Physapubescin I from husk tomato suppresses SW1990 cancer cell growth by targeting kidney-type glutaminase

Kidney-type glutaminase (KGA), catalyzing the hydrolysis of glutamine to glutamate for energy supply, is over-expressed in many cancers and has been regarded as a new therapeutic target for cancers. Physapubescin I was isolated from the fruits of the edible herb Physalis pubescens L., commonly named as “husk tomato or hairy groundcherry”, and was predicted to be a potential KGA inhibitor through structure-based virtual ligand screening. Enzyme inhibition assays, microscale thermophoresis (MST) and cellular thermal shift assay (CETSA) experiments have demonstrated the high efficiency and specificity of physapubescin I targeting KGA. EdU proliferation, Hoechst 33258 staining and cytotoxicity assays indicated that physapubescin I could inhibit cancer cell proliferation and promote apoptosis more effectively than the known KGA inhibitor, BPTES. Knockdown of KGA by siRNA reduced the inhibition of physapubescin I to SW1990 cells. Meanwhile, physapubescin I impaired glutamine metabolism in SW1990 cells with increasing intracellular level of glutamine, and correspondingly decreasing glutamate and its downstream metabolites, which may account for its inhibition of cancer cell proliferation and proapoptosis. Physapubescin I also showed significant tumor growth inhibition and low toxicity in a SW1990 xenograft mouse model. Collectively, physapubescin I may serve as a potential drug candidate or lead compound for cancer therapy by targeting KGA.     

Design and synthesis of biotinylated Hexylselen as a probe to identify KGA allosteric inhibitors by a convenient biomolecular interaction assay

Hexylselen is a novel submicromolar dual KGA/GDH inhibitor, which demonstrates potent inhibition of cancer cells with minimal toxicity. To further investigation its mechanism of action, we designed and synthesized its biotinylated derivative 2 as a novel probe. From commercially available starting material, 2 was obtained in 6 steps with 13.4% overall yield. It is notable that this practical synthetic route give a template for the preparation of unsymmetrical di-benzo[d][1,2]selenazol-3(2H)-ones. Based on probe 2, we developed a novel biomolecular interaction assay for convenient and reliable test of KGA allosteric inhibitors and confirmed that hexylselen as an allosteric inhibitor of KGA sharing the same binding pocket with BPTES but not with Ebselen via competitive experiments.     

Continuous conversion of rice starch hydrolysate to 2-keto-D-gluconic acid by Arthrobacter globiformis C224

A continuous conversion process of rice starch hydrolysate to 2-keto-D-gluconic acid (2KGA) by Arthrobacter globiformis C224 was developed. Its feasibility for industrial application was also evaluated. Results showed that the initial cell concentration exceeding 1.25 g/L met the continuous 2KGA production at a stable dilution rate and media composition, while the dilution rate and feeding glucose concentration had a significant effect on 2KGA production performance. The optimal operating parameters were obtained as: 0.090/h of dilution rate and 171.0 g/L of feeding glucose concentration. Under these conditions, the steady state had a produced 2KGA concentration of 124.74 g/L, average volumetric productivity of 11.23 g/L/h, and yield of 0.97 g/g. In conclusion, continuous 2KGA production by the A. globiformis C224 strain would be a superior industrial process for the production of 2KGA in terms of its high 2KGA productivity and yield.     

Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica

To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP⁺-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19 % more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95 % producing up to 186 g l^-1 KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.     

Production of 5-keto-d-gluconate by acetic acid bacteria is catalyzed by pyrroloquinoline quinone (PQQ)-dependent membrane-bound d-gluconate dehydrogenase

Gluconobacter suboxydans IFO 12528 was selected as the best strain for 5-keto-d-gluconate (5KGA) production by oxidative fermentation. 5KGA was markedly accumulated by the strain during cultivation in a medium containing d-glucose and/or d-gluconate. The resting cells and the membrane fraction also catalyzed 5KGA formation with a minimal formation of 2-keto-d-gluconate (2KGA), an alternative keto-d-gluconate from d-gluconate. The membrane fraction of the organism was confirmed to contain a membrane-bound d-gluconate dehydrogenase (GADH) catalyzing d-gluconate oxidation to 5KGA of which optimum pH and temperature were found at pH 4 and 15°C, respectively. After treating the membrane fraction with EDTA allowing conversion from holo-GADH to the apoenzyme, 5KGA-forming GADH was confirmed to be a pyrroloquinoline quinone (PQQ)-dependent enzyme by the fact that the enzyme activity was restored by the addition of CaCl₂ and PQQ. The 5KGA-forming GADH was totally distinct from 2KGA-forming GADH in which a covalently bound FAD functions as coenzyme. 5KGA-forming GADH was well solubilized from the membrane fraction with n-octyl-β-d-thioglucoside and 5KGA formation was favourably catalyzed at relatively lower temperature, while 2KGA-forming enzyme was solubilized with Triton X-100 and relatively higher temperatures was optimum for 2KGA formation. These results are completely discrepant from the conclusion proposed by Klasen et al. [R. Klasen, S. Bringer-Mayer, H. Sahm, J. Bacteriol., 177, 1995, 2637] claiming that 5KGA was produced by d-gluconate oxidation catalyzed by NADP-dependent cytoplasmic 5KGA reductase from Gluconobacter species at fairly alkaline pH such as 10.     

α-Ketoglutaric acid production by Yarrowia lipolytica and its regulation

The yeast Yarrowia lipolytica VKM Y-2412 was selected as a prospective producer of ??-ketoglutaric acid (KGA) from ethanol. The following peculiarities were found: (1) the intensive KGA production occurred only under the limitation of cell growth by thiamine and the excess of ethanol and nitrogen, (2) the production of KGA from ethanol required increased amount of zinc and iron ions, and (3) KGA production increased significantly with a high aeration at pH medium equal to 3.5. Under optimal conditions, the Y. lipolytica VKM Y-2412 produced up to 172?g?l^?1 of KGA with the mass yield coefficient of 0.70?g?g^?1.     

Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway

In previous research, a thiamine-auxotrophic yeast for alpha-ketoglutaric acid (KGA) overproduction was screened in our laboratory and named Yarrowia lipolytica WSH-Z06 (CCTCC no. M207143). However, the high concentration of by-products (mainly pyruvate) limited its application on an industrial scale. To enhance KGA production and reduce pyruvate (PA) accumulation, the pyruvate carboxylation pathway was regulated. By overexpressing the pyruvate carboxylase genes ScPYC1 from Saccharomyces cerevisiae and RoPYC2 from Rhizopus oryzae in Y. lipolytica WSH-Z06, the yields of KGA in Y. lipolytica-ScPYC1 and Y. lipolytica-RoPYC2 increased by 24.5 and 35.3?%, and the yields of PA decreased by 51.9 and 69.8?% in shake flasks, respectively. These changes in the expression levels and activities of key intracellular enzymes showed that enhancing the pyruvate carboxylation pathway had successfully redistributed the carbon flux from PA to KGA. Finally, by controlling the pH in a 3-L fermenter, the maximum concentration of KGA in Y. lipolytica-RoPYC2 reached 62.5?g?L^?1 with an evident decrease in PA yield from 35.2 to 13.5?g?L^?1.     

Enhanced α-ketoglutaric acid production and recovery in Yarrowia lipolytica yeast by effective pH controlling

The replacement of chemical synthesis by environmentally friendly energy-efficient technologies for production of valuable metabolites is a principal strategy of developing biotechnological industry all over the world. In the present study, we develop a method for α-ketoglutaric acid (KGA) production from rapeseed oil with the use of Yarrowia lipolytica yeast. Sixty strains of Y. lipolytica yeasts were tested for their ability to produce KGA, and the strain Y. lipolytica 212 (Y. lipolytica VKM Y-2412) was selected as a promising KGA producer. Using a three-stage pH controlling, in which pH was 4.5 in the growth phase, then since 72 to 144 h, pH was maintained at 3.5 and in the later phase of acid production, the titration by KOH was switch off, selected strain produced 106.5 g l^?1 of KGA with mass yield of 0.95 g g^?1. KGA in the form of monopotassium salt was isolated from the culture broth and purified. The isolation procedure involved separation of biomass, extraction of residual triglycerides, filtrate bleaching, and acidification with mineral acid (to pH 2.8–3.4), concentration, precipitation of mineral salts, and crystallization of the product. The purity of KGA isolated from the culture filtrate reached 99.1 %.     

Screening of Thermotolerant Gluconobacter Strains for Production of 5-Keto-d-Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing d-Gluconate Dehydrogenase

We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-d-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl₂ in vitro when cells were grown at 37°C. Addition of 5 mM CaCl₂ to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production.Gluconobacter is a genus of acetic acid bacteria that are able to oxidize a broad range of sugars, sugar alcohols, and sugar acids, and large amounts of the corresponding oxidized products accumulate in the culture medium. Such “incomplete” oxidation is carried out by membrane-bound enzymes, whose catalytic sites face the periplasm. These enzymes catalyze the dehydrogenization of d-glucose, d-sorbitol, d-mannitol, glycerol, d-gluconate, and the keto-d-gluconates. All of these enzymes are firmly attached to the cytoplasmic membrane, and the electrons abstracted from the substrates are passed on to ubiquinone and then to terminal ubiquinol oxidases, forming simple respiratory chains which create the membrane potential necessary to produce biological energy for these microorganisms.The oxidation of d-glucose to ketogluconates is known to be catalyzed by a series of enzymes. Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase oxidizes d-glucose to glucono-δ-lactone, and then gluconolactonase converts the glucono-δ-lactone to d-gluconate. The formation of ketogluconates in Gluconobacter strains has been reported to be catalyzed by two types of membrane-bound gluconate dehydrogenases (GADH) (10). One type is flavin adenine dinucleotide (FAD)-GADH, an FAD-containing, 2-keto-d-gluconate (2KGA)-producing enzyme, and the other type is a PQQ-containing, 5-keto-d-gluconate (5KGA)-producing enzyme. The former enzyme has three subunits: an FAD-containing dehydrogenase, a c-type cytochrome subunit containing three hemes, and a small subunit of unknown function (17). The latter enzyme, which produces 5KGA, is identical to the PQQ-containing polyol dehydrogenase (9), which is known as d-arabitol dehydrogenase (1), d-sorbitol dehydrogenase (20), or PQQ-dependent glycerol dehydrogenase (PQQ-GLDH) (2). PQQ-GLDH has broad substrate specificity but high regio- and stereospecificity, and it catalyzes reactions as predicted by the Bertrand-Hudson rule. This enzyme can oxidize d-gluconate only at the C-5 position to produce 5KGA from d-gluconate; however, the affinity of the enzyme for d-gluconate is quite low. The gene encoding this enzyme was cloned from Gluconobacter suboxydans IFO 3255 (11), and two open reading frames (ORFs) were found. One of these ORFs is believed to encode a hydrophobic protein with five membrane-spanning regions, and the other encodes a dehydrogenase subunit similar to that found in several PQQ-dependent enzymes, particularly the PQQ domain of membrane-bound glucose dehydrogenase. In contrast, 2KGA reductase and 5KGA reductase, the NADPH-dependent enzymes located in the cytoplasm, are thought to be involved in gluconate metabolism in the assimilation of 2KGA and 5KGA.5KGA is a useful raw material for the production of tartaric acid and xylaric acid and is used as a precursor for the synthesis of a number of flavor compounds, including 4-hydroxy-5- methyl-2,3-dihydrofuranone-3 (15). Moreover, it has been reported that 5KGA can be used to produce vitamin C by Gray''s method (6, 7), which is different from Reichstein''s method, which is now commonly used in industry. Reichstein''s method requires the use of high temperatures and an organic solvent in processing; however, Gray''s method does not.Most Gluconobacter strains produce both 2KGA and 5KGA from d-gluconate. Thus, production of 5KGA by Gluconobacter species generates 2KGA as a major by-product, and production of the two ketogluconates is competitive in vivo. Recently, an FAD-GADH-defective mutant strain of Gluconobacter oxydans 621 H which produced almost exclusively 5KGA from d-glucose was discovered (5). However, the optimum temperature for production of 5KGA in this mesophilic strain was around 20°C (19). For cost-effective industrial synthesis of 5KGA, we sought to develop a Gluconobacter strain which is able to produce 5KGA at higher temperatures, such as 37°C, in order to reduce the cost of cooling during fermentation.We successfully isolated thermotolerant Gluconobacter strains that are able to produce 5KGA at 37°C. We cloned the FAD-GADH gene and constructed FAD-GADH-defective mutants that produced almost exclusively 5KGA from d-gluconate at both ambient temperatures and higher temperatures up to 37°C. We believe that the thermotolerant strains reported in this study should be useful for industrial 5KGA production.     

α-Ketoglutaric acid production from rapeseed oil by Yarrowia lipolytica yeast

The possibility of using rapeseed oil as a carbon source for microbiological production of α-ketoglutaric acid (KGA) has been studied. Acid formation on the selective media has been tested in 26 strains of Yarrowia lipolytica yeast, and the strain Y. lipolytica VKM Y-2412 was selected as a prospective producer of KGA from rapeseed oil. KGA production by the selected strain was studied in dependence on thiamine concentration, medium pH, temperature, aeration, and concentration of oil. Under optimal conditions (thiamine concentration of 0.063 μg?g cells^?1, pH?3.5, 30 °C, high dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and oil concentration in a range from 20 to 60 g?l^?1), Y. lipolytica VKM Y-2412 produced up to 102.5 g?l^?1 of KGA with the mass yield coefficient of 0.95 g?g^?1 and the volumetric KGA productivity (Q _KGA) of 0.8 g?l^?1?h^?1.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

Overproduction and secretion of α-ketoglutaric acid by microorganisms

This mini-review presents a summary of research results of biotechnological production of alpha-ketoglutaric acid (KGA) by bacteria and yeasts. KGA is of particular industrial interest due to its broad application scope, e.g., as building block chemical for the chemical synthesis of heterocycles, dietary supplement, component of infusion solutions and wound healing compounds, or as main component of new elastomers with a wide range of interesting mechanical and chemical properties. Currently KGA is produced via different chemical pathways, which have a lot of disadvantages. As an alternative several bacteria and yeasts have already been studied for their ability to produce KGA as well as for conditions of overproduction and secretion of this intermediate of the tricarboxylic acid cycle. The aim of this mini-review was to summarize the known data and to discuss the potentials of biotechnological processes of KGA production.     

碳源和氮源对5-酮基-葡萄糖酸生成的影响

氧化葡萄糖杆菌Gluconobacter oxydans可以将葡萄糖氧化成葡萄糖酸,并进一步氧化成2-酮基-葡萄糖酸(2KGA)和5-酮基-葡萄糖酸(5KGA),其中5KGA在催化剂的作用下能够转化为L(+)-酒石酸。为了提高5-酮基-葡萄糖酸产量,以仅生成5KGA的氧化葡萄糖杆菌Gluconobacter oxydans HGI-1为出发菌株,研究不同碳源(蔗糖、乳糖、麦芽糖、淀粉、葡萄糖)和有机氮源(酵母浸粉、鱼粉、玉米浆、黄豆饼粉、棉籽饼粉)对5KGA产量的影响。500 mL摇瓶试验结果表明,当葡萄糖浓度为100 g/L时,5KGA产量最高为98.20 g/L;当有机氮源为酵母浸粉、鱼粉和玉米浆,其添加量的蛋白含量为1.60%时,5KGA产量分别为100.20 g/L、109.10 g/L和99.83 g/L,其中,使用鱼粉的5KGA产量最高,使用玉米浆的5KGA产量比酵母浸粉略低。出于经济考虑,文中选择玉米浆作有机氮源,并在5 L发酵罐中进行分批发酵放大试验,5KGA的产量为93.80 g/L,最大生成速率为3.48 g/(L·h),平均生成速率为1.56 g/(L·h)。结果表明,葡萄糖和玉米浆分别为Gluconobacter oxydans HGI-1规模化生产5KGA的最适碳源和氮源,可利用葡萄糖几乎全部(85.93%)转化为5KGA。     

Preparation of Enzymes Required for Enzymatic Quantification of 5-Keto-D-gluconate and 2-Keto-D-gluconate

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.     

Bmcc1s interacts with the phosphate-activated glutaminase in the brain

Bmcc1s, a brain-enriched short isoform of the BCH-domain containing molecule Bmcc1, has recently been shown to interact with the microtubule-associated protein MAP6 and to regulate cell morphology. Here we identified kidney-type glutaminase (KGA), the mitochondrial enzyme responsible for the conversion of glutamine to glutamate in neurons, as a novel partner of Bmcc1s. Co-immunoprecipitation experiments confirmed that Bmcc1s and KGA form a physiological complex in the brain, whereas binding and modeling studies showed that they interact with each other. Overexpression of Bmcc1s in mouse primary cortical neurons impaired proper mitochondrial targeting of KGA leading to its accumulation within the cytoplasm. Thus, Bmcc1s may control the trafficking of KGA to the mitochondria.     

Development of a stable shuttle vector and a conjugative transfer system for Gluconobacter oxydans

A shuttle vector pGE1 (11.9 kb) which can replicate both in Gluconobacter oxydans and Escherichia coli was constructed from the cryptic Gluconobacter plasmid pGO3293S (9.9 kb, relaxed type) and E. coli plasmid pSUP301 (5 kb, Km^r, Ap^r, relaxed type). The plasmid pGO3293S is one of the endogenous plasmids of G. oxydans IFO 3293 which converts l-sorbose to 2-keto-l-gulonic acid (2KGA), an intermediate of vitamin C synthesis. The other plasmid, pSUP301, is a conjugative plasmid which contains pACYC177 and the mob region from plasmid RP4. The plasmid pGE1 could be transferred into G. oxydans IFO 3293 with a high frequency (10⁻¹ transconjugants/recipient) by a conjugal transfer system, and maintained very stably without antibiotic selection. pGE1 can be introduced and maintained in other acetic acid bacteria including Gluconobacter and Acetobacter. The presence of pGE1 did not inhibit the growth or 2KGA productivity of 2KGA-producing strains derived from G. oxydans IFO 3293. The usefulness of pGE1 as a vector was confirmed by subcloning the membrane-bound l-sorbosone dehydrogenase gene of A. liquefaciens IFO 12258 in G. oxydans IFO 3293 derivatives; in this subcloning, pGE1 could be further shortened to the 9.8 kb plasmid, pGE2.     

Synthesis of α-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol

The ability of yeast to synthesize α-ketoglutaric acid (KGA) from ethanol has been studied. Thiamine-auxotrophic yeasts of different genera and species may be able to produce KGA; the main condition of synthesis is growth limitation by thiamine. Using a model culture, mutant Yarrowia lipolytica N 1, the principal conditions affecting KGA oversynthesis were identified. These were: thiamine concentration in medium and in cells, nitrogen and oxygen concentration in medium, and pH level. A KGA concentration of 49 g/l and a yield from ethanol consumed of 42% were achieved. Based on the results of the analysis of the activities of the key enzymes participating in ethanol metabolism and KGA synthesis, a concept of the mechanism of KGA biosynthesis by Y. lipolytica yeast is suggested and discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 June 1999     

Preparation of enzymes required for enzymatic quantification of 5-keto-D-gluconate and 2-keto-D-gluconate

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.     

Relative Expression of mRNAS Coding for Glutaminase Isoforms in CNS Tissues and CNS Tumors

Glutaminase (GA) in mammalian tissues occurs in three isoforms: LGA (liver-type), KGA (kidney-type) and GAC (a KGA variant). Our previous study showed that human malignant gliomas (WHO grades III and IV) lack expression of LGA mRNA but are enriched in GAC mRNA relative to KGA mRNA. Here we analyzed the expression of mRNAs coding for the three isoforms in the biopsy material derived from other central nervous system tumors of WHO grades I–III. Non-neoplastic resective epileptic surgery samples served as control, as did cultured rat astrocytes and neurons. The GAC mRNA/KGA mRNA expression ratio was as a rule higher in the neoplastic than in control tissues, irrespective of the cell type dominating in the tumor or tumor malignancy. LGA mRNA expression was relatively very low in cultured astrocytes, and very low to absent in astrocytoma pilocyticum, ependymoma and subependymal giant cell astrocytoma (SEGA), tumors of astrocytic origin. LGA mRNA expression was almost as high as that of KGA and GAC mRNA in cultured neurons and epileptic surgery samples which were enriched in neurons. LGA mRNA was also relatively high in ganglioglioma which contains a discernable proportion of neuronal cells, and in oligodendroglioma. The results show that low expression of LGA mRNA is a feature common to normal astrocytes and astroglia-derived tumor cells or ependymomas and can be considered as a cell-type, rather than a malignancy marker.     

Selective,High Conversion of D-Glucose to 5-Keto-D-gluoconate by Gluconobacter suboxydans

Selective, high-yield production of 5-keto-D-gluconate (5KGA) from D-glucose by Gluconobacter was achieved without genetic modification. 5KGA production by Gluconobacter suffers byproduct formation of 2-keto-D-gluconate (2KGA). By controlling the medium pH strictly in a range of pH 3.5–4.0, 5KGA was accumulated with 87% conversion yield from D-glucose. The pH dependency of 5KGA formation appeared to be related to that of gluconate oxidizing activity.