The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found when the cells were grown for more than 14 h. The E2 produced was partially active, varying 10 and 90% in different experiments. By limited proteolysis of the protein it was shown that the catalytic domain was incorrectly folded, caused by formation of intermolecular or intramolecular S-S bridges. The enzyme was fully activated after unfolding in 2.5 M guanidine hydrochloride containing 2 mM dithiothreitol, followed by refolding by dialysis. Active E2 was isolated in a simple three-step procedure. It possessed a specific activity in the same order as that found after isolation of E2 from purified pyruvate dehydrogenase complex from A. vinelandii. Active E2 comprises about 7% of the total soluble cellular protein in the E. coli clone. By genetic manipulation, deletion mutants of E2 were created, one encoding the lipoyl domain and the N-terminal half of the pyruvate-dehydrogenase (E1)- and lipoamide-dehydrogenase (E3)-binding domain, the other encoding the catalytic domain and the C-terminal half of the E1- and E3-binding domain. In E. coli expression of both mutants was observed. 相似文献
Oestradiol has been conjugated to allylamine-dUTP with an 11-atom spacer to allow enzymatic incorporation of the label into
DNA sequences. In a comparative DNA and mRNA FISH study we have used DNA probes that were either labelled with digoxigenin,
biotin or oestradiol. Results show that oestradiol-labelled probes can detect DNA and RNA sequences in FISH equally well as
digoxigenin- and biotin-labelled probes. Further, no crossreactivity between the various hapten-specific antibodies and the
three haptens were observed. Binding of the rabbit anti-oestradiol antibody to endogenous oestrogen in various tissues was
not observed under the conditions tested. In view of the increasing demands for multi-colour DNA and mRNA FISH applications,
oestradiol is a welcome addition to the collection of haptens employed in FISH.
Accepted 20 June 1997 相似文献
Summary The role of pyrimidine dimers in mutagenesis by ultraviolet light was examined by measuring the UV-induced reversion of six different bacteriophage M13 amber mutants for which the neighboring DNA sequences are known. The mutational response at amber (TAG) codons preceded by a guanine or adenine (where no pyrimidine dimer can be formed) were compared with those preceded by thymine or cytosine (where dimer formation is possible). Equivalent levels of UV-induced mutagenesis were observed at both kinds of sites. This observation demonstrates that there is no requirement for a pyrimidine dimer directly at the site of UV-induced mutation in this single-stranded DNA phage. UV irradiation of the phage was also performed in the presence of Ag+ ions, which specifically sensitize the DNA to dimer formation. The two methods of irradiation, when compared at equal survival levels (and presumably equal dimer frequencies), produced equivalent frequencies of reversion of the amber phage. We believe these results indicate that while the presence of pyrimidine dimers may be a prerequisite for UV mutagenesis, the actual mutagenic event can occur at a site some distance removed from a dimer. 相似文献
Summary Exposure of HTC cells to sodium butyrate caused inhibition of growth. The site of growth inhibition was studied by time-lapse
cinematography and [3H]thymidine incorporation studies. Evidence is presented that sodiunm butyrate affected the cell cycle at a specific point
immediately after mitosis. Inasmuch as it does not modify the interphase duration after its removal, butyrate may be used
for HTC synchronization.
This work was supported by l'Institut Nationale de la Santé et de la Recherche Médicale and la Centre Nationale de la Recherche
Scientifique (L. T. and J. K.). 相似文献
Summary A floating collagen matrix culture of neonatal rat heart myocardial cells shows rhythmic contractions which are dependent
on localization of cells, cell density, and collagen concentration. The rhythmic contractions of the collagen matrix can be
registered by a device scanning the optical density at the edge of the gel and have been observed over a temperature range
from 9° to 40° C. The results of the present study underline the usefulness of myocardial cell populated collagen matrixes
for studies on coherent contractions of heart cell cultures. 相似文献
The inbred strains GRS/A and LIS/A carry the haplotypeH-2dx, which had earlier been shown to have theKd,If,Sf, andGf alleles and a previously unknownD region allele,Ddx. We show here that theDdx allele determines a new private specificity, H-2.63, is H-2.28 negative, and determines at least one public specificity of the H-2.1 family. It is thus a second example (afterDk) of a H-2.1-positive H-2.28-negativeD region allele. Capping experiments show that the Ddx product comprises two molecules: H-2Ddx bearing the private specificity H-2.63, and H-2Ldx, which is H-2.63-negative but reacts with sera against the H-2.1 family of specificities. SDS gel electrophoresis of detergent-solubilized immunoprecipitated Ddx products shows that the H-2Ldx antigen has a molecular weight of approximately 45,000 daltons and is associated with a smaller polypeptide (mol. wt. 12,000). 相似文献
Brachyuran crabs are an important ecological and economical, yet often unstudied aspect of intertidal mudflats of the Arabian Peninsula. Here we provide baseline density estimates of crabs at the relatively pristine intertidal mudflats of Barr Al Hikman (Sultanate of Oman) and provide information on their life cycle and habitat preference. Across the winters of 2012–2015 crabs were sampled on a grid covering the entire intertidal depth gradient. 29 species were found and average densities varied between 12 and 54 crabs/m2. Deposit-feeding and herbivorous crabs were the most abundant species across all winters. Size frequency data and the presence of ovigerous females show that most crabs species reproduce in the intertidal area. P. segnis, the most important crab for local fisheries, was found to use the intertidal area as a nursery ground. We analysed the relationships between the two most abundant crab species, Macrophthalmus sulcatus and Thalamita poissonii and the environmental variables: seagrass density, tidal elevation, median grain size and sediment depth using Random Forest models. The predictive capacity of the models and the relative importance of the environmental predictors varied between years, but crab densities in general were positively associated with seagrass density, presumably because seagrass offers feeding habitat.
Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention. 相似文献
Statins are thought to reduce vascular inflammation through lipid independent mechanisms. Evaluation of such an effect in atherosclerotic disease is complicated by simultaneous effects on lipid metabolism. Abdominal aortic aneurysms (AAA) are part of the atherosclerotic spectrum of diseases. Unlike atherosclerotic occlusive disease, AAA is not lipid driven, thus allowing direct evaluation of putative anti-inflammatory effects. The anti-inflammatory potency of increasing doses (0, 20 or 40 mg/day) simvastatin or atorvastatin was evaluated in 63 patients that were at least 6 weeks on statin therapy and who underwent open AAA repair. A comprehensive analysis using immunohistochemistry, mRNA and protein analyses was applied on aortic wall samples collected during surgery. The effect of statins on AAA growth was analyzed in a separate prospective study in incorporating 142 patients. Both statins equally effectively and dose-dependently reduced aortic wall expression of NFκB regulated mediators (i.e. IL-6 (P<0.001) and MCP-1 (P<0.001)); shifted macrophage polarization towards a M2 phenotype (P<0.0003); selectively reduced macrophage-related markers such as cathepsin K and S (P<0.009 and 0.0027 respectively), and ALOX5 (P<0.0009), and reduced vascular wall NFκB activity (40 mg/day group, P<0.016). No effect was found on other cell types. Evaluation of the clinical efficacy of statins to reduce AAA progression did not indicate an effect of statins on aneurysm growth (P<0.337). Hence, in the context of AAA the clinical relevance of statins pleiotropy appears minimal. 相似文献