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891.
In SJL mice, growth of RcsX lymphoma cells induces an inflammatory response by stimulating V(beta)16+ T cells. During inflammation, various serum protein levels can increase (e.g., acute phase reactants) or decrease (e.g., albumin), and most of these altered proteins are thus potential biomarkers. Although blood plasma is a valuable and promising sample for biomarker discovery for diseases or for novel drug targets, its proteome is complex. To address this, we have focused on a comprehensive comparison of the plasma proteomes from normal and RcsX-tumor-bearing SJL mice using the 1D-Gel-LC-MS/MS method after removing albumin and immunoglobulins. This analysis resulted in the identification of a total of 1079 nonredundant mouse plasma proteins; more than 480 in normal and 790 in RcsX-tumor-bearing SJL mouse plasma. Of these, only 191 proteins were found in common. The molecular weights ranged from 2 to 876 kDa, covering the pI values between 4.22 and 12.09, and included proteins with predicted transmembrane domains. By comparing the plasma proteomic profile of normal and RcsX-tumor-bearing SJL mice, we found significant changes in the levels of many proteins in RcsX-tumor-bearing mouse plasma. Most of the up-regulated proteins were identified as acute-phase proteins (APPs). Also, several unique proteins i.e., haptoglobin, proteosome subunits, fetuin-B, 14-3-3 zeta, MAGE-B4 antigen, etc, were found only in the tumor-bearing mouse plasma; either secreted, shed by membrane vesicles, or externalized due to cell death. These results affirm the effectiveness of this approach for protein identification from small samples, and for comparative proteomics in potential animal models of human disorders. 相似文献
892.
The rate of fluid secretion in vitro by Malpighian tubules of 3-day-old adult blowflies (Calliphora vicina) is increased by more than 8 times when exposed to an extract of the fly's thoracic ganglion and abdominal nerves. Smaller amounts of stimulatory activity are also retrievable from other body tissues: some activity is present in the brain, but other tissue activity is consistent with the presence of the stimulatory factor in fine branches of the abdominal nerves. It is suggested that this factor is a diuretic hormone.Maximal stimulation of fluid secretion is achieved within 2–3 min of exposure to the hormone, but the rate of secretion decays only slowly when the hormone is removed ().The hormone is stable to boiling and freezing, and enzyme experiments suggest it is a polypeptide.The hormone is inactivated when incubated in the presence of haemolymph, but the Malpighian tubules themselves do not appear to break down or excrete the hormone. 相似文献
893.
894.
Parametric methods for identifying laterally transferred genes exploit the directional mutational biases unique to each genome. Yet the development of new, more robust methods—as well as the evaluation and proper implementation of existing methods—relies on an arbitrary assessment of performance using real genomes, where the evolutionary histories of genes are not known. We have used the framework of a generalized hidden Markov model to create artificial genomes modeled after genuine genomes. To model a genome, “core” genes—those displaying patterns of mutational biases shared among large numbers of genes—are identified by a novel gene clustering approach based on the Akaike information criterion. Gene models derived from multiple “core” gene clusters are used to generate an artificial genome that models the properties of a genuine genome. Chimeric artificial genomes—representing those having experienced lateral gene transfer—were created by combining genes from multiple artificial genomes, and the performance of the parametric methods for identifying “atypical” genes was assessed directly. We found that a hidden Markov model that included multiple gene models, each trained on sets of genes representing the range of genotypic variability within a genome, could produce artificial genomes that mimicked the properties of genuine genomes. Moreover, different methods for detecting foreign genes performed differently—i.e., they had different sets of strengths and weaknesses—when identifying atypical genes within chimeric artificial genomes. 相似文献
895.
DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity. 相似文献
896.
Harris PK Yeoh S Dluzewski AR O'Donnell RA Withers-Martinez C Hackett F Bannister LH Mitchell GH Blackman MJ 《PLoS pathogens》2005,1(3):241-251
Proteolytic shedding of surface proteins during invasion by apicomplexan parasites is a widespread phenomenon, thought to represent a mechanism by which the parasites disengage adhesin-receptor complexes in order to gain entry into their host cell. Erythrocyte invasion by merozoites of the malaria parasite Plasmodium falciparum requires the shedding of ectodomain components of two essential surface proteins, called MSP1 and AMA1. Both are released by the same merozoite surface "sheddase," but the molecular identity and mode of action of this protease is unknown. Here we identify it as PfSUB2, an integral membrane subtilisin-like protease (subtilase). We show that PfSUB2 is stored in apical secretory organelles called micronemes. Upon merozoite release it is secreted onto the parasite surface and translocates to its posterior pole in an actin-dependent manner, a trafficking pattern predicted of the sheddase. Subtilase propeptides are usually selective inhibitors of their cognate protease, and the PfSUB2 propeptide is no exception; we show that recombinant PfSUB2 propeptide binds specifically to mature parasite-derived PfSUB2 and is a potent, selective inhibitor of MSP1 and AMA1 shedding, directly establishing PfSUB2 as the sheddase. PfSUB2 is a new potential target for drugs designed to prevent erythrocyte invasion by the malaria parasite. 相似文献
897.
898.
Reynolds TH Pak Y Harris TE Manchester J Barrett EJ Lawrence JC 《The Journal of biological chemistry》2005,280(7):5510-5515
UDP-glucose (UDP-Glc) and glycogen levels in skeletal muscle fibers of defined fiber type were measured using microanalytical methods. Infusing rats with insulin increased glycogen in both Type I and Type II fibers. Insulin was without effect on UDP-Glc in Type I fibers but decreased UDP-Glc by 35-40% in Type IIA/D and Type IIB fibers. The reduction in UDP-Glc suggested that UDP-Glc pyrophosphorylase (PPL) activity might limit glycogen synthesis in response to insulin. To explore this possibility, we generated mice overexpressing a UDP-Glc PPL transgene in skeletal muscle. The transgene increased both UDP-Glc PPL activity and levels of UDP-Glc in skeletal muscles by approximately 3-fold. However, overexpression of UDP-Glc PPL was without effect on either the levels of skeletal muscle glycogen or glucose tolerance in vivo. The transgene was also without effect on either control or insulin-stimulated rates of (14)C-glucose incorporation into glycogen in muscles incubated in vitro. The results indicate that UDP-Glc PPL activity is not limiting for glycogen synthesis. 相似文献
899.
Nogami K Zhou Q Myles T Leung LL Wakabayashi H Fay PJ 《The Journal of biological chemistry》2005,280(18):18476-18487
Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser(205) --> Ala mutant indicated a high affinity site in the A1 subunit (K(d) approximately 5 nm) that was dependent upon the Na(+)-bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K(d) approximately 100 nm) was observed for both Na(+)-bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719-740, blocked thrombin cleavage of the isolated heavy chain at Arg(372) and Arg(740) and inhibited A2 binding to thrombin Ser(205) --> Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp(392) --> Ala/Asp(394) --> Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg(740), whereas cleavage at Arg(372) was not affected. These data suggest the acidic region comprising residues 389-394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation. 相似文献
900.
Baggio R Carven GJ Chiulli A Palmer M Stern LJ Arenas JE 《The Journal of biological chemistry》2005,280(6):4188-4194
Class II major histocompatibility complex proteins bind peptides for presentation to T-cells as part of the immune response process. Monoclonal antibody MEM-265 recognizes the peptide-free conformation of the major histocompatibility complex class II protein HLA-DR1 through specific binding to an epitope contained between residues 50-67 of the beta-chain. In previous work using alanine scanning (1), we identified residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 as essential for specific recognition by MEM-265. The spacing of these residues approximates a 3.5-residue repeat, suggesting that MEM-265 may recognize the epitope in an alpha-helical conformation. In the folded, peptide-loaded DR1 structure, the beta-chain residues 50-67 contain a kinked alpha-helical segment spanning Glu-52-Ser-63 (2). However, the conformation of this segment in the peptide-free form is unknown. We have used a new surface plasmon resonance approach in a SpotMatrix format to compare the kinetic rates and affinities for 18 alanine scanning mutants comprising epitope residues 50-67. In addition to the six essential residues described previously, we found two additional residues, Glu-52 and Gln-64, that contribute by enhancing MEM-265 binding. By contrast, mutation of either Gly-54 or Pro-56 to an alanine actually improved binding to MEM-265. In essentially all cases peptide substitutions that either improve or reduce MEM-265 recognition could be traced to differences in the dissociation rate (k off). The kinetic details of the present study support the presence of a structural component in the antigenic epitope recognized by MEM-265 in the peptide-free form of major histocompatibility complex II DR1 beta-chain. 相似文献