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)

Quantitation of mRNA content in samples of total cellular RNA is required for the analysis of Northern blot hybridization to estimate the relative level of specific gene expression. Commonly used methods based on UV absorbance and dye staining measure only total RNA, and mRNA normalization by probing for mRNA levels of housekeeping genes, such as beta-actin and glyceraldehyde-3-phosphate dehydrogenase, assumes a constant level of their expression, which, in fact, may vary as a function of cell proliferation and differentiation. We describe here a nonradioactive, slot-blotting method for quantifying eukaryotic mRNA levels using a biotinylated oligo(dT) probe, which hybridizes directly to the 3'-polyadenylated sequence of eukaryotic mRNAs. The method provides a more accurate estimation of mRNA content in total RNA samples and should be applicable for quantitative Northern analysis.
Mol Biotechnol 1996 Dec
PMID:Hybridization of biotinylated oligo(dT) for eukaryotic mRNA quantitation. 906 71

We have reported that overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is involved in age-induced apoptosis of the cultured cerebellar granule cells that grow in a depolarizing concentration (25 mM) of KCI. The present study was undertaken to investigate whether GAPDH overexpression also occurs and participates in apoptosis of the cerebellar granule cells that result from switching the culturing conditions from high (25 mM) to low (5 mM) concentrations of KCl. We found that exposure of granule cells to low potassium (K+) for 24 hr induces not only apoptosis but also necrotic damage. The latter is supported by the morphological observations that a subpopulation of neurons showed cell swelling, extensive cytoplasmic vacuolization, damaged mitochondria, and apparently intact nuclei. Treatments with two antisense but not sense oligodeoxyribonucleotides directed against GAPDH attenuated low K+-induced neuronal death by approximately 50%. Morphological inspection revealed that GAPDH antisense oligonucleotides preferentially blocked low K+-induced apoptosis with little or no effect on necrotic damage. Similar to antisense oligonucleotides, actinomycin-D partially inhibited low K+-induced death of granule cells with a predominant effect on apoptosis. In contrast, cycloheximide almost completely blocked low K+-induced neuronal death and seemed to prevent both apoptotic and necrotic damage. The levels of GAPDH mRNA and protein were markedly increased in a time-dependent manner after low K+ exposure. The overexpression of GAPDH mRNA and protein was completely blocked by cycloheximide, actinomycin-D, and its antisense but not sense oligonucleotides. Taken together, these results lend credence to the view that exposure of cerebellar granule cells to low K+ induces both apoptosis and necrosis and that only the apoptotic component involves overexpression of GAPDH.
Mol Pharmacol 1997 Apr
PMID:Overexpression of glyceraldehyde-3-phosphate dehydrogenase is involved in low K+-induced apoptosis but not necrosis of cultured cerebellar granule cells. 910 17

Mutations have been introduced in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus in order to convert its cofactor selectivity from a specificity towards NAD into a preference for NADP. In the B-S mutant, five mutations (L33T, T34G, D35G, L187A, P188S) were selected on the basis of a sequence alignment with NADP-dependent chloroplastic GAPDHs. In the D32G-S mutant, two of the five mutations mentioned above (L187A, P188S) have been used in combination with another one designed from electrostatic considerations (D32G). Both mutants exhibit a dual-cofactor selectivity at the advantage of either NAD (B-S) or NADP (D32G-S). In order to analyse the cofactor-binding site plasticity at the molecular level, crystal structures of these mutants have been solved, when complexed with either NAD+ (D32G-Sn, resolution 2.5 A, R = 13.9%; B-Sn, 2.45 A, 19.3%) or NADP+ (D32G-Sp, 2.2 A, 19.2%; B-Sp, 2.5 A, 14.4%). The four refined models are very similar to that of the wild-type GAPDH and as expected resemble more closely the holo form than the apo form. In the B-S mutant, the wild-type low affinity for NADP+ seems to be essentially retained because of repulsive electrostatic contacts between the extra 2'-phosphate and the unchanged carboxylate group of residue D32. Such an antideterminant effect is not well compensated by putative attractive interactions which had been expected to arise from the newly-introduced side-chains. In this mutant, recognition of NAD+ is slightly affected with respect to that known on the wild-type, because mutations only weakly destabilize hydrogen bonds and van der Waals contacts originally present in the natural enzyme. Thus, the B-S mutant does not mimic efficiently the chloroplastic GAPDHs, and long-range and/or second-layer effects, not easily predictable from visual inspection of three-dimensional structures, need to be taken into account for designing a true "chloroplastic-like" mutant of cytosolic GAPDH. In the case of the D32G-S mutant, the dissociation constants for NAD+ and NADP+ are practically reversed with respect to those of the wild-type. The strong alteration of the affinity for NAD+ obviously proceeds from the suppression of the two wild-type hydrogen bonds between the adenosine 2'- and 3'-hydroxyl positions and the D32 carboxylate group. As expected, the efficient recognition of NADP+ is partly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S). Another interesting feature of the reshaped NADP+-binding site is provided by the local stabilization of the extra 2'-phosphate which forms a hydrogen bond with the side-chain hydroxyl group of the newly-introduced S188. When compared to the presently known natural NADP-binding clefts, this result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor selectivity from NAD to NADP. In fact, as it is the case in this mutant, only a moderately polar hydrogen bond can be sufficient to make the extra 2'-phosphate of NADP+ well recognized by a protein environment.
J Mol Biol 1997 May 16
PMID:A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+. 917 58

In situ hybridization analysis provides a means to qualitatively study the heterogeneity of primary tumors and metastases based on the types of genes transcribed. In this study, we have tested some parameters for quantitative analysis of in situ hybridizations with paraffin-embedded human breast tumors and measured mRNA levels for the angiogenic protein, vascular endothelial growth factor (VEGF). VEGF mRNAs were highly tumor specific, with the highest levels near necrotic regions within the tissues (0.1 to 2.7 dpm/mm2). Normal cells within the tissue sections did not have detectable levels of VEGF mRNA. For comparison, tumor levels of c-myc (4 to 46 dpm/mm2) and glyceraldehyde-3-phosphate dehydrogenase mRNAs (48 to 214 dpm/mm2) were measured. The mRNAs for both of these genes were more broadly expressed across the tissue sections. The hybridization pattern for VEGF mRNAs was consistent with hypoxia-induced VEGF mRNA steady-state levels and supports the hypothesis that oxidative stress regulates VEGF expression in breast tumors.
Exp Mol Pathol 1997 Feb
PMID:Quantitation of vascular endothelial growth factor mRNA levels in human breast tumors and metastatic lymph nodes. 920 8

A gel penetration technique, that measures the dilution undergone by protein equilibrium on a short tightly packed gel column, has been employed to determine the molecular masses of aldolase (160 kDa), glyceraldehyde-3-phosphate dehydrogenase (GPDH; 145 kDa) in the absence and presence of each other and of other proteins. The dilution factor (concentration of protein applied/concentration of protein after equilibration) was found to be inversely related to the molecular mass of the protein. In equimolar mixtures of aldolase and GPDH, 0.5-2.5 microM each, the two enzymes exhibited a common molecular mass value of 309-316 kDa. These enzymes did not undergo any self association or disassociation in this concentration range. Moreover, their molecular masses were unaffected by the presence of other proteins tested. When the concentration of one of these enzymes (aldolase or GPDH) was held constant and that of the other varied, the dilution factor of the former was decreased as the concentration of the latter was increased until it corresponded to a molecular mass of ca. 310 kDa at equimolar concentrations of the two enzymes. Further increase in the concentration of the variable enzyme had no effect. It has been suggested that aldolase and GPDH form a 1:1 complex of dissociation constant equal to or less than 5 x 10(-8) M. The complex was found to dissociate in the presence of KCl, (NH4)2SO4, ATP and NADH whereas its formation was favoured by fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, NAD+, ADP, AMP and phosphate ions.
Biochem Mol Biol Int 1997 Jul
PMID:Interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase: molecular mass studies. 924 8

The supramolecular structure of oligomeric enzymes can be specifically regulated by changing the size of an inner cavity of Aerosol OT reversed micelles in octane. Both D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) reveal an ability to exist and function in monomeric, dimeric and tetrameric forms (homooligomers). Various heterooligomeric complexes, in particular, GAPDH monomer--LDH monomer, GAPDH dimer--LDH tetramer were detected in reversed micelles.
Biochem Mol Biol Int 1997 Jul
PMID:Formation of homo- and heterooligomeric supramolecular structures by D-glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase in reversed micelles of aerosol OT in octane. 924 10

The generation of 13C-labeled D-glucose isotopomers by rat hepatocytes incubated for 30 or 120 min in the presence of 10 mM [3-(13)C]pyruvate was assessed by 13C NMR. The amount of C1-labeled D-glucose exceeded that of C2-labeled hexose, which was itself higher than that of C3-labeled D-glucose. A comparable hierarchy was observed in the C6-C5-C4 moiety of the hexose. The latter moiety of D-glucose was more efficiently labeled, however, than the C3-C2-C1 moiety. This finding is similar to that both previously reported and again observed in the present study when hepatocytes were exposed to [2(-13)C]pyruvate. These converging observations thus support the concept of enzyme-to-enzyme channeling of D-glyceraldehyde 3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phospho-fructoaldolase.
Biochem Mol Med 1997 Aug
PMID:Asymmetrical labeling of D-glucose generated from [3(-13)C]pyruvate in rat hepatocytes. 925 88

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.
Mol Biol Cell 1997 Sep
PMID:Flight muscle function in Drosophila requires colocalization of glycolytic enzymes. 930 64

Combination of the targeted amplification of nuclear introns and the analysis of single-stranded conformational polymorphisms has the potential to provide an inexpensive, rapid, versatile and sensitive genetic assay for evolutionary studies and conservation. We are developing primers and protocols to analyse nuclear introns in vertebrates, and are testing them in a population genetic study of marbled murrelets Brachyramphus marmoratus. Here we present protocols and results for introns for aldolase B, alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase and lamin A. Results suggest that this approach presents a potentially powerful method for detecting genetic variation within and among local populations and species of animals: (i) a variety of genes can be surveyed, including genes of special interest such as those involved in disease resistance; (ii) assays are rapid and relatively inexpensive; (iii) large numbers of genes can be assayed, enabling accurate estimation of variation in the total genome; (iv) almost any mutation can be detected in the genes amplified; (v) the exact nature of variation can be investigated by sequence analysis if desired; (vi) statistical methods previously developed for proteins and/or sequence data can be used; (vii) protocols can be easily transferred to other species and other laboratories; and (viii) assays can be performed on old or degraded samples, blood or museum skins, so that animals need not be killed. Results of analyses for murrelets support earlier evidence that North American and Asiatic subspecies represent reproductively isolated species, and that genetic differences exist among murrelets from different sites within North America.
Mol Ecol 1997 Nov
PMID:Intron variation in marbled murrelets detected using analyses of single-stranded conformational polymorphisms. 939 63

The deposition of amyloid plaques in brain parenchyma is one of the major pathological hallmarks of Alzheimer's disease (AD). The amyloid in senile plaques is composed of the amyloid beta-peptide (A beta) of 39-43 amino acid residues derived from a larger beta-amyloid precursor protein (beta APP). Soluble derivatives of beta APP (sAPP) lacking the cytoplasmic tail, transmembrane domain, and a small portion of the extracellular domain are generated proteolytically by "secretases." Using cell cultures, the authors analyzed the level of sAPP in neuroblastoma and pheochromocytoma (PC12) cells by immunoblotting samples from conditioned media and cell lysates. Normal levels of secretion of sAPP into conditioned media were severely inhibited by treating cells with melatonin (3-4 mM). The inhibitory effect of melatonin on the secretion of sAPP can be reversed. When the cells that were pretreated with melatonin for 10 h were washed, the normal level of secretion of sAPP was restored. Northern blot analyses indicated that the treatment of PC12 cells with melatonin resulted in a significant decrease in the level of mRNA encoding beta APP, beta-actin, and glyceraldehyde-3-phosphate dehydrogenase, and that the treatment of a human neuroblastoma cell line with melatonin resulted in no change in levels of these messages. The secretion of sAPP into the conditioned medium was substantially reduced in the differentiated cells similar to reductions observed in melatonin-treated undifferentiated PC12 cells. Melatonin was found to potentiate the nerve growth factor-mediated differentiation in PC12 cells at 24 h. Taken together, these data suggest that melatonin regulates the metabolism of beta APP and other housekeeping genes in a cell-type specific manner, and that melatonin accelerates the early process of neuronal differentiation.
J Mol Neurosci 1997 Oct
PMID:Melatonin alters the metabolism of the beta-amyloid precursor protein in the neuroendocrine cell line PC12. 940 89


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