Enzymes Glutamate Dehydrogenase Research Articles

Abstract A17: Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth

Abstract How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1), commonly upregulated in human cancers, is predominantly important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate (a-KG) and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a ROS scavenging enzyme glutathione peroxidase 1 (GPx1) to regulate redox homeostasis, which provides a proliferative advantage to cancer cells and tumor growth. Our findings not only provide novel insights into understanding of the role of glutaminolysis in redox homeostasis but also suggest a novel signaling function of fumarate that regulates GPx1, allowing additional crosstalk between glutaminolysis, TCA cycle and redox status. Targeting GDH1 by shRNA or a newly identified small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth. Thus, our findings provide proof-of-principle suggesting GDH1 as a promising therapeutic target in the treatment of human cancers associated with elevated glutamine metabolism. Citation Format: Lingtao Jin, Dan Li, Gina Alesi, Jun Fan, Hee-Bum Kang, Lu Zhou, Titus Boggon, Peng Jin, Robert Egnatchik, Ralph DeBerardinis, Kelly Magliocca, Chuan He, Martha Arellano, Hanna Khoury, Dong Shin, Fadlo Khuri, Sumin Kang. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A17.

Abstract 3046: Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth

Abstract How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1), commonly upregulated in human cancers, is predominantly important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate (a-KG) and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a ROS scavenging enzyme glutathione peroxidase 1 (GPx1) to regulate redox homeostasis, which provides a proliferative advantage to cancer cells and tumor growth. Our findings not only provide novel insights into understanding of the role of glutaminolysis in redox homeostasis but also suggest a novel signaling function of fumarate that regulates GPx1, allowing additional crosstalk between glutaminolysis, TCA cycle and redox status. Targeting GDH1 by shRNA or a newly identified small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth. Thus, our findings provide proof-of-principle suggesting GDH1 as a promising therapeutic target in the treatment of human cancers associated with elevated glutamine metabolism. Citation Format: Lingtao Jin, Dan Li, Gina Alesi, Jun Fan, Hee-Bum Kang, Lu Zhou, Titus Boggon, Kelly Magliocca, Chuan He, Martha Arellano, Hanna Khoury, Dong Shin, Fadlo Khuri, Sumin Kang. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3046. doi:10.1158/1538-7445.AM2015-3046

Long-term reticuloruminal pH dynamics and markers of liver health in early-lactating cows of various parities fed diets differing in grain processing

The present study aimed to investigate the long-term effect of feeding barley grain steeped in lactic acid (La) with or without thermal treatment on reticuloruminal pH dynamics and metabolic activity of the liver in 12 primiparous and 18 multiparous early-lactating dairy cows. All cows were included on d 21 postpartum and sampled until d 90 postpartum. Cows were fed a diet based on differently processed ground barley grain: untreated grain (control diet, CON), or grain treated with 1% La alone for 24 h before feeding (La), or with an additional oven-heating at 55°C for 12 h (LaH). The reticuloruminal pH and temperature were measured via indwelling sensors that allowed for continuous (every 10min) and long-term measurement from d 21 to 80 postpartum. Blood samples were taken on d 21, 40, and 90 of lactation and analyzed for liver enzymes aspartate aminotransferase (AST), gamma-glutamyltransferase, and glutamate dehydrogenase, as well as bilirubin, bile acids, and serum amyloid A. Dry matter intake was higher in multiparous cows (20.7±0.27 kg/d) compared with primiparous cows (18.2±0.33 kg/d), but was not affected by dietary treatment. Overall, the relatively short duration (51±5min/d) of reticuloruminal pH <5.8 suggests low risk of subacute ruminal acidosis throughout the experiment. Results indicated that La treatment of barley, with or without heat, lowered the time duration of pH <5.8 compared with CON, but only in primiparous cows (from 118±13 to 46±11 and 25±11min/d for CON, La, and LaH, respectively). In multiparous cows, the opposite effect of feeding the La-treated barley on time duration of pH <5.8 (11±8 vs. 46±9 vs. 57±9min/d for CON, La, and LaH, respectively) was observed. Multiparous cows generally showed higher pH readings and shorter periods in which the ruminal pH dropped below the threshold of pH 5.8. The reticuloruminal temperature was not affected by dietary treatment, whereas parity affected the time duration of reticuloruminal temperature >39.5°C, being 60±19min/d shorter in primiparous cows. The measured activities of the liver enzymes AST, gamma-glutamyltransferase, and glutamate dehydrogenase, as well as bilirubin, bile acids, and the acute phase protein serum amyloid A, were not affected by grain feeding. Additionally, only one small effect of parity on investigated serum variables was noticed, showing slightly but significantly higher values of AST in multiparous (80.5±1.4 U/L) compared with primiparous cows (76.0±1.7 U/L). In conclusion, our results indicate greater risk for primiparous cows to develop subacute ruminal acidosis-like conditions during early lactation than multiparous cows. The study also suggests limited benefits of feeding processed barley grain with La with or without thermal treatment to modulate ruminal tolerance of grain feeding, whereby differing effects in primiparous cows were observed compared with multiparous cows.

Allosteric regulation: Illuminating allosteric conformational change with an environmentally sensitive fluorescent probe

Allosteric regulation was a hot topic in the 1960s, but there was very limited structural data on allosteric equilibria, and no solid information on the rates of allosteric conformational changes. In this Biochemical Journal Classic paper from 1969 George Radda and his first D.Phil. student, George Dodd determined the rate of allosteric transition in the regulatory enzyme glutamate dehydrogenase by a method new in the 1960s, the fluorescence of an environmentally sensitive extrinsic probe.

Development of a novel reagentless, screen-printed amperometric biosensor based on glutamate dehydrogenase and NAD+, integrated with multi-walled carbon nanotubes for the determination of glutamate in food and clinical applications

A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola's Blue (MB) has been investigated as the base transducer for a reagentless glutamate biosensor. The biopolymer chitosan (CHIT) and multiwalled carbon nanotubes (MWCNTs) were used to encapsulate the enzyme glutamate dehydrogenase (GLDH) and the co-factor nicotinamide adenine dinucleotide (NAD+).The biosensor was fabricated by sequentially depositing the components on the surface of the transducer (MB-SPCE) in a layer-by-layer process, details of which are included in the paper. Each layer was optimised to construct the reagentless device.The biosensor was used in conjunction with amperometry in stirred solution using an applied potential of +0.1V (vs. Ag/AgCl). Optimum conditions for the analysis of glutamate were found to be: temperature, 35°C; phosphate buffer, pH 7 (0.75mM, containing 0.05M NaCl).The linear range of the reagentless biosensor was found to be 7.5–105μM, and limit of detection was found to be 3μM (based on n=5, CV: 8.5% based on three times signal to noise) and the sensitivity was 0.39nA/μM (±0.025, coefficient of variation (CV) of 6.37%, n=5). The response time of the biosensor was 20–30s.A food sample was analysed for monosodium glutamate (MSG). The endogenous content of MSG was 90.56mg/g with a CV of 7.52%.The reagentless biosensor was also used to measure glutamate in serum. The endogenous concentration of glutamate was found to be 1.44mM (n=5), CV: 8.54%. The recovery of glutamate in fortified serum was 104% (n=5), CV of 2.91%.

Structure-assisted ligand-binding analysis using fluorogenic photoaffinity labeling.

Photoaffinity labeling (PAL) technique using a fluorogenic cross-linker is used to monitor the nucleotide-binding pocket within a protein. A coumarin fluorophore formed in the binding domain due to ultraviolet (UV) irradiation has been shown to accelerate the sequencing of the labeled peptide as well as identification of the labeled site by liquid chromatography (LC)-tandem mass spectrometry (MS), in addition to providing information on the ligand binding state. Selective monitoring of the predefined fluorescence peaks among the numerous digests obtained from high performance liquid chromatography (HPLC) clearly indicates the binding capability of the ligand to the entire protein as well as to the corresponding binding domain under various conditions. In the current study, ligand-binding analysis confirmed by the structural information of the binding state has been demonstrated using fluorogenic ATP/ADP photoactivatable probes under allosteric regulation of multiple substrates in the enzyme glutamate dehydrogenase (GDH).

Molecular mechanisms of congenital hyperinsulinism

Congenital hyperinsulinism (CHI) is a complex heterogeneous condition in which insulin secretion from pancreatic β-cells is unregulated and inappropriate for the level of blood glucose. The inappropriate insulin secretion drives glucose into the insulin-sensitive tissues, such as the muscle, liver and adipose tissue, leading to severe hyperinsulinaemic hypoglycaemia (HH). At a molecular level, genetic abnormalities in nine different genes (ABCC8, KCNJ11, GLUD1, GCK, HNF4A, HNF1A, SLC16A1, UCP2 and HADH) have been identified which cause CHI. Autosomal recessive and dominant mutations in ABCC8/KCNJ11 are the commonest cause of medically unresponsive CHI. Mutations in GLUD1 and HADH lead to leucine-induced HH, and these two genes encode the key enzymes glutamate dehydrogenase and short chain 3-hydroxyacyl-CoA dehydrogenase which play a key role in amino acid and fatty acid regulation of insulin secretion respectively. Genetic abnormalities in HNF4A and HNF1A lead to a dual phenotype of HH in the newborn period and maturity onset-diabetes later in life. This state of the art review provides an update on the molecular basis of CHI.

Glutamate Dehydrogenase 1 Signals through Antioxidant Glutathione Peroxidase 1 to Regulate Redox Homeostasis and Tumor Growth

How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a reactive oxygen species scavenging enzyme glutathione peroxidase 1. Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth.

Effects of ammonium salts on oleaster (Elaeagnus angustifolia)

Oleaster (Russian olive, Elaeagnus angustifolia) trees are highly tolerant against a variety of abiotic stresses (water, temperature, salt, and other chemicals). Therefore, they can be used for rehabilitation of contaminated and/or low quality soils (brownfields, dump sites, wastelands, etc.). In order to study responses of oleaster to environmental stress in vivo and in vitro, we successfully sterilized and initiated its callus cultures, regenerated shoots and roots and finally whole plants from the callus. Application of ammonium (in the form of sulfate salt) to the regenerated plantlets at concentrations higher than 10 mg L-1 inhibited root growth, reduced the leaf chlorophyll content and the activity of the enzyme glutamate dehydrogenase. At the same time, it induced activities of the stress marker enzyme glutathione S-transferase in the root and shoot tissues of the plant.

In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity

Glutamate dehydrogenase (GDH) is an NAD(H) dependent enzyme that catalyzes, in vitro, the reversible amination of glutamate. Here we describe how to determine spectrophotometrically GDH activity monitoring NADH evolution. This protocol is described here for Arabidopsis thaliana (A. thaliana) although it is also valid for other plant species. GDH protein is a hexamer composed, in the case of Arabidopsis, of a combination of GDHα, GDHβ and GDHγ subunits. Every combination of subunits is possible; however, it is still barely known whether different combinations affect the enzymatic properties of the hexamers. In other species, hexamers are a combination of GDHα and GDHβ but it cannot be discarded the existence of other genes since for instance GDHγ subunit in Arabidopsis was described in Fontaine et al. (2012).Glutamate + NAD+ + H+ → 2-Oxoglutarate + NADH + NH4+

Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris

Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris.

Interaction of islet α-cell and β-cell in the regulation of glucose homeostasis in HI/HA syndrome patients with the GDH(H454Y) mutation.

The hyperinsulinemia/hyperammonemia (HI/HA) syndrome—the secondmost common form of congenital hyperinsulinism—is a rare autosomal dominant disease manifested by hypoglycemic symptoms and elevated serum ammonia triggered by fasting or high-protein meals (1). In 1955, Cochrane et al. described a child and her father, both with hypoglycemia that was aggravated by consumption of a low-carbohydrate, high-protein diet (2). Subsequently, another group identified the gene GLUD1. This gene, located on chromosome 10q23.3, is composed of 13 exons and regulates mitochondrial enzyme glutamate dehydrogenase (GDH) (3). The GDH enzyme catalyzes glutamate metabolism and plays important roles in the regulation of amino acid−stimulated insulin secretion in β-cells, modulation of amino acid catabolism in hepatocytes, and ammoniagenesis in the brain (4). A total of 14 amino acid residues affected by GDH-activating mutations has been identified in patients with the HI/HA syndrome (5). GDH activity also is subject to complex regulation by GTP, ADP, and leucine (6). For example, the flux of glutamate into the tricarboxylic acid cycle for energy generation is modulated by the mitochondrial energy potential, which, in turn, is controlled by the ratio of GTP to ADP. When the energy potential is high, amino acid oxidation is not required, and GDH enzyme activity shuts down. When energy potential is low, GDH is activated to sustain energy generation through oxidation of amino acids (4). Interestingly, epigallocatechin gallate, a component of green tea, has been shown to be a potent allosteric inhibitor of GDH enzyme activity (7). Insulin secretion is upregulated through increased cellular phosphate energy potential, which is manifested as an increase in the ATP/ADP ratio. Elevated ATP/ADP concentrations …

Biocatalytic photosynthesis with water as an electron donor.

Efficient harvesting of unlimited solar energy and its conversion into valuable chemicals is one of the ultimate goals of scientists. With the ever-increasing concerns about sustainable growth and environmental issues, numerous efforts have been made to develop artificial photosynthetic process for the production of fuels and fine chemicals, thus mimicking natural photosynthesis. Despite the research progress made over the decades, the technology is still in its infancy because of the difficulties in kinetic coupling of whole photocatalytic cycles. Herein, we report a new type of artificial photosynthesis system that can avoid such problems by integrally coupling biocatalytic redox reactions with photocatalytic water splitting. We found that photocatalytic water splitting can be efficiently coupled with biocatalytic redox reactions by using tetracobalt polyoxometalate and Rh-based organometallic compound as hole and electron scavengers, respectively, for photoexcited [Ru(bpy)3](2+). Based on these results, we could successfully photosynthesize a model chiral compound (L-glutamate) using a model redox enzyme (glutamate dehydrogenase) upon in situ photoregeneration of cofactors.

Ammonium metabolism enzymes aid Helicobacter pylori acid resistance.

The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH3 that is immediately protonated to form NH4 (+). This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg(2+) at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium.

Mitochondrial SIRT3 and its target glutamate dehydrogenase are altered in follicular cells of women with reduced ovarian reserve or advanced maternal age.

Is the activity of sirtuin 3 (SIRT3) altered in granulosa and cumulus cells from young women with reduced ovarian reserve or women of advanced maternal age? SIRT3 mRNA and active protein in granulosa and cumulus cells were decreased in women with reduced ovarian reserve and advanced maternal age. Young women with reduced ovarian reserve or women of advanced maternal age have reduced oocyte viability, possibly due to altered granulosa and cumulus cell metabolism. The mitochondrial SIRT3 protein may be implicated in these processes as it is able to sense the metabolic state of the cell and alter mitochondrial protein function post-translationally. This is a prospective cohort study, in which women (n = 72) undergoing routine IVF/ICSI were recruited and allocated to one of three cohorts based on age and ovarian reserve (as assessed by serum anti-Mullerian hormone level). Women were classified as young (≤35 years) or of advanced maternal age (≥40 years). Granulosa and cumulus cells were collected. SIRT3 mRNA and protein levels and protein activity was analysed in granulosa and cumulus cells via quantitative PCR, immunohistochemistry and western blotting, and deacetylation activity, respectively. Activity of the glutamate dehydrogenase (GDH) enzyme, a known target of SIRT3, was assessed, and acetylated proteins in mitochondria isolated from granulosa and cumulus cells were separated by immunoprecipitation and acetylation of GDH assessed by western blotting. Data for women with good prognosis (young women with normal ovarian reserve) were compared with those from young women with reduced ovarian reserve and those of advanced maternal age. SIRT3 mRNA and active protein were present in granulosa and cumulus cells and co-localized to the mitochondria. SIRT3 mRNA in granulosa cells was decreased in young women with reduced ovarian reserve and advanced maternal age versus young women with normal ovarian reserve (P < 0.05). SIRT3 mRNA in cumulus cells was decreased in women of advanced maternal age versus young women with normal ovarian reserve only (P < 0.05). Granulosa cell GDH activity was decreased in young women with reduced ovarian reserve and in women of advanced maternal age (P < 0.05), whereas cumulus cell GDH activity was reduced in the advanced maternal age group only (P < 0.05). The acetylation profile of GDH in mitochondria revealed increased acetylation of GDH in granulosa and cumulus cells from women of advanced maternal age (P < 0.05) while young women with reduced ovarian reserve had increased GDH acetylation in granulosa cells only (P < 0.05). Although patients were allocated to groups based on maternal age and ovarian reserve and matched for BMI, other maternal factors may also alter the 'molecular health' of ovarian cells. Our data suggest that SIRT3 post-translational modification of mitochondrial enzymes in human granulosa and cumulus cells may regulate GDH activity, thus altering the metabolic milieu surrounding the developing oocyte. Owing to the association between the decline in oocyte quality and pregnancy rates in women of advanced maternal age and the possible association with reduced ovarian reserve, knowledge of perturbed SIRT3 function in granulosa and cumulus cells may lead to novel therapies to improve mitochondrial metabolism in the oocyte and follicular cells in women undergoing IVF treatment. No conflicts of interest to declare. Research was funded by an NHMRC project grant.

Differential contribution of the proline and glutamine pathways to glutamate biosynthesis and nitrogen assimilation in yeast lacking glutamate dehydrogenase

In Saccharomyces cerevisiae, the glutamate dehydrogenase (GDH) enzymes play a pivotal role in glutamate biosynthesis and nitrogen assimilation. It has been proposed that, in GDH-deficient yeast, either the proline utilization (PUT) or the glutamine synthetase–glutamate synthase (GS/GOGAT) pathway serves as the alternative pathway for glutamate production and nitrogen assimilation to the exclusion of the other. Using a gdh-null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway-specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). The GDH-null mutant was viable on all nitrogen sources tested, confirming that alternate pathways for nitrogen assimilation exist in the gdh-null strain. Enzyme assays point to GS/GOGAT as the primary alternative pathway on the preferred nitrogen sources ammonia and glutamine, whereas growth on proline required both the PUT and GS/GOGAT pathways. In contrast, growth on glucose–urea media elicited a decrease in GOGAT activity along with an increase in activity of the PUT pathway specific enzyme Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH). Together, these results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH-dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serves as the sole compensatory pathway.

An Unusual Case of Clostridium difficile Infection in Trinidad and Tobago

Clostridium difficile infection (CDI) is the leading cause of healthcare-associated diarrhea. Psuedomembraneous colitis is a major complication of CDI and its outbreaks with increased severity and mortality has been reported. In this study we report the first fatal case of a toxin A and B gene positive strain of C. difficile associated with pseudomembraneous colitis in a patient from Trinidad and Tobago. Patients admitted to tertiary hospitals with history of diarrhea were investigated and reviewed. A fatal case of a 47-year old Trinidadian of African descent who presented with diarrheal stool at a public hospital in Trinidad and Tobago was reviewed and is being reported. The identified patient had his stool sample analyzed using conventional and molecular microbiology procedures to identify the C. difficile bacteria, toxins and genes. The antimicrobial susceptibility profile was also determined using agar dilutions according to CLSI guidelines.This patient’s stool sample was positive for Clostridium difficile toxin and the enzyme glutamate dehydrogenase which were demonstrated using the Quick Check Complete® kit. The antimicrobial susceptibility test produced an unusual profile - being resistant to meropenem, ampicillin, ceftriaxone, cefotaxime and ciprofloxacin. PCR analysis demonstrated that the strain produced a 1266bp and 204bp DNA fragment corresponding to the toxin A and B gene respectively, based on the primers used. This organism was noted to be responsible for the development of psuedomembranous colitis that led to the demise of this patient. Surveillance work need to be instituted as well as antibiotics stewardship so as to prevent any more case of inflammatory bowel diseases prevalence in our country.

Molecular mechanisms of protein induced hyperinsulinaemic hypoglycaemia.

The interplay between glucose metabolism and that of the two other primary nutrient classes, amino acids and fatty acids is critical for regulated insulin secretion. Mitochondrial metabolism of glucose, amino acid and fatty acids generates metabolic coupling factors (such as ATP, NADPH, glutamate, long chain acyl-CoA and diacylglycerol) which trigger insulin secretion. The observation of protein induced hypoglycaemia in patients with mutations in GLUD1 gene, encoding the enzyme glutamate dehydrogenase (GDH) and HADH gene, encoding for the enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase has provided new mechanistic insights into the regulation of insulin secretion by amino acid and fatty acid metabolism. Metabolic signals arising from amino acid and fatty acid metabolism converge on the enzyme GDH which integrates both signals from both pathways and controls insulin secretion. Hence GDH seems to play a pivotal role in regulating both amino acid and fatty acid metabolism.

Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2

Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which converts l-glutamate into α-ketoglutarate through an irreversible reaction. The enzyme GDH is detected in the stool samples of patients with C. difficile-associated disease and serves as one of the diagnostic tools to detect C. difficile infection (CDI). We demonstrate here that supernatant fluids of C. difficile cultures contain GDH. To understand the role of GDH in the physiology of C. difficile, an isogenic insertional mutant of gluD was created in strain JIR8094. The mutant failed to produce and secrete GDH as shown by Western blot analysis. Various phenotypic assays were performed to understand the importance of GDH in C. difficile physiology. In TY (tryptose yeast extract) medium, the gluD mutant grew slower than the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the growth defect in TY medium. The presence of extracellular GDH may have a functional role in the pathogenesis of CDI. In support of this assumption we found higher sensitivity to H2O2 in the gluD mutant as compared to the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the H2O2 sensitivity.

Evaluation of sub-cellular distribution of glutamate dehydrogenase (GDH) in Drosophila melanogaster larvae

Glutamate dehydrogenase (GDH) enzyme was conventionally known as a mitochondrial marker. However, subsequently it was reported to be present in the nuclei as well. So far, the nuclear distribution of GDH has been reported in a number of organisms including yeast, rat, cow, chicken. However, the sub-cellular distribution of GDH, illustrated by in situ methods still remains elusive. Here, by assaying the GDH activity and by immuno-blotting using anti-GDH antibody in the fractionated nuclear and cytoplasmic fractions of Drosophila larvae, we demonstrate the cytoplasmic distribution of GDH. This observation was further supported by in situ immunostaining of salivary gland, Malpighian tubules and eye imaginal discs of Drosophila larvae. Collectively, our results demonstrate that in Drosophila larvae, GDH is not found in the nucleus, but is localized exclusively in the cytoplasm.