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1.
Abstract We examined the extremely thermophilic Gram-negative eubacterium Thermus aquaticus for the presence of plasmid DNA, and detected 5 species. The sizes were determined by electron microscopy and gel electrophoresis. The plasmids, pTA 1-pTA 5, were of respective length: 13.5, 12.4, 11.2, 9.6 and 8.3 kb. Clones were isolated which differed in plasmid distribution. No correlation could be observed between the phenotype or growth behaviour and the presence of plasmids. The function of the plasmid structures still needs to be elucidated. 相似文献
2.
Abstract We have recently demonstrated that the calmodulin antagonist trifluoperazine has antitubercular activity in vitro against Mycobacterium tuberculosis H37 Rv susceptible and resistant to isoniazid. It is shown that trifluoperazine at a concentration of 50 μ g ml−1 when added to the cells along with the labelled precursors inhibited the incorporation of [14 C]acetate into lipids (63%) and uptake of [14 C]glycine (74%) and [3H ]thymidine (52%) bu whole cells of M. tuberculosis H37 Rv by 6 h of exposure. After 48 h, the inhibition was 87%, 97% and 74%, respectively. However, when the drug was added to cells taking up and metabolizing the labelled precursors at a later point (3 h for [14 C]acetate and [3 H]thymidine and 12 h for [14 C]glycine) it inhibited completely the uptake of all the precursors, at least up to 24 h. The onset of inhibitory action was very rapid, i.e. 3 h. It is suggested that trifluoperazine has multiple sites of action and acts probably by affecting the synthesis of lipids, proteins and DNA. 相似文献
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Per-Ola Arvidsson Charlotte Eva Bratt Marie Carlsson Hans-Erik Åkerlund 《Photosynthesis research》1996,49(2):119-129
Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g–1 s–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b
6 and an internal sequence of polyphenol oxidase.Abbreviations DMSO
dimethylsulfoxid
- HIC
hydrophobic interaction chromatography
- MGDG
monogalactosyl diacylglycerol
- VDE
violaxanthin deepoxidase
A preliminary report of these results was presented at the Xth Int. Congress on Photosynthesis, Montpellier, France, 1995. 相似文献
5.
A mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase (EC 1.6.1.1) was isolated by assaying activity in clones of cells mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine. The mutant is missing both energy-independent and energy-dependent transhydrogenase, but has normal NADH dehydrogenase and ATPase activities. Compared to the parental strain, the mutant has normal growth rates with glucose, glycerol, or succinate aerobically and with glucose or glycerol plus fumarate anaerobically. The aerobic growth yield with limiting glucose concentrations is also normal. These growth properties indicate that the enzyme is not an essential source of NADPH or ATP in vivo. 相似文献
6.
Subdivision of equine Tf into H1 and H2 总被引:6,自引:0,他引:6
Subdivision of equine TfH into two variants, designated H1 (faster) and H2 (slower), has been accomplished by high voltage, thin layer polyacrylamide gel electrophoresis at pH 7.9. Transferrin H1 and H2 have been shown to be controlled by codominant alleles and gene frequencies of the Tf alleles have been determined in the Australian Thoroughbred, Standardbred. Quarter Horse and Arabian Horse breeds. 相似文献
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The soluble pyridine nucleotide transhydrogenase (STH) is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these two redox cofactors. The sth gene in Escherichia coli was cloned and expressed as a fused protein (EcSTH). The purified EcSTH displayed maximal activity at 35 °C, pH 7.5. Heat-inactivation studies showed that EcSTH retains 50% activity after 5 h at 50 °C. The enzyme was stable at 4 °C for 25 days. The apparent K(m) values of EcSTH were 68.29 μM for NADPH and 133.2 μM for thio-NAD(+) . The k(cat) /K(m) ratios showed that EcSTH had a 1.25-fold preference for NADPH over thio-NAD(+) . Product inhibition studies showed that EcSTH activity was strongly inhibited by excess NADPH, but not by thio-NAD(+) . EcSTH activity was enhanced by 2 mM adenine nucleotide and inhibited by divalent metal ions: Mn(2+) , Co(2+) , Zn(2+) , Ni(2+) and Cu(2+) . However, after preincubation for 30 min, most divalent metal ions had little effect on EcSTH activity, except Zn(2+) , Ni(2+) and Cu(2+) . The enzymatic analysis could provide the important basic knowledge for EcSTH utilizations. 相似文献
9.
Semi-solid medium was used to isolate an aerobic, N2 -fixing (C2 H2 -reducing), H2 -utilizing bacterium from the roots of kallar grass ( Leptochloa fusca ). The organism was identified by morphological, cultural and biochemical characteristics. The N2 -fixing, zoogloeal floc-forming isolate described here is a new species. 相似文献
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The annual cycles of CO2 and H2 O exchange over a northern mixed forest as observed from a very tall tower 总被引:1,自引:0,他引:1
Kenneth J. Davis Peter S. Bakwin† Chuixiang Yi Bradford W. Berger‡ Conglong Zhao§ Ronald M. Teclaw¶ J. G. Isebrands¶ 《Global Change Biology》2003,9(9):1278-1293
We present the annual patterns of net ecosystem‐atmosphere exchange (NEE) of CO2 and H2O observed from a 447 m tall tower sited within a mixed forest in northern Wisconsin, USA. The methodology for determining NEE from eddy‐covariance flux measurements at 30, 122 and 396 m above the ground, and from CO2 mixing ratio measurements at 11, 30, 76, 122, 244 and 396 m is described. The annual cycle of CO2 mixing ratio in the atmospheric boundary layer (ABL) is also discussed, and the influences of local NEE and large‐scale advection are estimated. During 1997 gross ecosystem productivity (947?18 g C m?2 yr?1), approximately balanced total ecosystem respiration (963±19 g C m?2 yr?1), and NEE of CO2 was close to zero (16±19 g C m?2 yr?1 emitted into the atmosphere). The error bars represent the standard error of the cumulative daily NEE values. Systematic errors are also assessed. The identified systematic uncertainties in NEE of CO2 are less than 60 g C m?2 yr?1. The seasonal pattern of NEE of CO2 was highly correlated with leaf‐out and leaf‐fall, and soil thaw and freeze, and was similar to purely deciduous forest sites. The mean daily NEE of CO2 during the growing season (June through August) was ?1.3 g C m?2 day?1, smaller than has been reported for other deciduous forest sites. NEE of water vapor largely followed the seasonal pattern of NEE of CO2, with a lag in the spring when water vapor fluxes increased before CO2 uptake. In general, the Bowen ratios were high during the dormant seasons and low during the growing season. Evapotranspiration normalized by potential evapotranspiration showed the opposite pattern. The seasonal course of the CO2 mixing ratio in the ABL at the tower led the seasonal pattern of NEE of CO2 in time: in spring, CO2 mixing ratios began to decrease prior to the onset of daily net uptake of CO2 by the forest, and in fall mixing ratios began to increase before the forest became a net source for CO2 to the atmosphere. Transport as well as local NEE of CO2 are shown to be important components of the ABL CO2 budget at all times of the year. 相似文献
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Plant responses to H2 S and SO2 fumigation. II. Differences in metabolism of H2 S and SO2 in spinach 总被引:1,自引:0,他引:1
Frank M. Maas Luit J. de Kok Willy Strik-Timmer Pieter J. C. Kuiper 《Physiologia plantarum》1987,70(4):722-728
Fumigation of spinach (Spinacia oleracea L. cvs Estivato and Monosa) with H2 S or SO, for 1 to 6 days resulted in accumulation of sulfhydryl (SH) compounds in the shoots of both H2 S- and SO2 -exposed plants. The sulfate concentration in shoots of SO2 -exposed plants increased linearly with time. SH accumulation showed saturation kinetics as a function of time as well as H2 S concentration, ascribed to the internal H2 S concentration in the plant and the availability of substrates for glutathione synthesis, respectively. SH compounds accumulated more at lower exposure temperatures, whereas sulfate accumulation was more pronounced at higher temperatures. These results are discussed in relation to the possible foliar uptake of H2 S and SO2 , the temperature dependence of uptake and the water solubility of these gases. The possibility of SO2 -induced H2 S emission rather than sulfate accumulation as a source for SH accumulation is also discussed. Cessation of fumigation resulted in a decrease in SH compounds and sulfate content that could be accounted for by sulfur metabolism and growth, respectively. 相似文献
13.
S Takahashi 《Journal of biochemistry》1967,61(2):258-259
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Abstract In situ concentrations of hydrogen and other metabolites involved in H2 -consuming and H2 -producing reactions were measured in anoxic methanogenic lake sediments, sewage sludge and fetid liquid of cottonwood. The data were used to calculate the Gibbs free energies of the metabolic reactions under the conditions prevailing in situ. The thermodynamics of most of the reactions studied were exergonic with Gibbs free energies being more negative for H2 -dependent sulfate reduction methanogenesis acetogenesis and for H2 -producing lactate fermentation ethanol fermentation. Butyrate and propionate fermentation, on the other hand, were endergonic under in situ conditions. This observation is interpreted by suggesting that butyrate and propionate is degraded within microbial clusters which shield the fermentating bacteria from the outside H2 (and acetate) pool. 相似文献
15.
MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% β-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB. 相似文献
16.
The cryptic-growth response of maize coleoptiles and its relationship to H2 O2 -dependent cell wall stiffening 总被引:1,自引:0,他引:1
Auxin-mediated elongation growth of maize ( Zea mays L.) coleoptile segments can be nullified by lowering the turgor pressure by 0.45 MPa. Under these conditions irreversible segment length (lin ) measured after freezing/thawing increases steadily over a period of 8 h although the in vivo length (ltot ) remains constant. This phenomenon, designated as 'cryptic growth', is an indication of a wall-stiffening process which appears to be an intrinsic component of irreversible cell wall extension. Using a range of metabolic inhibitors it is demonstrated that cryptic growth is caused by a temperature-sensitive biochemical process in the cell wall which depends on the presence of O2 and active peroxidase, but not on ATP and protein synthesis. Inhibition of cryptic growth by anaerobic conditions can be alleviated by extermal H2 O2 . Moreover, cryptic growth can be partially inhibited by the antioxidant ascorbate. It is concluded that cryptic growth represents a wall-stiffening reaction mediated by peroxidase-catalyzed, H2 O2 -dependent cross-linking of phenolic residues of wall polymers. The experimental demonstration of a wall-stiffening reaction in a rapidly growing organ supports the concept that irreversible cell elongation (growth) is caused by an interplay of two chemorheological reactions, a turgor-dependent wall-loosening reaction and a separate wall-stiffening reaction which fixes the viscoelastically extended wall structure through oxidative cross-linking and thus conferring irreversibility to wall extension. 相似文献
17.
Hans Thordal-Christensen Ziguo Zhang Yangdou Wei David B. Collinge 《The Plant journal : for cell and molecular biology》1997,11(6):1187-1194
Active oxygen species (AOS) are believed to have important roles in plants in general and in plant—pathogen interactions in particular. They are believed to be involved in signal transduction, cell wall reinforcement, hypersensitive response (HR) and phytoalexin production, and to have direct antimicrobial effects. Since current methods are inadequate for localizing AOS in intact plant tissue, most studies have been conducted using cell suspension culture/elicitors systems. 3,3-diaminobenzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H2 O2 in the presence of peroxidase, and it was found that, by allowing the leaf to take up this substrate, in-vivo and in-situ detection of H2 O2 can be made at subcellular levels. This method was successfully used to detect H2 O2 in developing papillae and surrounding haloes (cell wall appositions) and whole cells of barley leaves interacting with the powdery mildew fungus. Thus, H2 O2 can be detected in the epidermal cell wall subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium from 15 h. The earliest time point for observation of H2 O2 in relation to epidermal cells undergoing HR is 15 h after inoculation, first appearing in the zones of attachment to the mesophyll cells underneath, and eventually in the entire epidermal cell. Furthermore, it was observed that proteins in papillae and HR cells are cross-linked, a process believed to be fuelled by H2 O2 . This cross-linking reinforces the apposition, presumably assisting the arrest of the pathogen. 相似文献
18.
Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv 总被引:1,自引:0,他引:1
Protein phosphorylation-dephosphorylation is the principal mechanism for translation of external signals into cellular responses. Eukaryotic-like serine/threonine kinases have been reported to play important roles in bacterial development and/or virulence. The PknI protein is one of the 11 eukaryotic-like serine/threonine kinases in Mycobacterium tuberculosis H37Rv. From the bioinformatic studies, PknI protein has been shown to have an N-terminal cytoplasmic domain followed by a transmembrane region and an extracellular C-terminus suggestive of a sensor molecule. In this study, we have cloned, overexpressed, and characterized the entire coding region and the cytoplasmic domain of PknI as a fusion protein with an N-terminal histidine tag, and used immobilized metal affinity chromatography for purification of recombinant proteins. The purified recombinant proteins were found to be functionally active through in vitro phosphorylation assay and phosphoamino acid analysis. In vitro kinase assay of both proteins revealed that PknI is capable of autophosphorylation and showed manganese-dependent activity. Phosphoamino acid analysis indicated phosphorylation at serine and threonine residues. Southern blot analysis with genomic DNA highlighted the conserved nature of pknI among the various mycobacterial species. In silico analysis revealed a close homology of PknI to Stk1 from Streptococcus agalactiae, shown to have a role in virulence and cell segregation of the organism. 相似文献
19.
Annick Bernalier Marielle Lelait Violaine Rochet Jean-Philippe Grivet Glenn R. Gibson Michelle Durand 《FEMS microbiology ecology》1996,19(3):193-202
Abstract: The purpose of the study was to define the potential for reductive acetogenesis of colonic microflora from six non-methane- and four methane-excreting human subjects in relation to numbers of the different H2 -utilizing microorganisms. Faecal bacterial suspensions were incubated in the presence of NaH13 CO3 and under a gas phase composed of either 100% N2 (control) or 80% H2 –20% N2 . The effects of a specific methanogenesis inhibitor or of sulfate supplementation were also determined. Quantitative nuclear magnetic resonance showed the presence of both single- and double-labelled acetate in all incubations under hydrogen. H2 /CO2 -acetogenesis appears to be a quantitatively important activity only in the presence of very low numbers of methanogens. Inhibition of methanogenesis induced a large increase in 13 CO2 incorporation into acetate in CH4 -producing samples. These results showed that methanogens can efficiently outcompete acetogens in human colonic contents. In contrast, no clear-cut competition for H2 between acetogenesis and dissimilatory sulfate-reduction could be demonstrated. A slight reduction of the acetogenic activity was only observed at the highest sulfate addition (100 mM). 相似文献
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Ability of different adjuvants to promote cell mediated immune responses towards 30 kDa secretory protein of Mycobacterium tuberculosis H37Ra was monitored by assessing the lymphocyte proliferation and IgG1/IgG2a subclass profile in mouse model. Six formulations, viz. poly lactide-co-glycolide (PLG) microspheres, dimethyldioctadecyl ammoniumbromide (DDA), liposomes, liposomes containing monophosphoryl lipid A and coated with alum (L-LIPA-AL) or without alum (L-LIPA) were evaluated in comparison to standard Freund's incomplete adjuvant (FIA). Two adjuvant formulations of 30kDa-L-LIPA-AL and 30kDa-PLG showed maximum reactivity on VIIIth week post immunization (p.im) in terms of lymphoproliferation w.r.t. other adjuvant formulations. Both the vaccine formulations also exhibited a Th1 shift in terms of higher IgG2a response over IgGI. Flowcytometric analysis in the mesenteric lymph nodes (MLNs) of immunized animals revealed the capacity of 30kDa-PLG and 30kDa-L-LIPA-AL to activate T cell subsets like CD4 and CD8 T cells. The upregulation of B7 costimulatory molecules (B7-1 & B7-2) after immunization further proved the ability of the two vaccine formulations to activate antigen presenting cells. The immunostimulatory nature of the two formulations was also reflected in their capacity to reduce the bacilli load from the lungs of the experimentally infected mice. This study demonstrates PLG and L-LIPA-AL as potent adjuvants and their bioacceptibility and nontoxic nature make them suitable candidates for future subunit vaccine development against tuberculosis. 相似文献