Resistance to HIV-1 integrase (IN) inhibitor raltegravir (RAL), is encoded by mutations in the IN region of the pol gene. The emergence of the N155H mutation was replaced by a pattern including the Y143R/C/H mutations in three patients with anti-HIV treatment failure. Cloning analysis of the IN gene showed an independent selection of the mutations at loci 155 and 143. Characterization of the phenotypic evolution showed that the switch from N155H to Y143C/R was linked to an increase in resistance to RAL. Wild-type (WT) IN and IN with mutations Y143C or Y143R were assayed in vitro in 3′end-processing, strand transfer and concerted integration assays. Activities of mutants were moderately impaired for 3′end-processing and severely affected for strand transfer. Concerted integration assay demonstrated a decrease in mutant activities using an uncleaved substrate. With 3′end-processing assay, IC50 were 0.4 µM, 0.9 µM (FC = 2.25) and 1.2 µM (FC = 3) for WT, IN Y143C and IN Y143R, respectively. An FC of 2 was observed only for IN Y143R in the strand transfer assay. In concerted integration, integrases were less sensitive to RAL than in ST or 3′P but mutants were more resistant to RAL than WT. 相似文献
A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRYF109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.This paper is dedicated by G. S. to Professor Ulrich Wolf on the occasion of his 60th birthday 相似文献
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
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