Inhibition Of Glycolate Oxidase Research Articles

Himalayan Citrus jambhiri juice reduced renal crystallization in nephrolithiasis by possible inhibition of glycolate oxidase and matrix metalloproteinases.

Citrus fruits are a very rich source of electrolytes and citric acid. They have been used traditionally for treating urinary ailments and renal stones. Citrus jambhiri is indigenously used as a diuretic. Present study aimed at establishing the antiurolithiatic potential of the juice of Citrus jambhiri fruits along with the elucidation of the mechanism involved in the urolithiasis disease defying activity. The antiurolithiatic activity was established by means of nucleation, growth and aggregation assay in the in vitro settings and by means of ethylene glycol mediated calcium oxalate urolithiasis in the male Wistar rats. Docking studies were performed in an attempt to determine the mechanism of the antiurolithiatic action. Present study revealed the role of C. jambhiri fruit juice in reducing nucleation, growth and aggregation of calcium oxalate crystals by possible reduction in the urinary supersaturation relative to calcium oxalate and raising the zeta potential of the calcium oxalate crystals. C. jambhiri fruit juice treatment in experimental rats produced significant amelioration of hypercalciuria, hyperoxaluria, hyperphosphaturia, hyperproteinuria, hyperuricosuria, hypocitraturia and hypomagnesiuria and ion activity product of calcium oxalate. It exhibited nephroprotection against calcium oxalate crystals induced renal tubular dilation and renal tissue deterioration. Docking studies further revealed high binding potential of the phytoconstituents of C. jambhiri viz. narirutin, neohesperidin, hesperidin, rutin and citric acid with glycolate oxidase and matrix metalloproteinase-9. C. jambhiri fruit juice possesses excellent antiurolithiatic activity. The study reveals antiurolithiatic mechanism that involves restoration of equilibrium between the promoters and inhibitors of stone formation; and inhibition of matrix metalloproteinases and glycolate oxidase.

In vivo CRISPR-Cas9 inhibition of hepatic LDH as treatment of primary hyperoxaluria

Genome-editing strategies, especially CRISPR-Cas9 systems, have substantially increased the efficiency of innovative therapeutic approaches for monogenic diseases such as primary hyperoxalurias (PHs). We have previously demonstrated that inhibition of glycolate oxidase using CRISPR-Cas9 systems represents a promising therapeutic option for PH type I (PH1). Here, we extended our work evaluating the efficacy of liver-specific inhibition of lactate dehydrogenase (LDH), a key enzyme responsible for converting glyoxylate to oxalate; this strategy would not be limited to PH1, being applicable to other PH subtypes. In this work, we demonstrate a liver-specific inhibition of LDH that resulted in a drastic reduction of LDH levels in the liver of PH1 and PH3 mice after a single-dose delivery of AAV8 vectors expressing the CRISPR-Cas9 system, resulting in reduced urine oxalate levels and kidney damage without signs of toxicity. Deep sequencing analysis revealed that this approach was safe and specific, with no off-targets detected in the liver of treated animals and no on-target/off-tissue events. Altogether, our data provide evidence that in vivo genome editing using CRISPR-Cas9 systems would represent a valuable tool for improved therapeutic approaches for PH.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p < 0.05) as well as antibody-mediated cellular-dependent immune responses (p < 0.01) were significantly decreased. Silencing SLA class I expression did not increase susceptibility to NK-cell cytotoxicity. In stirred bioreactors, tissue engineered islets showing the typical 3D-structure and morphology of ICC were assembled from SLA-silenced pancreatic cell suspensions. These data shows the feasibility to generate low immunogenic porcine ICC from transgenic pigs after single cell engineering and post-transduction islet reassembling that might serve as a robust alternative to allogeneic pancreatic islet cell transplantation.

Inhibition of Glycolate Oxidase With Dicer-substrate siRNA Reduces Calcium Oxalate Deposition in a Mouse Model of Primary Hyperoxaluria Type 1

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure. Currently, the only treatment effective in reduction of oxalate production in patients who do not respond to high-dose vitamin B6 therapy is a combined liver/kidney transplant. We explored an alternative approach to prevent glyoxylate production using Dicer-substrate small interfering RNAs (DsiRNAs) targeting hydroxyacid oxidase 1 (HAO1) mRNA which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduces GO mRNA and protein in the livers of mice and nonhuman primates. Reduction of hepatic GO leads to normalization of urine oxalate levels and reduces CaOx deposition in a preclinical mouse model of PH1. Our results support the use of DsiRNA to reduce liver GO levels as a potential therapeutic approach to treat PH1.

Inhibition of endogenous oxalate production: biochemical considerations of the roles of glycollate oxidase and lactate dehydrogenase.

1. Both the peroxisomal, flavin-linked glycollate oxidase [(S)-2-hydroxy-acid oxidase; EC 1.1.3.15] and the cytosolic, nicotinamide-adenine dinucleotide (NAD)-linked lactate dehydrogenase (L-lactate dehydrogenase; EC 1.1.1.27) are thought to contribute to the formation of oxalate from its immediate precursors, glycollate and glyoxylate, but the relative contributions of each enzyme to endogenous oxalate production is not known. 2. In rat liver homogenates, [14C]oxalate production from labelled glycollate is halved and that from labelled glyoxylate is increased fourfold by the addition of either NAD or NADH. 3. In isolated rat hepatocytes, the 3-hydroxy-1H-pyrrole-2,5-dione derivatives of glycollate, which are specific inhibitors of glycollate oxidase, have a greater effect on glycollate metabolism than on glyoxylate metabolism. 4. These findings are consistent with an important role for lactate dehydrogenase in oxalate formation from glyoxylate. 5. With human and rat liver homogenates and with purified human liver glycollate oxidase and rabbit muscle lactate dehydrogenase, DL-phenyl-lactate (2 mmol/l) completely inhibits glycollate oxidase but has not effect on lactate dehydrogenase. On the other hand, the reduced form of a chemically synthesized, NAD-pyruvate adduct (1 mmol/l) almost completely inhibited lactate dehydrogenase but had no effect on glycollate oxidase. 6. Either alone or in combination, DL-phenyl-lactate and reduced NAD-pyruvate adduct reduce oxalate production from glycollate and glyoxylate in isolated rat hepatocytes, but do not abolish it completely. 7. These findings support a role for another enzyme, probably glycollate dehydrogenase (EC 1.1.99.14), in oxalate production in integrated cell metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycollate oxidase inhibition and its effect on photosynthesis and pigment formation in Hordeum vulgare

Investigations of the effect of 2-hydroxy-3-butynoic acid and its methyl ester on photosynthesis in Hordeum vulgare are reported. In the presence of either of these compounds the assimilation of 14CO 2 was greatly decreased. The labelling patterns showed massive accumulation of glycollate and greatly reduced incorporation into sucrose and other products of photosynthesis. The inhibition was specific for the S(+) enantiomers. In greening barely the S(+) enantiomers inhibited formation of chloroplast pigments, and this was paralleled by inhibition of glycollate oxidase. This was the only enzyme of the glycollate pathway whose activity was significantly decreased after inhibitor treatments. Of a range of metabolises tested, only supplementations with glycine and glutamate or glycine, serine and succinate fully restored greening.

Inhibition of glycolate oxidase by fatty acids.

Inhibition of glycolate oxidase by fatty acids.

Inhibition of glycollate oxidase from parsley leaves by HCO 3−

HCO 3 − is shown to be a noncompetitive inhibitor of glycollate oxidase from parsley leaves with respect ot glycolic acid. The K i is found to be 20 mM at pH 7,7.

Involvement of Hydrogen Peroxide in the Regulation of Senescence in Pear

Endogenous peroxide levels in pear fruit (Pyrus communis) were measured using a titanium assay method, and were found to increase during senescence in both Bartlett and Bosc varieties. Application of glycolic acid or xanthine, serving as substrates for the formation of H(2)O(2), increased the peroxide content of the tissue and accelerated the onset of ripening, as measured by increased softening and ethylene evolution. Application of ethylene also induced increased peroxide levels. Ripening processes were similarly promoted when peroxides were conserved by inhibiting the activity of catalase with hydroxylamine or potassium cyanide. By comparison, the inhibition of glycolate oxidase with alphahydroxy-2-pyridinemethanesulfonic acid decreased the peroxide content of the tissue and delayed the onset of ripening. These results indicate that the onset of ripening correlates with the peroxide content of fruit tissues as occurring under normal conditions or as influenced by the treatments. Hydrogen peroxide may be involved in oxidative processes required in the initiation and the promotion of ripening.

Inhibition of glycollate oxidase from pea leaves

Inhibition of glycollate oxidase from pea leaves

Inhibition of glycollate oxidase as a rational way of designing a herbicide

Inhibitors of glycollate oxidase (E.C. 1.1.3.1.) might be useful herbicides. An attempt was therefore made to improve on the known aldehyde bisulphite addition compounds as inhibitors. Unfortunately, it appears that, with the exception of the formaldehyde bisulphite addition compound, all these structures inhibit by reacting with enzymatically produced glyoxylate to give glyoxylate bisulphite. A wide range of other potential inhibitors was then tested on the enzyme. Some aminomethanesulphonates are good inhibitors, but they again are unstable, and probably work by conversion to the formaldehyde bisulphite addition compound. None of the inhibitors show useful activity on a general screen against higher plants and fungi.

Photorespiration and Glycolate Metabolism in Tobacco Leaves1

The stimulating effect of light on the evolution of CO2 from tobacco leaves into CO2‐free air was suppressed by inhibition of glycolate oxidase with α‐hydroxy‐2‐pyridinemethanesulfonic acid. The inhibitor strongly inhibited photosynthesis at both 25 and 35 C. The CO2 compensation concentration increased indefinitely for leaves treated with inhibitor. Glycolate accumulation in leaves treated with inhibitor was strongly dependent on inhibitor concentration and light intensity and was linear with time up to one hour. Temperature for growth or during the experiments had little effect on accumulation.The possible internal CO2 from the oxidation of glycolate calculated on the basis of glycolate accumulation under optimal conditions indicates that photorespiration from glycolate as a substrate may be at least 2‐fold dark respiration. Although the possible rate of respiration from glycolate metabolism was large, it was not sufficient to account for the differences in photosynthesis between maize and tobacco on the assumption that this difference was due to internal recycling of CO2 in tobacco.

Increased Rate of Net Photosynthetic Carbon Dioxide Uptake Caused by the Inhibition of Glycolate Oxidase

There is considerable variation among species in their rate of photorespiration, and photorespiration increases greatly at higher temperatures. The addition of an inhibitor of glycolate oxidase, alpha-hydroxy-2-pyridinemethanesulfonic acid, to tobacco leaf disks at 35 degrees stimulated photosynthetic (14)CO(2) uptake at least 3-fold, but (14)CO(2) uptake was not changed by the inhibitor at 25 degrees . The inhibitor did not increase photosynthesis in maize leaf disks at either temperature.The evolution of CO(2) from glycolate was greatly enhanced in tobacco at 35 degrees compared with 25 degrees . Labeling of the glycolate of tobacco with glycolate-1-(14)C and -2-(14)C showed that the increased CO(2) evolved in the light (photorespiration) arose specifically from the carboxyl-carbon atom of glycolate. Maize, a species known to have a negligible photorespiration, produced (14)CO(2) poorly from glycolate-1-(14)C in comparison to tobacco.Acetate-1-(14)C, a substrate metabolized by dark respiration, produced similar amounts of (14)CO(2) in the light in both tobacco and maize. This respiration was changed little relative to photosynthesis by increasing temperature.Most plants, such as tobacco, have a high photorespiration. The loss of fixed carbon causes an increase in the internal concentration of CO(2) especially at higher temperatures, and results in a lower CO(2) concentration gradient and therefore a lower net photosynthetic CO(2) uptake. Some species, like maize, have a negligible photorespiration and are thus more efficient photosynthetically. The use of an inhibitor of the oxidation of glycolate, the substrate for photorespiration, changed tobacco so that it behaved photosynthetically like maize. Thus high rates of photorespiration may limit the net CO(2) uptake in many plant species.