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The effect of training and detraining on several enzymes in horse skeletal muscle.

Training and detraining had little effect on the activity of glycogen synthase, hexokinase, glycerol 3-phosphate dehydrogenase or total protein. The activity of 3-hydroxyacyl-CoA dehydrogenase increased markedly during training. After 5 weeks of detraining, the activity of 3-hydroxyacyl-CoA dehydrogenase was returning to pre-training values, whilst by 10-week detraining, the levels were increasing again.

3-Hydroxyacyl CoA Dehydrogenases↗

Isolation and properties of cytoplasmic alpha-glycerol 3-phosphate dehydrogenase from the pectoral muscle of the fruit bat, Eidolon helvum.

Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.

Adenosine Triphosphate↗

Localization of adenosine A2a receptor in retinal development and oxygen-induced retinopathy.

PURPOSE: To investigate the association of adenosine A2a receptors (A2aR) with retinal vasculogenesis and angiogenesis that occurs in the canine model of oxygen-induced retinopathy (OIR). METHODS: One-day-old dogs were exposed to 100/o oxygen for 4 days and killed in oxygen (5 days old) and at 3, 10, 17, and 23 days after exposure to hyperoxia. Room air control animals were killed at 1, 5, 8, 15, 22, and 28 days of age. Immunolocalization of A2aR was performed on frozen sections, and reaction product density was quantified using microdensitometry. Cell types were identified in serial sections using antibodies against von Willebrand factor (endothelial cells) and GFAP (astrocytes), and enzyme histochemistry for menadione-dependent a-glycerophosphate dehydrogenase (M-a-GPDH) (to label angioblasts and developing blood vessels). RESULTS: A2aR immunoreactivity was associated with forming blood vessels and angioblasts in the nerve fiber layer (NFL) of peripheral retina. As development progressed, vascular labeling decreased, whereas labeling of neuronal elements increased. In OIR, A2aR immunoreactivity in the NFL was reduced after exposure to hyperoxia and significantly elevated in the inner retina throughout vascularized retina and in advance of forming vasculature in all oxygen-treated animals returned to room air. A2aR immunoreactivity was also prominent in fronds of intravitreal neovascularization. CONCLUSIONS: A2aR immunoreactivity was associated with developing retinal vessels. As development progressed, vascular-associated A2aR labeling decreased and, concomitantly, labeling of neuronal elements increased. A2aR immunoreactivity was significantly elevated at the edge of forming vasculature in all animals returned to room air after hyperoxia and in intravitreal neovas cular formations. These results provide additional evidence for the importance of A2aR and its ligand adenosine in retinal vascular development and in the vasoproliferative stage of canine OIR.

Animals↗

Cloning and sequence analysis of Gpdh in Callosobruchus chinensis (Coleoptera: Bruchidae).

The Sn-Glycerol-3-phosphate dehydrogenase (GPDH: NAD+ 2-oxidoreductase, EC 1.1.1.8) gene of C. chinensis was cloned and its nucleotide sequence was analyzed. The gene was obtained by screening a genomic library with Drosophila melanogaster Gpdh and PCR amplification. The 5,126 bp gene obtained is comprised of one 5' untranslated region, eight exons, seven introns, and three 3' untranslated regions. Comparison of Gpdh of D. melanogaster with that of C. chinensis showed a 89.9% identity in the coding region, 70% in the intron, 79% in the entire nucleotide sequence, and 83.2% in the deduced amino acid sequence. The transcription initiation site is located 33 nucleotides upstream of the initiation codon, and the sequence analysis of the promoter region showed TATA and CAAT boxes at the 5' end. The stop codon (TAA) and polyadenylation signal (AATAAA) are located at the 3' end of each of the exons 6 to 8. These findings show that GPDH isozymes in C. chinensis are produced by the alternative processing of 3' exons. The occurrence of the three transcripts was proven by RT-PCR using synthetic oligonucleotides complementary to the predicted unique 3' regions. Compared to the D. melanogaster GPDH isozymes, GPDH-1, -2, and -3, C. chinensis GPDH showed 83.6%, 83%, and 84% identities, respectively.

Amino Acid Sequence↗

Plasticity of skeletal muscle fibres in space-flown primates.

Skeletal muscle atrophy and fibre type transitions were observed as a rule in rats exposed to micro- and zero-gravity, flown on boards of biosatellites and space shuttle ships. Much less is known about the spaceflight-induced muscle events in primates. The latter are animals of special interest since pattern of their onground motor activities works in ways alike to the human one, though the opportunities of studies are much wider. One of the targets of the study was to investigate the influence of spaceflight conditions on tissue morphology in monkey skeletal muscles of different functional and structural organization.

Animals↗

Muscle fiber composition in patients with traumatic cord lesion.

Muscle fiber composition and oxidative and glycolytic enzymatic activity have been studied with complete traumatic transection of the spinal cord and spastic paralysis of the lower extremities. Muscle sample were taken by means of needle biopsy from the vastus lateralis, gastrocnemius, and soleus muscles. Biopsies were also taken for comparison from the deltoid muscle. Fibers staining darkly for alkaline stable myofibrillar ATP-ase (type II) dominated or were the only fibers identified in the paralysed muscles. The deltoid muscles of the same patients had a rather even mixture of type I and II fibers. Staining pattern was reversed after acid preincubation (pH 4.3). Mean diameters in the paralysed muscles were reduced for both fiber types. All fibers stained relatively weakly for NADH-diaphorase. Succinyldehydrogenase activity was low and phosphofructokinase activity usually moderately reduced. The findings imply that neuronal influence on the muscular fibers had led to a change in the staining characteristics of the muscle fibers. Such a change migh indicate altered contractile characteristics, though the detailed nature of the observed findings in still unclear.

Adenosine Triphosphatases↗

[Comparative evaluation of carbohydrate metabolism in perch (Perca fluviatilis L.) from water bodies with different contents of humic acids].

Characteristic features of energy metabolism, related to the adaptation of fish to adverse environmental conditions, were detected in perch (Perca fluviatilis L.) inhabiting the lake with a high humus content, compared to perch from the control lake. We studied a complex of enzymes involved in carbohydrate metabolism and assessed the relationship between metabolic pathways using correlation analysis. The intensity of oxidative metabolism was greater in the gulls. The activation of energy metabolism in the liver was characterized by an increased consumption of carbohydrates. In addition, the role of the liver-specific lactate dehydrogenase (LDH) isoform D4 also increased. The activity of enzymes involved in carbohydrate metabolism decreased most strongly in white skeletal muscles, whereas the role of the pentose-phosphate pathway (along with glycolysis) in the production of muscle lactate increased. The adaptation of fish to a high-humus aquatic environment was accompanied by a slight decrease in their fatness.

Animals↗

[Functional morphology of the heart conduction system in acute myocardial infarct].

The results of histochemical, histoenzymological, and fluorescence microscopy studies of the conductive tissue of the heart in acute myocardial infarction in 30 fatal cases are presented. Focal decrease in redox and hydrolytic enzymes, monoaminoxidase, and cholinesterase in elementa of the conduction system as well as changes in the pattern of fluorescence of the specific muscle cells were found. The intensity of metabolic lesions was more pronounced at early stages of myocardial infarction in those cases where the conducting pathways were proximal to the foci of necrosis.

Acid Phosphatase↗

Different activities of energy metabolism enzymes in children's cardiac atria and ventricles.

Tissue samples from the right atrium and ventricle of the same heart were obtained during surgery on 45 children operated on for congenital heart disease (tetralogy of Fallot, ventricular septal defect). Significant enzyme activity differences were found between atrial and ventricular muscle. Aerobic metabolism enzymes (citrate synthase, malate dehydrogenase), with lactate metabolism (lactate dehydrogenase) and fatty acid oxidation (hydroxyacyl-SoA dehydrogenase) showed significantly higher activities in ventricular muscle tissue. In contrast, hexokinase, the enzyme responsible for glucose phosphorylation was significantly higher in the atria. Hence, the right ventricle can utilize and oxidize to the full all the main nutrients (fatty acids, glucose and lactate) while the right atrium utilizes primarily glucose. These atrio-ventricular differences are independent of the type of congenital heart disease and their existence can be presumed in healthy persons, too.

3-Hydroxyacyl CoA Dehydrogenases↗

Glucocorticoids regulate the transcription of glycerol phosphate dehydrogenase in cultured glial cells.

Utilizing a glycerol phosphate dehydrogenase cDNA clone from the rat glioma C6 cell line, we report that the glucocorticoid regulation of glycerol phosphate dehydrogenase gene expression in glial cells occurs at the transcriptional level. Furthermore, our transcription results in vitro, as well as Northern blot analysis, show that the short-chain fatty acid, sodium butyrate, totally blocks this hydrocortisone-induced transcription of glycerol phosphate dehydrogenase, demonstrating that the site of action of this fatty acid resides in the cell nucleus. The C6 glycerol phosphate dehydrogenase cDNA clone (pGPDH-1,1800 base pairs) used in these studies was selected from a C6 library generated from polysomal poly(A)+ RNA. pGPDH-1 hybridized to a 4.7-kilobase RNA from rat muscle and brain, mouse liver, and C6 cells; this mRNA is at least four times larger than the required coding sequence for glycerol phosphate dehydrogenase. Northern blot analysis of developing rat brain reveals a striking increase in glycerol phosphate dehydrogenase transcripts during the period most associated with peak myelination.

Animals↗

[The function of shuttle systems of liver extramitochondrial hydrogen transport in experimental atherosclerosis].

An enzymatic activity of shuttle systems for reduction equivalents transport (malate-aspartate, glycerophosphate, lactate, glutamate and beta-hydroxybutyrate dehydrogenase) was studied spectrophotometrically in liver tissue of intact rabbits and animals with experimental atherosclerosis. Content of malate, oxaloacetate, glutamate, alpha-ketoglutarate, alpha-glycerophosphate, dihydroacetone phosphate, lactate, pyruvate, beta-hydroxybutyrate and acetoacetate were studied. In experimental atherosclerosis coordinated functioning of the enzymes, which participate in the utilization of the cytoplasmic NAD-H2 and the alteration in the ratio of reduced and oxidized metabolites in the systems, were found to be impaired.

Acetoacetates↗

Solubilization and immunochemical identification of mitochondrial glycerol-3-phosphate dehydrogenase.

Lysophosphatidylcholine (contrary to Lubrol WX, Triton X-100, digitonine and deoxycholate) solubilizes hamster brown fat mitochondrial glycerol-3-phosphate dehydrogenase without inactivation. Optimal ratio of lysophosphatidylcholine and membrane protein for solubilization of the enzyme was found to be 0.25 mg of lysophosphatidylcholine per mg protein. The activity of solubilized enzyme, however, was not affected by low concentrations of Lubrol WX, Triton X-100, digitonine, Zwittergent TM 314. Deoxycholate exhibited a pronounced inactivating effect. One-dimensional immunoelectrophoresis of the solubilized membrane proteins revealed 10 protein bands, 3-4 of which exhibited the enzyme activity. Two-dimensional immunoelectrophoresis revealed only a single main band of glycerol-3-phosphate dehydrogenase. This technique thus appears to be the best means for the identification of glycerol-3-phosphate dehydrogenase in the mixture of solubilized membrane proteins and for concentration of the enzyme activity in one major precipitating band.

Adipose Tissue, Brown↗

[Properties of muscle glycerol-3-phosphate dehydrogenase in rabbits with Brown-Pearce carcinoma].

Specific activity of glycerol-3-phosphate dehydrogenase, estimated in direct and reverse reactions, was decreased, as compared with normal state, without any alterations in the ratio of activities, in muscle homogenates of rabbits with Brown-Pearce carcinoma. The enzymatic activity was decreased by an order of magnitude in homogenates of the tumor as compared with muscle homogenates of the tumor-bearing animals simultaneously with relative stimulation of the reverse reaction. Specific activity of the crystalline enzyme from muscles of tumor-bearing animals was decreased as compared with normal tissues but the ratio of activities was 2-fold increased due to the reverse reaction. In the carcinoma activity of one of the three enzyme fractions, obtained after isoelectrofocusing, was similar to the control values; in the second fraction the ratio of activities was decreased due to inhibition of the direct reaction, the third fraction was inactive. Amino acid composition of crystalline glycerol-3-phosphate dehydrogenase, isolated from tumor-bearing animals, was different in content of 5 amino acids from the enzyme amino acid composition of controls, thus suggesting the structural differences of the proteins from these fractions.

Amino Acids↗