Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have demonstrated in rat adrenal (Natarajan, R.D. and Harding, B.W. (1985) J. Biol. Chem. 260, 3902-3905) that NADH-semidehydroascorbate reductase and ascorbate participate in an electron transport pathway (ETP) supplying reducing equivalents from NADH to cytochrome P-450scc. Here, we demonstrate that this ascorbate dependent ETP also supplies reducing equivalents to cytochrome P-450(11 beta/18) in both rat adrenal and bovine adrenal cortex. The activity is dependent upon addition of catalase or upon 'cold shock' treatment of isolated mitochondria. Comparison of the rates of 11 beta- and 18-hydroxylation supported by this ETP and by the classical pathway supported by various TCA cycle intermediates suggests that in vivo the ascorbate dependent pathway may be essential for maximal flow of reducing equivalents to the mitochondrial hydroxylases. Partial reconstitution of the ascorbate dependent 11 beta/18-hydroxylase activity was achieved with purified bovine outer mitochondrial and inner mitochondrial membranes fortified with supernatant from sonified mitochondria all preincubated with phosphatidyl choline. These preparations no longer require catalase or 'cold shock' treatment. Ascorbate and NADH-semidehydroascorbate reductase are unable to support 17 alpha- or 21-hydroxylase activity in isolated bovine adrenal cortical microsomes whether incubated with purified outer mitochondrial membranes or not.
Mol Cell Endocrinol 1987 Sep
PMID:The function of NADH-semidehydroascorbate reductase and ascorbic acid in corticosteroid hydroxylation. 366 95

The permeability characteristics of Plasmodium falciparum-infected human erythrocytes to various 3H-labelled solutes were measured during the maturation of the parasites in sorbitol-synchronised cultures. Using [14C]inulin as the extracellular marker, estimates were made of the influx kinetics of [3H]amino acids into trichloroacetic acid (TCA)-soluble pools within the erythrocyte and concomitant incorporation into TCA-precipitable material. These measurements provided values of the rates of protein synthesis by the parasite and the initial influx rates for the transport of precursor amino acids into the erythrocyte. For about 12-15 h after parasitisation, the influx of L-[3H]glutamine remained at a low level comparable to that in the uninfected cell (2-9 nmol g-1 cells min-1). As pigment appeared in the trophozoite, the initial rate of influx of L-glutamine increased to a value up to 100-fold higher than in the uninfected erythrocyte. The increase in permeability affected only the parasitised cells in a culture of partially infected erythrocytes, and was selective with respect to substrate since the influx kinetics for both [3H]isoleucine and [3H]arginine were not affected by parasitisation. The permeability changes occurred mainly over a 4-8 h period in the development of the young trophozoite, during which time [3H]glycine influx was enhanced by a factor of 3-10, with a comparable increase in the uptake of myo-[3H]inositol. L-[3H]glutamate, which did not penetrate significantly into uninfected erythrocytes, entered red cells infected with mature trophozoites at a rate which was much less than 1% of the parasite-induced-L-glutamine influx. At the stages when the permeability to L-glutamine was markedly enhanced, parasitised cells remained impermeable to [3H]sucrose. An analysis of the relative 3H activities in glutathione and free amino acid pools indicated that, if L-glutamine permeation did not increase during parasite maturation beyond the ring stage, or was blocked by a potential antimalarial compound, an insufficient supply of L-glutamine would be available for the increased rates of parasite protein synthesis and glutathione turnover within the red cell.
Mol Biochem Parasitol 1985 Jun
PMID:Selective stage-specific changes in the permeability to small hydrophilic solutes of human erythrocytes infected with Plasmodium falciparum. 389 58

The conventional assay procedure for cathepsin D (E.C.3.4.23.5) activity in tissue homogenates and subcellular fractions requires incubation with hemoglobin as substrate. Cathepsin D (CD) activity is calculated by determining the increase in absorbance at 280 nm after precipitation of all proteins with trichloroacetic acid. This increase in absorbance (presumably due to the release of tyrosine residues from hemoglobin) is converted to arbitrary CD activity units. Homogenization and fractionation of cardiac tissue frequently requires that ethylenediamine tetraacetic acid (EDTA) be included in the homogenization medium. We have observed that subcellular fractions of cardiac tissue prepared in the presence of EDTA demonstrate residual CD activity despite either quantitative removal of all CD protein by immunoprecipitation or complete inhibition of CD by pepstatin. The present study demonstrates that this 'apparent' CD activity (residual increase in absorbance at 280 nm) is due to the formation of an Fe-EDTA complex which absorbs at 280 nm. Data are presented which demonstrates that the EDTA of the medium complexes with non-heme iron which contaminates commercially available hemoglobin. A method for preparing hemoglobin free of contaminant non-heme iron is described for use in studies of CD metabolism when EDTA is present in the homogenization buffer.
J Mol Cell Cardiol 1985 Oct
PMID:Identification of artifactual cathepsin D activity in cardiac subcellular fractions related to formation of an iron-EDTA complex. 393 91

300 micrograms of total RNA was extracted from 1 ml of packed Toxocara canis larvae by centrifugation through a 5.7 M cesium chloride cushion. 60 micrograms of polyadenylated messenger RNA was separated from 300 micrograms of total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from T. canis larvae, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of [35S]methionine into trichloroacetic acid precipitable material in the lysate containing mRNA was 4-5 times greater than that of control. Translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides ranging in molecular weight from 10000 to 100000 were synthesised in the lysate. A T. canis positive human serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by the autoradiography of SDS-PAGE. Three antigenic polypeptides with molecular weights of 49000, 27000 and 22000 which reacted specifically with IgG antibody in T. canis positive human serum, were demonstrated. The 27000 MW polypeptide reacted particularly strongly with the IgG antibody.
Mol Biochem Parasitol 1985 Mar
PMID:In vitro translation of mRNA from Toxocara canis larvae. 399 Jul 7

The specific retrograde axonal transport of free glycine within the identified neurons R3-14 of Aplysia californica was studied. The soma of the R3-14 neurons are located in the parietovisceral ganglion and their axons project down the branchial nerve to end in a large peripheral field. Using a double-chambered apparatus, the peripheral tissue was incubated in medium containing a 3H-amino acid for 4-48 hr, while the nerve and ganglion were isolated and perfused with plain or chemically altered medium. The nerve and ganglion were then either rapidly frozen for scintillation counting or fixed for autoradiography. When 3H-glycine was used, radioactivity entered the nerve rapidly, reached the ganglion in 3 hr, and was transported largely (greater than 80%) in the free amino acid form [trichloroacetic acid (TCA) soluble]. The right parietovisceral hemiganglion accumulated up to nine times more radioactivity than the left hemiganglion, reflecting the presence of the R3-14 axons and soma. Two phases of radioactivity were observed, a fast component moving at about 3 mm/hr and a slower (but larger) component moving at about 0.4 mm/hr. Light microscope autoradiography on nerves containing 3H-glycine revealed that the R3-14 axons accounted for more than 30% of the total label in the nerve but occupied less than 7% of the total cross-sectional area of the axonal core. Electron microscope autoradiography showed a close association of silver grains and dense core vesicles in the R3-14 axons. Retrograde axonal transport of free glycine was inhibited by (in decreasing order of effectiveness) mercuric chloride, vinblastine, colchicine, Nocodazole, and 2,4-dinitrophenol (2,4-DNP). Comparative studies of other amino acids [3H-leucine, 3H-serine, 3H-glutamic acid, 3H-gamma-aminobutyric acid (3H-GABA), and 3H-alanine] showed that 3H-glycine is the only amino acid that is rapidly axonally transported in large quantities within the R3-14 axons. This work demonstrates, for the first time, that a free amino acid, glycine, is transported in the retrograde direction within a select group of axons. The significance of this transport of glycine is discussed in relation to its use as a neural messenger by neurons R3-14.
Cell Mol Neurobiol 1984 Sep
PMID:Selective retrograde axonal transport of free glycine in identified neurons of Aplysia. 608 51

Treatment of intact Chinese hamster ovary cells with HgCl2 produced a rapid, concentration-dependent induction of DNA single-strand breaks (SSB) as revealed by alkaline elution analysis. Direct addition of HgCl2 to cell lysates did not result in DNA strand breaks. HgCl2 treatment of cells also caused a rapid leakage of superoxide radicals that were detected in their media by measurement of the reduction of exogenously added cytochrome c. There was a linear relationship between the production of radicals and the induction of DNA strand breaks, and there were also excellent temporal correlations in these parameters. Addition of oxygen radical scavengers, such as the enzymes superoxide dismutase and catalase, to the extracellular media significantly reduced the extent of DNA damage caused by HgCl2 without a similar attenuation of its uptake into cells, as did the autoclaved enzymes. Similarly, addition of radical scavengers such as glycerol or ascorbate inhibited the DNA damage but also reduced the uptake of the metal by almost the same degree. Thus, because of secondary effects on uptake of the metal, the radical scavenger experiments could not address the importance of oxygen radicals in the DNA damage caused by HgCl2. SSB were enhanced when cells were treated with HgCl2 and diethylmaleate or diethyldithiocarbamate, agents that deplete cellular reduced glutathione or inhibit the intracellular activity of superoxide dismutase, respectively. Thus, DNA damage in cells rendered sensitive to radicals was greater when these cultures were subsequently treated with HgCl2. The binding of 203HgCl2 to the DNA of intact Chinese hamster ovary cells was also studied. These studies were made possible by the relatively high stability of Hg(II) interaction with DNA and by utilizing a gentle method of DNA isolation that minimized redistribution of intracellular Hg(II) complexes after cells were lysed. The amount of Hg(II) bound to DNA varied from approximately 7 to 35 Hg atoms per 10(4) base pairs (bp) at concentrations of HgCl2 that have been previously shown to produce between 1 SSB/10(7) bp and 1 SSB/10(6) bp. The Hg(II)-DNA adducts were relatively stable complexes, since they resisted treatment with 0.1 M EDTA and 1 M NaCl and were stable to precipitation of the DNA with ethanol and trichloroacetic acid. However, the Hg(II) was released from the DNA when it was degraded enzymatically to mononucleosides, suggesting that the Hg(II)-DNA bonds formed in the cell were not truly covalent and that the strength of Hg(II) binding to DNA depended upon polynucleotide structure.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1984 Sep
PMID:Mechanism of HgCl2 cytotoxicity in cultured mammalian cells. 609 Aug 87

Total RNA was extracted from mature and juvenile Fasciola hepatica by homogenizing in 5.0 M guanidine thiocyanate and centrifugation through a 5.7 M CsCl cushion. Yields of 2 mg/g and 1 mg/g wet weight starting material were obtained, respectively. Messenger RNA was separated from the bulk extracted RNA by binding to oligo(dT)-cellulose. About 25% of the extracted RNA bound in both adult and juvenile cases. This material was subsequently tested in a rabbit reticulocyte cell-free translation system. Up to a 12-fold stimulation of incorporation of [35S]methionine into trichloroacetic acid-precipitable material was observed over that where no message was added. When the in vitro translation products were analysed by autoradiography of SDS-polyacrylamide gradient gels, polypeptides ranging in apparent molecular weight from about 10,000 to 100,000 were observed. Several minor differences in the electrophoretic patterns obtained from juvenile and adult mRNA were observed.
Mol Biochem Parasitol 1981 Dec 31
PMID:Preparation and in vitro translation of mRNA from Fasciola hepatica. 612 Dec 88

Transglutaminase, purified from guinea pig liver, was used to catalyze the incorporation of [14C]putrescine into exposed surface proteins of intact mouse neuroblastoma cells. This method specifically labeled two surface proteins (Mr = 92 000 and 76 000) in the N-18 mouse neuroblastoma cells and three surface proteins (Mr = 92 000, 76 000, and 72 000) in the NB-15 mouse neuroblastoma cells. In addition, transglutaminase also catalyzed cross-linking reactions of exposed surface proteins. In both the N-18 and NB-15 cells, differentiation was accompanied by a 2-fold increase of specific radioactivity incorporated into trichloroacetic acid insoluble cellular material, suggesting that the differentiated mouse neuroblastoma cells may possess greater amount of accessible peptide-bound glutaminyl residues on their surface than their malignant counterparts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic method revealed that while the [14C]putrescine-labeled protein patterns of undifferentiated and differentiated mouse neuroblastoma cells were similar, the intensity of labeling of individual bands was specifically modulated by cell differentiation.
Mol Cell Biochem 1984
PMID:Transglutaminase catalyzed incorporation of putrescine into surface proteins of mouse neuroblastoma cells. 614 57

Niridazole, an antischistosomal nitrothiazole derivative, is metabolized by adult Schistosoma mansoni to one or more reactive intermediates, as evidenced by extensive covalent binding of [14C]niridazole to parasite macromolecules. When worm pairs were incubated for 16 hr in culture medium containing 70 microM [14C]niridazole, 26-34% of the total parasite-associated radioactivity was irreversibly bound to trichloroacetic acid-precipitable material. Drug binding was both time- and [14C]niridazole concentration-dependent. Of the bound drug fraction, 85-90% was associated with parasite proteins, 3-5% with RNA and 4-7% with DNA. When schistosomes were recovered from infected mice, treated with periodic doses of [14C]niridazole, over 40% of the total parasite-associated radioactivity was bound to macromolecules. Niridazole caused up to a 40% decrease in the concentration of total nonprotein thiols in intact schistosomes incubated with the drug over an 8-hr period. Under strictly anaerobic conditions, cell-free schistosome preparations catalyzed a reduced pyridine nucleotide-dependent reduction of niridazole's essential nitro group, as evidenced by disappearance of absorption at 400 nm. Net nitroreduction did not occur under aerobic conditions, although the drug did stimulate oxidation of the pyridine nucleotide cofactor. Covalent binding of [14C]niridazole also took place in this cell-free system, with requirements identical with those needed for enzymatic nitroreduction. Covalent drug binding, but not nitroreduction, was inhibited up to 80-85% by 2 mM L-cysteine, N-acetyl-L-cysteine, or glutathione; S-carboxymethyl-L-cysteine, which has no free sulfhydryl group, was not inhibitory. [14C]4'-Methylniridazole, a nonschistosomicidal analogue of niridazole, was taken up by intact schistosomes in vitro, but was not metabolized and did not bind covalently to parasite macromolecules. Furthermore, 4'-methylniridazole did not affect the concentration of nonprotein thiols in intact parasites and did not serve as a substrate for schistosomal nitroreductase in vitro. These results indicate a positive correlation between proximal metabolic activation of niridazole within these facultative anaerobic organisms and its antiparasitic activity.
Mol Pharmacol 1983 Sep
PMID:Reductive metabolism of niridazole by adult Schistosoma mansoni. Correlation with covalent drug binding to parasite macromolecules. 619 6

The effect of steroids on the growth of AtT-20 mouse pituitary tumor cells was investigated. It was found that under certain conditions glucocorticoids inhibit growth. Specificity studies indicated that only glucocorticoids caused this effect. Biopotency studies indicated that dexamethasone had its mid-maximal effect around 5 nM. At least part of this inhibition was caused by a decrease in the cell's ability to synthesize DNA; glucocorticoids inhibit the rate at which tritiated thymidine is incorporated into TCA-precipitable material. The magnitude of the growth-inhibiting effect depended upon the prior culture history of the cell. The effect was least on cells derived from exponentially growing cultures and most effective on cells derived from cultures that were approaching their density limit. It is concluded that glucocorticoids inhibit AtT-20 cell growth by interfering with their ability to transform from confluent cultures to ones that grow exponentially. The significance of this finding is that now two mechanisms must be considered when investigating the pathways through which glucocorticoids decrease the ACTH production of the AtT-20 cell.
Mol Cell Endocrinol 1984 Apr
PMID:Glucocorticoids inhibit the growth of AtT-20 mouse pituitary tumor cells. 632 77


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