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251.
The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   
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Endoreplication Controls Cell Fate Maintenance   总被引:1,自引:0,他引:1  
Cell-fate specification is typically thought to precede and determine cell-cycle regulation during differentiation. Here we show that endoreplication, also known as endoreduplication, a specialized cell-cycle variant often associated with cell differentiation but also frequently occurring in malignant cells, plays a role in maintaining cell fate. For our study we have used Arabidopsis trichomes as a model system and have manipulated endoreplication levels via mutants of cell-cycle regulators and overexpression of cell-cycle inhibitors under a trichome-specific promoter. Strikingly, a reduction of endoreplication resulted in reduced trichome numbers and caused trichomes to lose their identity. Live observations of young Arabidopsis leaves revealed that dedifferentiating trichomes re-entered mitosis and were re-integrated into the epidermal pavement-cell layer, acquiring the typical characteristics of the surrounding epidermal cells. Conversely, when we promoted endoreplication in glabrous patterning mutants, trichome fate could be restored, demonstrating that endoreplication is an important determinant of cell identity. Our data lead to a new model of cell-fate control and tissue integrity during development by revealing a cell-fate quality control system at the tissue level.  相似文献   
255.
The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.  相似文献   
256.
Although Nitrosomonas europaea lacks measurable alpha-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the sucA gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of ammonia monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.  相似文献   
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The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H(+)-dependent F(0)F(1) type, one Na(+)-dependent V type).  相似文献   
259.
We determined whether distinct subclasses of dendritic cells (DC) could polarize cytokine production and regulate the pattern of xenograft rejection. C57BL/6 recipients, transplanted with Lewis rat hearts, exhibited a predominantly CD11c(+)CD8alpha(+) splenic DC population and an intragraft cytokine profile characteristic of a Th1-dominant response. In contrast, BALB/c recipients of Lewis rat heart xenografts displayed a predominantly CD11c(+)CD8alpha(-) splenic DC population and IL-4 intragraft expression characteristic of a Th2 response. In addition, the CD11c(+)IL-12(+) splenic DC population in C57BL/6 recipients was significantly higher than that in BALB/c recipients. Adoptive transfer of syngeneic CD8alpha(-) bone marrow-derived DC shifted a Th1-dominant, slow cell-mediated rejection to a Th2-dominant, aggressive acute vascular rejection (AVR) in C57BL/6 mice. This was associated with a cytokine shift from Th1 to Th2 in these mice. In contrast, transfer of CD8alpha(+) bone marrow-derived DC shifted AVR to cell-mediated rejection in BALB/c mice and significantly prolonged graft survival time from 6.0 +/- 0.6 days to 14.2 +/- 0.8 days. CD8alpha(+) DC transfer rendered BALB/c mice susceptible to cyclosporine therapy, thereby facilitating long-term graft survival. Furthermore, CD8alpha(+) DC transfer in IL-12-deficient mice reconstituted IL-12 expression, induced Th1 response, and attenuated AVR. Our data suggest that the pattern of acute xenogeneic rejection can be regulated by distinct DC subsets.  相似文献   
260.
We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium “Pseudomonas butanovora.” Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ~20 nmol mg protein−1 of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein−1. Oxidation of similar amounts of each DCE isomer (~20 nmol mg protein−1) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown “P. butanovora” did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.  相似文献   
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