiments were performed in a thin channel crossflow membrane apparatus(TCF-2,Amicon,Danvers,MA).The polysulfone membranes were received from the manufacturer already wetted in an aqueous solution containing glycerol,propionic acid,and caustic soda. The membranes were cut from the sheet and placed in deionized water in the refrigerator for12h to ensure complete wetting. The membrane cell was cleaned with1L of sodium hydroxide solution(pH12),1L of hydrochloric acid solution(pH2),1L of sodium hydroxide solution(pH12),and2L of deionized water. After the cell was cleaned,the membrane was inserted into the cell and the buffer at which the experiment was to be run was passed through the membrane under0.3MPa of N2pressure for 5h with a1m/s crossflow velocity until the steady state water flux,J w1,was achieved.The water was then removed from the cell and10mL of the buffer was added followed by200mL of the protein solution(50mg/L)under crossflow.The cell was once again placed under0.3MPa of N2pressure until50mL of permeate was collected.Less than50mL of permeate was collected for the cases where the membrane was extremely fouled. For these cases,a filtration time was used similar to that for the case where50mL of permeate was collected.The permeate and solute fluxes at the end of the protein filtration period are denoted by J p and J s,respectively.The cell was drained and rinsed twice with the buffer solution and then filled again with the buffer to determine the recovery of the initial flux.The steady state flux measurement for this time period is denoted by J w2.A typical run denoting the different flux values is given in Figure1.ResultsCharacterization of Polymer Surfaces.In order to compare the force-distance measurements using polysul-(26)Hamilton,W. C.A technique for the characterization of hydrophilic surfaces,J.Colloid Interface Sci.1972,40,219-222.(27)Israelachvili,J.N.;Adams,G.E.Measurement of forces between two mica surfaces in aqueous electrolyte solutions in the range1-100 nm.J.Chem.Soc.,Faraday Trans.1978,74,975.(28)Ke´kicheff,P.;Ninham,B.W.The double layer interaction in asymmetric electrolytes.Europhys.Lett.1990,12,471.(29)Ducker,W.A.;Xu,Z.;Clarke,D.R.;Israelachvili,J.N.Forces between alumina surfaces in salt solutions:non-DLVO forces and the implications for colloidal processing.J.Am.Ceram.Soc.1994,77,437.(30)Afshar-Rad,T.;Bailey,A.I.;Luckham,P.F.;MacNaughtan, W.;Chapman,D.Forces between proteins and model polypeptides adsorbed on mica surfaces.Biochim.Biophys.Acta1987,915,101.(31)Lee,C.S.;Belfort,G.Changing activity of ribonuclease a during adsorption:a molecular explanation.Proc.Natl.Acad.Sci.U.S.A.1989, 86,8392.(32)Belfort,G.;Lee,C.S.Attractive and repulsive interactions between and within adsorbed ribonuclease a layers.Proc.Natl.Acad. Sci.U.S.A.1991,88,9146.(33)Luckham,P.F.;Ansarifar,M.A.Biomedical aspects of the direct measurement of the forces between adsorbed polymers and proteins. Br.Polym.J.1990,22,233.(34)Gallinet,J.-P.;Gauthier-Manuel,B.Structural transitions of concanavalin A adsorbed onto bare mica plates:surface force measure-ments.Eur.Biophys.J.1993,22,195.(35)Blomberg, E.;Claesson,P.M.;Tilton,R. D.Short-range interaction between adsorbed layers of human serum albumin.J.Colloid Interface Sci.1994,166,427.(36)Leckband,D.E.;Schmitt,F.-J.;Israelachvili,J.N.;Knoll,W. Direct force measurements of specific and nonspecific protein interac-tions.Biochemistry1994,33,4611.(37)Nylander,T.;Ke´kicheff,P.;Ninham,B.W.The effect of solution behavior of insulin on interactions between adsorbed layers of insulin. J.Colloid Interface Sci.1994,164,136.(38)Pincet,F.;Perez,E.;Belfort,G.Do denatured proteins behave like polymers?Macromolecules1994,27,3424.(39)Pincet,F.;Perez,E.;Belfort,G.Molecular interactions between proteins and synthetic membrane polymer ngmuir1995,11, 1229.(40)Christenson,H.K.;Claesson,P.M.;Parker,J.L.Hydrophobic attraction:A reexamination of electrolyte effects.J.Phys.Chem.1992, 96,6725.(41)Tsao,Y.-H.;Wennerstro¨m,H.;Evans,D.F.Long-range attraction between a hydrophobic surface and a polar surface is stronger than that between two hydrophobic ngmuir1993,9,779.(42)Waltermo,A.;Sjo¨berg,M.;Anhede,B.;Claesson,P.M.Adsorption of an ethoxylated amine surfactant on mica and its effect on the surface forces.J.Colloid Interface Sci.1993,156,365.(43)Mao,G.;Tsao,Y.-H.;Tirrell,M.;Hessel,V.;van Esch,J.; Ringsdorf,H.;Davis,H.T.Interactions,structure,and stability of photoreactive bolaform amphiphile ngmuir1995,11, 942.(44)Argillier,J.-F.;Ramachandran,R.;Harris,W.C.;Tirrell,M. Polymer-surfactant interactions studied with the surface force ap-paratus.J.Colloid Interface Sci.1991,146,242.(45)Kuhl,T.L.;Leckband,D.E.;Lasic,D.D.;Israelachvili,J.N. Modulation of interaction forces between bilayers exposing short-chained ethylene oxide headgroups.Biophys.J.1994,66,1479.(46)Mangipudi,V.;Pocius,A.V.;Tirrell,M.Direct measurement of the surface energy of corona-treated polyethylene using the surface forces ngmuir1995,11,19.(47)Luckham,P.;Wood,J.;Swart,R.The surface properties of gangliosides:ii.direct measurement of the interaction between bilayers deposited on mica surfaces.J.Colloid Interface Sci.1993,156,173.(48)Leckband,D.E.;Kuhl,T.;Wang,H.K.;Herron,J.;Mu¨ller,W.; Ringsdorf,H.4-4-20Anti-fluorescyl igg fab’recognition of membrane bound hapten:direct evidence for the role of protein and interfacial structure.Biochem.1995,34,11467.(49)Wood,J.;Sharma,R.Interaction forces between hydrophobic mica surfaces.J.Adhesion Sci.Technol.1995,9,1075.(50)Wood,J.;Sharma,R.How long is the long-range hydrophobic attraction?Langmuir1995,11,4797.(51)Ducker,W.A.;Xu,Z.;Israelachvili,J.N.Measurements of hydrophobic and dlvo forces in bubble-surfactant interactions in aqueous ngmuir1994,10,3279.(52)Claesson,P.M.;Christenson,H.K.Very long range attractive forces between uncharged hydrocarbon and fluorocarbon surfaces in water.J.Phys.Chem.1988,92,1650.(53)Herder,P. C.Forces between hydrophobed mica surfaces immersed in dodecylammonium chloride solution.J.Colloid Interface Sci.1990,134,336.(54)Brash,J.L.;Lyman,D.J.Adsorption of plasma proteins in solution to uncharged,hydrophobic polymer surfaces.J.Biomed.Mater. Res.1969,3,175.(55)Giroux,T. A.;Cooper,S.L.FTIR/ATR studies of human fibronectin adsorption onto plasma derivatized polystyrene.J.Colloid Interface Sci.1990,139,351.(56)Fu,F.-N.;Fuller,M.P.;Singh,e of fourier transform infrared/attenuated total reflectance spectroscopy for the study of surface adsorption of proteins.Appl.Spectrosc.1993,47,98.4164Langmuir,Vol.13,No.15,1997Koehler et al.fone films with the filtration results using commercial polysulfone membranes,it is necessary to show that the films and membranes are similar in chemical composition.To this end,we used the ATR-FTIR technique to probe the chemical nature of the surfaces at penetration depths of approximately 0.5-1.0µm.Gross estimates of the surface polarity were determined by contact angle mea-surements.The ATR-FTIR scans for the polysulfone membrane and film are shown in Figure 2.The two scans illustrate how similar the two materials were.The aliphatic CH 3stretching is seen at 3000-2800cm -1,and the aromatic CH stretching appears at 3100-3000cm -1.The strong aromatic ether peak can been seen at ca.1240cm -1.The symmetric and asymmetric sulfone stretches can be seen at ca.1160and ca.1328cm -1,respectively.The fingerprint region (1600-800cm -1)also shows good correlation between the two samples.Figure 3a shows the AFM scan of the polysulfone film that was spun on a flat piece of mica.The film had an average roughness of 2.1Åand a RMS (root mean square)roughness of 2.6Åwith a mean height of 15Å.These values were smooth enough for SFA measurements.The AFM image of the surface of the gr81pp polysulfone membrane is shown in Figure 3b.The membrane had an average roughness of 7.6Åand an RMS roughness of 9.6Åwith a mean height of 67Å.Both surfaces were also characterized using captive bubble contact angle measurements with a bubble of air in water (Table 1).The values of the contact angles were similar,and the hystereses or differences between the advancing and receding contact angles were close to 20°.The closeness of these values is not surprising,since the AFM images have shown that both the film and membrane are fairly smooth and the ATR-FTIR images have shown that they have similar chemistry.The contact angle for mica exhibited almost no hysteresis,as expected.It is expected that the static angle values are about halfway between the values of the advancing and receding angles,and this was found to be so.These results indicate that the film and membrane surfaces are similar.Therefore,it is possible to compare the intermolecular forces between the polysulfone film and the adsorbed Lz layer with the ultrafiltration measurements of Lz solutions.In the SFA,the contact area was about 1µm by 1µm;these dimensions were much larger than the height fluctuations of the surface as measured by theAFM.Figure 1.Typical flux -time curve,showing flux designations used for filtration experiments.J w1is the steady state buffer flux (b )after 5h at a 1m/s crossflow at 0.3MPa of pressure;J p is the steady state protein solution flux (9)after 50mL of 50mg/L solution permeated the membrane at a 1m/s crossflow at 0.3MPa of pressure;J w2is the steady state buffer flux (2)after about 100min at a 1m/s crossflow at 0.3MPa of pressure.All these parameters are read on the left hand ordinate.The calculated solute flux (0)estimated from the solution flux,the feed concentration,and the solute rejection is read on the right handordinate.Figure 2.ATR-FTIR spectra of (a)the polysulfone film (Udel 3500,Union Carbide,Danbury,CT)and (b)the polysulfone membrane (gr81pp,Dow Dansk,Naskov,Denmark).Spectra were taken with a 45°Gecrystal.Figure 3.AFM scans of film and membrane taken in noncontact mode.The cantilever was a 0.2µm ultralever.The probe tip was Si 3N 4and had a tip diameter of approximately 100Å.(a,top)Polysulfone film:median height 15Å,RMS roughness 2.6Å,average roughness 2.1Å.(b,bottom)Polysul-fone membrane:median height 67Å,RMS roughness 9.6Å,average roughness 7.6Å.Table 1.Captive Air Bubble Contact AngleMeasurements in Watercaptive bubble contact angles (air/water)material static Θ(deg)receding Θ(deg)advancing Θ(deg)cleaved mica21(121(119(1PSU (membrane)99(4105(482(4PSU (film)95(1106(288(2Forces between Proteins and Polymer Films Langmuir,Vol.13,No.15,19974165Lz-Mica Adsorption Kinetics and Amount.From the kinetic experiments,the amount of adsorbed Lz reached a plateau(maximum)in less than30min for all four pH values(6.6,11.0,11.6,and12.0).The time frame of4h for the adsorption of the Lz in the SFA experiments was well above the time required to reach a plateau value. Similarly,all the filtration experiments with protein in the feed were longer than2h.Above the p I of Lz(pH 12.0),the maximum adsorbed amount was less than half (54%drop)that at the p I.This was probably due to the repulsive nature between the negatively charged mica and the net negatively charged Lz.The maximum adsorbed amount of Lz on mica was at the p I of the protein, possibly due to the hydrophobic interactions.Below the pI of Lz,adsorption was about17%less than that at the p I but still larger than that above the p I.This was likely due to the attractive electrostatic interactions between the negatively charged mica and the positively charged Lz.The maximum adsorbed amounts(equilibrium)for all four pH values together with their initial diffusion coefficients are summarized in Table2.The diffusion coefficients were obtained from a linear plot of adsorbed amount,A(t),versus the square root of time,according to the following equation:57where C0and D are the solution concentration of protein (mg/L)and the mutual diffusion coefficient(cm2/s), respectively.Since all the diffusivity values reported in Table2are below the single molecule diffusion coefficient of11.3×10-7cm2/s,57we suspect aggregation to have occurred.Adsorption and desorption of Lz on silica surfaces does not change the molecule’s conformation greatly.59The adsorption of Lz onto graphite showed an ordered two-dimensional array of adsorbed lysozyme,whereas adsorp-tion of Lz on mica was seen to exhibit two-dimensional structures at low lysozyme concentrations60and three-dimensional aggregation at higher concentrations.61,62PSU-Lz Force Measurements.Before the PSU-Lz forces were measured,it was necessary to determine the stability of the film in the SFA in aqueous solution and whether it remained adhered to the curved mica surface.Thus,the interaction between the PSU film and mica was measured.Since the slopes of the compression and decompression curves followed a straight line on a semilog plot and were similar and hysteresis between the curves was absent for all four pH values(two above the p I,one at the p I,and one below the p I;data not shown), the film was considered stable and well formed.The repulsion curves were linear for long distances,suggesting electrostatic repulsion between the two layers.Since the DLVO theory is not applicable for dissimilar surfaces and since we did not know the potential or charge at each of the surfaces,nor did we know if the system was operating at constant charge or potential,a nonlinear Poisson-Boltzmann theory for calculating the electric double-layer force and interaction free energy between dissimilar charged surfaces was not used.63Next,Lz was adsorbed onto the mica of one surface at the respective pH values and the PSU-Lz interactions were measured(Figure4).In all cases,a small attractive interaction(<450µN/m)was seen when the surfaces were brought into contact.They began at a large separation (about2000Å).For the case of pH11.8(above the pI of Lz),a repulsion was seen at about250Åon the first compression.This was reduced to about150Åon the successive compressions.At the pI of Lz,a repulsion was seen at500Åon the first compression,which was reduced to about150Åin the following compressions.Below the p I of the protein,the repulsion was evident at about400-500Åand was not reduced on the second compression. This indicated some rearrangement of the surfaces (possibly aggregate compression)at the two high pH values.The striking difference between all of the cases is seen in the adhesion(maximum attractive force during decompression)between the two layers.The average of the adhesion values for each pH is given in Table3.The strongest adhesive interaction was seen below the p I, where the two surfaces(PSU-Lz)were of opposite charge and where denaturation of Lz(due to surface-induced interactions)was most likely.Of the three remaining pH values,the adhesion was higher at the p I of Lz than that above the p I.Lz-Lz Force Measurements.During filtration with protein solutions,after the initial adhesion of Lz molecules onto the exposed membrane surface,further interactions were between previously adsorbed and freely dissolved Lz molecules.Clearly,these latter interactions needed to be accounted for in order to correlate the force and filtration measurements.We simulate these interactions by measuring the intermolecular forces between two previously adsorbed Lz layers in solution at various pH values.The Lz-Lz interactions are shown in Figure5. The curves all show that an attractive interaction is absent for all of the pH values,unlike the case for the polymer-protein interactions.Another difference is the fact that the repulsive interactions between the two surfaces on the first compression is evident at large separations only for the case when the pH value was lower than the p I value.The repulsive interaction starts at about600Åon the first compression and is reduced to400Åon each successive compression.The average value of the adhesion for each pH is also included in Table3.Qualitatively,the values inversely follow the filtration experiments,with(57)Amiel,C.;Sikka,M.;Schneider,J.W.;Tsao,Y.H.;Tirrell,M.; Mays,J.W.Adsorption of hydrophilic-hydrophobic block copolymers on silica from aqueous solutions.Macromolecules1995,28,3125.(58)Creighton,T.E.Proteins:Structures and Molecular Properties, 2nd ed.;W.H.Freeman&Co.:New York,1993;p266.(59)Norde,W.;Favier,J.P.Structure of adsorbed and desorbed proteins,Colloid Surf.1992,64,87.(60)Haggerty,L.;Lenhoff,A.M.Analysis of ordered arrays of adsorbed lysozyme by scanning tunneling microscopy.Biophys.J.1993, 64,886.(61)Tilton,R.D.;Blomberg,E.;Claesson,P.M.Effect of anionic surfactant on interactions between lysozyme layers adsorbed on mica.Langmuir1993,9,2102.(62)Blomberg,E.;Claesson,P.M.;Fro¨berg,J.C.;Tilton,R.D. Interaction between adsorbed layers of lysozyme studied with the surface force ngmuir1994,10,2325.(63)McCormack,D.;Carnie,S.L.;Chan,D.Y.C.Calculations of electric-layer force and interaction free energy between dissimilar surfaces.J.Colloid Interface Sci.1995,169,177.Table2.Maximum Adsorbed Amounts of Lysozyme on Freshly Cleaved Mica and Early-Time Diffusion Coefficients of Lysozyme in Solution apH maximum adsorbedamount(mg/m2)measured diffusioncoefficient b(×10-7cm2/s)6.60.830.84811.0(p I) 1.00 3.2811.60.799.0812.00.46(5) 4.56a Lysozyme concentration:50mg/L.T)21°C.10-2M KOH/ HNO3buffer.b Creighton58reports a value of11.3×10-7cm2/s for the diffusion coefficient.The diffusion coefficients were obtained from the slopes of the kinetic data(not shown),as described by Amiel et al.57A(t))2π1/2C(Dt)1/2(1)4166Langmuir,Vol.13,No.15,1997Koehler et al.。
酸-热诱导罗非鱼肌肉蛋白凝胶形成及机理的研究
II
Study on acid-heat-induced gel formation and
2、采用葡萄糖酸内酯（GDL）建立模拟酸化体系，研究 GDL 添加量、蛋白质 浓度和加盐量对酸诱导罗非鱼肌肉蛋白凝胶形成过程的影响。结果表明，在罗非鱼 肌肉蛋白中添加 GDL，4℃放置 24h，GDL 缓慢水解生成葡萄糖酸，使体系 pH 值 在 2h 内迅速下降，12h 后达到稳定；在蛋白浓度 8%和加盐量 3%时，添加 1% GDL 酸化处理的罗非鱼肌肉蛋白凝胶特性最好；酸化过程中蛋白分子中总巯基含量和表 面疏水性均随酸化时间的延长而减小，采用四种变性剂依次对酸诱导体系进行处 理，酸化过程中蛋白质溶解度在 S3 中最大，表明酸诱导凝胶形成过程中起主要作 用的是蛋白分子间疏水相互作用。
酸-热诱导罗非鱼肌肉蛋白凝胶形成及机理的研究
摘要
以罗非鱼为原料加工的鱼糜及鱼糜制品深受广大消费者的欢迎。但工业化加工 鱼糜的方式都以热处理为主，由于加热速度慢、物料温度梯度大和加热时间长，易 引起凝胶劣化而导致鱼糜制品品质下降。针对热诱导凝胶的凝胶特性差，营养损失 大和不易保藏等问题，国内外研究了将高压处理、酶法交联和酸处理等非热处理方 法用于was used, the best gel properties of Tilapia muscle protein was obtained, the total sulphydryl group of the protein molecule and the surface hydrophobicity would reduced with increasing acidification time. Four denaturant were used to dissolute acid-induced gel, the highest protein solubility was obtained in S3, which showed that hydrophobic interactions between protein molecules played a major role in the acid-induced gel process.
兴奋收缩耦联和心力衰竭的治疗
Bers DM. Nature, 2002, 415(6868): 198-205.
Excitation
The cardiac action potential
● A notable difference between skeletal and cardiac myocytes is how each elevates the myoplasmic Ca2+ to induce contraction.
Inotropic Agents for HF
Inotropic Agents and β-blocker
● Digitalis ● Phosphodiesterase inhibitor ● β- adrenoceptor blocker
Digitalis (﹥200 years)
Digilis purpurea Purple foxglove
TOPICS
Excitation-contraction (EC) coupling
➢ Excitation ➢ Calcium Cycling ➢ Contraction
Alterations of E-C coupling in HF Inotropic agents for HF
Excitation-contraction coupling
Jeffery D Molkentin. Nature Medicine 11, 1284 - 1285 (2005)
TGF-β Signaling Controls Embryo Development in the Parasitic Flatworm Schistosoma mansoni
TGF-b Signaling Controls Embryo Development in the Parasitic Flatworm Schistosoma mansoni Tori C.Freitas,Euihye Jung,Edward J.Pearce*Department of Pathobiology,School of Veterinary Medicine,University of Pennsylvania,Philadelphia,Pennsylvania,United States of AmericaOver200million people have,and another600million are at risk of contracting,schistosomiasis,one of the major neglected tropical diseases.Transmission of this infection,which is caused by helminth parasites of the genus Schistosoma,depends upon the release of parasite eggs from the human host.However,approximately50%of eggs produced by schistosomes fail to reach the external environment,but instead become trapped in host tissues where pathological changes caused by the immune responses to secreted egg antigens precipitate disease.Despite the central importance of egg production in transmission and disease,relatively little is understood of the molecular processes underlying the development of this key life stage in schistosomes.Here,we describe a novel parasite-encoded TGF-b superfamily member,Schistosoma mansoni Inhibin/Activin(SmInAct),which is key to this process.In situ hybridization localizes SmInAct expression to the reproductive tissues of the adult female,and real-time RT-PCR analyses indicate that SmInAct is abundantly expressed in ovipositing females and the eggs they produce.Based on real-time RT-PCR analyses,SmInAct transcription continues,albeit at a reduced level,both in adult worms isolated from single-sex infections,where reproduction is absent,and in parasites from IL-7RÀ/Àmice,in which viable egg production is severely compromised.Nevertheless,Western analyses demonstrate that SmInAct protein is undetectable in parasites from single-sex infections and from infections of IL-7RÀ/Àmice,suggesting that SmInAct expression is tightly linked to the reproductive potential of the worms.A crucial role for SmInAct in successful embryogenesis is indicated by the finding that RNA interference–mediated knockdown of SmInAct expression in eggs aborts their development. Our results demonstrate that TGF-b signaling plays a major role in the embryogenesis of a metazoan parasite,and have implications for the development of new strategies for the treatment and prevention of an important and neglected human disease.Citation:Freitas TC,Jung E,Pearce EJ(2007)TGF-b signaling controls embryo development in the parasitic flatworm Schistosoma mansoni.PLoS Pathog3(4):e52.doi:10. 1371/journal.ppat.0030052IntroductionAmongst the Bilateria,transforming growth factor–b(TGF-b)signaling is recognized as playing an essential role in embryogenesis in deuterostomes and in arthropod proto-stomes,but its role in lophotrochozoan protostomes is unclear[1].Schistosomes,the causative agents of schistoso-miasis,one of the major neglected tropical diseases[2,3],are metazoan parasites that belong to the lophotrochozoan phylum Platyhelminthes.Components of TGF-b signaling have been molecularly characterized in metazoans throughout the animal kingdom. Activation of this pathway begins at the cell surface when a dimeric ligand binds a complex consisting of types I and II receptor serine/threonine kinases[4].Upon ligand binding, the constitutively active type II receptor phosphorylates and activates the type I receptor,which then phosphorylates cytoplasmic Smad proteins that translocate to the nucleus, where they mediate gene expression[4].Components of a functional TGF-b pathway(s),including one type I receptor [5](Schistosoma mansoni receptor kinase-1[SmRK1],S.mansoni transforming growth factor–b type I receptor[SmT b RI]),one type II receptor[6,7](SmRK2,SmT b RII),and three Smads [8–10],have been identified in S.mansoni,with nearly all components localized to either the surface of the worm or reproductive tissues of the female[5–9,11].Nevertheless, while nearly the entire transcriptome of S.mansoni has been examined with the identification of163,000expressed sequence tags(ESTs)[12],a ligand of parasite origin for the TGF-b pathway(s)has remained elusive.This has led to the hypothesis that the ligands for schistosome TGF-b receptors are of host origin[5,13,14],and a suggestion that host TGF-b, signaling through SmRK2,plays a role in the pairing of male and female parasites[7].Sexually mature S.mansoni live within the mesenteric vasculature,where each female produces approximately300 eggs each day.Transmission of schistosomiasis depends upon the release of parasite eggs from the human host.Develop-ment of an immature egg into a mature egg containing a miracidium,the stage of the parasite that invades the intermediate fresh water snail host,occurs outside of the female worm,and takes approximately5d.Many of the eggs produced by schistosomes fail to reach the external environ-ment,but instead become trapped in host tissues,where Editor:Rick M.Maizels,University of Edinburgh,United KingdomReceived October9,2006;Accepted February20,2007;Published April6,2007Copyright:Ó2007Freitas et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Abbreviations:ARE,adenosine-and uridine-rich element;ARE-BP,adenosine-and uridine-rich element–binding protein;bp,base pairs;BMP,bone morphogenetic protein;dsRNA,double-stranded RNA;IL-7RÀ/À,interleukin-7receptor knockout; RNAi,RNA interference;RT-PCR,reverse transcriptase–polymerase chain reaction; SmCB1,S.mansoni cathepsin B1;SmInAct,S.mansoni Inhibin/Activin;s.d.,standard deviation;TGF-b,transforming growth factor–b;UTR,untranslated region*To whom correspondence should be addressed.E-mail:ejpearce@mail.med. pathological changes caused by the immune responses to secreted egg antigens cause disease[15].Despite the central importance of egg production in transmission and disease, and recent advances in proteomics and transcriptomics [12,16–18],essentially nothing is known of the molecular pathways involved in embryogenesis in schistosomes.In this study,we describe the cloning and characterization of a S.mansoni TGF-b homolog,S.mansoni Inhibin/Activin (SmInAct).Although we found SmInAct to be expressed in adult male and female parasites,and in eggs,the localization of SmInAct expression to the reproductive organs of female parasites focused our attention on the role of this gene in egg production.A role for SmInAct in reproduction was supported by analyses of female parasites recovered from infertile infections,in which we found that SmInAct protein was undetectable.Confirmation of the importance of this TGF-b superfamily member in the reproductive process was obtained from RNA interference(RNAi)studies,in which targeted knockdown of SmInAct in female worms or directly in the eggs that they produce resulted in a marked cessation of embryogenesis.ResultsCloning and Sequence Analysis of SmInActSmInAct was identified through a t blast n search of the Wellcome Trust’s Sanger Institute’s S.mansoni genome sequence using the C-terminal region of the Drosophila melanogaster dActivin sequence.We were unable to identify SmInAct in EST databases regardless of whether we searched using the coding or39–untranslated region(UTR)sequences. The59and39ends of SmInAct were amplified via rapid amplification of cDNA ends(RACE)using primers designed from within putative coding sequence and adult S.mansoni cDNA as template.The1.3-kb,full-length SmInAct transcript contains10base pairs(bp)of59UTR,808bp of39UTR,and a poly-A tail.The deduced amino acid sequence of SmInAct is 161residues long and contains many of the molecular hallmarks for a TGF-b,including a putative basic proteolytic cleavage site located at position32as RQRR where the bioactive,C-terminal domain(126amino acids)is enzymati-cally separated from the N-terminal pro-domain.Nine invariant cysteine moieties,and invariant proline and glycine residues(Figure1A)essential for the proper dimerization and tertiary structure of a TGF-b homolog,are all predicted in SmInAct.The deduced amino acid sequence of SmInAct contains one putative N-linked glycosylation site at position 110.Within the bioactive domain,SmInAct is27%identical to both DAF-7from Caenorhabditis elegans and dActivin from D.melanogaster,and29%identical to human TGF-b1(Figure 1A).Phylogenetic analysis of SmInAct among other TGF-b superfamily members groups this homolog with members of the TGF-b/Activin subfamily(Figure1B),and further clusters SmInAct phylogenetically with TGF-b homologs from the free-living nematode C.elegans(DAF-7)and the parasitic nematodes Brugia malayi(Bm-TGH-2)and Strongyloides stercor-alis(Ss-TGH-1).SmInAct Transcript and Protein Expression and LocalizationTo determine the expression of SmInAct at the transcript level,real-time reverse transcriptase–polymerase chain reac-tion(RT-PCR)was performed on cDNA from eggs,adult male parasites,and adult female parasites from mixed-sex infec-tions.As seen in Figure2A,SmInAct is expressed in all stages tested at relatively similar levels.Western analyses using polyclonal antibodies against recombinant SmInAct were used to determine the protein expression profile of SmInAct. The anti-SmInAct serum recognized a28-kDa protein in egg antigen extracts and a doublet(32kDa and28kDa)in adult male and female extracts(Figure2B,lanes1–3);these bands presumably represent the unprocessed(32kDa)inactive and processed(28kDa)active forms of the molecule.The relative molecular weights of the two bands recognized by anti-SmInAct antiserum in parasite extracts are larger than that predicted by the sequence,presumably due to detergent and reducing agent-resistant dimerization,and/or to glycosylation at amino acid110.Glycosylation plays an important role in the solubility and secretion of other members of the TGF-b superfamily[19,20].Eggs appear to contain only the lower molecular weight,putatively active form of SmInAct.To localize SmInAct within the parasite,we performed in situ hybridization on sections of adult worms.Anti-sense probes localized SmInAct transcripts to the reproductive tissues of the adult female,with strong signals in the vitellaria and ovary (Figure2C),whereas in adult males,SmInAct transcripts localized to various subtegumental regions(Figure2D).The expression pattern in the female suggested a role for SmInAct in egg production.We focused on this possibility,and reasoned that if this were the case,SmInAct expression might be diminished in unfertile females.In vivo,successful oogenesis requires the presence of male schistosomes[21], and,for reasons that have remained unclear,an intact CD4þT lymphocyte compartment within the host[22].Therefore, we analyzed SmInAct expression in female parasites from mice harboring single-sex infections,and in parasites from severely lymphopenic interleukin-7receptor knockout(IL-7RÀ/À)mice carrying mixed-sex infections,which produce a significant number of dead eggs[23,24].Real-time RT-PCR demonstra-ted that SmInAct mRNA levels were significantly decreased,Author SummarySchistosomes are parasitic worms that infect hundreds of millions of people in developing countries.They cause disease by virtue of the fact that the eggs that they produce,which are intended for release from the host in order to allow transmission of infection,can become trapped in target organs such as the liver,where they induce damaging inflammation.Egg production by female schisto-somes is critically dependent on the presence of male parasites, without which females never fully develop,and(counterintuitively) on the contribution of signals from the host’s immune system.Very little is understood about the molecular basis of these interactions. Here,we describe a newly discovered schistosome gene,which is expressed in the reproductive tract of the female parasite and in parasite eggs.The protein encoded by this gene is made only when females are paired with males in an immunologically competent ing recently developed tools that allow gene function to be inhibited in schistosomes,we show that the product of this gene plays a crucial role in egg development.Examining how the expression of this gene is controlled has the potential to provide insight into the molecular nature of the interactions between male and female parasites and their hosts.Moreover,the pivotal role of this gene in the egg makes it a potential target for blocking transmission and disease development.but not absent,in females from these infections (Figure 2E).Of particular interest,SmInAct protein was undetectable by Western analyses in females from single-sex infections as well as from infections of IL-7R À/Àmice (Figure 2B).While the localization of SmInAct transcripts to the male subtegumental region is not immediately informative in terms of function in the male,we nevertheless noted that male parasites recovered from infertile infections in IL-7R À/Àmice were similar to female parasites in terms of transcrip-tional and post-transcriptional regulation of SmInAct ex-pression (Figure 2B and 2F).Moreover,this was also the casefor male parasites recovered from male single-sex infections (Figure 2B and 2F).RNAi-Mediated Knockdown of SmInAct ExpressionTo gain a better understanding of the function of SmInAct and the signaling pathway it activates,this TGF-b homolog was targeted for knockdown via RNAi [25–27].Pairs of adult males and females recovered from infected mice were soaked in double-stranded RNA (dsRNA)corresponding to SmInAct (1l g/ml)or an irrelevant control dsRNA (luciferase)for 1wk in vitro,followed by RNA extraction and real-time RT-PCR analyses.SmInAct dsRNA–treated worms showed aconsistentFigure 1.SmInAct Is a Member of the TGF-b /Activin Subfamily(A)ClustalW alignment of the C-terminal domain of the SmInAct protein with three other members of the TGF-b /Activin subfamily.Amino acids identical to the SmInAct sequence are shaded gray.Numbers to the right indicate position of the last amino acid in the row within each respective full-length sequence.Stars indicate invariant amino acid residues in TGF-b homologs.(B)Phylogenetic dendrogram demonstrating that SmInAct is a member of the TGF-b superfamily.SmInAct (red)is shown clustering among members of the TGF-b /Activin subfamily (solid line),and not with members of the BMP/growth differentiation factor subfamily (dashed line).Conserved residues in the C-terminal region of each homolog (final 94–106amino acids)were used in the analysis.Percentages at branch points are based on 1,000bootstrap runs.doi:10.1371/journal.ppat.0030052.g001and significant decrease in SmInAct expression of .40%when compared to SmInAct expression in worms soaked in the irrelevant control dsRNA (Figure 3A).No consistently significant difference in the numbers of eggs produced by control versus SmInAct dsRNA–treated worm pairs was observed,suggesting that SmInAct is not important for egg production per se.However,in examining these cultures,wenoted that eggs produced by SmInAct dsRNA–treated para-sites failed to develop (unpublished data).To specifically address the role of SmInAct in egg development,we treated eggs directly with SmInAct dsRNA.Approximately 20%of eggs laid by adult parasites during the first 2d of in vitro culture will develop over the ensuing 5d to contain miracidia [28],with a typical progression of development throughsixFigure 2.SmInAct Expression Is Linked to Reproductive Capability(A)SmInAct is expressed in the egg,adult male,and adult female.The RNA tested is indicated on the x -axis,and the y -axis represents the ratio of SmInAct cDNA relative to a -tubulin cDNA (reference gene),as determined by real time RT-PCR.Data are presented as mean ratios (þ/Àstandard deviation [s.d.])from three separate experiments.There is no significant difference in SmInAct expression among the stages tested.(B)SmInAct protein is detectable in eggs,adult males,and adult females from mixed-sex infections in wild-type mice,but is not detectable in females or males from single-sex infections,or in females or males from mixed-sex infections of IL-7R À/Àmice.(C)SmInAct transcript is localized to the reproductive tissues of the adult female,including the ovary (O)and vitellaria (V)(left panel,in situ hybridization with anti-sense probe)A serial section was probed with sense-strand SmInAct and over-developed (right panel).G,gut.Scale bar ¼110l m.(D)SmInAct transcript is localized to subtegumental regions of the adult male,with concentrations of expression around the oral sucker (O.S.)and ventral sucker (V.S.)(left panel,in situ hybridization with anti-sense probe).A serial section was probed with sense-strand SmInAct and over-developed (right panel).Scale bar ¼110l m.(E)SmInAct mRNA levels are significantly lower in females isolated from single-sex infections or from IL-7R À/Àmice than in females isolated from infected wild-type mice.SmInAct mRNA levels were measured by real-time RT-PCR.Data are presented as mean fold change in expression (þ/Às.d.)from two RNA extractions.(F)SmInAct mRNA levels in males isolated from single-sex infections or from IL-7R À/Àmice compared to mRNA levels in wild-type mice.SmInAct mRNA levels were measured by real-time RT-PCR.Data are presented as mean fold change in expression (þ/Às.d.)from two RNA extractions.There is no significant difference in SmInAct expression in males from single-sex infections or from IL-7R À/Àmice versus males isolated from mixed-sex infection of wild-type mice.doi:10.1371/journal.ppat.0030052.g002stages illustrated in Figure 3B.Therefore,eggs produced by worm pairs for the first 2d ex vivo were collected and soaked in dsRNA (1l g/ml)corresponding to SmInAct or an irrelevant dsRNA for 5d,and their development was scored.Relative to eggs soaked in an irrelevant dsRNA,where ;20%of the eggs developed through stage 6,eggs treated with SmInAct dsRNA aborted development at stage 2(Figure 3C and 3D).An absence of SmInAct transcripts (Figure S1),and a nearly 10-fold decrease in SmInAct protein (Figure 3E),were associated with the failure of SmInAct dsRNA–treated eggs to develop.This phenotype was not observed when eggs were treated with dsRNA corresponding to luciferase,a sequence not encoded in the schistosome genome (Figure 3C and 3D),or toS.mansoni cathepsin B1(SmCB1),a cathepsin B detectable in eggs (Table 1).DiscussionMultiple components of a TGF-b signaling pathway have been characterized in S.mansoni,but a ligand of parasite origin for the pathway has remained elusive.Additionally,while functions in host–parasite interactions have been proposed based on the expression of receptors on the parasite surface,and on the responsiveness of the parasite receptors to host TGF-b [5–7,14],the function that TGF-b signaling plays in S.mansoni has remained unclear.In this study,we report the expression of SmInAct,a TGF-b–likeFigure 3.SmInAct Is Essential for Egg Development(A)Treatment of adult parasites with dsRNA corresponding to SmInAct led to a 40%reduction in SmInAct mRNA levels.dsRNA treatment is indicated on the x -axis,where control worms were treated with luciferase dsRNA.Data are presented as the mean fold change in SmInAct expression (þ/Às.d.)from three separate experiments,as determined by real-time RT-PCR using paramyosin as a reference gene for expression.(B)Developmental progression of eggs laid in vitro.Eggs produced by paired males and females during the first 48h ex vivo were cultured in vitro for 5d,and an egg from the developing majority was photographed.Stages of development approximate progressive 20-h periods.Scale bar ¼110l m.(C)Immature eggs produced by adult parasites ex vivo and soaked in SmInAct dsRNA failed to develop into miracidia.Eggs soaked in an irrelevant control dsRNA (luciferase,1l g/ml)developed through stage 6within 5d (left)while eggs soaked in SmInAct dsRNA (1l g/ml)for the same period halted development at stage 2(right).Main scale bar ¼210l m.Inset scale bar ¼110l m.(D)Quantitative analysis of the SmInAct dsRNA–induced developmental phenotype.Control-or SmInAct dsRNA–treated eggs were examined microscopically and scored as either developed or undeveloped based on the presence or absence of a miracidium.Data are presented as mean percent developed (þ/Às.d.)from four separate experiments.(E)SmInAct protein levels are decreased by approximately 10-fold following treatment with SmInAct dsRNA.Protein extacts from 350control or SmInAct dsRNA–treated eggs were separated via SDS-PAGE in 10-fold serial dilutions,blotted,and probed with anti-SmInAct antiserum.A silver-stained sister SDS-PAGE gel is shown to confirm protein loading.doi:10.1371/journal.ppat.0030052.g003ligand in the parasiticflatworm S.mansoni,the production of which is coupled to the reproductive potential of the worms. We provide evidence that SmInAct plays a crucial role in embryogenesis.Understanding of the developmental processes regulated by TGF-b in invertebrates is based largely on data from the model organisms D.melanogaster and C.elegans.Decapentaplegic, a bone morphogenetic protein(BMP)–like homolog in D. melanogaster,acts as a morphogen by determining cell fate along the dorsal–ventral axis in a gradient-dependent manner[29].Also in D.melanogaster,a type I receptor,baboon, stimulates cellular proliferation and is essential for normal embryonic development[30].Presumably,SmInAct could be fulfilling functions in the schistosome egg analogous to these known roles for decapentaplegic and/or baboon.None of the three characterized TGF-b homologs in C.elegans are important for patterning or growth of the embryo[31–33]; however,two TGF-b homologs have yet to be examined(tig-2 and Y46E12BL.1),and,intriguingly,serial analysis of gene expression(SAGE)tags for both homologs have been found in the C.elegans embryo[34].Like the other C.elegans TGF-b homologs that are resistant to RNAi affects,tig-2and Y46E12BL.1have no phenotype in genome-wide RNAi screens[35,36];therefore,direct mutagenesis will likely be required to determine the function of these genes.The identification of SmInAct,a TGF-b superfamily member,as a key component of egg development in S. mansoni,a member of the Platyhelminthes,the earliest branch of the Bilateria[37],underscores the central role played by this pathway in embryogenesis.While one type I and one type II TGF-b receptor have been characterized for S.mansoni, there appears to be at least three type I receptors and two type II receptors present in the genome based on a preliminary blast search for homologs.It will be important to delineate which of the S.mansoni type I and type II TGF-b receptors are involved in SmInAct signaling and to identify the Smads important for transmitting the signal induced by this growth factor.Furthermore,identifying the genes regulated by SmInAct signaling will provide information regarding the precise function that this growth factor serves in egg maturation,as well as the functions the pathway may serve in other life stages of the parasite,including the adult male.SmInAct protein was not detectable in infertile females recovered from single-sex infections or from IL-7RÀ/Àmice, despite the fact that these parasites contained SmInAct transcripts(although at lower levels than in fecund parasites). This strongly indicates that SmInAct is both transcriptionally and post-transcriptionally regulated by worms of the oppo-site sex as well as by signals from the host.It is well established that parasites recovered from hosts lacking CD4þT cells are developmentally stunted and produce significantly fewer fertile eggs than those recovered from mixed-sex infections of immunocompetent hosts.Translation of SmInAct mRNA is thefirst identified molecular process downstream of the effect of the host immune system on schistosome develop-ment[22–24],and as such,could open the way towards an increased understanding of this unusual feature of schisto-some biology.Thefinding that the production of SmInAct in males is under the same constraints as in females is curious and perhaps indicates an additional function(s)for SmInAct in S.mansoni.We are unaware of a link between the site of expression of SmInAct in the male schistosome and repro-ductive events,and further work is required to elucidate the function of SmInAct in male worms.In other settings,the uncoupling of transcription and translation is linked to the activation of the integrated stress response[38–41].This mechanism,conserved in eukaryotes, re-programs cells to conserve energy in response to stress signals such as amino acid deficiency and oxidative stress by restricting the translation of transcripts requiring an active translation initiation complex[38–41].Limited cellular energy is then used for the expression of genes necessary to maintain cell viability[42].In this context,parasites in single-sex infections and in mice lacking CD4þT cells may be considered stressed due to the lack of signals received from the opposite sex and immunocompetent host,thereby restricting the translation of non-essential transcripts.SmI-nAct protein expression may be considered expendable considering the role it plays in embryogenesis rather than in crucial cellular functions linked to the survival of the adult worm.A more thorough investigation of the S.mansoni homologs of translation factors involved in the stress response and of the regulation of other transcripts and protein expression will be required to evaluate this possi-bility.Post-transcription regulation of eukaryotic transcripts is controlled in part by the39UTR[43].This region can bind elements(including microRNAs and proteins)that inhibit the translation and/or decrease mRNA stability.For example, 39UTRs of several mammalian cytokines contain adenosine-and uridine-rich elements(AREs)that bind ARE-binding proteins(ARE-BPs)(reviewed in[44]).The binding of ARE-BPs to these transcripts causes either rapid decay or inhibits their translation.While AREs are somewhat divergent in sequence,they often contain the consensus‘‘AUUUA’’andparsion of the Development of Eggs Soaked in SmInAct dsRNA versus SmCB1dsRNAdsRNA Treatment Developed Eggs a(number)Undeveloped Eggs b(number)Percent Developed Significance c Control7134217.2SmInAct49721 6.4p0.0001 SmCB112452019.3p¼0.446a The number of eggs that reached stage6of development(Figure3B).b The number of eggs that failed to develop past stage2(Figure3B).c p-values based on Yates’chi-square between SmInAct or SmCB1dsRNA–treated eggs and control.doi:10.1371/journal.ppat.0030052.t001are found in a uridine-rich environment.Interestingly,the long39UTR of SmInAct has two exact repeats of‘‘UUUC-TAUUUA’’that contain the consensus‘‘AUUUA’’ARE (underlined).Furthermore,the39UTR of SmInAct is U-rich (43%uridines).It will be interesting to determine whether these repeats,or other regions of the long39UTR,play a role in the post-transcriptional regulation of SmInAct expression. It is of interest when considering the relationship of schistosomes with their mammalian hosts to note that in other systems,TGF-b superfamily members have been shown to function across phylum boundaries[45,46].For example, the Drosophila BMP homologs DPP and60A are able to induce bone development when injected into the skin of rats[45], and mammalian BMP-4can rescue Drosophila DPP mutants [46].Consequently,we believe that it is feasible that SmInAct could act as a ligand to initiate signaling in host cells.It is clear that proteins produced by eggs have distinct immuno-modulatory functions[47],and SmInAct could conceivably participate in these effects if secreted/excreted from the schistosome egg.Our identification of SmInAct as a cytokine that is molecularly conserved between host and parasite, coupled with the description of an effective method for altering gene expression in the schistosome egg,allows these and other issues to now be addressed.Despite recent advances in vaccine design[48],a solution for schistosomiasis remains an elusive goal.Current attempts to control schistosomiasis depend on repeated administration of one drug,praziquantel,with no replacements waiting in the wings should resistance develop.Understanding how schistosome eggs develop could provide targets for intervention in the schistosome life cycle and for blocking disease progression. Materials and MethodsParasites and animals.The Puerto Rican/NMRI strain of S.mansoni was used in all experiments.Adult schistosomes were recovered by hepatic-portal perfusion from C57BL/6female mice or B6IL-7RÀ/À(The Jackson Laboratory,)that had each been percutaneously exposed to;60cercariae8wk earlier.Adult parasites and eggs laid were maintained in vitro in M199(Gibco,http:// ),10%fetal calf serum,1%Antibiotic/Antimy-cotic(Gibco),and1%HEPES in a378C/5%CO2atmosphere as previously described[11,28].Isolation of full-length SmInAct cDNA from S.mansoni.The C-terminal,translated region of the Drosophila activin homolog (dActivin)(amino acids565–669)was used to search the Wellcome Trust’s Sanger Institute’s S.mansoni genome assembly using the t blast n algorithm.A contig(0020320)with significant similarity to dActivin was identified.Full-length cDNA corresponding to SmInAct was isolated using total RNA(1l g)from adult parasites and the SuperScript III GeneRacer59and39RACE kit(Invitrogen,http:// )as per manufacturer’s instructions.Gene-specific primers were designed for isolation of the59-end(59-G G T T C A A A A C T T T T C G G G T G T A-39)a n d39-e n d(59-AATCTTGTTGTCATCCAACTCAA-39)of SmInAct and used in RT-PCR with GeneRacer59and39primers according to manufacturer’s suggestions.Resulting amplicons were cloned into the TOPO cloning vector(Invitrogen)and sequenced.To verify the full-length sequence of SmInAct,primers designed from the59and39ends of the transcript were used in RT-PCR,and the resulting fragment was cloned and sequenced.Sequence analysis.Sequence similarities between the deduced amino acid sequence of SmInAct and other members of the TGF-b superfamily were determined through multiple sequence alignments using the ClustalW algorithm,as well as the Align2sequences(bl2seq) program at the National Center for Biotechnology Information ().An unrooted phylogram was drawn using amino acids within the conserved C-terminal domain of SmInAct,and known TGF-b superfamily members and distances were drawn using the Dayhoff Pam matrix and neighbor-joining algorithm in the PHYLIP software package developed by J. Felsenstein,University of Washington,Seattle,Washington,United States(/phylip.html).Percen-tages at branch points are based on1,000bootstrap runs.Real-time RT-PCR.Total RNA was extracted from parasites using Qiagen’s RNeasy Mini kit(),and contaminating genomic DNA was removed by DNase treatment using the Turbo DNA-free endonuclease(Ambion,).First-strand cDNA was synthesized using500ng of RNA,SuperScript II reverse transcriptase(Invitrogen),and oligo dT as a primer.RT-minus controls were performed to confirm the absence of genomic DNA(unpublished data).SmInAct transcript levels in egg and adult stages were quantified relative to a-tubulin using Applied Biosystems’7500real-time PCR system and SYBR green PCR Master Mix(Applied Biosystems,http:// ).Total reaction volume was10l l with 300nM of each primer,5l l of SYBR green PCR Master Mix,and0.5 l l of cDNA as template(or water as a negative control).SmInAct primers were:forward59-AATCTTGTTGTCATCCAACTCAA-39and reverse59-AACTACAAGCACATCCTAAAACAA-39.a-Tubulin pri-mers were:forward59-CCAGCAAATCAGATGGTGAA-39and reverse 59-TTGACATCCTTGGGGACAAC-39.PCR efficiency(E)was deter-mined for both primer sets by plotting cycle thresholds from a10-fold serial dilution of cDNA and inputting the slope in the equation E ¼10(À1/slope).For expression analyses,quantification of SmInAct transcript relative to a-tubulin was calculated using the equation: ratio¼(E Sm a-tubulin)CT/(E SmInAct)CT where E Sm a-tubulin is the PCR efficiency of the reference gene,E SmInAct is the PCR efficiency of target gene,and CT is the cycle threshold.For analysis of RNAi-induced knockdown,quantification of SmInAct transcript relative to p a r a m y o s i n(p a r a m y o s i n p r i m e r s w e r e:f o r w a r d59-CGTGAAGGTCGTCGTATGGT-39and reverse59-GACGTT-CAAATTTACGTGCTTG-39)was calculated using the2ÀDD Ct method. Dissociation curves were generated for each real-time RT-PCR to verify the amplification of only one product.Recombinant SmInAct expression,antiserum production,and Western analyses.Eco RI(forward)and Xho I(reverse)tagged primers were designed to amplify the C-terminal bioactive region of SmInAct(forward59-GGAATTCTCATTAACTAAAGGAGATGA-3 and reverse59-CCGCTCGAGTTAACTACAAGCACATCCTA-39). The amplified product was cloned into the expression vector pET28aþ(Novagen,)and sequenced to verify the absence of any mutations.Expression of recombinant SmInAct was induced in Escherichia coli BL21(DE3)by addition of1 mM IPTG when cultures reached an OD600of0.5at378C,followed by 3hours of shaking at room temperature.Recombinant SmInAct was expressed in bacteria as insoluble inclusion bodies.Exhaustive attempts to refold the protein using gluathione and reduced glutathione proved unsuccessful.We therefore purified the protein via nickel column chromatography under denaturing conditions(6M urea)as per the manufacturer’s protocol(Novagen).Antiserum was generated by Cocalico Biologicals(http://www.cocalicobiologicals. com)through subcutaneous inoculation of a rabbit with100l g of purified protein in complete Freund’s adjuvant,followed by three boosts of50l g in incomplete Freund’s adjuvant on days14,21,and 49,followed by exsanguinations on day64.For detection of SmInAct protein,10l g of protein extracted from eggs,adult males,and adult females via Dounce homogenizing in lysis buffer(1%Triton-X100,20mM HEPES,10%glycerol,150mM NaCl) supplemented with a protease inhibitor cocktail(Sigma,http://www. )were separated by SDS-PAGE,electroblotted,and probed with anti-SmInAct antiserum(1:10,000),pre-immune serum (1:10,000),or a monoclonal antibody(4B1)against paramyosin. Affinity purified HRP-conjugated goat anti-rabbit IgG(Cell Signaling Technology,)was used to detect bound rabbit antibodies,while an affinity purified HRP-conjugated horse anti-mouse IgG(Cell Signaling Technology)was used to detect the anti-paramyosin monoclonal antibody.The secondary antibodies were detected using ECL reagents as per manufacturer’s instructions (GE Healthcare,).In situ hybridization.Localization of SmInAct in5-l m sections of adult S.mansoni was performed as previously described[49].DIG-labeled sense and anti-sense transcripts were generated using Roche’s DIG RNA labeling mix()as per manufacturer’s instructions with T7-tagged amplicons as template(sense:forward59-TAATACGACTCACTATAGGGTTGATCCAAAAAAGGTTGT-TATGG-39,reverse59-TTAACTACAAGCAGCTCCTA-39;anti-sense: forward59-ATAATATGTAATAATTGTGA-39reverse59-TAATAC-GACTCACTATAGGGAACTACAAGCACATCCTAAAACAA-39).The hybridized DIG-probes were detected using an alkaline-phosphatase。
分子互作实验的英文
分子互作实验的英文Molecular Interaction ExperimentMolecular interaction experiments are a type of scientific study that aims to investigate the interactions between molecules. These experiments are crucial in understanding various biological processes, drug discovery, and the development of new materials.The experimental process typically involves the use of techniques such as spectroscopy, chromatography, and microscopy to observe and analyze the interactions between molecules. By studying these interactions, researchers can gain insights into the mechanisms underlying biological processes, the binding affinity and kinetics of molecules, and the effects of molecular modifications on interaction properties.Molecular interaction experiments can be conducted at the cellular, molecular, or atomic level. They help researchers understand the behavior of molecules within living systems, as well as their roles in diseases and therapeutic interventions. For example, in drug discovery, molecular interaction experiments can aid in the identification of potential drug targets and the development of novel therapeutic agents.Furthermore, these experiments also contribute to the field of materials science by investigating the interactions between molecules in the development of new materials with desired properties. Understanding molecular interactions is essential for the design of advanced materials, catalysts, and sensors.In conclusion, molecular interaction experiments play a vital role in scientific research, providing valuable insights into biological processes, drug discovery, and materials development. Through these experiments, researchers can unravel the complexities of molecular interactions and drive advancements in various scientific disciplines.。
化学chemistry
化学chemistryChemistryChemistry is the scientific discipline involved with elements, compounds, and molecules and their interactions with one another and with other forms of energy. It is a branch of physical science that studies the composition, structure, properties and changes of matter. Chemistry is divided into several branches, including organic chemistry, inorganic chemistry, physical chemistry, analytical chemistry, biochemistry and medicinal chemistry.The study of chemistry has several goals. The first is to understand the behavior of the elements and compounds, and the second is to understand how and why they interact with one another. As a result, chemists use a variety of tools to gain a better understanding of the behavior of matter, from the most basic laboratory equipment to sophisticated computer models. Chemists use the laws of atomic theory and quantum mechanics to explain the behavior of matter. They also use the tools of mathematics, such as calculus and linear algebra, to develop models of chemical interactions. These models help chemists describe and predict the behavior of substances and molecular structures.The field of chemistry has many practical applications. It is used to manufacture a wide variety of products, from food additives and medicines to fuel and building materials. It also has important implications for our environment and human health. For example, chemists can use their knowledge of the properties of materials to create safer and more efficient products, such as cleaner fuels, better medical treatments, and more efficient heating and cooling systems.。
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Chii-Shen Yang, Ravi Iyengar, Richard J. Miller, Lily Y. Jan, Robert J. Lefkowitz, Heidi E. Hamm†
To determine whether purified G1H6␥1 mutants could form heterotrimers, we mea-
dent signaling, suggesting that G␥ may use
a common binding surface for interaction Fig. 1. Effects of G1H6␥1 on heterotrimer A
with G␣ and with its diverse effectors. Two regions on G␥ that interact with G␣ have
quired for activation of each effector and provides evidence for partially overlapping ceptors that catalyze the exchange of GDP
domains on G for regulation of these effectors. This organization of interaction regions for guanosine triphosphate (GTP) on the
G␣ subunits (G␣-GDP) can compete with with G␣, to be activated by rhodopsin, porting receptor-catalyzed nucleotide ex-
G␥ effectors and deactivate G␥-depen-
(A) The ability of recombinant G1H6␥1 and G1 mutants to assemble into hetero-
face (G residues 57, 59, 98, 99, 101, 117, trimers with Gt␣ was determined by testing
gesting a common interface on G␥ subunits for G␣ binding and effector interaction. The Ala80 (I80A), K89A, L117A, and W332A
molecular basis for interaction of G␥ with effectors was characterized by mutational (11) showed reduced ability to form hetero-
Upon receptor activation, G proteins dis- and 89). Each of these residues on retinal Y59A) and the NH2-terminal interface mu-
sociate into free G␣ and G␥ subunits that G (G1) was substituted with alanine, tants I80A and K89A were defective in for-
dinucleotide (10). The G residue mutated
C. E. Ford, N. P. Skiba, H. Bae, C.-S. Yang, H. E. Hamm, to alanine is indicated by a number be-
Institute for Neuroscience and Department of Molecular neath each bar in the figure. Clear bar (C)
assembly and receptor interaction. The data are the normalized percentage of wild-type (WT) recombinant G␥ activity.
been defined by the crystal structures of heterotrimeric G␣␥ (8), the switch inter-
represents the basal amount of ADP-ribosylation of Gt␣ that occurred in absence of G␥. (B) The ability of recombinant
kinases (5) and phosphoinositide 3-kinases tionally modified appropriately, and were binding to Gt␣. The switch interface mu-
(6). Other potential G␥ effectors incluห้องสมุดไป่ตู้e expressed at similar amounts as in the wild tants (S98A, W99A, M101A, N143A, and
pholipases (2), adenylyl cyclases (3), ion G␥ subunit. All mutated G1␥1 dimers assays, indicating that residues 80, 89, 117,
channels (4), G protein–coupled receptor were folded properly, were post-transla- and 332 of G are the major determinants of
dynamin I and the nonreceptor protein ty- type (9). The G␥ mutants were tested for D186A) were normal in heterotrimer assem-
rosine kinases Btk and Tsk (7). GDP-bound their ability to assemble into heterotrimers bly, but were impaired functionally in sup-
the G mutants to support light-activated
rhodopsin-catalyzed nucleotide exchange on
the ␣ subunit of transducin (Gt␣) (12). All
switch interface mutants (except K57A and
sured the ability of the G␥ mutants to
facilitate pertussis toxin–catalyzed adeno-
Both the ␣ and ␥ subunits of heterotrimeric guanine nucleotide–binding proteins (G sine diphosphate (ADP) ribosylation of
analysis of G residues that make contact with G␣-GDP. Analysis of the ability of these trimers (Fig. 1A).
mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2,
on G for different effectors and G␣ explains why subunit dissociation is crucial for signal G␣ subunit, we also measured the ability of
transmission through G␥ subunits.
adenylyl cyclase 2 (AC2), muscarinic potas-
Carolyn E. Ford,* Nikolai P. Skiba,* Hyunsu Bae, Yehia Daaka, sium channel (GIRK1/GIRK4), and the cal-
Eitan Reuveny, Lee R. Shekter, Ramon Rosal, Gezhi Weng, cium channel ␣1B subunit (CC␣1B).
REPORTS
Molecular Basis for Interactions of G Protein ␥ and to interact with G␥ downstream sig-
Subunits with Effectors
naling partners: -adrenergic receptor kinase (ARK), phospholipase C-2 (PLC-2),
ADP-Ribosylation (% WT)
119, 143, 186, 228, and 332) and the NH2- whether pertussis toxin could ADP-ribosyterminal interface (G residues 55, 78, 80 late Gt␣ with [32P]nicotinamide adenine
Because G␥ is essential for functional