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991.
Natarajan V. Vepa S. Shamlal R. Al-Hassani M. Ramasarma T. Ravishankar H.N. Scribner W.M. 《Molecular and cellular biochemistry》1998,183(1-2):113-124
Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC and , the major isotypes of PKC in BPAECs, by TPA ( 100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation. 相似文献
992.
Celler Jakub W. Luo Xinmei Böhmer Frank D. 《Molecular and cellular biochemistry》1998,178(1-2):157-162
The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 µg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or non-degenerate quantitative RT-PCR. 相似文献
993.
K. Oami 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1998,182(4):403-409
The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior
end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the
depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration
applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing
one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external
Ca2+ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing
external K+ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation,
and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear
relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase
during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of
Ca2+ channels, and the hyperpolarizing quinine receptor potential by an activation of K+ channels.
Accepted: 3 October 1997 相似文献
994.
DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (~55 pmol/min/mg protein for tyrosine; ~40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of ~10 M for either amino acid. The Km for either amino acid was about 1 M (medium concentration). At tyrosine concentrations above 30 M, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (300 M). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the Km for either amino acid is 20–30 M; maximal synthesis occurred at 120–140 M. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 M), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 M). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine. 相似文献
995.
An eightfold auxotrophic strain of Bacillus subtilis was constructed. It was about equally well transformable for all markers. When this strain was used as recipient in transformation, single marker transformation frequencies of 0.5–2.0% were obtained. The markers were located relatively to each other using marker frequency analysis. Two UV-sensitive derivatives, equally well transformable as the parental multiple auxotroph, were isolated. One was highly sensitive to UV irradiation, was host cell reactivation-negative and did not show DNA breakdown or recovery of DNA synthesis after exposure to UV. Using UV-inactivated transforming DNA, this strain's transformability was strongly reduced as compared with both the UV-resistant parental strain and the other, moderately UV-sensitive, strain. 相似文献
996.
Ultraviolet inactivation and excision-repair in Bacillus subtilis. II. Differential inactivation and differential repair of transforming markers 总被引:6,自引:0,他引:6
Ultraviolet inactivation of transforming Bacillus subtilis markers was studied with the aid of an eightfold auxotrophic recipient and its excision-repair-deficient derivative. The results allow the following conclusions. (i) Wild-type B. subtilis cells are able to repair approx. 80% of the UV-induced lesions causing inactivation of transforming activity in UV-sensitive recipients; (ii) Saturating amounts of donor DNA increase the apparent marker sensitivities. This phenomenon is most pronounced in transformation of UV-sensitive recipients; (iii) various markers are inactivated to different degrees, both when assayed on the wild-type as well as on the UV-sensitive strain; (iv) Various markers are repaired to different degrees in the wild-type recipient. 相似文献
997.
Studies on the comparative utilization of tyrosine for protein and alkaloid biosynthesis indicate that this amino acid is incorporated into peyote alkaloids at three times the rate at which it is incorporated into protein. In addition, the biosynthetic pathway for tyrosine formation appears to be compartmented into two channels; one supplying the needs for alkaloid biosynthesis and the other providing tyrosine for protein biosynthesis. The latter compartment is possibly under a negative feedback control mechanism. 相似文献
998.
Miwa Sugiura Tania Tibiletti Itsuki Takachi Yuya Hara Shin Kanawaku Julien Sellés Alain Boussac 《BBA》2018,1859(12):1259-1273
In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824–6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV–visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking TyrZ and the halide. The V185 contributes to the stabilization of S2 into the low spin (LS), S?=?1/2, configuration. Indeed, in the V185T mutant a high proportion of S2 exhibits a high spin (HS), S?=?5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S1TyrZ?→?S2HSTyrZ transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S2LSTyrZ?→?S3TyrZ transition was observed and we propose that it occurs in the S2LSTyrZ?→?S2HSTyrZ intermediate step before the S2HSTyrZ?→?S3TyrZ transition occurs. The dramatic slowdown of the S3TyrZ?→?S0TyrZ transition in the V185T mutant does not originate from a structural modification of the Mn4CaO5 cluster since the spin S?=?3?S3 EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein. 相似文献
999.
1000.
Michio Hiroshima Chan-gi Pack Kazunari Kaizu Koichi Takahashi Masahiro Ueda Yasushi Sako 《Journal of molecular biology》2018,430(9):1386-1401
Cell signaling depends on spatiotemporally regulated molecular interactions. Although the movements of signaling proteins have been analyzed with various technologies, how spatial dynamics influence the molecular interactions that transduce signals is unclear. Here, we developed a single-molecule method to analyze the spatiotemporal coupling between motility, clustering, and signaling. The analysis was performed with the epidermal growth factor receptor (EGFR), which triggers signaling through its dimerization and phosphorylation after association with EGF. Our results show that the few EGFRs isolated in membrane subdomains were released by an EGF-dependent increase in their diffusion area, facilitating molecular associations and producing immobile clusters. Using a two-color single-molecule analysis, we found that the EGF-induced state transition alters the properties of the immobile clusters, allowing them to interact for extended periods with the cytoplasmic protein, GRB2. Our study reveals a novel correlation between this molecular interaction and its mesoscale dynamics, providing the initial signaling node. 相似文献