Gelatin degradation at elevated temperature

Four gelatin types (A, B, C and AB), two different samples of each, were subjected to temperature treatments with the incubation temperature, incubation time, gelatin concentration and solvent (type and concentration of salt ions and pH) as variables. Degradation was studied by means of fast protein liquid chromatography and sodium dodecylsulphate polyacrylamide gel electrophoresis. All the variables tested seemed to be critical.

Addition of a protease inhibitor cocktail confirmed that the observed degradation was not due to the action of proteases.

Fluorescence measurements indicated that during the temperature treatment pentosidine and pyridinoline cross-links can be broken, while the cleavage of peptide bonds was verified by ninhydrin tests and N-terminal amino-acid analyses with phenyl isothiocyanate.     

Characterization of the stability of a fully human monoclonal IgG after prolonged incubation at elevated temperature

The susceptible degradation sites of therapeutic proteins are routinely assessed under accelerated conditions such as exposure to chemicals or incubation at elevated temperature or a combination of both. A fully human monoclonal IgG(1) antibody was characterized after incubation at 40 degrees C for 6 months by employing mass spectrometry and chromatography analyses. It was found that deamidation, fragmentation and N-terminal glutamate cyclization to form pyroglutamate are the major degradation pathways. Three major deamidation sites were identified and one site in a small tryptic peptide accounted for more than 80% of the total. Peptide cleavage was observed at several positions between different pairs of amino acids. Most of the cleavage sites were located in the hinge or other flexible regions of the IgG molecule.     

Hydrogen bonding at a conserved threonine in lentivirus capsid is required for virus replication

The N terminus of the capsid protein (CA) undergoes a considerable conformational change when the human immunodeficiency virus (HIV) protease cleaves it free from the Pr55(Gag) polyprotein. This rearrangement is thought to facilitate the establishment of specific CA-CA interactions that are required for the formation of the mature viral core. Substitution of amino acids that are critical for this refolding of the N terminus is generally detrimental to virus replication and mature virion core morphology. Here, we identify a conserved threonine in simian immunodeficiency virus (SIV) CA, T(47)(CA), that is requisite for viral replication. Replacement of T(47)(CA) in the infectious viral clone SIVmac239 with amino acids with different hydrogen-bonding capabilities and analysis of the effects of these substitutions at key steps in the viral life cycle demonstrate that hydrogen bonding at this position is important for virus infectivity and virion release. In the HIV-based homology model of the mature SIV CA N terminus presented in this study, T(47)(CA) forms several hydrogen bonds with a proximal aspartate, D(50)(CA). This model, coupled with strong phenotypic similarities between viral substitution mutants of each of these two residues in all of the virological assays described herein, indicates that hydrogen bonding between T(47)(CA) and D(50)(CA) is likely required for viral replication. As hydrogen bonding between these two residues is present in HIV CA as well, this interaction presents a potential target for antiviral drug design.     

Competition limits adaptation and productivity in a photosynthetic alga at elevated CO2

When competitive exclusion between lineages and genetic adaptation within lineages occur on the same timescale, the two processes have the potential to interact. I use experimental microbial evolution where strains of a photosynthetic microbe that differ in their physiological response to CO₂ enrichment are grown either alone or in communities for hundreds of generations under CO₂ enrichment. After about 300 generations of growth, strains that experienced competition while adapting to environmental change are both less productive and less fit than corresponding strains that adapted to that same environmental change in the absence of competitors. In addition, I find that excluding competitors not only limits that strain''s adaptive response to abiotic change, but also decreases community productivity; I quantify this effect using the Price equation. Finally, these data allow me to empirically test the common hypothesis that phytoplankton that are most able to take advantage of carbon enrichment in single-strain populations over the short term will increase in frequency within multi-strain communities over longer timescales.     

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 degrees C and a shift of 10 degrees C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29-42 degrees C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature.     

Differential stability of photosynthetic membranes and fatty acid composition at elevated temperature in Symbiodinium

Coral reefs are threatened by increasing surface seawater temperatures resulting from climate change. Reef-building corals symbiotic with dinoflagellates in the genus Symbiodinium experience dramatic reductions in algal densities when exposed to temperatures above the long-term local summer average, leading to a phenomenon called coral bleaching. Although the temperature-dependent loss in photosynthetic function of the algal symbionts has been widely recognized as one of the early events leading to coral bleaching, there is considerable debate regarding the actual damage site. We have tested the relative thermal stability and composition of membranes in Symbiodinium exposed to high temperature. Our results show that melting curves of photosynthetic membranes from different symbiotic dinoflagellates substantiate a species-specific sensitivity to high temperature, while variations in fatty acid composition under high temperature rather suggest a complex process in which various modifications in lipid composition may be involved. Our results do not support the role of unsaturation of fatty acids of the thylakoid membrane as being mechanistically involved in bleaching nor as being a dependable tool for the diagnosis of thermal susceptibility of symbiotic reef corals.     

Effects of point mutations in the major capsid protein of beet western yellows virus on capsid formation,virus accumulation,and aphid transmission

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.     

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (～3.0?Å) and size (～310.0?Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508?Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9?Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.     

Operational stability of copolymerized enzymes at elevated temperatures

The copolymerization method of immobilization was used to obtain preparations of enzymes covalently incorporated in polyacrylamide gel. They possess properties making them suitable for practical use. First, the preparations are hundreds of times more stable against irreversible thermoinactivation than native enzymes. Second, on immobilization, the reversible conformational changes which also lower enzyme activity at elevated temperatures are completely suppressed. As a result, the temperatures of maximum activity for trypsin and alpha-chymotrypsin covalently entrapped in polyacrylamide gel are 75 and 70 degrees C, respectively-25 and 30 degrees C higher than the corresponding values for the native enzymes. Therefore, the copolymerized enzyme preparations have a high operational stability at elevated temperatures.     

Methionine-mediated lethality in yeast cells at elevated temperature.

Saccharomyces cerevisiae cells grown at 30 degrees C in minimal medium containing methionine lose viability upon transfer to 45 degrees C, whereas cells grown in the absence of methionine survive. Cellular levels of two intermediates in the sulfate assimilation pathway, adenosine 5'-phosphosulfate (APS) and adenosine 5'-phosphosulfate 3'-phosphate, are increased by a posttranslational mechanism after sudden elevation of temperature in yeast cultures grown in the absence of methionine. Yeast cells unable to synthesize APS because of repression by methionine or mutation of the MET3 gene do not survive the temperature shift. Thus, methionine-mediated lethality at elevated temperature is linked to the inability to synthesize APS. The results demonstrate that APS plays an important role in thermotolerance.     

风疹病毒松叶株衣壳蛋白C基因稳定性研究

目的研究风疹病毒(Rubella virus,RV)疫苗松叶株(Matsuba)衣壳蛋白(Capsid Protein)的基因稳定性及遗传特征,探讨其功能结构及生物学活性在病毒传代过程中的变化特点。方法利用RT-PCR方法扩增风疹病毒Matsuba株14、16、17、18、23代次C基因序列,测序后进行序列比对分析,并将各代次病毒的C基因与风疹病毒疫苗株Matsuba(GenBank登陆号:AB588193)及其他风疹病毒株C基因序列进行同源性分析。结果风疹病毒Mat-suba株传代病毒C基因在传代过程中核苷酸及氨基酸均未发生变异;各代次病毒与AB588193核苷酸及氨基酸序列完全一致,各关键功能区未发生变异;Matsuba株与18株风疹病毒核苷酸相似性在90.2%～99.9%之间。结论风疹病毒Matsuba疫苗株C基因遗传特性非常稳定,与生物学作用相关的区域未发生传代改变。从分子水平证明Matsuba疫苗株毒种及其生产的疫苗具有安全性。     

Multihost experimental evolution of a plant RNA virus reveals local adaptation and host-specific mutations

For multihost pathogens, adaptation to multiple hosts has important implications for both applied and basic research. At the applied level, it is one of the main factors determining the probability and the severity of emerging disease outbreaks. At the basic level, it is thought to be a key mechanism for the maintenance of genetic diversity both in host and pathogen species. Using Tobacco etch potyvirus (TEV) and four natural hosts, we have designed an evolution experiment whose strength and novelty are the use of complex multicellular host organism as hosts and a high level of replication of different evolutionary histories and lineages. A pattern of local adaptation, characterized by a higher infectivity and virulence on host(s) encountered during the experimental evolution was found. Local adaptation only had a cost in terms of performance on other hosts in some cases. We could not verify the existence of a cost for generalists, as expected to arise from antagonistic pleiotropy and other genetic mechanisms generating a fitness trade-off between hosts. This observation confirms that this classical theoretical prediction lacks empirical support. We discuss the reasons for this discrepancy between theory and experiment in the light of our results. The analysis of full genome consensus sequences of the evolved lineages established that all mutations shared between lineages were host specific. A low degree of parallel evolution was observed, possibly reflecting the various adaptive pathways available for TEV in each host. Altogether, these results reveal a strong adaptive potential of TEV to new hosts without severe evolutionary constraints.     

Genomic adaptation of prokaryotic organisms at high temperature

One of the central issues of evolutionary genomics is to find out the adaptive strategies of microorganisms to stabilize nucleic acid molecules under high temperature. Thermal adaptation hypothesis gives a link between G+C content and growth temperature if there is a considerable variation of guanine and cytosine content between species. However, there has been a long-standing debate regarding the correlations between genomic GC content and optimal growth temperature (Topt). We urged that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting: (i) nucleic acid thermostability and (ii) stability of codon-anticodon interactions. Moreover, in Bacillaceae family we have demonstrated that a higher genomic GC level do not have any role in stabilizing mRNA secondary structure at high growth temperature. Comparative analysis between homologous sequences of thermophilic Thermus thermophilus and mesophilic Deinococcus radiodurans suggests that increased levels of GC contents in the coding sequence corresponding to strand structure of Thermus thermophilus genes have stabilizing effect on the mRNA secondary structure, whereas increased levels of GC contents in coding sequences corresponding to aperiodic structure have destabilizing effect on the mRNA secondary structure. In this perspective, a critical review of thermal adaptation hypothesis is further advocated.     

Internal point mutations of the capsid modify the serotype of Rice yellow mottle virus

Rice yellow mottle virus is classified in five major serotypes; the molecular diversity of the coat protein (CP) is well established, but the amino acids involved in the recognition by discriminant monoclonal antibodies (MAbs) remain unknown. Reconstruction of a phylogenetic tree and sequence alignment of the CP gene of a sample representative of the continental-large diversity were used to identify 10 serospecific amino acids (i.e., conserved in all isolates belonging to the same serotype and distinct in other serotypes). Positions occupied by serospecific residues were localized on the crystal structure of the CP monomer and on modeled capsomers. Structural, molecular, and serological properties of each serotype were analyzed, and subsequently, hypotheses on the potential role of amino acids in discriminating reactions with antibodies were formulated. The residues 114 and 115 (serospecific of Sr1) and 190 (serospecific of Sr2) were localized on the outer surface of the capsid and might be directly involved in the immunoreactivity with MAb D and MAb A, respectively. In contrast, residues 180 (Sr3) and 178 (Sr5) lay within the inner surface of the capsid. To understand the role of these internal positions in the recognition with the antibodies, two substitutions (T180K and G178D) were introduced in the CP of an infectious clone. These mutations modified the antigenicity with MAb G and MAb E discriminating Sr3 and Sr5, respectively, while the reaction with MAb D remained unaffected. This result suggests an indirect effect of these two internal mutations on local immunostructure while the global structure was maintained.     

Stability of xanthan in aqueous sodium chloride at elevated temperature

Aqueous NaCl solutions of dimerized Na xanthan with salt concentrations of 0.005, 0.01 and 0.1 were exposed to 80°C for different time periods t, and their viscosities were determined as a function of t. The measured relative viscosities decreased markedly with t, suggesting that Na xanthan denatured at 80°C undergoes some conformation changes or degradation. The molecular weights of the test samples recovered at different t were estimated by viscometry in cadoxen, a single-coil solvent for xanthan, and were found to decrease monotonically with t. Thus, it was concluded that the observed decreases in relative viscosity are due primarily to degradation of Na xanthan.     

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid resistance

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH(4)Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.     

Reversible pachytene arrest of Saccharomyces cerevisiae at elevated temperature

Summary The temperature sensitivity of sporulation in a well-characterized yeast strain lacking any known temperature sensitive genes has been investigated. Cytological observations by electron microscopy demonstrate that cells incubated in sporulation medium at a temperature inhibitory to sporulation became arrested in meiotic prophase. The stage of arrest was identified as pachytene by the presence of duplicated (but unseparated) spindle pole bodies and synaptonemal complex. Transfer of the arrested culture to lower temperature permitted resumption of meiosis and sporulation; transfer to vegetative medium resulted in reversion to mitotic division. Genetic analysis of cells that had reverted to mitosis revealed that commitment to intragenic recombination had occurred by the time of arrest. Prolonged incubation at the elevated temperature resulted in the enhancement of intragenic recombination above normal levels, suggesting that some aspect of recombination continued to occur during the pachytene arrest. Evidence is presented that DNA replication, although depressed overall in the arrested cultures, had occurred to completion in many arrested cells.