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991.
Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. 总被引:14,自引:2,他引:12 下载免费PDF全文
We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride. By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield. 相似文献
992.
The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions. 相似文献
993.
994.
Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis 总被引:1,自引:0,他引:1
Yoshinori Akiyama Kunio Koshimura Tetsuya Ohue Ken Lee Soichi Miwa Sen Yamagata Haruhiko Kikuchi 《Journal of neurochemistry》1991,57(3):997-1002
The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihy-droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O2 in N2) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism. 相似文献
995.
In this article, we demonstrate that an increase in intracellular Ca2+ concentration may represent a specific common step(s) in the mechanism(s) of action of glutamate (Glu) and depolarizing agents on formation of inositol phosphates (IPs) in 8-day-old rat forebrain synaptoneurosomes. In fact, A23187, a Ca2+ ionophore, induces a dose-dependent accumulation of IPs, which is not additive with that evoked by Glu and K+ but is slightly synergistic with that induced by carbachol. In addition, Glu and K+ augment the intracellular Ca2+ concentration in synaptoneurosome preparations as measured by the fura-2 assay. The absence of external Ca2+ decreases basal and Glu-, and K(+)-stimulated formation of IPs. Cd2+ (100 microM) fully inhibits both Glu- and K(+)-evoked formation of IPs without affecting the carbachol-elicited response of IPs. Zn2+ inhibits Glu- and K(+)-stimulated accumulation of IPs (IC50 approximately 0.4 mM) but with a lower affinity than Cd2+ (IC50 approximately 0.035 mM). The organic Ca2+ channel blockers verapamil (10 microM), nifedipine (10 microM), omega-conotoxin (2 microM), and amiloride (10 microM) as well as the inorganic blockers Co2+ (100 microM) and La3+ (100 microM) block neither Glu- nor K(+)-evoked formation of IPs, a result suggesting that the opening of the L-, T-, N-, or P-type Ca2+ channels does not participate in these responses. All these data suggest that an increase in intracellular Ca2+ concentration resulting from an influx of Ca2+, sensitive to Cd2+ but not to other classical Ca2+ antagonists, may play a key role in the transduction mechanism activated by Glu or depolarizing agents. 相似文献
996.
Vitamin D receptor (VDR) is a nuclear protein which mediates the physiological actions of its hormone ligand, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). While it appears that the receptor-hormone complex regulates the expression of hormone-dependent genes involved in mineral homeostasis, its role in induction of differentiation of leukaemic cells is less clear. We have studied the expression of the VDR gene in several sublines of HL-60 leukaemic cells with varying responsiveness to 1,25(OH)2D3. Sublines which rapidly differentiated to monocytic forms were shown to contain elevated steady-state levels of VDR mRNA within 1 h of exposure to high concentration of 1,25(OH)2D3. This up-regulation of the expression of VDR was not apparent in sublines in which monocytic differentiation occurred after a delay of several days. Beginning at approximately 3 h after exposure to 1,25(OH)2D3 in most cases, there was a gradual decline in VDR mRNA levels. Measurement of steady-state levels of mRNA for c-myc and c-fos showed that in sublines of HL-60 cells which respond rapidly to 1,25(OH)2D3, elevation of VDR mRNA is evident prior to the changes in proto-oncogene expression. These data are consistent with the hypothesis that a change in VDR gene expression is one of the steps that promote monocytic differentiation. 相似文献
997.
ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite. 总被引:4,自引:1,他引:3 下载免费PDF全文
Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase. 相似文献
998.
Carr vegetation was once extensive in New Zealand. It can be divided into Cordyline australis I Carex secta carr, comprising an open wood with scattered large herbs and an abundance of Carex species, and podocarp carr, dominated by tall conifers. Both have been almost eliminated by agricultural development. We studied a remnant of Cordyline australis I Carex secta carr in South Island, which graded at one end into salt marsh. Eight communities were recognized, including one pure salt marsh and two more with saline influence. Near the stream that provided moisture and alluvium for the area were herbaceous communities. In the poorly-drained area away from the stream were communities dominated by shrubs and small trees. The soil environment is similar to that of many European carrs, though generally less organic. A number of the exotic species are also found in European carrs. 相似文献
999.
Jerry Lee Lovelace Jozef J. Kusmierz Dominic M. Desiderio 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,562(1-2)
Methionine enkephalin (ME = YGGFM) was measured in five individual human post-mortem pituitaries using four different analytical methods, with the objective of comparing the molecular specificities of the methods. Radioreceptor assay (RRA) used a receptor-rich preparation from brain and [3H]etorphine as radioligand to determine ME-like receptoractivity (ME-LR). Radioimmunoassay (RIA) measured ME-like immunoreactivity (ME-LI). Pituitary samples analyzed by RRA and RIA were purified first with a high-performance liquid chromatography (HPLC) gradient on a polymer analytical column. Fast atom bombardment mass spectrometry (FAB-MS) in two different detection modes quantified ME using the protonated molecular ion MH+ of ME at 574 a.m.u. and B/E linked-field selected reaction monitoring (SRM) to monitor the specific unimolecular metastable transition that produced the unique amino acid sequence-determining tetrapeptide fragment ion YGGF+ from the MH+ precursor ion. Both FAB-MS methods used the deuterated internal standard YGG[2H5-F]M. Samples analyzed with FAB-MS were purified first with multi-dimensional reversed-phase HPLC. The first dimension was an ODS gradient, and the second dimension was a polymer isocratic elution. The following ME amounts were measured (mean ± standard error of the mean): ME-LR, 7.0 ± 1.9 μg g−1 tissue; ME-LI, 1.8 ± 0.7 μg g−1 tissue; MH+, 2.7 ± 0.6 μg g−1 tissue; SRM, 3.0 ± 0.8 μg g−1 tissue. The FAB SRM method provided the highest level of molecular specificity amount these four analytical methods used to measure picomole amounts of endogenous ME in a human pituitary. 相似文献
1000.