利用绿色荧光蛋白基因gfp研究芽胞杆菌的启动子活性

利用绿色荧光蛋白基因gfpmut3,分别标记苏云金芽胞杆菌(Bacillus thuringiensis)的cry3A启动子Pcry3A、BtI_BtII启动子PBtI_BtII和来自蜡状芽胞杆菌特异启动子P44-12以研究其表达差异。其中,Pcry3A和PBtI_BtII分别与gfpmut3构成融合基因,以调控gfpmut3在苏云金芽胞杆菌中的表达。将重组质粒pGFP_304(含P44-12)、pGFPExpA(含Pcry3A_ gfpmut3融合基因)和pGFPExpB(含PBtI_BtII_ gfpmut3融合基因)分别导入大肠杆菌(Escherichia coli)和苏云金芽胞杆菌后发现,P44-12和PBtI_BtII在大肠杆菌与苏云金芽胞杆菌中均可表达gfpmut3,其中PBtI_BtII在大肠杆菌中具有极强的启动基因表达的能力。而Pcry3A不能启动gfpmut3在大肠杆菌中表达,在苏云金芽胞杆菌中启动的gfpmut3表达的荧光强度也较弱。进一步通过荧光显微镜和生物活性检测器对含重组质粒pGFP_304、pGFPExpA和pGFPExpB的转化子分别进行荧光检测及微量热检测。结果表明,3种启动子驱动下的gfpmut3基因均可在苏云金芽胞杆菌无晶体突变株BMB171中表达并检测得到不同的发光类型。微量热法检测发现P44_12和PBtI_BtII启动gfpmut3表达的代谢热低于Pcry3A驱动gfpmut3表达的代谢热。     

Estimating Orientation Factors in the FRET Theory of Fluorescent Proteins: The TagRFP-KFP Pair and Beyond

The orientation factor κ², one of the key parameters defining Förster resonance energy transfer efficiency, is determined by the transition dipole moment orientations of the donor and acceptor species. Using the results of quantum chemical and quantum mechanical/molecular mechanical calculations for the chromophore-containing pockets in selected colored proteins of the green fluorescent protein family, we derived transition dipole moments corresponding to the S_0,min → S₁ excitation for green fluorescent protein, red fluorescent protein (TagRFP), and kindling fluorescent protein, and the S_1,min → S₀ emission for TagRFP. These data allowed us to estimate κ² values for the TagRFP-linker-kindling fluorescent protein tetrameric complex required for constructing novel sensors.     

Heterogeneity in Thymic Emigrants: Implications for Thymectomy and Immunosenescence

The development of mature, antigen-inexperienced (naive) T cells begins in the thymus and continues after export into the periphery. Post-thymic maturation of naive T cells, in humans, coincides with the progressive loss of markers such as protein tyrosine kinase 7 (PTK7) and platelet endothelial cell adhesion molecule-1 (CD31). As a consequence, subpopulations of naive T cells can be recognised raising questions about the processes that give rise to the loss of these markers and their exact relationship to recent thymic emigrants (RTE). Here, we combine a mathematical survival analysis approach and data from healthy and thymectomised humans to understand the apparent persistence of populations of ‘veteran’ PTK7⁺T cells in thymectomised individuals. We show that a model of heterogeneity in rates of maturation, possibly linked to natural variation in TCR signalling thresholds or affinity for self-antigens, can explain the data. This model of maturation predicts that the average post-thymic age of PTK7⁺T cells will increase linearly with the age of the host suggesting that, despite the immature phenotype, PTK7⁺cells do not necessarily represent a population of RTE. Further, the model predicts an accelerated increase in the average post-thymic age of residual PTK7⁺T cells following thymectomy and may also explain in part the prematurely aged phenotype of the naive T cell pool in individuals thymectomised early in life.     

A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid β-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.Peroxisomes are single membrane-bound organelles harboring at least one H₂O₂-generating oxidase and one H₂O₂-decomposing catalase, and they are present in virtually all eukaryotic cells, from yeast to mammals. The most conserved activity of peroxisomal metabolism is the β-oxidation of fatty acids (27).Peroxisome assembly requires more than 20 PEX gene products, termed peroxins, in any given organism (5). The impairment of peroxisomal protein transport caused by mutations in PEX genes causes fatal human peroxisome biogenesis disorders (PBDs) (34). In the cells of such PBD patients, essential enzymes normally localized to peroxisomes are found mostly in the cytosol. Mammalian cell lines harboring mutations in peroxins (including fibroblasts from PBD patients) grow well in cell culture. On the other hand, pex mutants of the methylotrophic yeast Pichia pastoris can grow normally on glucose but not oleate or methanol (37).Peroxisomal metabolic pathways can generate a high level of reactive oxygen species (ROS) (32). Therefore, peroxisomal disorders have been studied with a focus on the generation of ROS. However, the relationship between PBDs and the intracellular redox state is unclear (13, 32).Peroxisomes have long been thought to be in a more highly oxidized state than the cytosol due to this generation of ROS. However, there is no reported experimental evidence supporting this notion. We previously identified a 20-kDa peroxisomal membrane protein, named Pmp20, in methanol-induced peroxisomes of methylotrophic yeasts. Pmp20 had a glutathione (GSH) peroxidase activity, suggesting the presence of glutathione within the peroxisomes (9). However, we and other groups of investigators have been unable to determine the levels of the reduced and oxidized forms of glutathione due to technical difficulties and therefore have been unable to assess the redox state within peroxisomes by conventional biochemical methods.In general, the intracellular redox state is determined by the levels of redox-related metabolites that are generated by multiple metabolic pathways. (We herein refer to the “redox state” as an intracellular environment at steady state, which is distinct from oxidative stress or ROS, which functions as a signal for further intracellular events such as apoptosis.) Therefore, the redox state is considered to reflect the overall metabolic status. While the standard redox potential (E₀′) is a general index used to express the redox state of a compound, it cannot be used to describe the intracellular redox state because it does not take into account various physiological considerations, such as the cytosol, where many compounds coexist in a mixture of various redox states (14). Therefore, the equilibrium redox state in living cells has been estimated from indices such as the ratio of oxidized and reduced forms of glutathione, from indirect indices of the redox state, such as the NAD(P)H ratio (12, 40), or from the level of the expression of antioxidant enzymes. However, the measurement of these indices often yields contradictory results, making it difficult to evaluate the physiological redox state using any single index. This situation might have led to misunderstanding the redox state in cells from patients with PBDs. Reductive conditions could occur during conditions of oxidative stress, when the ROS defense system is functioning normally.With the aim of determining the intracellular redox state directly, we developed a fluorescent redox probe, Redoxfluor, with a novel sensing mechanism. Several green fluorescent protein (GFP) variants that report the in vivo redox state (roGFP [4, 7], rxYFP [18, 24, 25]) or H₂O₂ level (HyPer [3]) have been developed since the start of our research. However, none of these reporters have been used to visualize the redox state in mammalian cytosol, and differences in the redox potential between normal and pathological states have not been reported.In the present work, we developed a Redoxfluor that discriminates the redox state of peroxisome assembly mutant cell lines (34) from that of the normal cell line. Our findings shed light on how to tackle problems with monitoring the spatiotemporal dynamics of the redox state within living mammalian cells and also should pave the way for the development of a screen for drugs that can affect various metabolic disorders with abnormal redox state.     

The Impact of Sediment Characteristics on PCB-dechlorinating Cultures: Implications for Bioaugmentation

Polychlorinated Biphenyl (PCB)-dechlorinating cultures with complimentary activities, previously derived from estuarine Baltimore Harbor (B), marine Palos Verdes (P), and riverine Hudson River (H) sediments, were mixed and then inoculated into sterile sediments from the same sources. In the treatments containing sterile B sediment, the different inocula had limited impact on the bacterial community development and on dechlorination patterns, all of which were similar. In treatments containing sterile P or H sediment, however, different inocula resulted in significantly different PCB dechlorination patterns and bacterial communities. The B sediment appeared to support not only the most extensive and rapid dechlorination of the three sediments, but also supported a more diverse bacterial community. This was thought to be a result of nutritional richness, as it was high in organic carbon and micronutrients such as zinc and cobalt. Although mixing three PCB-dechlorinating cultures was able to produce a culture capable of enhanced PCB-dechlorinating activity as compared to single cultures, some activities were lost upon culture transfer. This indicates that care must be taken to establish robust PCB-dechlorinating cultures capable of extensive dechlorination prior to pursuing bioaugmentation. In addition, our results indicate that the concentration and availability of macro-and micronutrients could have a significant impact on the microbial community structure, and thus a thorough characterization of the sediment at contaminated sites is essential for implementing bioaugmentation for PCB bioremediation.     

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.     

Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4–6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.     

Malaria Drug Resistance: The Impact of Human Movement and Spatial Heterogeneity

Human habitat connectivity, movement rates, and spatial heterogeneity have tremendous impact on malaria transmission. In this paper, a deterministic system of differential equations for malaria transmission incorporating human movements and the development of drug resistance malaria in an \(n\) patch system is presented. The disease-free equilibrium of the model is globally asymptotically stable when the associated reproduction number is less than unity. For a two patch case, the boundary equilibria (drug sensitive-only and drug resistance-only boundary equilibria) when there is no movement between the patches are shown to be locally asymptotically stable when they exist; the co-existence equilibrium is locally asymptotically stable whenever the reproduction number for the drug sensitive malaria is greater than the reproduction number for the resistance malaria. Furthermore, numerical simulations of the connected two patch model (when there is movement between the patches) suggest that co-existence or competitive exclusion of the two strains can occur when the respective reproduction numbers of the two strains exceed unity. With slow movement (or low migration) between the patches, the drug sensitive strain dominates the drug resistance strain. However, with fast movement (or high migration) between the patches, the drug resistance strain dominates the drug sensitive strain.     

Impact of Emission Anisotropy on Fluorescence Spectroscopy and FRET Distance Measurements

The objective of this report is to provide a practical and improved method for estimating Förster resonance energy transfer distance measurement error due to unknown angles in the dipole orientation factor based on emission anisotropy measurements. We improve on the method of Dale et al. (1979), which has minor mistakes and is frequently interpreted in overly optimistic ways in the literature. To facilitate proper fluorescence intensity measurements, we also evaluated instrument parameters that could impact the measurement. The apparent fluorescence intensity of isotropic samples depends on the sample emission anisotropy, fluorometer geometry, and optical apertures. We separate parameters of the sample, and those of the cylindrically symmetric illumination source and detector in the equations describing results of unpolarized and polarized fluorescence intensity measurements. This approach greatly simplifies calculations compared with the more universal method of Axelrod (1989). We provide a full computational method for calculating the Förster resonance energy transfer distance error and present a graph describing distance error in the simplest case.     

The Impact of Using Alternative Forages on the Nutrient Value within Slurry and Its Implications for Forage Productivity in Agricultural Systems

Alternative forages can be used to provide valuable home-grown feed for ruminant livestock. Utilising these different forages could affect the manure value and the implications of incorporating these forages into farming systems, needs to be better understood. An experiment tested the hypothesis that applying slurries from ruminants, fed ensiled red clover (Trifolium pratense), lucerne (Medicago sativa) or kale (Brassica oleracea) would improve the yield of hybrid ryegrass (Lolium hybridicum), compared with applying slurries from ruminants fed ensiled hybrid ryegrass, or applying inorganic N alone. Slurries from sheep offered one of four silages were applied to ryegrass plots (at 35 t ha⁻¹) with 100 kg N ha⁻¹ inorganic fertiliser; dry matter (DM) yield was compared to plots only receiving ammonium nitrate at rates of 0, 100 and 250 kg N ha⁻¹ year⁻¹. The DM yield of plots treated with 250 kg N, lucerne or red clover slurry was significantly higher than other treatments (P<0.001). The estimated relative fertiliser N equivalence (FNE) (fertiliser-N needed to produce same yield as slurry N), was greatest for lucerne (114 kg) >red clover (81 kg) >kale (44 kg) >ryegrass (26 kg ha⁻¹ yr⁻¹). These FNE values represent relative efficiencies of 22% (ryegrass), 52% (kale), 47% (red clover) and 60% for lucerne slurry, with the ryegrass slurry efficiency being lowest (P = 0.005). Soil magnesium levels in plots treated with legume slurry were higher than other treatments (P<0.001). Overall, slurries from ruminants fed alternative ensiled forages increased soil nutrient status, forage productivity and better N efficiency than slurries from ruminants fed ryegrass silage. The efficiency of fertiliser use is one of the major factors influencing the sustainability of farming systems, these findings highlight the cascade in benefits from feeding ruminants alternative forages, and the need to ensure their value is effectively captured to reduce environmental risks.     

Investigation of Endoglin Wild-Type and Missense Mutant Protein Heterodimerisation Using Fluorescence Microscopy Based IF,BiFC and FRET Analyses

The homodimeric transmembrane receptor endoglin (CD105) plays an important role in angiogenesis. This is highlighted by mutations in its gene, causing the vascular disorder HHT1. The main role of endoglin function has been assigned to the modulation of transforming growth factor β and bone morphogenetic protein signalling in endothelial cells. Nevertheless, other functions of endoglin have been revealed to be involved in different cellular functions and in other cell types than endothelial cells. Compared to the exploration of its natural function, little experimental data have been gathered about the mode of action of endoglin HHT mutations at the cellular level, especially missense mutations, and to what degree these might interfere with normal endoglin function. In this paper, we have used fluorescence-based microscopic techniques, such as bimolecular fluorescence complementation (BiFC), immunofluorescence staining with the endoglin specific monoclonal antibody SN6, and protein interaction studies by Förster Resonance Energy Transfer (FRET) to investigate the formation and cellular localisation of possible homo- and heterodimers composed of endoglin wild-type and endoglin missense mutant proteins. The results show that all of the investigated missense mutants dimerise with themselves, as well as with wild-type endoglin, and localise, depending on the position of the affected amino acid, either in the rough endoplasmic reticulum (rER) or in the plasma membrane of the cells. We show that the rER retained mutants reduce the amount of endogenous wild-type endoglin on the plasma membrane through interception in the rER when transiently or stably expressed in HMEC-1 endothelial cells. As a result of this, endoglin modulated TGF-β1 signal transduction is also abrogated, which is not due to TGF-β receptor ER trafficking interference. Protein interaction analyses by FRET show that rER located endoglin missense mutants do not perturb protein processing of other membrane receptors, such as TβRII, ALK5 or ALK1.     

Alkyl Chain Length Effects of Polymer Donors on the Morphology and Device Performance of Polymer Solar Cells with Different Acceptors

The development of nonfullerene acceptors has brought polymer solar cells into a new era. Maximizing the performance of nonfullerene solar cells needs appropriate polymer donors that match with the acceptors in both electrical and morphological properties. So far, the design rationales for polymer donors are mainly borrowed from fullerene‐based solar cells, which are not necessarily applicable to nonfullerene solar cells. In this work, the influence of side chain length of polymer donors based on a set of random terpolymers PTAZ‐TPD10‐Cn on the device performance of polymer solar cells is investigated with three different acceptor materials, i.e., a fullerene acceptor [70]PCBM, a polymer acceptor N2200, and a fused‐ring molecular acceptor ITIC. Shortening the side chains of polymer donors improves the device performance of [70]PCBM‐based devices, but deteriorates the N2200‐ and ITIC‐based devices. Morphology studies unveil that the miscibility between donor and acceptor in blend films depends on the side chain length of polymer donors. Upon shortening the side chains of the polymer donors, the miscibility between the donor and acceptor increases for the [70]PCBM‐based blends, but decreases for the N2200‐ and ITIC‐based blends. These findings provide new guidelines for the development of polymer donors to match with emerging nonfullerene acceptors.     

Functional Heterogeneity of Arcuate Nucleus Pro-Opiomelanocortin Neurons: Implications for Diverging Melanocortin Pathways

Arcuate nucleus (ARC) pro-opiomelanocortin (POMC) neurons are essential regulators of food intake, energy expenditure, and glucose homeostasis. POMC neurons integrate several key metabolic signals that include neurotransmitters and hormones. The change in activity of POMC neurons is relayed to melanocortin receptors in distinct regions of the central nervous system. This review will summarize the role of leptin and serotonin receptors in regulating the activity of POMC neurons and provide a model in which different melanocortin pathways regulate energy and glucose homeostasis.     

New Anthracene‐Fused Nonfullerene Acceptors for High‐Efficiency Organic Solar Cells: Energy Level Modulations Enabling Match of Donor and Acceptor

Two new nonfullerene small molecule acceptors (NF‐SMAs) AT‐NC and AT‐4Cl based on heptacyclic anthracene(cyclopentadithiophene) (AT) core and different electron‐withdrawing end groups are designed and synthesized. Although the two new acceptor molecules use two different end groups, naphthyl‐fused indanone (NINCN) and chlorinated INCN (INCN‐2Cl) demonstrate similar light absorption. AT‐4Cl with chlorinated INCN as end groups are shifted significantly due to the strong electron‐withdrawing ability of chlorine atoms. Thus, desirable V_oc and photovoltaic performance are expected to be achieved when polymer PBDB‐T is used as the electron donor with AT‐NC as the acceptor, and fluorinated analog PBDB‐TF with down‐shifted energy levels is selected to blend with AT‐4Cl. Consequently, the device based on PBDB‐TF:AT‐4Cl yields a high power conversion efficiency of 13.27% with a slightly lower V_oc of 0.901 V, significantly enhanced J_sc of 19.52 mA cm^?2 and fill factor of 75.5% relative to the values based on PBDB‐T:AT‐NC. These results demonstrate that the use of a new electron‐rich AT core, together with energy levels modulations by end‐group optimizations enabling the match with polymer donors, is a successful strategy to construct high‐performance NF‐SMAs.     

绿色荧光蛋白的特性及其在信号转导中的应用

细胞和分子生物学的最终目标是 ,明确细胞内的各种事件是如何发生的 ,明了细胞内复杂的动态变化的生化机制。绿色荧光蛋白 (ｇｒｅｅｎｆｌｕｏｒｅｓｃｅｎｔｐｒｏｔｅｉｎ ,ＧＦＰ)自从克隆、表达之后 ,以其良好的物理特性及荧光特性而成为良好的报告基因和荧光标记分子 ,并在探索生命现象过程中得到了非常广泛的应用。ＧＦＰ作为报告基因 ,可用在活细胞中直接观察蛋白质向细胞器 ,如细胞核、内质网中运动 ;作为荧光标记分子 ,ＧＦＰ既具有敏感的标记检测率 ,又没有放射性的危害 ;最近又发现ＧＦＰ是一个良好的细胞间信号传递的动态标记…     

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Background

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.

Methodology

Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.

Conclusions

All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.     

FRET Spectrometry: A New Tool for the Determination of Protein Quaternary Structure in Living Cells

Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in the kinetic theory of FRET, provide the necessary tool kits for determining the stoichiometry and relative disposition of the protomers within protein complexes (i.e., quaternary structure) of membrane receptors and transporters in living cells. In contrast to standard average-based methods, this method relies on the analysis of distributions of apparent FRET efficiencies, E_app, across the image pixels of individual cells expressing proteins of interest. The most probable quaternary structure of the complex is identified from the number of peaks in the E_app distribution and their dependence on a single parameter, termed pairwise FRET efficiency. Such peaks collectively create a unique FRET spectrum corresponding to each oligomeric configuration of the protein. Therefore, FRET could quite literally become a spectrometric method—akin to that of mass spectrometry—for sorting protein complexes according to their size and shape.     

Homepeptide Repeats:Implications for Protein Structure,Function and Evolution

Analysis of protein sequences from Mycobacterium tuberculosis H37Rv(Mtb H37Rv) was performed to identify homopeptide repeatcontaining proteins(HRCPs).Functional annotation of the HRCPs showed that they are preferentially involved in cellular metabolism.Furthermore,these homopeptide repeats might play some specific roles in protein-protein interaction.Repeat length differences among Bacteria,Archaea and Eukaryotes were calculated in order to identify the conservation of the repeats in these divergent kingdoms.From the results,it was evident that these repeats have a higher degree of conservation in Bacteria and Archaea than in Eukaryotes.In addition,there seems to be a direct correlation between the repeat length difference and the degree of divergence between the species.Our study supports the hypothesis that the presence of homopeptide repeats influences the rate of evolution of the protein sequences in which they are embedded.Thus,homopeptide repeat may have structural,functional and evolutionary implications on proteins.     

Challenges in the Testing of Non-Heart-Beating Cadavers for Viral Markers: Implications for the Safety of Tissue Donors

Natural changes that occur in blood and tissue after death may result in false positive results in antigen and antibody detection tests performed to identify markers of viral infection in potential tissue donors. Such tissue, which might otherwise be acceptable for therapeutic purposes, would not meet current standards for safe tissue banking. This is especially important in the context of insufficiency in the tissue supply. In this study, a series of blood samples collected during routine post-mortem examination was assayed using a range of commercially available kits for the detection of HBsAg, anti-HCV and anti-HIV 1 + 2 antibody/antigen. Results of tests on 104 samples collected from 97 individuals indicate that some kits result in a higher number of initial reactive samples than others. Approximately 40% of samples were reactive in one or more HBsAg assay, less than 10% in at least one anti-HIV kit and only 1 sample at low level on an anti-HCV kit. Liver or lymph node samples from individuals whose serum sample gave reactive results in antigen/antibody assays were tested for viral nucleic acid in the corresponding nucleic acid amplification test. Only one individual’s sample was confirmed to test positive for HBsAg in a confirmatory neutralisation test and by nucleic acid amplification technology, and a second individual whose serum was scored reactive for anti-HCV, but negative for HBsAg, had a liver sample which was HBV DNA positive and HCV RNA negative. The results of the study indicate that antibody/antigen assays are not as specific as NAT using state of the art DNA extraction techniques. Both types of assay complement each other and used together will help assure the safety of tissues for transplantation.     

In Vivo Study of Trichoderma-Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.