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991.
Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells. 相似文献
992.
Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris 总被引:1,自引:0,他引:1
Ramchuran SO Vargas VA Hatti-Kaul R Karlsson EN 《Applied microbiology and biotechnology》2006,71(4):463-472
A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZαB vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (α-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a “batch-induced” cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate. 相似文献
993.
The main step in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conformational change of the normal cellular prion protein (PrP(C)) into the abnormal isoform, named prion (PrP(Sc)). Since PrP is a highly conserved protein, the production of monoclonal antibodies (mAbs) of high specificity and affinity to PrP is a difficult task. In the present study we show that it is possible to overcome the unresponsiveness of the immune system by immunizing wild-type BALB/c mice with a 13 amino acid PrP peptide from the C-terminal part of PrP, bound to the keyhole limpet hemocyanin (KLH). Immunization induced predominantly anti-PrP(Sc) humoral immune response. Furthermore, we were able to obtain a panel of mAbs of IgG class specific for different non-self-conformations of PrP, with anti-PrP(Sc)-specific mAbs being the most abundant. 相似文献
994.
Szappanos H Szigeti GP Pál B Rusznák Z Szucs G Rajnavölgyi E Balla J Balla G Nagy E Leiter E Pócsi I Hagen S Meyer V Csernoch L 《Peptides》2006,27(7):1717-1725
The antifungal protein AFP is a small, cystein-rich protein secreted by the imperfect ascomycete Aspergillus giganteus. The protein efficiently inhibits the growth of filamentous fungi, including a variety of serious human and plant pathogens mainly of the genera Aspergillus and Fusarium, whereas AFP does not affect the growth of yeast and bacteria. This restricted susceptibility range makes it very attractive for medical or biotechnological use to combat fungal infection and contamination. We, therefore, analyzed whether AFP affects the growth or function of a number of mammalian cells. Here we show that the protein neither provokes any cytotoxic effects on human endothelial cells isolated from the umbilical vein nor activates the immune system. Moreover, potassium currents of neurons and astrocytes do not change in the presence of AFP and neither excitatory processes nor the intracellular calcium homeostasis of cultured skeletal muscle myotubes are affected by AFP. Our data, therefore, suggest that AFP is indeed a promising candidate for the therapeutic or biotechnological use as a potential antifungal agent. 相似文献
995.
Erythroid protein 4.1 (4.1R) stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of non-erythroid protein 4.1R, we used sedimentation, pull-down and co-immunoprecipitation assays to investigate the ability of protein 4.1R to establish inter-/intra-molecular associations. We demonstrated that the small 4.1R isoforms of 60 kDa (4.1R60), but not the larger isoforms of 80 and 135 kDa (4.1R80 and 4.1R135), were self-associated, and that a domain contained in all 4.1R isoforms, the core region, was responsible for 4.1R self-association. Results from denaturing-renaturing experiments, in which an initially non-self-associated 4.1R80 isoform became self-associated, suggested that an initially hidden core region was subsequently exposed. This hypothesis was supported by results from pull-down assays, which showed that the core region interacted with the N-terminal end of the FERM (4.1, ezrin, radixin, moesin) domain that is present in 4.1R80 and 4.1R135 isoforms but absent from 4.1R60 isoforms. Consistently, 4.1R80 isoforms bound neither to each other nor to 4.1R60 isoforms. We propose that 4.1R60 isoforms are constitutively self-associated, whereas 4.1R80 and 4.1R135 self-association is prevented by intramolecular interactions. 相似文献
996.
997.
Warda M Toida T Zhang F Sun P Munoz E Xie J Linhardt RJ 《Glycoconjugate journal》2006,23(7-8):555-563
Heparan sulfate (HS), is a proteoglycan (PG) found both in the extracellular matrix and on cell surface. It may represent
one of the most biologically important glycoconjugates, playing an essential role in a variety of different events at molecular
level. The publication of the mouse genome, and the intensive investigations aimed at understanding the proteome it encodes,
has motivated us to initiate studies in mouse glycomics focused on HS. The current study is aimed at determining the quantitative
and qualitative organ distribution of HS in mice. HS from brain, eyes, heart, lung, liver, kidney, spleen, intestine and skin
was purified from 6–8 week old male and female mice. The recovered yield of HS from these organs is compared with the recovered
whole body yield of HS. Structural characterization of the resulting HS relied on disaccharide analysis and 1H-NMR spectroscopy. Different organs revealed a characteristic HS structure. These data begin to provide a structural understanding
of the role of HS in cell-cell interactions, cell signaling and sub-cellular protein trafficking as well as a fundamental
understanding of certain aspects of protein-carbohydrate interactions. 相似文献
998.
Forward genetic analysis of the circadian clock separates the multiple functions of ZEITLUPE
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Kevei E Gyula P Hall A Kozma-Bognár L Kim WY Eriksson ME Tóth R Hanano S Fehér B Southern MM Bastow RM Viczián A Hibberd V Davis SJ Somers DE Nagy F Millar AJ 《Plant physiology》2006,140(3):933-945
The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in dark-grown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci. 相似文献
999.
Wölfling J Oravecz EA Ondré D Mernyák E Schneider G Tóth I Szécsi M Julesz J 《Steroids》2006,71(9):809-816
17beta-Dihydrooxazinyl steroids 5a-l and 6a-l were synthetized. The acid-catalyzed reactions of 21-azidomethyl-20-hydroxy- and 21-hydroxymethyl-20-azidosteroids with substituted aromatic aldehydes led to the formation of androst-5-en-3beta-ols substituted in position 17beta with dihydrooxazine residues. The inhibitory effects of these compounds on rat testicular C(17,20)-lyase were investigated with an in vitro radioincubation technique. 相似文献
1000.
In the dog, unlike most other domestic animal species, corpus luteum (CL) life span is not affected by hysterectomy. Only in pregnant dogs, during the immediate prepartum decline of progesterone, does PGF2alpha clearly seem to act as an endogenous luteolytic agent. Whether endogenous PGF2alpha plays a role in the slow regression of the corpora lutea of the nonpregnant cycle is not known. To test for possible paracrine/autocrine effects of locally produced PGF2alpha, luteal expression of the key rate-limiting enzymes in prostaglandin biosynthesis, i.e. cyclooxygenase 1 and 2 (Cox1 and Cox2), was examined in dogs during diestrus, including the periods of CL formation, as well as early and late CL regression. Corpora lutea were collected by ovariohysterectomy from nonpregnant bitches 5, 15, 25, 35, 45 and 65 days after ovulation. On the mRNA-level, expression of Cox1 and Cox2 was tested by qualitative and quantitative, Real Time (Taq Man) RT-PCR; on the protein level, expression of Cox2 was studied by immunohistochemistry. The mRNA for Cox1 and Cox2 were detected at all stages of diestrus. Expression of Cox1 was lowest on Day 5 (ovulation = Day 0) and higher and nearly constant thereafter. Expression of Cox2-mRNA was distinctly cycle related and highest on Day 5; it decreased by Day 15 and remained constantly low until Day 65. Immunohistochemistry localized expression of Cox2 in the cytoplasm of luteal cells. Staining was restricted to Days 5 and 15, with stronger signals on Day 5. These data suggested that increased expression of Cox2 is associated with luteal growth and development and not luteal regression. Furthermore, the expression of Cox1 more likely reflected activity of a housekeeping gene. 相似文献