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41.
Impedance aggregometry was used to evaluate the potency of anti-platelet agents on Platelet Activating Factor (PAF)--induced platelet aggregation in citrated human whole blood. Drugs were tested for ability to inhibit maximum aggregation to PAF. Dose response curves were obtained and the concentration of drug producing 50% inhibition of maximum aggregation (ED50) determined. ED50's (microM) for specific PAF antagonists WEB 2086, Ro 19-3704, FR-900452, BN 52021, L-652,731, CV 3988, WEB 2118 and 48740 RP are: 0.39, 2.4, 4.7, 19.5, 21.0, 5.32, 161.0, 924.0, respectively. ED50's for non-specific PAF antagonists, diltiazem, propranolol, ketotifen, procaine HCL, and lidocaine HCL are: 38.0, 56.0, 250.0, 513.0 and 768.0, respectively. Ibuprofen was inactive at 2300 microM. Results are consistent with concept that there are specific receptors on platelets mediating PAF-induced aggregation in whole blood. Aggregation is inhibited potently by specific and competitive PAF receptor antagonists. Whole blood aggregometry may be a valid method for predicting in vivo activity of PAF antagonists. 相似文献
42.
Vidadi M. Yusibov Pak Chun II Viacheslav M. Andrianov Eleonora S. Piruzian 《Plant molecular biology》1991,17(4):825-836
The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress. 相似文献
43.
Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent- and detergent-treated microbes 总被引:1,自引:0,他引:1
Err-Cheng Chan Jimmy Kuo Hsiou-Ping Lin Duen-Gang Mou 《Applied microbiology and biotechnology》1991,34(6):772-777
Summary A wild-type strain of Cryptococcus neoformans and Pseudomonas aeruginosa were used to convert n-pentadecane to the corresponding dioic acid, tridecane 1,13-dicarboxylic acid (DC-15). Altering the cell permeability by treating C. neoformans with 1% (v/v) toluene or 7% (v/v) Triton X-100 stimulated production of DC-15 by 1.5-fold and fourfold, respectively. Furthermore, DC-15 productivity was increased from 2.5 mg/l per hour to 18 or 30 mg/l per hour, respectively. If 10% (v/v) hexane was used to treat the yeast culture, stimulation of DC-15 production could reach 200% and more viable cells remained compared to the toluene-treated culture. Data from the organic solvent treatment experiment indicated that the solvent with a higher polarity showed a more adverse effect on DC-15 production. P. aeruginosa was vulnerable to most organic solvents; however, Tween 80 could greatly stimulate the conversion of n-pentadecane to DC-15. Although organic solvents and non-ionic detergents could enhance DC-15 formation by microbial conversion, it was inhibited by elevated levels of DC-15.Offprint requests to: E.-C. Chan 相似文献
44.
L V Krasheninnikova G V Rassadina S N Kirsanova L M Khromova V M Iusibov I r Pak Chun V M Andrianov E S Piruzian 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1991,(11):17-20
The genetic constructions based on integrative vector pGV3850 were used to introduce bacterial genes xyl and T-cyt into potato cells. The transformation was carried out using the leaf-disc method with modifications. A special system for obtaining regenerants from explants of potato in vitro plants or calli has been designed that permitted the selection of transgenic shoots. The presence of the genes in potato genome has been proved by testing the NPTII and glucoisomerase activities. The transgenic plants expressing T-cyt gene differed from the wild type in sharp decrease of the apical dominance. 相似文献
45.
P F Chen Y L Marcel C Y Yang A M Gotto R W Milne J T Sparrow L Chan 《European journal of biochemistry》1988,175(1):111-118
Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined. 相似文献
46.
We measured the effect of repeated haemorrhagic stress, performed on four consecutive days in conscious adult sheep, on the plasma concentrations of cortisol and ACTH-related peptides to determine whether the pituitary-adrenal response was altered by stress repetition. Peptides from the C-terminus of the ACTH pro-hormone was measured by beta-endorphin RIA. Glycopeptides derived from the N-terminus of the ACTH pro-hormone were measured by tau 3-MSH RIA. The immunoreactive tau 3-MSH in sheep plasma was found to have an apparent molecular weight of approximately 10,000 by gel chromatography through Sephadex G-75, which is similar to the size of the major circulating form of pro-tau-MSH found in human and rat plasma. Daily haemorrhage consistently elevated plasma concentrations of cortisol and pro-tau-MSH. There was no significant difference in the daily responses of either cortisol or pro-tau-MSH when considered individually. However, there was a significant change over the four days in the relationship between the cortisol and pro-tau-MSH responses, as judged by analysis of variance of the difference in daily z-scores of cortisol and pro-tau-MSH. This trend indicated a relative increase in the secretion of pro-tau-MSH from the pituitary compared to the cortisol response, and suggested that repeated exposure to stressful stimuli may alter the pituitary-adrenal-axis. 相似文献
47.
When the low-potential metal centers of cytochrome c oxidase are reduced, the enzyme undergoes a conformational transition that shifts the fluorescence maximum of the emitting tryptophan residues from 329 to 345 nm. At pH 7.4, the change in this tryptophan fluorescence intensity is a nonlinear function of the electron equivalents added to the cyanide-inhibited enzyme. This nonlinear behavior is a result of the difference in redox potential between cytochrome a and CuA, which, at equilibrium, favors electron occupancy at cytochrome a. Studies on the cyanide-inhibited enzyme suggest that the conformational change is associated with reduction of CuA [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. In this work we present tryptophan fluorescence data for the cyanide-inhibited enzyme at pH 8.9. Because of the pH dependence of the midpoint potential of cytochrome a in this form of the enzyme, the two low-potential centers become virtually isopotential at pH 8.9. The results obtained confirm our earlier conclusion that the observed conformational change is linked to the reduction of CuA only, rather than to the redox activity of both low-potential metal centers. We find that, in partially reduced cyanide-inhibited oxidase, raising the pH from 7.4 to 8.9 results in an intensification and red shift of the enzyme's tryptophan emission as the electron occupancy redistributes from cytochrome a to CuA. Moreover, when the fluorescence change is plotted as a function of the number of electrons added to the enzyme at pH 8.9, the data fit the nearly linear function expected for a conformational change triggered by reduction of CuA exclusively. 相似文献
48.
M?ssbauer spectra of 57Fe-enriched NADH-reduced yeast cytochrome c oxidase reveal two quadrupole doublets of unequal intensity; one (approximately 33%) is typical of high-spin ferrous heme with histidine coordination and is assigned to heme a3, while the other (approximately 67%) is typical of low-spin heme with two nitrogeneous axial ligands as expected from heme a. The excess intensity (approximately 17%) of the low-spin doublet must therefore be assigned to heme a3 in a modified environment. The M?ssbauer spectra of the same sample exposed to CO show that 50% of the heme iron forms a CO adduct, consistent with heme a3 being inhibited by CO. While low-spin hem a has the same M?ssbauer parameters as in the reduced sample, its intensity has dropped to 35%. A distinctly new high-spin species (approximately 15%) is observed and assigned to heme a in a modified environment. The comparable size of the unexpected high-spin heme a fraction in the CO adduct and the low-spin heme a3 fraction in the reduced enzyme suggest that they arise from the same material. This material is likely to be the inactive fraction that has been found in all preparations of resting yeast cytochrome c oxidase (Siedow, J.N., Miller, S., and Palmer, G. (1981) J. Bioenerg. Biomembr. 14, 171-179). The kinetics of CO recombination following photolysis of the CO complex further confirms the coexistence of two distinct fractions associated with active and inactive protein. The majority (approximately 74%), presumably active protein, recombines exponentially from 160 to 270 K following an Arrhenius law. The large activation enthalpy, delta H approximately 35 kJ/mol, is comparable to that found in the beef heart enzyme, suggesting that the flashed-off CO is bound by the nearby CuB as in the mammalian system (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 250, 1639-1650). In the minority, presumably inactive, fraction the CO recombination has fast nonexponential kinetics with a distribution of activation enthalpies peaking near delta Hp = 13 kJ/mol reminiscent of CO binding to myoglobin. In this inactive fraction CuB is apparently not accessible to the flashed-off CO. 相似文献
49.
Y Endo Y L Chan A Lin K Tsurugi I G Wool 《The Journal of biological chemistry》1988,263(17):7917-7920
An oligoribonucleotide (35-mer) that mimics the alpha-sarcin and the ricin region of eukaryotic 28 S rRNA was transcribed in vitro from a synthetic template with T7 RNA polymerase and was used to test whether the specificity of the hydrolysis by the toxins was retained. alpha-Sarcin, at a low concentration, cleaved a single phosphodiester bond on the 3' side of a guanosine residue in the synthetic oligomer that corresponds to G-4325 in 28 S rRNA, the site of action of the toxin in intact ribosomes. At a high concentration of alpha-sarcin, the substrate (35-mer) was hydrolyzed after each of its purines. alpha-Sarcin was without an effect on a synthetic RNA (20-mer) that reproduces the near universal sequence of nucleotides in the loop, but lacks the stem, of the toxin's domain. Thus, the specificity of the attack of alpha-sarcin on a precise region of 28 S rRNA appears to be contingent on the sequence of the nucleotides and the structure of the domain. Ricin depurinated a nucleotide in the synthetic oligomer (35-mer), and in the presence of aniline the phosphoribose backbone was cleaved at a position that conforms to A-4324 in 28 S rRNA, the site of action of the toxin in vivo. 相似文献
50.
The proton-pumping site of cytochrome c oxidase: a model of its structure and mechanism 总被引:8,自引:0,他引:8
Cytochrome c oxidase is an electron-transfer driven proton pump. In this paper, we propose a complete chemical mechanism for the enzyme's proton-pumping site. The mechanism achieves pumping with chemical reaction steps localized at a redox center within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes previously determined (Blair, D.F., Gelles, J. and Chan, S.I. (1986) Biophys. J. 50, 713-733) to be needed to accomplish redox-linked proton pumping. These processes include: (1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; (2) control of the rates of the pump's redox reactions with its electron-transfer partners during the turnover cycle (gating of electrons); and (3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and sufficiently specific that a realistic assessment can be made of how well the model pump would function as a redox-linked free-energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the model would function as an efficient pump and that its behavior would be very similar to that observed of cytochrome oxidase both in the mitochondrion and in purified preparations. The analysis presented here leads to the following important general conclusions regarding the mechanistic features of the oxidase proton pump. (1) A workable proton-pump mechanism does not require large protein conformational changes. (2) A redox-linked proton pump need not display a pH-dependent midpoint potential, as has frequently been assumed. (3) Mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping. 相似文献