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51.
In all organisms the fourth catalytic step of the pyrimidine biosynthesis is driven by the flavoenzyme dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11). Cytosolic DHODH of the established model organism Saccharomyces cerevisiae catalyses the oxidation of dihydroorotate to orotate and the reduction of fumarate to succinate. Here, we investigate the structure and mechanism of DHODH from S. cerevisiae and show that the recombinant ScDHODH exists as a homodimeric enzyme in vitro. Inhibition of ScDHODH by the reaction product was observed and kinetic studies disclosed affinity for orotate (K(ic)=7.7 microM; K(ic) is the competitive inhibition constant). The binding constant for orotate was measured through comparison of UV-visible spectra of the bound and unbound recombinant enzyme. The midpoint reduction potential of DHODH-bound flavine mononucleotide determined from analysis of spectral changes was -242 mV (vs. NHE) under anaerobic conditions. A search for alternative electron acceptors revealed that homologues such as mesaconate can be used as electron acceptors. 相似文献
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Valerie Vreeland Suzanne R. Morse Robert H. Robichaux Kathleen L. Miller Sui-Sheng T. Hua Watson M. Laetsch 《Planta》1989,177(4):435-446
Carbohydrate-hybridization probes (Vreeland and Laetsch, 1989, Planta (177, 423–434) were used to localize the homogalacturonan (pectate) component of pectins in the cell walls of leaves and soybean root nodules. Leaves of two species of the dicotyledon Dubautia were compared; these species contain much pectin but differ in their tissue water relations with respect to their cell-wall properties. Maturation of the primary cell walls in nodules was studied in the Bradyrhizobium japonicum-Glycine max symbiosis. Probe labelling was based on the divalent-cation-mediated association between pectate in tissue sections and fluorescein-conjugated pectate fragments. Pectate was also labelled by mixed-dimer formation with fluorescent polyguluronate derived from alginate. The specificity of the probe for unesterified polygalacturonate was indicated by increased cell-wall labelling after chemical or enzymatic deesterification of tissue sections, in contrast to elimination of labelling by chemical esterification. Postfixation of tissue sections improved retention of soluble pectate. Pectate differences were found in the leaves among cell types, in degree of esterification, and between plant species. The cell walls of soybean nodules were strongly labelled by the pectate probe in nodules one week and three weeks after infection. Pectate was more highly esterified in the central infected zone than in the surrouding cortex. Within the infected zone, walls of uninfected cells and infected cells were similarly labelled by the pectate probe. The results indicate that the pectate molecular probe provides detailed information on pectate distribution at the cellular level for investigations of cell-wall structure, development and physiology.Abbreviations EDTA
ethylenedinitrilotetraacetic acid (ethylenediaminetetraacetic acid)
- NMR
nuclear magnetic resonance spectroscopy
- TTB
1,3,5-triazido-2,4,6-trinitrobenene 相似文献
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Janice A. Williams Simon Y. Long Xiankun Zeng Kathleen Kuehl April M. Babka Neil M. Davis Jun Liu John C. Trefry Sharon Daye Paul R. Facemire Patrick L. Iversen Sina Bavari Margaret L. Pitt Farooq Nasar 《PLoS neglected tropical diseases》2022,16(5)
Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61–68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure. 相似文献
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The stalk of Colacium mucronatum Bourr. & Chad. is composed primarily of a cylindrical shaft with a lightly staining inner core and diffuse peripheral cortex. The shaft and cortex arise from a ring-shaped region around the canal opening whereas the core appears continuous with the canal which may be associated with initial cell attachment. All parts of the stalk, as well as the lining and contents of the reservoir, canal and flagellum exhibit stain reactions associated with neutral or mildly acidic carbohydrate with widely spaced anionic groups in low concentration. 相似文献
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Abstract An isoprotein of enolase from the yeast Saccharomyces cerevisiae was reported to be a heat shock protein. The possible role of the C. albicans enolase as a heat shock protein was therefore investigated. The de novo synthesis of C. albicans enolase protein and mRNA did not increase during heat stress, but remained constitutively expressed. Amino acid similarity to the heat shock proteins suggests that although the C. albicans enolase is not a classical heat shock protein, it may be a memberof a group of constitutively expressed, structurally related proteins, the heat shock cognate proteins. 相似文献
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Unilateral amygdala electrodes were implanted in male Sprague-Dawley rats stimulated once daily with a 200 μamp pulse of 500 millisecond duration to produce kindling. Forty-six percent (12 of 26) of the animals that eventually developed after-discharges demonstrated rhythmic oscillations in after-discharge duration. The presence or absence of generalized bilateral clonic seizures also showed rhythmic oscillations in close association with after-discharge duration. It is suggested that during kindling some animals, independent of electrode placement, develop rhythmic oscillations in excitability of the amygdala. This model may represent a means of experimentally eliciting or uncovering neuronal substrates which show regular alterations in excitability and may be relevant to the oscillations in mood and behavior observed in the affective disorders. 相似文献
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