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61.
Antonietta Bernardo Mario Patrizio Giulio Levi Tamara C. Petrucci 《Cellular and molecular neurobiology》1994,14(2):159-173
Summary 1. We have previously shown that acute exposure to the HIV coat protein gp120 interferes with the -adrenergic regulation of astroglial and microglial cells (Leviet al., 1993). In particular, exposure to 100 pM gp120 for 30 min depressed the phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) induced by isoproterenol in rat cortical astrocyte cultures. In the present study we have extended our analysis on the effects of gp120 on astroglial protein phosphorylation.2. We found that chronic (3-day) treatment of the cells with 100 pM gp120 before exposure to isoproterenol was substantially more effective than acute treatment in depressing the stimulatory effect of the -adrenergic agonist on vimentin and GFAP phosphorylation.3. Even after chronic treatment with gp120, no differences were found in the levels and solubility of these proteins.4. Besides stimulating the phosphorylation of intermediate filament proteins, isoproterenol inhibited the incorporation of32P into a soluble acidic protein of 80,000M
r
, which was only minimally present in Triton X-100-insoluble extracts.5. Treatment of astrocytes with a phorbol ester or exposure to3H-myristic acid indicated that the acidic 80,000M
r
protein is a substrate for protein kinase C (PKC) and is myristoylated, thus suggesting that it is related to the MARCKS family of PKC substrates.6. Acute (30-min) treatment with 100 pM gp120 totally prevented the inhibitory effect of isoproterenol on the phorphorylation of the 80,000M
r
MARCKS-like protein.7. Our studies corroborate the hypothesis that viral components may contribute to the neuropathological changes observed in AIDS through the alteration of signal transduction systems in glial cells. 相似文献
62.
Purified thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster have been compared with the two common enzyme forms ADH-F and ADH-S. Enzyme kinetic parameters for various primary and secondary alcohols were determined under standard conditions used previously. Both ADH-71k and ADH-FCh.D. show ADH-S-like reaction kinetics andK
m values, due to retrograde evolution at site 214, Pro Ser. Inhibition studies with alcohol dehydrogenase inhibitors pyrazole, 4-methylpyrazole, and cibacron blue 3GA were also performed. Activity measurements on crude extracts of larvae and flies from isogenic lines of ADH-FCh.D. revealed a consistently higher activity than in ADH-71k-containing strains, in contrast to the original strains.K.Th.E is indebted to the Royal Norwegian Council for Technological and Scientific Research for their postdoctoral fellowship. Prof. J. S. McKinley-McKee gave me the opportunity to work in his laboratory. I thank Dr. Knut Sletten of the Biochemical Institute for the kind gift of 2-methoxyethanol and amino acid analysis of some samples. The Biological Institute, Oslo, Section of General Genetics, is gratefully acknowledged for enabling me to use their fly-breeding facilities. Dr. John B. Gibson provided us with a sample of FCh.D. flies for the construction of isogenic lines in which Dr. Johan Hageman participated, owing to Postdoctoral Grant 436-931-P from the Foundation of Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO). J. H. and Paula Truyens were involved in the measurements on the crude extracts. Work at Victoria University was supported by the VUW Internal Grant Committee. 相似文献
63.
Summary Spectrophotometric peptide probes, derivatized at the C-terminus, are conveniently prepared by means of an Fmoc solid-phase strategy. Using a resin such as Sasrin, the fully protected peptide can be cleaved from the resin with hydrazine, yielding the protected peptide-hydrazide which is subsequently oxidized to the azide. An amino-containing chromophore or fluorophore such as 5-[(2-aminoethyl)-amino]-naphthalene sulfonic acid (EDANS) can be coupled directly to this activated carboxyl group. This allows for specific placement of the fluorophore at the C-terminal carboxyl group in the presence of trifunctional amino acids. 相似文献
64.
Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity. 总被引:1,自引:0,他引:1
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A. M. Dhalla B. Li M. F. Alibhai K. J. Yost J. M. Hemmingsen W. M. Atkins J. Schineller J. J. Villafranca 《Protein science : a publication of the Protein Society》1994,3(3):476-481
In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
65.
Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.
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T. Kitlar F. Dring D. F. Diedrich R. Frank H. Wallmeier R. K. Kinne J. Deutscher 《Protein science : a publication of the Protein Society》1994,3(4):696-700
Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity. 相似文献
66.
Sandra L. Gage Stanley R. Keim Jay R. Simon Walter C. Low 《Neurochemical research》1994,19(11):1379-1386
The cholinergic projections from basal forebrain nuclei to the retrosplenial cortex (RSC) have previously been studied using a variety of histological approaches. Studies using acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) immunocytochemistry have demonstrated that this projection travels via the cingulum on route to the RSC. Preliminary studies from our laboratory, however, have shown that the fornix may also be involved in this projection. The present study uses the combination of pathway lesions, and the analysis of cholinergic neurochemical markers in the RSC to determine the role of the fornix in the cholinergic projection to the RSC. High affinity choline uptake (HACU) and ChAT activity were measured in the RSC of control rats, animals with cingulate lesions, and animals with fornix plus cingulate lesions. Fornix plus cingulate lesions resulted in significant deceases in HACU and ChAT activity in comparison to cingulate lesions alone. Muscarinic receptor binding was also evaluated in combination with the various lesions, and a significant increase in retrosplenial receptor binding was noted following fornix lesions. Together, these results support the concept of a fornix-mediated cholinergic pathway to the RSC. 相似文献
67.
Martin M. Lee Francis H.Y. Green W.Michael Schoel Samuel Schürch 《生物化学与生物物理学报:疾病的分子基础》1994,1226(2):151-162
Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays. 相似文献
68.
S. H. Ogo C. F. Bernardes M. L. Glass M. A. Torsoni A. E. Vercesi 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(7):614-619
Digitonin was applied to permeabilize the plasma membrane of Bothrops alternatus erythrocytes to study respiration, oxidative phosphorylation and Ca2+ transport by mitochondria in situ. These mitochondria oxidized added NAD-linked substrates, succinate and N,N,N, N-tetramethyl-p-phenylenediamine. Respiration was sensitive to rotenone and cyanide but not to antimycin A. This indicates that Bothrops mitochondria possess the respiratory complexes NADH-ubiquinone, succinate-ubiquinone, and ferrocytochrome c-oxygen oxidoreductases, although the lack of sensitivity to antimycin A raises doubt about the composition of the ubiquinol cytochrome c-reductase complex. An ability to build up and sustain a membrane potential was documented by their capacity to accumulate tetraphenylphosphonium and Ca2+ through an uncoupler-sensitive mechanism. Addition of ADP caused a transient decrease in the membrane potential, indicating that this is the predominant driving force for ATP synthesis as in most types of mitochondria. Uncoupling of phosphorylation from the oxidative process increased hemoglobin O2 affinity, which suggests that ATP production by mitochondria may participate in modulation of O2 transport by hemoglobin.Abbreviations
membrane potential
- BAE
Bothrops alternatus erythrocytes
- DNP
2,4-dinitrophenol
- DPG
2,3-diphosphoglycerate
- EGTA
ethyleneglycol tetra-acetic acid
- FCCP
carbonylcyanide p-trifloromethoxyphenylhydrazone
- TMPD
N,N,N,N-tetramethyl-p-phenylenediamine
- TPP+
tetraphenylphosphonium
- TRIS
tris-(hydroxymethyl)aminomethane 相似文献
69.
Different experiments using Mucor miehei CBS 370.65 were carried out to study the effect of agitation speed on the production of the mold acid protease. The experiments were conducted in shake flasks at a fixed substrate concentration of 58 g l−1 of total carbohydrates and at shaker speeds from 80 to 380 rev min−1. Enzyme production was found to be directly proportional to the shaker speeds, with the highest concentration of enzyme of 1,400 Soxhlet Rennet units (SU) ml−1 obtained at 380 rev min−1. The yield of product to substrate at 380 rev min−1 was determined to be 27,081.0 SU g−1 substrate and the productivity of the process was 221 SU g−1 h−1. Enzyme production was partially growth associated, and glucose supported both cell growth and enzyme production. Product formation and cell concentration were directly related to the rate of substrate consumption. The rate of product formation decreased when product started to accumulate, suggesting that the process was affected by feedback repression. 相似文献
70.
A chemically reactive analog of the phytotropin N-1-naphthylphthalamic acid (NPA) was synthesized and evaluated as a site-directed irreversible ligand for the NPA receptor. The NPA analog (5-isothiocyanato-N-1-naphthylphthalamic acid; NCS-NPA) was synthesized in two steps. Pretreatment of etiolated Helianthus hypocotyl segments with NCS-NPA at concentrations in excess of 1 M resulted in a dose-dependent inhibition of basipetal [14C]IAA transport. Net uptake of IAA by hypocotyl segments was stimulated by NCS-NPA at concentrations of 1 M or greater. NCS-NPA inhibited the saturable binding of [3H]NPA in Helianthus microsomes in a dose-dependent fashion with 50% inhibition occurring at NCS-NPA concentrations of 3 to 10 nM. The binding affinity of [3H]NPA in microsomes pretreated with NCS-NPA followed by extensive washing was substantially reduced. These results demonstrate that NCS-NPA is a site-directed irreversible ligand for the NPA receptor and suggest that it may be of use in the purification and characterization of this biologically important receptor.Abbreviations ANPA
5-amino-naphthylphthalamic acid
- IAA
indole-3-acetic acid
- NCS-NPA
5-isothiocyanato-N-1-naphthylphthalamic acid
- NPA
N-1-naphthylphthalamic acid
- TLC
thin-layer chromatography 相似文献