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Isozyme characterization of cattle (Bos taurus) and American buffalo (Bison bison) cell cultures.

Four Bovidae cell lines (BEK-1, MDBK, Bu and EBTr) were characterized by means of enzymatic biochemical markers. Out of 15 enzymatic systems, 3--adenosine deaminase (Ada), phosphoglucomutase (Pgm) and nucleoside phosphorylase (Np)--were found to be polymorphic and quite suitable for biochemical identification of each cell line. The Bu cell line has shown a Np phenotypic pattern which could be distinctive of the Bison bison species.

Adenosine Deaminase↗

In vivo differentiation of leukaemic blasts and effect of low dose Ara-C in a marrow grafted patient with leukaemic relapse.

The possible induction of differentiation of leukaemic cells by low dose cytosine arabinoside (Ara-C) was studied in an human chimaera with leukaemic relapse. A 35-year-old woman had been grafted with bone marrow from her HLA identical sister and developed a leukaemic relapse of the host type demonstrated by typing phosphoglucomutase 1 isoenzymes. Before treatment with low dose Ara-C only leukaemic cells and a part of the mature granulocytes were of host type. Lymphocytes, platelets and erythrocytes were still of donor type. The leukaemic cells and the mature host type granulocytes disappeared and the patient went into complete remission after treatment with low dose Ara-C. The results indicate that (a) the leukaemic blasts could differentiate in vivo into mature granulocytes, (b) leukaemic transformation was limited to granulopoiesis, and (c) low dose Ara-C acted as a cytotoxic drug instead of inducing differentiation of host type granulopoiesis.

Adult↗

Location of genes on chromosome arms in the Anopheles gambiae group of species and their correlation to linkage data for other anopheline mosquitoes.

The use of paracentric inversions as genetic markers in the Anopheles gambiae group of mosquitoes is described. The gene for dieldrin resistance is assigned to chromosome 2 which in turn is correlated to the previous assignment of the gene to linkage group II. The locus of the enzyme phosphoglucomutase 2 (Pgm 2) is similarly assigned to chromosome 2 and evidence is presented for possible linkage between Pgm 2 and dieldrin resistance. There was no linkage or correlation of chromosome 2 and loci of the enzymes superoxide dismutase (Sod) and octanol dehydrogenase (Odh). These genes are therefore assumed to be on chromosome 3 (linkage group III). Evidence that such gene linkage group/chromosome correlations may extend to other species for which chromosome maps and homologies have been worked out is discussed.

Alcohol Oxidoreductases↗

The distribution of polymorphic traits in atopic asthmatic patients.

The phenotypic distribution and gene frequencies of haptoglobin (Hp), transferrin (Tf), group specific component (Gc), cholinesterase (Cho E2), and alpha1-antitrypsin (Pi) in plasma proteins, and phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase ((6-PGD), esterase D (Es D), phosphohexose isomerase (PHI), adenosine deaminase (ADA) and acid phosphatase (AcP) in red cells were studied in 127 atopic, asthmatic patients. The gene frequencies were compared with normal groups. The phenotypic distribution of the Pi system in atopic patients was somewhat different from the normal. No significant differences were found between the two groups in protein systems or in enzyme systems, except Pi systems. In conclusion, except for the Pi system, no definite association between polymorphic traits and atopic asthma was found in this study.

Acid Phosphatase↗

Orientation of major histocompatibility (MHC) genes relative to the centromere of human chromosome 6.

Linkage analysis of the data obtained from a three-generation Dutch family segregating for genetic variants of centromeric heterochromatic region in the band p11 on chromosome 6 (6ph), major histocompatibility (MHC) genes HLA-A, HLA-B and HLA-C, glyoxalase I (GLO) and phosphoglucomutase-3 (PGM3) showed that the genetic distance between the HLA gene cluster and 6ph is about 6 cM (at an estimated peak lod score of 3.466), that GLO is closer than HLA to the centromere and that PGM3 is probably not situated on the same chromosomal arm as HLA.

Centromere↗

Application of a computer program for the mapping of a gene locus to the disputed PGM3 localization on human chromosome 6.

The program GENEMAP, which performs the computations of a model for the mapping of a gene locus (Baur & Spitzer 1978), is applied to the localization of the phosphoglucomutase 3 with family data as published by Lamm et al. (1972), Rittner & Kalbheim (1975), Albert et al. (1977) and Kömpf et al. (1977). On the basis of Lamm's recombination fractions the combined odds were 13 : 1 in favor of an orientation of PGM3 to the HLA--B-side.

Chromosome Mapping↗

Synthesis of deoxyglucose-1-phosphate, deoxyglucose-1,6-bisphosphate, and other metabolites of 2-deoxy-D-[14C]glucose in rat brain in vivo: influence of time and tissue glucose level.

When the kinetics of interconversion of deoxy[14C]glucose ([14C]DG) and [14C]DG-6-phosphate ([14C]DG-6-P) in brain in vivo are estimated by direct chemical measurement of precursor and products in acid extracts of brain, the predicted rate of product formation exceeds the experimentally measured rate. This discrepancy is due, in part, to the fact that acid extraction regenerates [14C]DG from unidentified labeled metabolites in vitro. In the present study, we have attempted to identify the 14C-labeled compounds in ethanol extracts of brains of rats given [14C]DG. Six 14C-labeled metabolites, in addition to [14C]DG-6-P, were detected and separated. The major acid-labile derivatives, DG-1-phosphate (DG-1-P) and DG-1,6-bisphosphate (DG-1,6-P2), comprised approximately 5 and approximately 10-15%, respectively, of the total 14C in the brain 45 min after a pulse or square-wave infusion of [14C]DG, and their levels were influenced by tissue glucose concentration. Both of these acid-labile compounds could be synthesized from DG-6-P by phosphoglucomutase in vitro. DG-6-P, DG-1-P, DG-1,6-P2, and ethanol-insoluble compounds were rapidly labeled after a pulse of [14C]DG, whereas there was a 10-30-min lag before there was significant labeling of minor labeled derivatives. During the time when there was net loss of [14C]DG-6-P from the brain (i.e., between 60 and 180 min after the pulse), there was also further metabolism of [14C]DG-6-P into other ethanol-soluble and ethanol-insoluble 14C-labeled compounds. These results demonstrate that DG is more extensively metabolized in rat brain than commonly recognized and that hydrolysis of [14C]DG-1-P can explain the overestimation of the [14C]DG content and underestimation of the metabolite pools of acid extracts of brain. Further metabolism of DG does not interfere with the autoradiographic DG method.

Animals↗

Electrophoretic isoenzyme patterns of Entamoeba histolytica and Entamoeba chattoni in a primate survey.

Stocks of Entamoeba histolytica grown in a monoxenic culture system from the feces of nonhuman primates are compared with the eleven zymodemes of E. histolytica so far demonstrated from man. In a similar fashion, Entamoeba chattoni has also been grown and identified. Both E. histolytica and E. chattoni have been demonstrated in keepers of the primate collections. Comparisons have been made using the electrophoretic patterns of three enzymes: glucosephosphate isomerase [(GPI) E.C.5.3.1.9], phosphoglucomutase [(PGM) E.C.2.7.5.1], and L-malate--NADP+ oxidoreductase (oxaloacetate-decarboxylating) [(ME) E.C.1.1.1.40]. Enzyme patterns of E. histolytica from the apes were found to be identical with three of those already demonstrated from man. The enzyme pattern of E. chattoni was distinctly different from that of any of the E. histolytica zymodemes. Other protozoa found in the single fecal sample examined from each subject are also listed.

Animals↗

Trypanosoma cruzi from the Paraguayan Chaco: isoenzyme profiles of strains isolated at Makthlawaiya.

At Makthlawaiya, in the Paraguayan Chaco, the prevalence of Trypanosoma (Schizotrypanum) cruzi infection among both domestic Triatoma infestans and domestic dogs was 38%, and IgG anti-T. cruzi antibody was detected by the quantitative enzyme-linked immunosorbent assay (ELISA) in 80% (105/133) of human sera. Ninety percent (25/28) of T. cruzi strains isolated from both T. infestans and dogs showed heterozygous isoenzyme profiles for glucose phosphate isomerase, phosphoglucomutase and 6-phosphogluconate dehydrogenase. These strains appeared to be closely related to Bolivian zymodeme 2. Three Paraguayan T. cruzi strains showed homozygous isoenzyme profiles, similar to those of major Brazilian zymodemes. It was concluded that T. cruzi strains with heterozygous isoenzyme profiles predominate in domestic transmission cycles in this highly endemic area of the Paraguayan Chaco.

Animals↗

A fast electrophoretic isoenzyme technique for the identification of invasive and non-invasive Entamoeba histolytica and "E. histolytica-like" organisms.

Cellulose acetate electrophoresis (CAE) was used to separate glucosephosphate isomerase, hexokinase, malic enzyme, and phosphoglucomutase extracted from invasive and non-invasive Entamoeba histolytica and "E. histolytica-like" organisms. Each of these morphologically similar organisms possessed a unique CAE isoenzyme profile that can be used as an aid in their identification. The CAE technique used to obtain these isoenzyme profiles is rapid, simple, and economical, and it requires neither specialized training nor elaborate equipment.

Animals↗

Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae.

Yeast cells lacking UDP-Glc pyrophosphorylase (UGPase) encoded by UGPI are not viable. Two strategies were developed to drastically reduce the intracellular concentration of UDP-Glc in order to study the consequences of this metabolic engineering on physiology and morphology. Firstly, UGP1 was placed under the strongly regulatable THI4 promoter. This resulted in a 95% reduction of UGPase activity in the presence of thiamine. The phenotypic effects of this reduction were slightly stronger than those of glucose on the GALI0/CYC1-UGP1 gene fusion [Daran et al. (1995) Eur. J. Biochem. 230, 520-530]. A further reduction of flux towards UDP-Glc was achieved by deletion of the two phosphoglucomutase genes in the ugp1 conditional strain. The growth of this new mutant strain was hardly affected, while it was extremely sensitive to cell wall interfering drugs. Surprisingly, UDP-Glc levels were reduced only by 5-fold, causing a proportional decrease in both glycogen and beta-glucans. Taken altogether, these results indicate that a few percent of enzymatic activities leading to the formation of UDP-Glc appears sufficient to provide the UDP-Glc demands required for cell viability, and that the loss of function of UGP1 is lethal mainly because of the inability of yeast cells to properly form the cell wall.

Cell Wall↗

The Pseudomonas aeruginosa algC gene product participates in rhamnolipid biosynthesis.

Pseudomonas aeruginosa produces exoproducts correlated with its pathogenicity. One of these virulence-associated traits is the surfactant rhamnolipid. The production of alginate and lipopolysaccharide (LPS) are also of importance for P. aeruginosa virulence. The product of the algC gene (which is involved in alginate production through its phosphomannomutase activity and in LPS synthesis through its phosphoglucomutase activity) participates in rhamnolipid production, presumably catalyzing the first step in the deoxy-thymidine-diphospho-L-rhamnose (dTDP-L-rhamnose) pathway, the conversion of glucose-6-phosphate to glucose-1-phosphate. Other structural alg genes, encoded in the alg operon, are not involved in rhamnolipid nor LPS production. These results show that the AlgC protein plays a central role in the production of the three P. aeruginosa virulence-associated saccharides: alginate, LPS and rhamnolipid.

Glycolipids↗

Activity of the enzymes involved in the synthesis of exopolysaccharide precursors in an overproducing mutant ropy strain of Streptococcus thermophilus.

The activities of some enzymes belonging to the Leloir pathway, phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase and galactose 1-P uridyl transferase, were studied in a wild ropy, a non-ropy and an overproducing mutant ropy strain of Streptococcus thermophilus. These activities were assayed over successive culture transfers along with exocellular polysaccharide (EPS) production. The overproducing mutant ropy strain showed increments in polysaccharide production over successive culture transfers, as opposed to reductions in production by the wild ropy strain. The observed variations among strains in the enzyme activities that were analysed in relation to EPS production suggest their involvement in the synthesis of sugar-nucleotide EPS precursors.

Gene Expression Regulation, Bacterial↗

The pentacovalent phosphorus intermediate of a phosphoryl transfer reaction.

Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated beta-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme-Mg(II)-glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate.

Binding Sites↗

HeLa cultures defined.

A list is presented of references to all known publications on properties which have served to relate strains of HeLa cells to each other as well as to indict other purported human cell lines as HeLa cell contaminants. Eleven additional cell lines not previously indicted are described. When they exhibit (i) type A (fast) mobility for glucose-6-phosphate dehydrogenase, (ii) phosphoglucomutase type 1 at locus 1 and locus 3, (iii) absence of a Y chromosome by fluorescent staining, and (iv) possession of a complex of trypsin-Giemsa banded marker chromosomes present in known HeLa cells, then cell substrates regardless of designation should be considered de facto strains of HeLa.

Cell Line↗

Genetic mapping of the ecotropic murine leukemia virus-inducing locus of BALB/c mouse to chromosome 5.

By means of an approach that combined the techniques of somatic cell genetics and Mendelian breeding studies, the inducibility locus, designated Cv, for ecotropic murine leukemia virus in BALB/c mice, was mapped to chromosome 5, 23 units from the locus for phosphoglucomutase-1, with gene order Cv-Pgm-1-Gus. This low-efficiency inducibility locus is therefore not allelic with the chromosome 7 loci previously described for two other mouse strains with high virus inducibility. These studies provide further evidence that endogenous ecotropic viruses represent viral genomes inserted at different chromosomal sites in the various mouse strains.

Animals↗

Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor.

The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in Streptomyces coelicolor. Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by zwf2 plays a more important role than that encoded by zwf1 in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The pgm-deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in S. coelicolor.

Acetyl-CoA Carboxylase↗

Enhanced exopolysaccharide production by metabolic engineering of Streptococcus thermophilus.

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal(+)) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal(+) strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal(+) strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal(+) strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.

Base Sequence↗