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991.
The effect of infusion of nerve growth factor (NGF) into the rat testis on the expression of androgen-binding protein (ABP) mRNA was studied. A major 1.7-kb and a minor 3.7-kb ABP mRNA were present at all stages of the seminiferous epithelium with maximal levels at stages VIII-XI and the lowest levels at stages IV-VI. Infusion of 15 ng/h of NGF with a mini-osmotic pump for 14 days resulted in a 2-fold increase of ABP mRNA as revealed by Northern blots, whereas the mRNA level of another Sertoli cell protein, urokinase-type plasminogen activator, remained unchanged. Image analysis of autoradiograms obtained by in situ hybridization of sections from treated testes showed a similar increase in APB mRNA compared to noninfused or PBS-infused testes. However, at the cellular level the labeling intensity for ABP mRNA over Sertoli cells of different stages of the seminiferous epithelium was the same in NGF-infused and control testes. This suggests that the increase of ABP mRNA in NGF-infused testes was caused by prolongation of stages VII-VIII with maximal ABP mRNA expression; the suggestion is supported by an increase of 30 percent in frequency of these stages in histological sections from NGF-infused testes. 相似文献
992.
V Divoky E Bissé J B Wilson L H Gu H Wieland I Heinrichs J F Prior T H Huisman 《Biochimica et biophysica acta》1992,1180(2):173-179
We have analyzed the hemoglobins of a young German patient with beta-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the beta-globin genes through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the GTG-->GGG mutation at codon 126 leading to the synthesis of Hb Dhonburi or alpha 2 beta (2)126(H4)Val-->Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G-->C) mutation that leads to a rather severe beta(+)-thalassemia and the GTG-->ATG mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered beta-chain variant, named Hb Baden, was present for only 2-3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G-->C) thalassemic mutation. The chromosome with the codon 18 (GTG-->ATG) and the IVS-I-5 (G-->C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G-->C) mutation living in different countries failed to detect the codon 18 (GTG-->ATG) change. 相似文献
993.
H Thomassin L Ménard C Hengartner J B Kirkland G G Poirier 《Biochimica et biophysica acta》1992,1137(2):171-181
This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations. 相似文献
994.
Dictyostelium myosin II heavy-chain kinase A (MHCK A) is activated by autophosphorylation. Heparin and DNA, as well as vesicles composed of phosphatidylserine or phosphatidylinositol, were found to increase the initial rate of MHCK A autophosphorylation 5-10-fold in a Ca(2+)-independent manner. The negatively charged molecules also increased the activity of the autophosphorylated MHCK A by about 2-fold. In contrast, positively charged polypeptides such as poly(D-lysine), poly(L-lysine), poly(L-arginine) and histones strongly inhibited (IC50 of 0.5 micrograms/ml) the activity of the active, autophosphorylated MHCK A. Similar levels of inhibition, on a weight basis, were observed for poly(L-lysine) fractions with molecular weights from 3800 to 150,000-300,000. The inhibition was competitive with respect to peptide substrate and mixed with respect to ATP. At much higher concentrations poly(L-lysine) also inhibited the ability of MHCK A to autophosphorylate. It is proposed that negatively charged compounds and autophosphorylation increase the activity of MHCK A by weakening the interaction between the catalytic domain and a positively charged autoinhibitory domain. 相似文献
995.
Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii. 总被引:1,自引:0,他引:1
R Pérez-Vicente J M Alamillo J Cárdenas M Pineda 《Biochimica et biophysica acta》1992,1117(2):159-166
Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form. 相似文献
996.
M Delaforge P Ladam G Bouillé J G Benarous M Jaouen J P Girault 《Chemico-biological interactions》1992,85(2-3):215-227
The effects of pH on access to the cytochrome P-450 active site, N-demethylation and formation of the cytochrome P-450 Fe(II)-RNO metabolite complex for a series of erythromycin derivatives were examined. Studies were performed with dexamethasone-treated rat liver microsomes containing large amounts of cytochrome P-450 3A isozymes. In addition to factors such as hydrophobicity or hindrance around the dimethyl-amino function, the ionisation state of the N(CH3)2 group played an important role in the recognition and metabolism of the substrate by cytochrome P-450. Esterification of the desosamine in the beta position of the N(CH3)2 group leads to lower pKa values for the R--N+ H(CH3)2 <--> [R--N (CH3)2] + H+ equilibrium. At physiological pH, the amine group is mainly in the unprotonated form. Consequently, easier access to the protein active site and significant formation of cytochrome P-450 Fe(II)-RNO metabolite complex are observed for these derivatives. These results led us to interpret the formation of cytochrome P-450 Fe(II)-RNO metabolite complex as a series of multiple steps equilibria depending on the ionisation state of the N(CH3)2 group, the partition coefficient of the substrate between the microsomal layer and the aqueous media and a series of metabolic reactions leading partially to the final inhibitory nitrosoalkane-cytochrome P-450 Fe(II) complex. 相似文献
997.
M E Ludérus F Kesbeke M L Knetsch R Van Driel C D Reymond B E Snaar-Jagalska 《European journal of biochemistry》1992,208(2):235-240
Drug-resistance selection in Dictyostelium discoideum transformants resulted in up to eight-times-higher ras protein levels. Over-production of the wild-type ras protein did not lead to an aberrant phenotype. Increased levels of the mutated [G12T]ras protein, however, were correlated with severe deficiencies in aggregation and development. This aberrant phenotype is associated with reduced cAMP binding, due to a lower number of cell-surface receptors. We show that both RNA and cAMP-receptor-protein levels are reduced. These results indicate that ras in Dictyostelium discoideum seems to be involved in regulating cAMP-receptor-gene expression. 相似文献
998.
I Lascu A Chaffotte B Limbourg-Bouchon M Véron 《The Journal of biological chemistry》1992,267(18):12775-12781
Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure. 相似文献
999.
C Kahan K Seuwen S Meloche J Pouysségur 《The Journal of biological chemistry》1992,267(19):13369-13375
In CCL39 cells transfected with m1-muscarinic receptors, carbachol stimulates phosphoinositide turnover and early events associated with mitogenesis as efficiently as thrombin but, in contrast to thrombin, fails to induce cell proliferation (Seuwen, K., Kahan, C., Hartmann, T., and Pouysségur, J. (1990) J. Biol. Chem. 265, 22292-22299). We show here that the action of the two agents can be dissociated at the level of S6 kinase and mitogen-activated protein kinase (MAP kinase) activation. Mitogenic concentrations of thrombin and basic FGF were found to stimulate S6 kinase activity, measured in whole cell lysates, with a biphasic time course; an early peak of activity is induced 10 min following stimulation and a sustained phase of activity can be measured over several hours. A very similar profile emerged for p44 MAP kinase (p44mapk), assayed in immunoprecipitates. In this case, the activity first peaks at 6-8 min, preceding S6 kinase. In contrast to thrombin and FGF, carbachol stimulates S6 kinase and MAP kinase only transiently, corresponding to the first peak of activity, but the sustained phase is not observed. Similarly, phorbol dibutyrate induces an early phase of activity only. Pertussis toxin (PTX), which is known to block thrombin mitogenicity efficiently, inhibited the first peak of thrombin-induced S6 kinase and MAP kinase activity only partially, but totally blocked the sustained phase. The toxin had no effect on FGF-induced kinase activities. The cAMP elevating hormone PGE1 did not inhibit p44mapk or S6 kinase activation by thrombin or FGF, demonstrating that the PTX-sensitive signal generated by thrombin does not depend on a Gi-mediated sustained inhibition of adenylylcyclase. Surprisingly, PGE1 was found to stimulate sustained phase S6 kinase activity both alone and in synergy with FGF or thrombin. This result, as well as the biphasic activation of S6 kinase by thrombin, could be qualitatively reproduced in immunocomplex kinase assays using an antiserum immunoprecipitating p70 S6 kinase (p70S6k). Our data show that activation of phosphoinositide turnover and PKC does not quantitatively explain thrombin action, in particular the sustained phase of kinase activities, which critically depends on a PTX-sensitive signal different from adenylylcyclase inhibition. We postulate that this signal does not exclusively originate from the recently identified G protein-coupled thrombin receptor. 相似文献
1000.