Descripción: The relationship between antidopaminergic drugs and glucose has not been extensively studied, even though chronic neuroleptic treatment causes hyperinsulinemia in normal subjects or is associated with diabetes in psychiatric patients. We sought to evaluate dopamine D2 receptor (D2R) participation in pancreatic function. Glucose homeostasis was studied in D2R knockout mice (Drd2-/-) mice and in isolated islets from wild-type and Drd2-/- mice, using different pharmacological tools. Pancreas immunohistochemistry was performed. Drd2-/- male mice exhibited an impairment of insulin response to glucose and high fasting glucose levels and were glucose intolerant. Glucose intolerance resulted from a blunted insulin secretory response, rather than insulin resistance, as shown by glucose-stimulated insulin secretion tests (GSIS) in vivo and in vitro and by a conserved insulin tolerance test in vivo. On the other hand, short-term treatment with cabergoline, a dopamine agonist, resulted in glucose intolerance and decreased insulin response to glucose in wild-type but not in Drd2 -/- mice; this effect was partially prevented by haloperidol, a D2R antagonist. In vitro results indicated that GSIS was impaired in islets from Drd2-/- mice and that only in wild-type islets did dopamine inhibit GSIS, an effect that was blocked by a D2R but not a D1R antagonist. Finally, immunohistochemistry showed a diminished pancreatic β-cell mass in Drd2-/-mice and decreasedβ-cell replication in 2-month-old Drd2-/- mice. Pancreatic D2Rs inhibit glucose-stimulated insulin release. Lack of dopaminergic inhibition throughout development may exert a gradual deteriorating effect on insulin homeostasis, so that eventually glucose intolerance develops. Copyright © 2010 by The Endocrine Society.

Descripción: Whereas biosensors have been usually proposed as analytical tools, used to investigate the surrounding media pursuing an analytical answer, we have used a biosensor-like device to characterize the microbial cells immobilized on it. We have studied the kinetics of transport and degradation of glucose at different concentrations and temperatures. When glucose concentrations of 15 and 1.5 mM were assayed, calculated activation energies were 25.2 and 18.4 kcal mol-1, respectively, in good agreement with previously published data. The opportunity and convenience of using Arrhenius plots to estimate the activation energy in metabolic-related processes is also discussed. © 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.

Descripción: Fil:Flexer, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Descripción: Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Descripción: Strains derived from HfrH carrying the arcA2 null mutation exhibit a higher respiratory rate, enhanced glucose consumption, and a more-reduced intracellular redox state than arcA deletion mutants of a different lineage. The phenotype of the arcA2 mutants was due to the presence of a creC constitutive mutation introduced by P1 transduction. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Descripción: 4-O-Substituted D-glucuronic acid derivatives were synthesized from D-glucose in order to study the regioselectivity of sulfation.

Descripción: Asr (for ABA, stress, ripening) genes are exclusively found in the genomes of higher plants, and the encoded proteins have been found localized both to the nucleus and cytoplasm. However, before the mechanisms underlying the activity of ASR proteins can be determined, the role of these proteins in planta should be deciphered. Results from this study suggest that ASR is positioned within the signaling cascade of interactions among glucose, abscisic acid, and gibberellins. Tobacco (Nicotiana tabacum) transgenic lines with reduced levels of ASR protein showed impaired glucose metabolism and altered abscisic acid and gibberellin levels. These changes were associated with dwarfism, reduced carbon dioxide assimilation, and accelerated leaf senescence as a consequence of a fine regulation exerted by ASR to the glucose metabolism. This regulation resulted in an impact on glucose signaling mediated by Hexokinase1 and Snf1-related kinase, which would subsequently have been responsible for photosynthesis, leaf senescence, and hormone level alterations. It thus can be postulated that ASR is not only involved in the control of hexose uptake in heterotrophic organs, as we have previously reported, but also in the control of carbon fixation by the leaves mediated by a similar mechanism. © 2013 American Society of Plant Biologists. All Rights Reserved.

Descripción: Potato (Solanum tuberosum L.) tuber UDP-glucose:protein transglucosylase (UPTG) (EC 2.4.1.112) is involved in the first of a two-step mechanism proposed for protein-bound α-glucan synthesis by catalyzing the covalent attachment of a single glucose residue to an acceptor protein. The resulting glucosylated 38-kilodalton polypeptide would then serve as a primer for enzymic glucan chain elongation during the second step. In the present report, we describe the fast protein liquid chromatography purification of UPTG from a membrane pellet of potato tuber. An apparently close association of UPTG, phosphorylase, and starch synthase was observed under native conditions during different purification steps. Enrichment of a 38-kilodalton polypeptide was found throughout enzyme purification. It is now shown that the purified UPTG, with an apparent molecular mass of 38 kilodaltons, undergoes self-glucosylation in a UDP-glucose- and Mn2+-dependent reaction. Therefore, it is concluded that UPTG is the enzyme and at the same time the priming protein required for the biogenesis of protein-bound α-glucan in potato tuber.

Descripción: The endosperm of the seed of Gleditsia triacanthos L. contains 18.55% of its dry weight as nonreserve, cell-wall carbohydrates. Of this carbohydrate material, comprising mainly mannose, galactose, and glucose, 76.1% was of low-molecular weight or highly hydrophilic. Mannose, galactose, and glucose were also the major sugar components of the polysaccharides extracted with alkali (23.1% of the cell-wall), while the same sugars, with minor amounts of arabinose, form the residues. Methylation analysis of the polysaccharides and the borate-sodium hydroxide residue indicate that the cell walls are built up on a network of galactomannans, with high Man/Gal ratios, reinforced with minor amounts of cellulose.

Descripción: The Saccharomyces cerevisiae UGA4 gene, which encodes the γ-aminobutyric acid (GABA) and δ-aminolaevulinic acid (ALA) permease, is well known to be regulated by the nitrogen source. Its expression levels are low in the presence of a rich nitrogen source but are higher when a poor nitrogen source is used. In addition, GABA can induce UGA4 expression when cells are grown with proline but not when they are grown with ammonium. Although vast amounts of evidence have been gathered about UGA4 regulation by nitrogen, little is known about its regulation by the carbon source. Using glucose and acetate as rich and poor carbon source respectively, this work aimed to shed light on hitherto unclear aspects of the regulation of this gene. In poor nitrogen conditions, cells grown with acetate were found to have higher UGA4 basal expression levels than those grown with glucose, and did not show UGA4 induction in response to GABA. Analysis of the expression and subcellular localization of the transcription factors that regulate UGA4 as well as partial deletions and site-directed mutations of the UGA4 promoter region suggested that there are two parallel pathways that act in regulating this gene by the carbon source. Furthermore, the results demonstrate the existence of a new factor operating in UGA4 regulation. © 2007 SGM.

Descripción: Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating malts which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490- 2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non- gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.

Descripción: Three wild type strains of Rhizobium fredii, USDA 191, USDA 257 and HH 303, do not synthesize in vivo or in vitro beta(1-3), beta(1-6) cyclic glucans, all strains form in vitro and in vivo cyclic beta(1-2) glucans. Approximately 80% of the recovered R. fredii cellular cyclic beta(1-2) glucans were anionic and the substituent was identified as phosphoglycerol. Inner membranes prepared from these R. fredii strains have a beta(1-2) glucan-intermediate-protein with apparent molecular mass undistinguishable from Agrobacterium tumefaciens beta(1-2) glucan intermediate protein. Studies of the degree of polymerization of the oligosaccharides recovered from the protein-intermediate after short pulse incubations with UDP-14C-glucose suggested that the rate limiting step in the biosynthesis of cyclic glucan is cyclization. Kinetic studies revealed that the K(m) for UDP-glucose was 0.33 mM. No difference was detected between the K(m) for initiation/elongation and cyclization reactions. Nodulation studies of a ndvB R. fredii mutant with Mc Call and Peking soybean cultivars, revealed that beta(1-2) glucans do not seem to be required for normal nodule invasion of these soybean cultivars.

Descripción: Pseudomonas sp. 14-3 accumulates polyhydroxybutyrate (PHB) from octanoate, but not from glucose. To elucidate this unusual phenotype, genes responsible for the synthesis of PHB were cloned and analyzed. A PHB polymerase gene (phaC) was found downstream from genes coding for a β-ketothiolase (phaA), an acetoacetyl-coenzyme A reductase (phaB) and a putative transcriptional regulator (phaR). All genes were similar to pha genes from several related species, but differences were observed in the distal region of phaA. Complementation with heterologous β-ketothiolase genes from Azotobacter sp. FA8 or Pseudomonas putida GPp104 restored the capability of Pseudomonas sp. 14-3 to synthesize PHB from glucose, demonstrating that its β-ketothiolase was nonfunctional. Analysis of the genome sequences of other Pseudomonas species has revealed the existence of putative β-ketothiolase genes. The functionality of one of these thiolase genes, belonging to P. putida GPp104, was experimentally demonstrated. Pseudomonas sp. 14-3 is the first natural phaA mutant described, that despite this mutation accumulates high amounts of PHB when growing on fatty acids. © 2006 Federation of European Microbiological Societies.

Descripción: Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O2 and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ΔarcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of D-glucose catabolic fluxes, different from that observed in the ΔarcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, D-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD+ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes. © 2012, American Society for Microbiology.

Descripción: Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Descripción: UDP-Glucose:glycoprotein glucosyltransferase (UGGT) is a central component of the endoplasmic reticulum (ER) glycoprotein-folding quality control system, which prevents the exit of partially folded species. UGGT activity can be regulated by the accumulation of misfolded proteins in the ER, a stimulus that triggers a complex signaling pathway known as unfolded protein response (UPR) which is closely associated with inflammation and disease. In this work, we investigated the effect of progesterone (P4) on the expression and activity of UGGT in a mouse hybridoma. We detected the expression of two UGGT isoforms, UGGT1 and UGGT2, and demonstrated that both isoforms are active in these cells. Interestingly, the expression of each isoform is regulated by high physiological P4 concentrations. This work provides the first evidence of a hormonal regulation of UGGT isoform expression and activity, which might influence the glycoprotein quality control mechanism. These findings could contribute to the study of pathologies triggered by the accumulation of misfolded proteins. © 2013 Elsevier B.V.

Descripción: Single-cell cytoplasm sap (1-10 pL) was extracted by using a pressure probe glass microcapillary tip from tulip leaf and bulb and analyzed by UV-MALDI-TOF MS for free underivatized carbohydrate content. Three matrices including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and carbon nanotubes (CNTs) in positive ion mode were selected for analysis because of acceptable carbohydrate-related signal reproducibility. Disaccharide and oligosaccharide (up to 15 Hex when THAP was used, 11 Hex with DHB, and 7 Hex with CNTs) were detected in tulip bulb cell cytoplasm sample. When DHB was used as matrix, neutral carbohydrates were more abundantly detected as sodiated cations; the sugar-related signals, however, appeared as dominant potassiated cations when THAP and CNTs were used. Small amount of monosaccharide was also detected in bulb cell cytoplasm with CNTs as matrix. UV-MALDI-TOF MS of leaf cell extract resulted in high-resolution detection of hexose and disaccharide with DHB, THAP, and CNTs. © 2008 American Society for Mass Spectrometry.

Descripción: Although the existence of O-linked oligosaccharide residues in glycoproteins of Plasmodium falciparum has been shown, the existence of N- linked glycoproteins is still a matter of controversy and skepticism. This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase®, oligosaccharides were released. It was possible to identify an N-linked glycoprotein of >200 kDa in the ring stage and also N-linked glycoproteins in the range of 200-30 kDa in the trophozoite stage. Treatment of trophozoites with 12 μM tunicamycin inhibited differentiation to the schizont stage. To our knowledge, this is the first report in the literature unequivocally showing N-linked glycoproteins in trophozoites of P. falciparum as well as their importance for the differentiation of the intraerythrocytic stages of this parasite.

Descripción: Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for β-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Raktonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or oclanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chuin-length hydroxyalkanoates into PHB.