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111.
Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system 总被引:3,自引:0,他引:3
Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or "retrotranslocated" into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-alpha factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61alpha. In addition, pro-alpha factor photocrosslinked derlin-1, but not Sec61alpha. Thus, derlin-1 appears to be involved in pro-alpha factor retrotranslocation. 相似文献
112.
Trefzer A Jungmann V Molnár I Botejue A Buckel D Frey G Hill DS Jörg M Ligon JM Mason D Moore D Pachlatko JP Richardson TH Spangenberg P Wall MA Zirkle R Stege JT 《Applied and environmental microbiology》2007,73(13):4317-4325
Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4'-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4'-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin. 相似文献
113.
From the petroleum ether extract of the rhizomes of Tamus communis, the 7-hydroxy-2,3,4,8-tetramethoxyphenanthrene (1) was isolated, together with the known 2,3,4-trimethoxy-7,8-methylenedioxyphenanthrene (2), 3-hydroxy-2,4,-dimethoxy-7,8-methylenedioxyphenanthrene (3), 2-hydroxy-3,5,7-trimethoxyphenanthrene (4) and 2-hydroxy-3,5,7-trimethoxy-9,10-dihydrophenanthrene (5), through cytotoxic assay guidance. The structures were determined by means of HREIMS, (1)H NMR, JMOD and NOESY experiments. The cytotoxic effects of the isolated compounds were tested on cervix adenocarcinoma (HeLa) cells, with the MTT assay. The results demonstrated that, with the exception of 2, all these compounds displayed pronounced cytotoxic activity; especially 1 and 3 exhibited significant cell growth inhibitory effects, with IC(50)=8.52+/-0.70 and 3.64+/-0.12 microM, respectively. 相似文献
114.
Molnár K 《Diseases of aquatic organisms》2007,78(1):45-53
Myxozoans are common parasites of fish kidneys, with most having specific sites of development. Five specific sites of development include (1) the lumen of renal tubules, (2) the renal corpuscles followed by location in renal tubules, (3) intracellular location within the tubular epithelium followed by a stage in the lumen of the ducts, (4) haematopoietic tissue with dispersed trophozoites, and (5) haematopoietic tissue with large, localized plasmodia. A coelozoic development preceded by presporogonic multiplication characterises most Sphaerospora spp. Early plasmodial stages of Myxidium and Chloromyxum spp. are frequently found in the renal glomerules, while spores develop in the urinary channels in plasmodia released from the renal corpuscles. In Hoferellus and Myxobilatus spp., spores are formed in small plasmodia inside the lumen of the urinary ducts after several internal cleavages in the epithelium of renal tubules. The presence of dispersed trophozoites among haematopoietic tissue cells of the renal interstitium characterises the development of Sphaerospora tincae and Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD). Spores of S. tincae are formed at the place of plasmodial development, while spore formation of PKD is in the renal tubules. A large mass of spores, often surrounded by a connective tissue capsule, can appear in the renal interstitium during infections by several Myxobolus spp.; furthermore, a large number of these spores formed in plasmodia in distant tissues can also accumulate in melano-macrophage centres. 相似文献
115.
Luyt K Slade TP Dorward JJ Durant CF Wu Y Shigemoto R Mundell SJ Váradi A Molnár E 《Journal of neurochemistry》2007,100(3):822-840
GABA(B) receptors (GABA(B)Rs) are involved in early events during neuronal development. The presence of GABA(B)Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GABA(B)R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA(B1abcdf) and GABA(B2) subunits of the GABA(B)R. Using semiquantitative PCR analysis with GABA(B)R isoform-selective primers we found that the expression level of GABA(B1abd) was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA(B2) isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA(B1) and GABA(B2) subunits are under independent control during oligodendroglial development. Activation of GABA(B)Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA(B)R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA(B)Rs in oligodendrocyte development. The presence of GABA(B)Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury. 相似文献
116.
Málnási-Csizmadia A Tóth J Pearson DS Hetényi C Nyitray L Geeves MA Bagshaw CR Kovács M 《The Journal of biological chemistry》2007,282(24):17658-17664
After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release. 相似文献
117.
Heterosubtypic immunity to influenza A virus infection requires a properly diversified antibody repertoire
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Nguyen HH Zemlin M Ivanov II Andrasi J Zemlin C Vu HL Schelonka R Schroeder HW Mestecky J 《Journal of virology》2007,81(17):9331-9338
Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT(-/-)) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (D(H)) with an altered D(H) segment (namely, Delta D-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT(-/-) mice compared to those in Delta D-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses. 相似文献
118.
119.
Wittmann I Molnár GA Wagner L Köszegi T Wagner Z Laczy B Tamaskó M Markó L Mohás M Nagy J 《Acta physiologica Hungarica》2007,94(1-2):159-166
BACKGROUND: Anaemia in diabetes mellitus (DM) and/or chronic renal failure (CRF) may be caused by a decreased production of erythropoietin (EPO), EPO resistance, and by the lysis of the young circulating red blood cells (neocytolysis) induced by subclinical inflammation and low EPO level. Aims of this study were to detect EPO resistance in patients with DM and/or CRF and to prove, that acetylsalicylic acid (ASA) is able to improve the haemopoietic status by decreasing neocytolysis. METHODS: In a cross-sectional study, three groups of selected patients (patients with DM; patients with DM+CRF; patients with CRF without DM, n=15 each) and a group of controls (non-diabetic, nonazotemic subjects, n = 10) were compared. In the intervention part of the study, the effect of a single dose of 1 gram ASA on neocytolysis was investigated in a subgroup of these patients. RESULTS: Despite the similar EPO level (p = 1.000), all three patient groups had lower haemoglobin and haematocrit than control persons (p < 0.05 in all cases). Patients with DM+CRF had lower haemoglobin than patients with DM or CRF alone (p < 0.05). Single dose of ASA induced a fast increase in serum EPO level, a concomitant rise of the Rtc number and rate, red blood cell count, haematocrit and haemoglobin p < 0.01 for each). These changes were accompanied by a marked decrease in serum lactate dehydrogenase activity (p < 0.01). CONCLUSIONS: DM and CRF may induce erythropoietin resistance. In these patients, ASA treatment increases serum EPO level. The higher EPO level and the anti-inflammatory effect of ASA may decrease neocytolysis. 相似文献
120.
The bulk formation of yeast-like (arthrospore-like) cells were typical in carbon-depleted submerged cultures of the high beta-lactam producer Penicillium chrysogenum NCAIM 00237 strain independently of the nitrogen-content of the culture medium. This morphogenetic switch was still quite common in carbon-starving cultures of the low-penicillin-producer strain P. chrysogenum ATCC 28089 (Wis 54-1255) when the nitrogen-content of the medium was low but was a very rare event in wild-type P. chrysogenum cultures. The mycelium-->yeast-like cell transition correlated well with a relatively high glutathione concentration and a reductive glutathione/glutathione disulfite (GSH/GSSG) redox balance in autolysing cultures, which was a consequence of industrial strain development. Paradoxically, the development of high beta-lactam productivity resulted in a high intracellular GSH level and, concomitantly, in an increased y-glutamyltranspeptidase (i.e. GSH-decomposing) activity in the autolytic phase of growth of P. chrysogenum NCAIM 00237. The hypothesized causal connection between GSH metabolism and cell morphology, if verified, may help us in future metabolic engineering of industrially important filamentous fungi. 相似文献