136 resultados para trypsin-like serine protease


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A new completely integrable model of strongly correlated electrons is proposed which describes two competitive interactions: one is the correlated one-particle hopping, the other is the Hubbard-like interaction. The integrability follows from the fact that the Hamiltonian is derivable from a one-parameter family of commuting transfer matrices. The Bethe ansatz equations are derived by algebraic Bethe ansatz method.

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Calponins are proteins present in vertebrate smooth musculature where they occur in association with thin myofilaments. Calponins are not present in vertebrate or invertebrate striated muscles. The blood fluke Schistosoma japonicum expresses a 38.3-kDa protein that bears substantial homology with vertebrate calponin and occurs entirely within smooth musculature of adults. Calponin-like immunoreactivity has been demonstrated in smooth muscles of many invertebrate phyla. The Schistosoma japonicum calponin has been localised in smooth myofibrils of adults where it is associated with myofilaments and sarcoplasmic reticulum. In this study, the ultrastructural localisation of the protein in muscles of S. japonicum cercariae is described. The protein is present in smooth muscles of the forebody and the stratified muscle of the tail. Within the stratified layer, the protein occurs predominantly in transverse arrays of sarcoplasmic reticulum. The localisation data suggest that the calponin-like protein of S. japonicum is involved in contraction of the stratified tail muscle. Furthermore, the presence of a calponin system in the stratified muscle suggests that this muscle is simply a superior form of muscle, closely related to smooth muscles that use a caldesmin-calponin system in contraction. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

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The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

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The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

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[GRAPHICS] The stereocontrolled synthesis of (2S,4R,6R,8S,10S,1'R,1"R)-2(acetylhydroxymethyl)-4, 10-dimethyl-8(isopropenylhydroxymethyl)-1, 7-dioxaspiro[5,5]-undecane (4a) and its C1"-epimer (4b), the key mother spiroketals of the HIV-1 protease inhibitive didemnaketals from the ascidian Didemnum sp., has been carried out through multisteps from the natural (R)-(+)-pulegone, which involved the diastereoselective construction of four chiral carbon centers(C-2, C-6, C-8, and C-1') by intramolecular chiral induce.

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SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by H-1-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 Angstrom and 0.66 Angstrom, respectively. The structures consist of two short antiparallel beta -strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta -strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides. (C) 2001 Academic Press.

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Recombinant cathepsin D aspartic protease of Schistosoma japonicum cleaved human IgG in vitro in a time and dose-dependent manner. Optimal cleavage was seen at pH 3.6-4.5; modest cleavage remained at pH 5.0, and no cleavage was detected above pH 5.0. Amino terminal sequencing of the major cleavage fragments of human IgG identified a Fab fragment from the VH1 domain, and 2 cleavage sites in the CH2 domain below the hinge region. The P1 and P1' residues at the 2 CH2 cleavage sites were Phe254-Leu255 and Leu325-Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the immunoglobulin light chain was detected. In addition, the recombinant schistosome protease indiscriminately degraded the human serum proteins complement C3 and serum albumin into numerous small fragments. These results demonstrate specific cleavage of human IgG by the recombinant schistosome aspartic protease, and highlight the broad range digestive specificity of the enzyme which may play a role in the degradation of host serum proteins ingested as part of the schistosome bloodmeal.

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Mice were vaccinated with recombinant Schistosoma japonicum cathepsin D aspartic protease, expressed in both insect cells and bacteria, in order to evaluate the vaccine efficacy of the schistosome protease. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, we recorded increased egg output per female worm in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgG1, IgG2a and IgG2b isotypes), but there was no correlation between antibody levels and protective efficacy. Immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by the recombinant protease, and passive transfer of serum or antibodies from vaccinated animals, before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals. These results suggest that antibodies may not play a key role in the protective effect elicited, and that protection may be due to a combination of humoral and cell-mediated responses.

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Injection of particulate hepatitis B virus surface antigen (HBsAg) in mice leads to the induction of a HBsAg-specific class-I-restricted cytotoxic T lymphocyte (CTL) response. It is proposed that any protein internal to HBsAg will also be able to elicit a specific CTL response. In this study, several carboxy-terminal truncations of hepatitis C virus (HCV) core protein were fused to varying lengths of amino-terminal truncated large hepatitis delta antigen (L-HDAg). These constructs were analysed for their ability to be expressed and the particles secreted in the presence of HBsAg after transfection into HuH-7 cells. The secretion efficiency of the various HCV core-HDAg chimeric proteins was generally poor. Constructs containing full length HDAg appeared to be more stable than truncated versions and the length of the inserted protein was restricted to around 40 amino acids. Thus, the use of L-HDAg as a chimera to package foreign proteins is limited. Consequently, a polyepitope (polytope) containing a B-cell epitope from human papillomavirus (HPV 16) and multiple T-cell epitopes from the HCV polyprotein was used to create the construct, L-HDAg-polyB. This chimeric protein was shown to be reliant on the co-expression of HBsAg for secretion into the cell culture fluid and was secreted more efficiently than the previous HCV core-HDAg constructs. These L-HDAg-polyB virus-like particles (VLPs) had a buoyant density of similar to 1.2 g/cm(3) in caesium chloride and similar to 1.15 g/cm(3) in sucrose. The VLPs were also immunoprecipitated using an anti-HBs but not an anti-HD antibody. Thus, these recombinant VLPs have similar biophysical properties to L-HDAg VLPs.

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The pore-opening size of MCM-41 is tailored to be in the microporous region using a chemical vapor deposition technique for selective tailoring. Although the pore opening is narrowed, the internal pore body of MCM-41 remains unchanged so the pore volume retains a substantial portion (80%) of its original value. The adsorption equilibrium of nitrogen and benzene in the modified MCM-41 shows a type I isotherm, which significantly improves the adsorption performance of MCM-41 for low-concentration volatile organic compounds. The adsorption kinetics of benzene in the modified MCM-41 is also studied.

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Reef-building corals are renowned for their brilliant colours yet the biochemical basis for the pigmentation of corals is unknown. Here, we show that these colours are due to a family of GFP-like proteins that fluoresce under ultraviolet (UV) or visible light. Pigments from ten coral species were almost identical to pocilloporin (Dove et al. 1995) being dimers or trimers with approximately 28-kDa subunits. Degenerative primers made to common N-terminal sequences yielded a complete sequence from reef-building coral cDNA, which had 19.6% amino acid identity with green fluorescent protein (GFP). Molecular modelling revealed a 'beta -can' structure, like GFP, with 11 beta -strands and a completely solvent-inaccessible fluorophore composed of the modified residues Gln-61, Tyr-62 and Gly-63. The molecular properties of pocilloporins indicate a range of functions from the conversion of high-intensity UV radiation into photosynthetically active radiation (PAR) that can be regulated by the dinoflagellate peridinin-chlorophyll-protein (PCP) complex, to the shielding of the Soret and Q(x) bands of chlorophyll a and c from scattered high-intensity light. These properties of pocilloporin support its potential role in protecting the photosynthetic machinery of the symbiotic dinoflagellates of corals under high light conditions and in enhancing the availability of photosynthetic light under shade conditions.

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Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as,well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.