Gamma-glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the rate-limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4-5 times, due to the amplification of the gene encoding the catalytic subunit of GCS. These mutant cells contained a wild-type steady-state level of GSH and, after depletion, synthesized GSH at the same rate as wild-type cells because their rate of endogenous production of cysteine was limiting. An exogenous supply of cysteine expanded the pool of GSH in mutant cells by 80% but did not increase that of wild-type cells, and, in GSH-depleted cells, increased the rate of GSH biosynthesis by eight and 35-times in wild-type and mutant cells, respectively. These experiments indicated that GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cisplatin or nitrogen mustard. 相似文献
The HsdS and HsdM polypeptides of the type IC restriction enzyme EcoR124 have been purified independently and used in a set of gel retardation experiments to determine the minimum requirements for sequence-specific recognition of DNA by this enzyme. The HsdS polypeptide alone is able to bind to DNA in a sequence-specific manner. In addition, whilst the presence of the HsdM polypeptide gives rise to a stimulation of DNA binding by the HsdS subunit it is not clear whether, under the conditions of the experiments reported here, the HsdS subunit maintains the same interactions with the HsdM subunits observed in the absence of DNA. 相似文献
Abstract: Early in the course of productive Herpes simplex virus type 1 (HSV-1) infection of PC12 cells, activities of both acetylcholinesterase (AChE) and choline acetyltransferase (CAT) fell. Studies using metabolic inhibitors and a temperature-sensitive mutant of the virus suggested that the decline in activities of both enzymes was associated with events occurring early in the replicative cycle related to expression of the immediate-early (α) group of viral polypeptides. HSV-1 gene products thus may alter specialized cell functions well before the production of viral progeny and initiation of cell lysis. The early clinical manifestations of nervous system viral infection may reflect focal metabolic disturbance rather than, or in addition to, simple cell death. 相似文献
We have tested the hypothesis that cell lineage restriction boundaries define the borders between cytoarchitectonic areas in the cerebral cortex. Clonally related cells were identified using a retroviral marking technique, and the dispersion of neuronal clones was examined with respect to the transitions between cortical areas. We chose to study the hippocampal formation because we found that clones of hippocampal neurons, unlike those in neocortex, are compact and readily identifiable in the adult and that transitions between areas in the hippocampus are sharp relative to the spread of a typical clone. We conclude, contrary to the hypothesis, that clones of neurons transgress the boundaries between areas in the hippocampal formation, that border-crossing clones are observed as frequently as would be expected if clones spread freely over the hippocampus with no constraint imposed by area borders, and that different types of pyramidal neurons, characteristic of different areas, may appear to a single clone. different areas, may appear in a single clone. 相似文献
Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes. 相似文献
Some P-450 systems, notably aromatase and 14-demethylase catalyse not only the hydroxylate reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C---C bond cleavage process. All these reactions occur at the same active site. A somewhat analogous situation is noted with 17-hydroxylase-17,20-lyase that participates in hydroxylation as well as C---C bond cleavage process. The C---C bond cleavage reactions catalysed by the above enzymes conform to the general equation:
It is argued that all three types of reaction catalyzed by these enzymes may be viewed as variations on a common theme. In P-450 dependent hydroxylation the initially formed FeIII---O---O. species is converted into FeIII---O---OH and the heterolysis of the oxygen—oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example FeV = O ↔ (+.)FeIV = O ↔ FeIV---O.. One of these, FeIV---O. behaves like an alkoxyl radical and participates in hydrogen abstraction from C---H bond to produce FeIV---OH and carbon radical. The latter is then quenched by the delivery of hydroxyl radical from FeIV---OH. The latter species may thus be regarded as a carrier of hydroxyl radical. We have proposed that the C---C bond cleavage reaction occurs through the participation of the FeIII---O---OH species that is trapped by the electrophilic property of the carbonyl compound giving a peroxide adduct that fragments to produce an acyl—carbon cleavage. Scientific developments leading up to this conclusion are considered. In the first author's views,
“The study of mechanisms is not a scientific but a cultural activity. Mechanisms do not aim at an absolute truth but are intended to be a “running” commentary on the status of knowledge in a field. As the structural knowledge in a field advances Mechanisms evolve to take note of the new findings. Just as a constructive “running” commentary provides the stimulus for higher standards of performance, so Mechanisms call for better and firmer structural information from their practitioners”. 相似文献
Fractional anisotropy (FA) is an effective marker of motor outcome at the chronic stage of stroke yet proves to be less efficient at early time points. This study aims to determine which diffusion metric in which location is the best marker of long-term stroke outcome after thrombolysis with diffusion tensor imaging (DTI) at 24 hours post-stroke. Twenty-eight thrombolyzed patients underwent DTI at 24 hours post-stroke onset. Ipsilesional and contralesional FA, mean (MD), axial (AD), and radial (RD) diffusivities values were calculated in different Regions-of-Interest (ROIs): (1) the white matter underlying the precentral gyrus (M1), (2) the corona radiata (CoRad), (3) the posterior limb of the internal capsule (PLIC) and (4) the cerebral peduncles (CP). NIHSS scores were acquired at admission, day 1, and day 7; modified Rankin Scores (mRS) at 3 months. Significant decreases were found in FA, MD, and AD of the ipsilesional CoRad and M1. MD and AD were also significantly lower in the PLIC. The ratio of ipsi and contralesional AD of the CoRad (CoRad-rAD) was the strongest diffusion parameter correlated with motor NIHSS scores on day 7 and with the mRS at 3 months. A Receiver-Operator Curve analysis yielded a model for the CoRad-rAD to predict good outcome based on upper limb NIHSS motor scores and mRS with high specificity and sensitivity. FA values were not correlated with clinical outcome. In conclusion, axial diffusivity of the CoRad from clinical DTI at 24 hours post-stroke is the most appropriate diffusion metric for quantifying stroke damage to predict outcome, suggesting the importance of early axonal damage. 相似文献