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Metabolic effects of galactose on human HepG2 hepatoblastoma cells.

HepG2 cells were used as a model system to study the effects of galactose overload on the liver, a target organ of galactose toxicity in patients suffering from transferase-deficient galactosemia. In the presence of galactose, HepG2 cell growth was slow and the pattern of gene expression remained characteristic of liver cells (secretion of alpha-fetoprotein [AFP] albumin, and transferrin). Galactose-1-phosphate (Gal-1-P) accumulated, as it does in galactosemic cells, but did not affect the energetic status of the cells (no adenosine triphosphate [ATP] depletion). However, the substitution of galactose for glucose as the sole hexose in the medium affected the specific activities of the galactose-metabolizing enzymes. Galactokinase (GALK) activity was decreased, and those of galactose-1-phosphate uridyltransferase (GALT), phosphoglucomutase, and glucose-6-phosphate dehydrogenase (G6PDH) were increased. The conversion of radiolabeled galactose to glucose (CO2 production and glycogen level) was greater in galactose medium than in glucose medium after a 7-day culture. Therefore, the culture of HepG2 cells in galactose medium indicates that the enhanced utilization of this hexose is due to the increased enzyme activities regulating its own metabolism. Hence, HepG2 cells constitute a good model for the study of modulation of galactose-metabolizing enzymes by galactose.

Analysis of Variance↗

Electrophoretic isoenzyme patterns of Entamoeba histolytica and Entamoeba coli.

Cultures of 14 stocks of Entamoeba histolytica and one only of Entamoeba coli were compared by electrophoretic patterns of three enzymes: glucose-phosphate isomerase, phosphoglucomutase and L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating). Easily distinguished patterns divided E. histolytica into three groups, whilst a distinctly different pattern for E. coli was also seen.

Electrophoresis, Starch Gel↗

The differentiation of invasive and non-invasive Entamoeba histolytica by isoenzyme electrophoresis.

Cultures of 85 stocks of Entamoeba histolytica were compared by electrophoretic patterns of three enzymes: glucosephosphate isomerase, phosphoglucomutase and L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating). Four groups of E. histolytica stocks have been distinguished. 18 of these stocks were derived from individual patients with clinical amoebiasis and all of these were of one enzyme group, which did not occur among the other stocks.

Electrophoresis, Starch Gel↗

Enzyme analyses of Bulinus africanus group snails (Mollusca: Planorbidae) from Tanzania.

38 population samples of snails of the Bulinus africanus group, collected from three separate areas of Tanzania, have been examined. Enzymes in crude digestive gland extracts of individual snails have been analysed by isoelectric focusing in polyacrylamide gels. The enzymes studied were: malate dehydrogenase (MDH); phosphoglucomutase (PGM); glucosephosphate isomerase (GPI); acid phosphatase (AcP) and hydroxybutyrate dehydrogenase (HBDH). Samples of B. nasutus were clearly differentiated from other species and enzyme differences were apparent between samples from the lake and coastal areas. Similarly, although clear distinctions could not always be made, samples of B. africanus, B. globosus and B. ugandae were characterized by their enzyme types. Individual variation was detected within populations and the significance of enzyme polymorphisms in relation to identification has been considered. No correlation was found between snail enzyme type and susceptibility to Schistosoma haematobium or S. bovis.

Acid Phosphatase↗

Electrophoretic isoenzyme patterns of the pathogenic and non-pathogenic intestinal amoebae of man.

Cultured stocks of Entamoeba hartmanni, Endolimax nana, Iodamoeba buetschlli and Dientamoeba fragilis were compared with the four Entamoeba histolytical groups already described (SARGEAUNT et al., 1978), by the electrophoretic patterns of three enzymes: glucose phosphate isomerase (GPI), phosphoglucomutase (PGM) and L-malate: NADP+ oxidoreductase (oxalacetate-decarboxylating) (ME). All the species were easily distinguished by their characteristic patterns.

Amoeba↗

Influence of geographical factors in the distribution of pathogenic zymodemes of Entamoeba histolytica: identification of zymodeme XIV in India.

Stocks of Entamoeba histolytica isolated and maintained in a variety of culture media, both axenic and polyxenic, were compared with each other and with strains previously characterized. The present stocks were isolated from subjects living in various cities in India. Using the enzyme patterns of: E C 5319 glucose phosphate isomerase (GPI); E C 11140 L-malate: NADP+ oxidoreductase (oxaloacetate decarboxylating) (ME): E C 2751 phosphoglucomutase (PGM); and E C 2711 hexakinase (HK), developed after electrophoresis, three zymodemes were identified in 54 individual isolations of E. histolytica. Most importantly only one zymodeme associated with pathogenicity occurred, identified 28 times, among the collection, one of which was from a liver abscess.

Adult↗

Enzyme markers for Vibrio cholerae: identification of classical, El Tor and environmental strains.

Enzyme electrophoretic variants were studied in 49 strains of Vibrio cholerae using zymovar analysis. The following seven enzymes were selected for use: alanine dehydrogenase (ADH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucosephosphate isomerase (GPI), 6-phosphogluconate dehydrogenase (6PGDH) and glucose-6-phosphate dehydrogenase (G6PDH). The results indicated the presence of three main groups defined chiefly by their GPI and 6PGDH variants. The first group, defined by possessing the variants GPI-2 and 6PGDH-3, contained all the 01 serovar and E1T or biovar isolates from cholera cases. The second group, defined by possessing the variants GPI-3 and 6PGDH-2, contained all the 01 serovar and classical biovar isolates; the third group was heterogeneous and included the 01 serovar isolates from environmental sources as well as isolates of other serovars (the so called NAGs, non-agglutinable with 01 antisera or NCVs). It is thus now possible to separate the epidemic strains of 01 serovar from other members of this serovar isolated from the environment. Zymovar analysis deals with differences which are a direct expression of the genome and seems to be unaffected by gross phenotypic changes such as smooth-rough variation and phage resistance. It is a promising tool for investigating bacteriological and epidemiological questions, in particular the significance of an environmental reservoir of cholera.

Alanine Dehydrogenase↗

Giardia lamblia: isoenzyme analysis of 19 axenic strains isolated from symptomatic and asymptomatic patients in Mexico.

Infection of the small intestine of humans with the parasitic protozoon Giardia lamblia may have an asymptomatic course, or it may produce acute or chronic diarrhoea. In order to establish if the different clinical outcome of giardiasis in children could be due, at least partially, to strain differences, 19 isolates from asymptomatic and symptomatic cases studied in Mexico City were cultured under axenic conditions and the isoenzyme electrophoretic patterns of 10 different enzymes were compared. Strains from carriers and from symptomatic cases of giardiasis were equally amenable to isolation and axenization. Isoenzyme electrophoresis demonstrated remarkable homogeneity in 7 enzyme patterns for all 19 isolates, except for phosphoglucomutase, for which 3 different zymodemes were found. Therefore, these isolates of G. lamblia, obtained from a single geographical location, tended to be genetically homogeneous. In addition, there were no consistent zymodeme differences between isolates from symptomatic and asymptomatic human infections.

Animals↗

Association of HLA and other genetic markers in South Indian patients with pulmonary tuberculosis.

Histocompatibility antigens (A, B & C loci) and 23 other single gene characters were studied in 204 pulmonary tuberculosis patients belonging to a single endogamous group in South India. None of the previously reported associations with HLA antigens was confirmed, nor any new one found. The blood O and Rh negative associations were also not confirmed, although a new association with the Jk blood group system appears possible. Of particular interest is the association with the phosphoglucomutase (PGM1) system, which parallels that found in a different population located some 1000 km away. Relative risks were calculated to measure the resistance of individuals with the PGM1*2+ allele.

Blood Group Antigens↗

Biochemical polymorphism in Parascaris equorum, Toxocara canis and Toxocara cati.

Vertical starch gel electrophoresis was used to resolve proteins encoded by 18 gene loci in ascaridoid nematodes. Estimates of genetic variability were made from population samples of the dog ascarid (Toxocara canis), cat ascarid (Toxocara cati), and the horse ascarid (Parascaris equorum). Levels of polymorphism and mean heterozygosity were high, which is not consistent with the hypothesis that the intestinal environment selects for monomorphism among endoparasites. Most observed allele frequencies conformed to Hardy-Weinberg equilibrium expectations as tested by chi2 goodness-of-fit, suggesting that the proteins evaluated are inherited in a Mendelian fashion and that these nematodes are mating at random. Subunit structures of the following enzymes, deduced from electrophoretic phenotypes of heterozygotes, corresponded to those of vertebrates: lactate dehydrogenase; malate dehydrogenase; 6-phosphogluconate dehydrogenase; phosphoglucomutase; esterase D; peptidase B; peptidase D; and mannose-6-phosphate isomerase. This observation substantiates the conservative nature of polypeptide subunit number across phylogenetically diverse groups of organisms.

Alcohol Oxidoreductases↗

Enzyme activity in livers from chickens (Gallus domesticus) experimentally infected with the digenetic trematode, Amphimerus elongatus (Trematoda: Opisthorchiidae).

Day old Leghorn chickens were infected with the digenetic trematode Amphimerus elongatus. After different periods of growth, birds were sacrificed and liver enzyme activity determined for six enzymes. Specific activities of adenylate kinase, lactate dehydrogenase, malate dehydrogenase and phosphoglucomutase were significantly lower in infected chickens than in controls. Creatine kinase and glucosephosphate isomerase activities were unaffected by the parasite.

Adenylate Kinase↗

Low blood protein heterozygosity in zoo-bred hamadryas baboons.

An electrophoretic study of erythrocyte allozymes and serum proteins representing 32 genetic loci in 32 hamadryas baboons (Papio H.hamadryas) from Cologne and Frankfurt zoos revealed biallelic polymorphism in phosphoglucomutase (PGM), mannose phosphate isomerase (MPI) and transferrin (Tf). Polymorphism amounted to P = 0.097 over 31 loci and observed heterozygosity to H(o) = 0.011, indicating significant reduction from the respective gene diversity parameters encountered in free-living hamadryas baboons. Polygynous mating reduced numbers of heterozygotes at the Tf and the PGM-II loci significantly below Hardy-Weinberg expectations. This results support the importance of active genetic management in programmes for captive breeding and species conservation.

Alleles↗

Electrophoretic phenotyping of erythrocyte enzymes.

Erythrocyte acid phosphatase (EAP), esterase D (ESD) and phosphoglucomutase (PGM) phenotypes among the erythrocyte enzyme types of blood groups are surveyed and a modified cellulose acetate membrane isoelectric focusing (CAM-IEF) method for their exploration is described. The phenotyping procedures are usually classified as either equilibrium or non-equilibrium IEF. Equilibrium IEF, which is based on differences in pI values, includes three methods: (i) a narrow pH range of carrier ampholytes, (ii) a relatively narrow pH range of carrier ampholytes containing chemical separators and (iii) immobilized pH gradient gels. Among the three methods, immobilized pH gradients provides a better resolution of isozymes. Conversely, the disadvantages of immobilized pH gradients include longer focusing times and complex gel preparations. Moreover, immobilized pH gradients are unsuitable for stain analysis because of the insensitivity of PGM1 detection. A hybrid IEF system and a commercial immobilized pH gradient dry plate have overcome these problems. However, EAP typing is extremely expensive and ESD typing is not well distinguished by hybrid IEF. As each method has both merits and demerits, the most suitable technique should be selected based on the kind of erythrocyte enzyme types and sample conditions. On the other hand, non-equilibrium IEF is a rapid method because isozymes are detected on the basis of their charge differences under non-equilibrium conditions. Moreover, the appropriate addition separators increases the charge difference and provides a good resolution within a shorter time. Addition of more separators produces a narrow pH range in the gel and takes a substantially longer time to reach the optimum pH range for charge difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗

Detection of aldehyde dehydrogenase deficiency in Chachi Indians, Ecuador.

The activity of mitochondrial aldehyde dehydrogenase (ALDH2) was tested by isoelectric focusing of hair root extracts from 50 Chachi Indians (Ecuador). Quality of extracts and the intactness of cytoplasmic and mitochondrial enzymes were ascertained by assaying of phosphoglucomutase (PGM) and malate dehydrogenase (MDH) in the same extracts. Three of the 39 successfully assayed Chachi Indian samples showed virtual absence of the ALDH2 band on the isoelectropherogram, and the control enzymes were stained normally in these subjects. These data confirm the existence of a mitochondrial ALDH deficiency among South American Indians. The molecular origin of the ALDH2 deficiency in this population is unknown.

Adolescent↗

A malic enzyme probe detects cross-hybridizing sequences closely linked to loci encoding other metabolic enzymes.

The cytoplasmic malic enzyme (Mod-1) catalyzes the oxidative decarboxylation of malate: malate + NADP+----pyruvate + CO2 + NADPH + H+. Using a cDNA clone of Mod-1 as a probe, two new DNA markers not at the Mod-1 locus (restriction fragment length polymorphisms, RFLP) were detected by Southern blot analysis that showed extensive homology to Mod-1 sequences. Linkage of each restriction fragment length polymorphism to loci other than Mod-1 was assessed using the BXD (C57BL/6J x DBA/2J) recombinant inbred strains and confirmed by backcrosses. One polymorphic site, designated D9Rti1, was found to be closely linked to the phosphoglucomutase (Pgm-3) locus on Chromosome 9. The other hybridization site, designated D1Rti2, was closely linked to the isocitrate dehydrogenase (Idh-1) locus on Chromosome 1. The data presented imply that Mod-1 homologous sequences are tightly linked to three different metabolic enzymes.

Animals↗

Selective inhibition of metabolic enzymes by enzymatically synthesized D-glucal-6-phosphate.

Yeast hexokinase (EC 2.7.1.1) catalyzes the phosphorylation of D-glucal and methyl alpha- and beta-D-glucopyranosides at 1-5% of the rates of phosphorylation of D-glucose and 2-deoxy-D-glucose. Maltose, cellobiose, D-galactal and tetrahydropyran-2-methanol are not substrates of hexokinase. Enzymatically synthesized D-glucal-6-phosphate inhibits rabbit muscle phosphoglucose isomerase competitively (KI = 1.94 mM) and phosphoglucomutase noncompetitively (KI = 0.122 mM).

Animals↗

Protective effects of allozyme genotype during chemical exposure in the grass shrimp, Palaemonetes pugio.

The primary objective of this research was to determine if the genotype of the enzymes glucose phosphate isomerase (Gpi), phosphoglucomutase (Pgm), or mannose phosphate isomerase (Mpi) are factors affecting survival during acute endosulfan, fluoranthene, and chromium(VI) exposures. Palaemonetes pugio were exposed in the laboratory to 6.3 microg/L endosulfan, 100 mg/L chromium(VI), or 0.6 mg/L fluoranthene. Dead shrimp were removed at approximately 15-30 min intervals and the individual's genotypes for the Gpi, Mpi, and Pgm enzymes were determined. These data were used to establish whether allozyme genotype was related to tolerance as analyzed with non-parametric methods (i.e. development of survival curves using the Kaplan-Meier method of estimation) and parametric methods (i.e. proportional hazards models). Since the sex and/or size of the organism was expected to affect tolerance, these variables were accounted for in the proportional hazards models. Results indicated individuals that were heterozygous (MF) for the Gpi allozyme survived longer and had less overall mortality than the homozygous MM genotype when exposed to chromium(VI) and to fluoranthene. No allozyme genotypes were related to tolerance during the endosulfan exposure when sex was included as a covariate in the analysis. These results support the hypothesis that there is a genetic basis for tolerance in P. pugio during acute exposures to chromium(VI) and to fluoranthene. Although a relationship between Gpi genotype and contaminant tolerance has been identified in previous studies, no such relationship has been documented in P. pugio or with chromium(VI) as the contaminant.

Animals↗

Cellular signaling mediated by calphoglin-induced activation of IPP and PGM.

Universal protein networks conserved from bacteria to animals dictate the core functions of cells. Inorganic pyrophosphatase (IPP) is an essential enzyme that plays a pivotal role in a broad spectrum of cellular biosynthetic reactions such as amino acid, nucleotide, polysaccharide, and fatty acid biosynthesis. However, the in vivo cellular regulation mechanisms of IPP and another key metabolic enzyme, phosphoglucomutase (PGM), remain unknown. This study aimed to examine the universal protein regulatory network by utilizing genome sequences, yeast proteomic data, and phosphoryl-transfer experiments. Here we report a novel human protein, henceforth referred to as calphoglin, which interacts with IPP and activates it. Calphoglin enhances PGM activity through the activated IPP and more directly on its own. Protein structure and assembly, catalytic function, and ubiquitous cellular localization of the calphoglin (-IPP-PGM) complex were conserved among Escherichia coli, yeast, and mammals. In the rat brain, calphoglin mRNA was enriched in the hippocampus and the cerebellum. Further, the linkage of the calphoglin complex to calcium signaling was demonstrated by its interactive co-localization within the calmodulin/calcineurin signaling complex, by Ca(2+)-binding and Ca(2+)-controlled activity of calphoglin-IPP, and by calphoglin-induced enhancement of microsomal Ca(2+) uptake. Collectively, these results suggest that the calphoglin complex is a common mechanism utilized in mediating bacterial cell metabolism and Ca(2+)/calmodulin/calcineurin-dependent mammalian cell activation. This is the first report of an activator of IPP and PGM, a function novel to proteins.

Amino Acid Sequence↗