Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Nitric oxide (NO) has been suggested to act as a regulator of endogenous intracellular ADP-ribosylation, based on radiolabelling of proteins in tissue homogenates incubated with [32P]NAD and NO. After the NO-stimulated modification was replicated in a defined system containing only the purified acceptor protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the hypothesis of NO-stimulation of an endogenous ADP-ribosyltransferase became moot. The NO-stimulated, NAD-dependent modification of GAPDH was recently characterized as covalent binding of the whole NAD molecule to the enzyme, not ADP-ribosylation. With this result, along with the knowledge that GAPDH is stoichiometrically S-nitrosylated, the role of NO in protein modification with NAD may be viewed as the conferring of an unexpected chemical reactivity upon GAPDH, possibly due to nitrosylation of a cysteine in the enzyme active site.
Mol Cell Biochem 1994 Sep
PMID:Nitric oxide and NAD-dependent protein modification. 789 64

Tubulin and microtubules were modified with the protease, subtilisin. The modification reduced the length of alpha- or beta-tubulin by cleaving a peptide fragment from the C-terminals. Generation of alpha'beta'-tubulin, which is cleaved at both the alpha- and beta-subunit terminals, and alpha beta'-tubulin, which is cleaved at the beta-subunit C-terminal, have already been reported. In this work an isotype, alpha'beta-tubulin, was produced. The three modified tubulin isotypes were compared for their ability to interact with glycolytic enzymes. Cleavage of alpha led to a poorer interaction when tested via affinity chromatography. Tubulin also inhibits the activity of aldolase and glyceraldehyde 3-phosphate dehydrogenase. When the alpha-subunit C-terminal was intact, inhibition was greatest. These results imply that the C-terminal of the tubulin alpha-subunit is responsible for interactions with glycolytic enzymes.
J Mol Recognit 1993 Dec
PMID:Glycolytic enzyme-tubulin interactions: role of tubulin carboxy terminals. 791 12

We report the identification of a full-length cDNA clone encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in the desiccation-tolerant plant Craterostigma plantagineum. The DNA sequence of the cDNA clone is homologous to cytosolic GAPDH cDNAs from other higher plants. The GAPDH transcript increases rapidly in abundance during dehydration or abscisic acid (ABA) treatment. The increase in mRNA levels is directly correlated with higher protein and enzyme levels. These results imply that enhanced rates of glycolysis are one of the immediate cellular responses to water deficit. This may be a mechanism by which the plant cell prepares for a demand of ATP and NADH2 during recovery.
Plant Mol Biol 1994 Oct
PMID:Dehydration and ABA increase mRNA levels and enzyme activity of cytosolic GAPDH in the resurrection plant Craterostigma plantagineum. 794 5

A number of reports have indicated that RNA recovered from paraffin-embedded tissue can be used as a substrate in the polymerase chain reaction (PCR). Although it is established that RNA in paraffin-embedded tissue undergoes significant degradation, the specific contributions of different fixatives and fixation times to this degradation are not known. Mouse splenic tissue was harvested and fixed immediately for 2, 8, or 24 h in either formalin, Omnifix II, or Carnoy's fixative and then processed and embedded in paraffin. RNA was extracted from deparaffinized cubes of tissue using an adaptation of the technique described by Chomczynski and Sacchi. RNA was reverse transcribed using a random hexamer primed reaction. PCR amplification for cDNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) mRNAs was then performed. Although GAPDH amplification is used routinely on fresh and frozen tissues, we show that the presence of DNA contamination in the RNA preparations limits its usefulness in paraffin-embedded tissue. Amplifiable HPRT mRNA sequences were detected in nine of 12 samples fixed in Omnifix II, in four of 12 samples fixed in Carnoy's fixative, and in none of 12 formalin-fixed samples. Because of primer selection to preclude amplification of genomic HPRT, DNA contamination is not an issue when HPRT is amplified. Thus, HPRT represents the control system of choice for the evaluation of RNA in PET. The techniques described provide a rapid, uniform, and reproducible method of obtaining RNA from PET for molecular analysis, but they indicate limited utility for retrospective analysis of archival tissues.
Diagn Mol Pathol 1994 Sep
PMID:Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls. 752 44

We recently demonstrated that rat preoptic area and anterior hypothalamus, sites of GnRH neurons, contain receptors for LH/hCG. We investigated in the present study whether LH/hCG receptor and GnRH genes are coexpressed in the same neurons and whether LH/hCG can directly regulate GnRH gene expression in immortalized hypothalamic GT1-7 neurons. The immunostaining for both LH/hCG receptors and GnRH are present in the same neurons in rat preoptic area and the GT1-7 neurons. The reverse transcription-nested polymerase chain reaction generated an expected 255-basepair LH/hCG receptor fragment in GT1-7 neurons. Northern blotting showed the presence of a major 1.8-kilobase and minor 2.6- and 4.3-kilobase receptor transcripts. Immunoblotting detected an 80-kilodalton receptor protein. Covalent receptor cross-linking studies showed that [125I]hCG binds to an 80-kilodalton protein with a specificity expected of LH/hCG receptors. Scatchard plot analysis demonstrated that GT1-7 neurons contain a single class of high affinity (Kd = 3.8 x 10(-11) M) and low capacity (5000 sites/neuron) LH/hCG receptors. Culturing GT1-7 neurons with highly purified hCG resulted in a dose- and time-dependent decrease in steady state GnRH, but not glyceraldehyde-3-phosphate dehydrogenase, messenger RNA (mRNA) levels. Human and rat LH, but not hCG alpha or -beta, FSH, or TSH, mimicked the down-regulating action of hCG on GnRH mRNA levels. Pretreatment of GT1-7 neurons with LH/hCG receptor antisense, but not sense, phosphorothioate oligodeoxynucleotides for 48 h resulted in decreases in [125I]hCG binding and the GnRH mRNA response to exogenous hCG. The half-life of GnRH mRNA transcripts, as determined by blocking transcription by actinomycin-D, was 32.5 +/- 2.5 h. This half-life was virtually unchanged by treatment with 100 ng/ml hCG (30.5 +/- 3.5 h). Treatment of GT1-7 neurons with 100 ng/ml hCG resulted in a dramatic decrease in nuclear run-on transcription of GnRH, but not beta-actin, gene compared to that in the controls. The same hCG concentrations and time points that decreased steady state GnRH mRNA levels also decreased cellular GnRH protein levels. Paradoxically, hCG stimulated the secretion of preexisting GnRH until the levels were depleted. In summary, GnRH neurons in the rat preoptic area and GT1-7 neurons coexpress LH/hCG receptor gene. Treatment of GT1-7 neurons with LH/hCG results in a decrease in steady state GnRH mRNA levels. This decrease is dose and time dependent and hormone specific, and requires the presence of cellular LH/hCG receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Endocrinol 1994 Aug
PMID:Novel presence of luteinizing hormone/human chorionic gonadotropin (hCG) receptors and the down-regulating action of hCG on gonadotropin-releasing hormone gene expression in immortalized hypothalamic GT1-7 neurons. 799 35

In order to identify genes in the Prader-Willi/Angelman syndrome critical region, radiolabeled cDNA probes from poly(A)+ RNA from mouse tissues were used to identify potential exon-containing genomic DNA fragments in cosmid or phage clones from appropriate yeast artificial chromosomes, and these fragments were subsequently used to screen human cDNA libraries. A mouse brain cDNA probe was effective in detecting control genes of various abundance including small nuclear ribonucleoprotein polypeptide N (SNRPN), hypoxanthine-guanine phosphoribosyl transferase, glyceraldehyde-3-phosphate dehydrogenase, and beta-actin. Two genes mapping within the Angelman syndrome critical region were isolated. One gene was found to encode the E6-associated protein (E6-AP; gene symbol HPVE6A), a protein which interacts with the E6 protein of human papilloma virus. The other gene is previously uncharacterized and is designated PAR-2 (D15S225E) for Prader-Willi and Angelman region-gene 2. Imprinting analysis using reverse transcription-polymerase chain reaction of RNA from fibroblasts and lymphoblasts of deletion Prader-Willi and Angelman patients demonstrated imprinting of SNRPN with exclusive expression from the paternal allele, but E6-AP and PAR-2 were not imprinted in these cultured human cells. The ability to analyze for imprinting and expression of SNRPN and other genes in this region in cultured human cells will be a valuable tool for analyzing the molecular basis of the Prader-Willi and Angelman syndromes, although imprinting may differ between cultured cells and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
Hum Mol Genet 1994 Feb
PMID:Imprinting analysis of three genes in the Prader-Willi/Angelman region: SNRPN, E6-associated protein, and PAR-2 (D15S225E). 800

We have cloned and sequenced cDNAs for the glyceraldehyde-3-phosphate dehydrogenase of glycolysis, gapC, from a bryophyte, a gymnosperm, and three angiosperms. Phylogenetic analyses are presented for these data in the context of other gapC sequences and in parallel with published nucleotide sequences for the chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL). Relative-rate tests were performed for these genes in order to assess variation in substitution rate for coding regions, along individual plant lineages studied. The results of both gene analyses suggest that the deepest dichotomy within the angiosperms separates not magnoliids from remaining angiosperms, but monocotyledons from dicotyledons, in sharp contrast to prediction from the Euanthial theory for angiosperm evolution. Furthermore, these chloroplast and nuclear sequence data taken together suggest that the separation of monocotyledonous and dicotyledonous lineages took place in late Carboniferous times [approximately 300 Myr before the present (Mybp)]. This date would exceed but be compatible with the late-Triassic (approximately 220 Mybp) occurrence of fossil reproductive structures of the primitive angiosperm Sanmiguelia lewisii.
Mol Biol Evol 1993 Jan
PMID:Molecular phylogenies in angiosperm evolution. 809 91

The aim of the present study was to determine whether N-methyl-D-aspartate (NMDA) stimulates somatostatin gene function in primary cultures of hypothalamic neurons. Neurons were either shortly (for 3, 8, 24 and 72 h) or chronically (for 11 days) exposed to NMDA (20 microM). Medium and cellular somatostatin contents were determined by radioimmunoassay, and steady-state preprosomatostatin mRNA levels by Northern blot analysis with an oligonucleotide probe. DNA content was measured as a cellular viability control. After 8 h incubation, NMDA induced a significant 2-fold increase in somatostatin mRNA accumulation, with a maximal 4-fold increase after 24 h incubation. A significant and dose-dependent (1.7-fold and 2.5-fold at 20 and 100 microM, respectively) stimulatory effect was also observed after chronic treatment. The kinetic patterns for medium and cellular somatostatin contents were similar to those obtained for somatostatin mRNA levels. Total DNA content was not modified under any experimental condition. The augmentations in cellular somatostatin and somatostatin mRNA determined after 24 h or chronic exposure to NMDA were blocked by (+)-5-methyl-10.11-dihydro-5H-dibenzo(a,d')cyclohepten-5,10-imine hydrogen maleate (MK-801), an NMDA receptor antagonist. MK-801 alone significantly (P < 0.05) reduced somatostatin mRNA. The stimulatory effect of NMDA on somatostatin mRNA was specific since it was not accompanied by any change in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. After immunostaining with a specific antibody against somatostatin, no difference was observed in the number of immunostained neurons detected in control and NMDA exposed groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain Res Mol Brain Res 1993 Mar
PMID:Stimulatory effect of N-methyl-D-aspartate on somatostatin gene expression in cultured hypothalamic neurons. 809 1

Transcription of ten nuclear genes was analysed in the albostrians mutant of barley (Hordeum vulgare L.). The lack of plastid ribosomes in white seedlings of this mutant results in a complex alteration of nuclear gene expression at the transcriptional level. We found a strong reduction in the accumulation of mRNAs transcribed from nuclear genes encoding chloroplast enzymes involved in the Calvin cycle, the chlorophyll a/b binding protein, and the cytosolic enzyme nitrate reductase. In contrast, the levels of transcripts of the genes encoding the cytosolic glycolytic enzymes glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase were slightly enhanced. Accumulation of chalcone synthase mRNA even reaches much higher levels in white than in green leaves. Ribosome-deficient plastids were combined by crossing with a nuclear genotype heterozygous for the albostrians allele. Analysis of transcript levels in F1 plants having the same nuclear genotype and differing only with respect to their content of normally developed chloroplasts versus undifferentiated mutant plastids, provided strong genetic evidence for the plastid being the origin of a signal (chain) involved in regulation of nuclear gene expression. Results of run-on transcription in isolated nuclei demonstrated that the plastid signal acts at the level of transcription; it does not interfere with gene regulation in general. Mechanisms triggering nuclear gene expression in response to light operate in white mutant leaves: the very low levels of mRNAs derived from nuclear genes encoding chloroplast proteins and the strongly enhanced level of chalcone synthase mRNA were both light inducible. Also the negative regulation of leaf thionein gene expression by light is observed in white albostrians seedlings.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1994 Feb
PMID:Ribosome-deficient plastids affect transcription of light-induced nuclear genes: genetic evidence for a plastid-derived signal. 810 78

The tetrameric mung bean glyceraldehyde-3-phosphate dehydrogenase is found to bind approximately four moles substrate, glyceraldehyde-3-phosphate, per mole enzyme with Kdiss equal to or less than 9.6 microM at pH 7.3, showing a slight positive cooperativity. Addition of excess substrate to a solution of the enzyme and excess NAD+ leads to a "burst" of NADH formation followed by a slow linear increase (monitored spectrophotometrically). Amount of NADH formed in the burst phase is pH-dependent and is equal to 3.6 moles per mole enzyme at pH 8.6 and above. Presuming four equivalent and independent sites per enzyme molecule (i.e. D2-symmetry), consistent values were obtained for the equilibrium constant of the oxidation-reduction step at different pH and most substrate concentrations. At lower pH (7.3) and high [NAD+]/[substrate] ratios, favouring the C2- symmetry conformation of the enzyme, the magnitude of the burst phase was negligibly small; practically no oxidation reduction reaction took place. Combining these with earlier results on the group transfer step, it is suggested that the oxidation-reduction and group transfer steps of the reaction catalysed by this enzyme require the D2 and C2 symmetry conformations of the enzyme, respectively.
Biochem Mol Biol Int 1993 Nov
PMID:Functional significance of protein conformational isomerisation in the glyceraldehyde-3-phosphate dehydrogenase-catalysed reaction. 811 17


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