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61.
62.
Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae. 总被引:4,自引:0,他引:4 下载免费PDF全文
S Jensen A H Andersen E Kjeldsen H Biersack E H Olsen T B Andersen O Westergaard B K Jakobsen 《Molecular and cellular biology》1996,16(7):3866-3877
The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus. 相似文献
63.
Staphylococcal enterotoxins (SE’s) are a group of small exoproteins produced by some strains of Staphylococcus aureus. The SE’s, designated A to E according to their antigenic specificities, are important causes of food poisoning worldwide. Milk and dairy products are frequently associated with S. aureus enter-otoxin food poisoning, and it is supposed that infected milk from mastitic animals constitute the main source of enterotoxigenic S. aureus of animal origin (Bryon 1983, Gilmour & Harvey 1990, Bergdoll 1989). Indeed, S. aureus is the most common cause of bovine mastitis worldwide, and if mastitis strains produce SE this makes up an enormous reservoir of potential enterotoxin producers. The production of SE by S. aureus isolated from bovine mastitis have been investigated in several countries (Matsunaga et al. 1993, Kenny et al. 1993, Olson et al 1970, Orden et al. 1992, Olsvik et al. 1981, Adekeye 1980, Garcia et al. 1980, Abbar 1986, Harvey & Gilmour 1985). Since no studies have been performed on the prevalence of enterotoxigenic strains of S. aureus isolated from bovine mastitis in Denmark, a well characterized collection of S. aureus (Aarestrup et al. 1995) was investigated with respect to this property. 相似文献
64.
Evaluation of mass spectrometric techniques for charaterization of engineered proteins 总被引:7,自引:0,他引:7
Roepstorff Peter Schram Karl H. Andersen Jens S. Rafn Kate Baldursson Trausti Krøll Jenny Poulsen Kjeld Knudsen Jens Kristiansen Karsten 《Molecular biotechnology》1995,3(1):1-7
A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described.
The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used
as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation.
The various aspects of the procedure and its application is described in detail. 相似文献
65.
The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms. The general pathway consists of six enzymatic steps. In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated. The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined. One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis. It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors. We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes. Only the double mutant is pyrimidine auxotrophic. 相似文献
66.
Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior. 总被引:2,自引:0,他引:2 下载免费PDF全文
LamB, a sugar-specific channel of Escherichia coli outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated using fast Fourier transformation. The current noise through the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz. The spectral density of the noise of the open LamB channels was a quadratic function of the applied voltage. Its magnitude was not correlated to the number of channels in the lipid bilayer membrane. Upon addition of sugars to the aqueous phase the current decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the sugars with the binding site inside the channel. The frequency dependence of the spectral density was of Lorentzian type, although the power of its frequency dependence was not identical to -2. Analysis of the power density spectra using a previously proposed simple model (Benz, R., A. Schmid, and G. H. Vos-Scheperkeuter. 1987. J. Membr. Biol. 100: 12-29), allowed the evaluation of the on- and the off-rate constants for the maltopentaose binding to the binding site inside the LamB channels. This means also that the maltopentaose flux through the LamB channel could be estimated by assuming a simple one-site, two-barrier model for the sugar transport from the results of the noise analysis. 相似文献
67.
Michael S. Goligorsky Hirokazu Tsukahara Harold Magazine Thomas T. Andersen Asrar B. Malik Wadie F. Bahou 《Journal of cellular physiology》1994,158(3):485-494
Cellular mechanisms responsible for the termination of ET-1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET- 1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ETA receptor cDNA (CHO-ET) were studied. CHO-ET responded to ET-1 with robust [Ca2+], transients and developed a long-lasting homologous desensitization. Donors of nitric oxide (NO), 3-morpholino-sydnonimine HCl(SIN-1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca2+], recovery, and counteracted the development of homologous desensitization by a cyclic GMP-independent mechanism, suggesting an alternative mode for NO modulation of ET-1 responses. Stimulation of CHO-ET cells with mastoparan, a wasp venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO-induced modulation of [Ca2+] mobilization. Using a lys9-biotinylated ET-1 (ET-1 [BtK9]), binding sites were “mapped” in CHO-ET cells. Receptor-ligand complexes did not exhibit spontaneous dissociation during 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN-1 caused a rapid, concentration-dependent, and reversible dissociation of biotinylated ET- 1 from ETA receptor (EC50 = 75 μM and 6 μM, respectively), an effect that was not mimicked by 8-bromo-cyclic GMP. “Sandwich” co-culture of endothelial cells with CHO-ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET-1 [BtK9] from ETA receptors. We hypothesize that NO plays a role in physiological termination of ET-1 signalling by dual mechanisms: (1) displacement of bound ET-1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca2+] mobilization, hence inhibiting end-responses to ET-1. © 1994 Wiley-Liss, Inc. 相似文献
68.
Diurnal variations of amino acids and organic acids in xylem fluid from Lagerstroemia indica: an endogenous circadian rhythm 总被引:2,自引:0,他引:2
Diurnal variations in the concentrations of major organic compounds occurred in xylem fluid extracted from Lagerstroemia indica L. The concentration of amino acids and the N/C ratio was at a maximum and that of organic acids was at a minimum between 1230 and 2030 h. Since the concentrations of total organic nitrogen, total amino acids and most individual amino acids (but not organic acids or sugars) were also proportional to xylem tension two experiments were performed to discern whether variations in chemistry were a consequence of diurnal changes in moisture stress. In the first experiment, L. indica , exposed to variable levels of moisture stress during midday, manifested an increase in organic acids and a reduction in the N/C ratio. In the second experiment, chemical profiles of xylem fluid were collected and compared for plants exposed to a natural photoperiod, constant darkness or continuous light at noon and midnight. After 1 day amino acids increased in concentration during midday for all treatments; the variation was greatest (10-fold) for plants in constant darkness where xylem tension varied from 0.20 to 0.25 MPa. Only plants exposed to continuous light lost a diurnal rhythm after 3 days. Thus, the circadian rhythm was endogenous, terminated in continuous light and was not mediated by changes in moisture stress. Glutamine accounted for most of the diurnal variation in total amino acids, organic nitrogen and the N/C ratio in xylem fluid. 相似文献
69.
Xia Guoliang Anne Grete Byskov Claus Yding Andersen 《Molecular reproduction and development》1994,39(1):17-24
The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed. The GVBD of unstimulated DO also increased significantly when cocultured with cumulus cells from primed CEO. The percentage of GVBD in unprimed DO and in DO isolated from primed CEO was the same. We suggest that within 1–2 hr, forskolin and cAMP stimulate cumulus cells to produce a diffusible meiosis-inducing substance which overcomes HX-inhibition and induces oocyte maturation, including both GVBD and PB formation. The CEO must be primed for more than 2 hr before the resumption of meiosis in DO isolated from such CEO is induced. Oocyte-cumulus connections are crucial as far as initiating the production of a meiosis-inducing substance is concerned. Oocyte-cumulus connections are not needed for transferring this substance to the oocyte. © 1994 Wiley-Liss, Inc. 相似文献
70.
The principal sulfatide of virulentMycobacterium tuberculosis, sulfolipid-I (SL-I), both directly stimulates neutrophil superoxide (O
2
–
) release and, at substimulatory concentrations, primes these cells for markedly enhanced oxidative responsiveness to other stimuli. The present study was undertaken to clarify the priming mechanisms by comparing cellular events following priming doses of SL-I with those following priming with N-formyl-methionyl-leucyl-phenylalanine (FMLP). We compared the involvement of the calcium cation (Ca2+), as well as membrane protein kinase C (PKC) activity and the translocation of NADPH oxidase-cytosolic cofactor effected by priming levels of the two agonists. The investigation led to two important conclusions. First, we clearly demonstrate that priming by both SL-I and FMLP results from activation of cellular processes that are not involved in direct oxidative activation. For example, whereas direct induction of O
2
–
generation by FMLP and SL-I required increases in intracellular Ca2+, an increase in intracellular calcium concentration ([Ca2+]i) above basal levels was not required for priming. Second, we identified key differences in the cellular responses to priming doses of SL-I and FMLP. Whereas increased membrane PKC activity caused by priming doses of FMLP was only partially blocked by chelation of intracellular Ca2+, Ca2+ chelation completely inhibited the increase in membrane PKC activity caused by SL-I. NADPH oxidase-cytosolic factor translocation to plasma membranes was completely blocked by pertussis toxin when priming doses of SL-I were used. This guanine-nucleotide-binding protein inhibitor had no effect on FMLP-dependent translocation of the oxidase cofactors. The comparative approach introduced in this report provides a valuable and novel method to discern the complex interactions of various cellular processes that regulate the state of activation of stimulated cells. 相似文献