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1.
Essentially chlorophyll-free mitochondria were isolated from green leaves of spinach (Spinacia oleracea L. cv. Viking II). Uncoupled oxidation of exogenous NADPH (1 mM) to oxygen had an optimum at pH 6.0, and activity was relatively low at pH 7.0, even in the presence of 1 mM-CaCl2. There was a proportional increase in the apparent Km for NADPH with decreasing H+ concentrations, suggesting that NADPH protonated on the 2'-phosphate group was the true substrate. Exogenous NADH was oxidized by oxygen with an optimum at pH 6.9. Under low-cation conditions, EGTA or EDTA (both 1 mM) had no effect on the Vmax. of NADH oxidation, although the removal of bivalent cations from the membrane surface by the chelators could be observed by use of 9-aminoacridine fluorescence. In contrast, under high-cation conditions, chelators lowered the Vmax. by about 50%, probably due to a better approach of the negatively charged chelators to the negative membrane surface than under low-cation conditions. In a low-cation medium, the Vmax. of NADH oxidation was increased by about 50% by the addition of cations. This was caused by a lowering of the size of the negative surface potential through charge screening. In contrast with other cations, La3+ inhibited NADH oxidation, possibly through binding to lipids essential for NADH oxidation. The apparent Km for NADH varied 6-fold in response to changes in the size of the surface potential, suggesting that the approach of the negatively charged NADH to the active site is hampered by the negative surface potential. The results demonstrate that the spinach leaf cell can regulate the mitochondrial NAD(P)H oxidation through several mechanisms: the pH; the cation concentration in general; and the concentration of Ca2+ in particular. The results also emphasize the importance of electrostatic considerations when investigating the kinetic behaviour of membrane-bound enzymes.  相似文献   
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Extremophiles - We accessed the culturable mycobiota present in marine sediments at different depths in Antarctica Ocean. Acremonium fusidioides, Penicillium allii-sativi, Penicillium chrysogenum,...  相似文献   
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Isolated sheaths of Leptothrix discophora SP-6 (ATCC 51168) were tested for susceptibility to degradation by a variety of chemical denaturants and lytic enzymes and found to be resistant to many reagents and enzyme treatments. However, disulfide bond-reducing agents such as dithiothreitol (DTT), beta-mercaptoethanol, sodium cyanide, and sodium sulfite degraded the sheath, especially at elevated pH (pH 9) and temperature (50 degrees C). DTT and beta-mercaptoethanol caused more rapid degradation of the sheath than cyanide or sulfite. Treatment of the sheath with 1 N NaOH resulted in rapid breakdown, while treatment with 1 N HCl resulted in slow but significant hydrolysis. Transmission electron microscopy showed that the 6.5-nm fibrils previously shown to be an integral structural element of the sheath fabric (D. Emerson and W. C. Ghiorse, J. Bacteriol. 175:7808-7818, 1993) were progressively dissociated into random masses during DTT-induced degradation. Quantitation of disulfide bonds with DTT showed that the sheaths contained approximately 2.2 mumol of disulfides per mg of sheath protein. Reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) showed that sheaths also contained approximately 0.8 mumol of free sulfhydryls per mg of protein. A sulfhydryl-specific fluorescent probe (fluorescein 5-maleimide) showed that the free sulfhydryls in sheathed cell filaments were evenly distributed throughout the sheath. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of [14C]iodoacetamide-labeled sheaths and DTT-dissociated sheath fibril suspensions showed that the majority of 14C-labeled sulfhydryls in the sheaths did not enter the gel. However, low-molecular-mass silver-staining bands (14 to 45 kDa) did appear in the gels after iodoacetic acid or iodoacetamide alkylation of the dissociated fibrils. These bands did not stain with Coomassie blue. Their migration in gels was slightly affected by digestion with pronase. The fibrils contained 20 to 25% protein. These results confirm that the sheath fibrils consist of high molecular-weight heteropolysaccharide-protein complexes. We hypothesize that proteins in the fibril complexes provide interfibril cross-linking to maintain the structural integrity of the sheath.  相似文献   
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Structures resembling Metallogenium spp. were observed in agar and in liquid cultures of a Mn-oxidizing basidiomycetous fungus only when Mn2+ was oxidized. Fungal viability was necessary for formation of the structures; Mn2+ concentration and the presence or absence of agar in the medium were important factors determining their morphology. Slide cultures revealed no identifiable cells in any stage of development. Fluorescent dyes that stained nucleic acids and polysaccharides in the fungal hyphae did not stain the Metallogenium-like structures. Likewise, Rhodamine 123, a fluorescent probe for membrane potential, stained fungal mitochondria, but did not stain the structures. Thin sections through the structures showed no biological membranes or other cellular features. Only the characteristic ultrastructure of biological Mn oxides were observed in serial thin sections. In agar, unfixed structures disappeared permanently during reduction of Mn oxides with hydroxylamine. Glutaraldehyde fixation stabilized these structures. Fixed structures lost most of their original phase density during reduction with hydroxylamine, but continuous microscopic observations showed that their phase density could be restored by staining with Coomassie blue. Structures that formed in liquid medium did not require stabilization with glutaraldehyde during reduction of Mn oxides. They, too, lost their original phase density during reduction with hydroxylamine; phase density could be restored by staining with cationic colloidal iron or Coomassie blue. The results suggest that the Metallogenium-like structures were formed as a result of Mn oxidation associated with exopolymers produced by the fungus.Non-standard abbreviations HEPES (N-hydroxyethylpiperazine-N-2-ethane sulfonic acid) - DAPI (4,6-diamidino-2-phenylindole) - PIPES (piperazine-N,N-bis[2-ethane sulfonic acid])  相似文献   
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The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.  相似文献   
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Sometimes, valuable lessons from history are forgotten, remain unknown, or worse, are ignored. This article reminds us of the pioneering work of Angelo Celli at the end of the 19th century, who demonstrated that people could be protected from malaria by screening their homes against mosquitoes. Since then, public health scientists have continued to show that simple changes in house design have the potential for protecting people against this life-threatening disease. Yet today, this type of intervention remains virtually ignored. The literature reviewed here demonstrates the enormous potential of these methods to reduce malaria, in the hope that it will stimulate scientific debate and further research.  相似文献   
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