The effect of glutaminase inhibitor on polycystic kidney disease development

Aim To study if inhibition of glutaminase has effect on polycystic kidney disease （PKD）development and to further investigate the mechanism for the effect. Methods Human autosomal dominant PKD epithelial cells were used to form cysts which were treated with glutaminase specific inhibitors BPTES or CB839. The number and size of cysts were recorded. PKD mice were gavaged with CB839 for two weeks. Kidney morphology and renal func- tion were analyzed. Western blot and seahorse were used to investigate the mechanism. Results Glutaminase in- hibitor retarded the cysts development both in vitro and in vivo. The expression of glutaminase was higher in PKD mice compared to control mice. CB839 inhibited PKD development through reduction of mitrochondrial ATP pro- duction, then resulting into inhibiting proliferation and increasing apoptosis in abnormal renal epithelial cells which would form cysts. Conclusion Glutaminase inhibitor ameliorated the development of PKD. Glutaminase inhibitor might be developed as a candidate drug for PKD and can be combined with other drugs.

Study on Zymomonas Mobilis Growth and Its Relationship with Glutaminase Production by Using Statistical Tools

Glutaminase is used industrially to enhance flavor and aroma and to enrich foodstuffs nutritionally. It also has potential for pharmaceutical application as anti-leukemia agent. The bacteria of Zymomonas mobilis has been studied for ethanol production, however, there is no study regarding glutaminase production. The aim of the present study was to establish the influencing factors for the growth of Z mobilis and its relationship with glutaminase production using statistical tools. Analysis of variance in blocks was carried out in a complete block design and the Tukey test demonstrated the importance of the components of the culture medium, absence of agitation and fermentation time. Minimum culture medium was used in the optimization varying the glucose concentrations （10, 30, and 50 g/L）, glutamine （0, 0.5 and 1g/L） and culture time （18, 24 and 30 hours）. Maximum production was obtained at 8.86 U/L glutaminase. Optimized conditions were used in the growth kinetics, where typical exponential growth was observed. Glutaminase production was shown to be related to biomass production.

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na+. Moreover, the Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na+. These data demon-strate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na+ through in-creasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, △slr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2079 cells was higher than that of the wild-type. To further charac-terize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in △slr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in △slr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress re-sponse by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.

A facile and sensitive method of quantifying glutaminase binding to its ninhibitor CB-839 in tissues

Many cancer types reprogram their metabolism to become addicted to glutamine.One of the critical enzymes in the utilization of glutamine in these cells is glutaminase.CB-839(telaglenastat)is a drug that targets glutaminase that is currently being evaluated in many clinical trials for efficacy in various cancer types that are known to be driven by glutamine metabolism.Despite its use,there are limited assays available for testing the pharmacodynamic on-target effects of CB-839 on the limited,small-volume patient samples that are obtained in early-phase clinical trials.Thus,we developed an assay based on the cellular thermal shift assay technique using AlphalLlSA technology to show that CB-839 specifically engages glutaminase in colon cancer cell lines in vitro and in minute quantities of mouse xenograft tumors.Notably,we show that this assay detects CB-839 binding to glutaminase in platelets of patients collected while receiving CB-839 on a clinical trial.This assay may be used to study the pharmacodynamic profile of CB-839 in very small tissue samples obtained from patients on a clinical trial and may be useful infuture studies designed to screen other in hibitors of glutaminase.

Serial deletion reveals structural basis and stability for the core enzyme activity of human glutaminase 1 isoforms:relevance to excitotoxic neurodegeneration

Background:Glutaminase 1 is a phosphate-activated metabolic enzyme that catalyzes the first step of glutaminolysis,which converts glutamine into glutamate.Glutamate is the major neurotransmitter of excitatory synapses,executing important physiological functions in the central nervous system.There are two isoforms of glutaminase 1,KGA and GAC,both of which are generated through alternative splicing from the same gene.KGA and GAC both transcribe 1–14 exons in the N-terminal,but each has its unique C-terminal in the coding sequence.We have previously identified that KGA and GAC are differentially regulated during inflammatory stimulation and HIV infection.Furthermore,glutaminase 1 has been linked to brain diseases such as amyotrophic lateral sclerosis,Alzheimer’s disease,and hepatic encephalopathy.Core enzyme structure of KGA and GAC has been published recently.However,how other coding sequences affect their functional enzyme activity remains unclear.Methods:We cloned and performed serial deletions of human full-length KGA and GAC from the N-terminal and the C-terminal at an interval of approximately 100 amino acids(AAs).Prokaryotic expressions of the mutant glutaminase 1 protein and a glutaminase enzyme activity assay were used to determine if KGA and GAC have similar efficiency and efficacy to convert glutamine into glutamate.Results:When 110 AAs or 218 AAs were deleted from the N-terminal or when the unique portions of KGA and GAC that are beyond the 550 AA were deleted from the C-terminal,KGA and GAC retained enzyme activity comparable to the full length proteins.In contrast,deletion of 310 AAs or more from N-terminal or deletion of 450 AAs or more from C-terminal resulted in complete loss of enzyme activity for KGA/GAC.Consistently,when both Nand C-terminal of the KGA and GAC were removed,creating a truncated protein that expressed the central 219 AA-550 AA,the protein retained enzyme activity.Furthermore,expression of the core 219 AA-550 AA coding sequence in cells increased extracellular glutamate concentrations to levels comparable to those of full-length KGA and GAC expressions,suggesting that the core enzyme activity of the protein lies within the central 219 AA-550 AA.Full-length KGA and GAC retained enzyme activities when kept at 4°C.In contrast,219 AA-550 AA truncated protein lost glutaminase activities more readily compared with full-length KGA and GAC,suggesting that the Nterminal and C-terminal coding regions are required for the stability KGA and GAC.Conclusions:Glutaminase isoforms KGA and GAC have similar efficacy to catalyze the conversion of glutamine to glutamate.The core enzyme activity of glutaminase 1 protein is within the central 219 AA-550 AA.The N-terminal and C-terminal coding regions of KGA and GAC help maintain the long-term activities of the enzymes.

EFFECTS OF L-GLUTAMINE, GLUTAMINASE AND GLUTAMINE SYNTHETASE ON CAP THRESHOLD OF COCHLEAR NERVE OF GUINEA PIG

Negative direct current (__DC 300 μA) stimulation was applied to the round window ofthe guinea pig cochlea to exhaust the pre-synaptic intracellular reserves of the transmitter inhair cells, and then the scala tympani was perfused respectively with L- glutamine, glutaminesynthetase and glutaminase. Experimental results showed that the negative DC electricalstimulation applied to the round window elevated the CAP threshold of the cochlear nervein the basal turn of the cochlea, which recovered over a period of approximately 17- 39 min.The perfusion of L- glutamine apparently elevated the CAP threshold. The recovery of theCAP threshold following electrical stimulation, however, was accelerated by the perfusion of10 mmol/L L- glutamine. The time for recovery only took about 5- 6 min. The perfusion ofenzyme glutamine synthetase elevated the CAP threshold by 50 dB, while glutaminase hadlittle effect. These results suggest that the effect of L- glutamine on the CAP threshold inthe cocblea of the guinea pig appears to be that of a potent depolarizing agent which acceler-ates the recovery of the CAP threshold during the depletion of the transmitter, and L-glutamine may be the candidate for the afferent excitatory transmitter.

The effect of rehabilitation exercise on the expression of glutaminase and cardiopulmonary remodeling in pulmonary hypertension

Pulmonary hypertension(PH)is featured by pulmonary vascular and cardiac remodeling.Rehabilitation exercise can improve patients’quality of life.We previously pinpointed a potential glutamine metabolism dysfunction in PH.Hence,we aim to investigate whether rehabilitation exercise could mitigate right ventricular and pulmonary vascular remodeling and its effect on glutaminase(GLS).We collected clinical indicators of PH patients and analyzed their correlation with GLS.Rehabilitation exercise(moderate intensity swimming exercise)was performed in monocrotaline-induced PH(MCT-PH)rats.We found that plasma GLS1 level in patients was lower than healthy subjects,and it was negatively correlated with end-systolic stage eccentricity index,right atrial transverse dimension and right atrial longitudinal dimension.MCT-PH rats displayed pulmonary vascular remodeling and right ventricular hypertrophy.Compared to control rats,higher levels of GLS1 and GLS2 mRNA in lung and lower levels of these two isoforms of GLS in right ventricle(RV)were displayed in MCT-PH rats.After swimming exercise,GLS mRNA levels in the lung and RV were significantly upregulated,and the cross-sectional area of right ventricular cardiomyocytes was significantly decreased although the percentage of pulmonary arteriolar medial wall thickness was not significantly changed.Therefore,we hold the opinion that plasma GLS1 level was decreased in PH.The transcriptional levels of GLS1 and GLS2 were increased in the lung tissues in PH,but were decreased in the RV tissues.Meanwhile,the changes of GLS levels indicated the pulmonary vascular and right ventricular remodeling.Whereas moderate intensity swimming exercise might improve the right ventricular remodeling by regulating the levels of GLS.

The Effect of Glycyl-Glutamine Dipeptide Concentration on Enzyme Activity, Cell Proliferation and Apoptosis of Jejunal Tissues from Weaned Piglets

An experiment was conducted in a singly factorial design to study the effect of glycyl-glutamine dipeptide on enzyme activity, cell proliferation and apoptosis of jejunal tissues from weaned piglets at different glycyl-glutamine concentration levels of 2, 4, 10, 20, and 30 mmol L-1, respectively. The glutaminase activity, diamine oxidase （DAO） activity, cell peoliferation, apoptosis, and perotein metabolism were measured by the tissue culture method in vitro using jejunal tissues. The immunohistochemical method was used to study the cell proliferation and apoptosis of jejunal tissues. The results showed that compared to the blank control, the percentage and MOD value of BrdU-positicve cells incubated with glycyl-glutamine dipeptide solution were significantly （P0.05） increased. Accordingly, the percentage and MOD value of caspase-3-positive cells from tissue incubated with glycyl-glutamine dipeptide were notably lower （P0.05） than that from the control treatment. The glycyl-glutamine dipeptide increased the glutaminase activity, DAO activity and protein content of jejunal tissues, as the dipeptide concentration was on the rise （P0.05）. These results indicated that glycyl-glutamine dipeptide affected the jejunum development and adaptation of weaned piglets, and the function might be fulfilled by enhancing the glutamine-related enzyme activity, thereby increasing the consumption of glutamine, and then improving the jejunal cell proliferation and suppressing cell apoptosis. The effects of glycyl-glutamine dipeptide relied in a dose-dependent manner, and the maximum effect was achieved at 20-30 mmol L-1 glycyl-glutamine dipeptide.

Alteration of mitochondrial protein succinylation against cellular oxidative stress in cancer

Dear Editor,Lysine residue succinylation is a novel post-translational modification that recently attracted extensive attention.Succinylation is achieved by non-enzymatic processes or by a series of enzymes[like p300,lysine acetyltransferase 2A(KAT2A)]that transfer the succinyl groups from succinyl-coenzyme A(CoA)to the specific lysine,modulating protein function in various physiological processes[1].As a high-energy metabolite,succinyl-CoA is mainly produced within the mitochondrial matrix and peroxisomes.Its high-rate generation in the tricarboxylic acid(TCA)cycle and its impermeability across the mitochondrial inner membrane(due to its negative charge property)enhance succinyl-CoA accumulation within mitochondria.

ALTERATION IN ENTEROCYTE GENE EXPRESSION MAY EXPLAIN STRUCTURAL AND FUNCTIONAL CHANGES FOLLOWING GLUTAMINE SUPPLEMENTED PARENTERAL NUTRITION

Following extensive bowel resection, the intestinal tract undergoes a variety of adaptive responses to enhance bowel function. The purpose of this study was to determine the effect of glutamine-supplemented parenteral nutrition on mucosal cellularity and gut function. In addition, enterocyte gene expression of two relevant systems was also characterized and related to the structural and functional changes that occurred. Male Wistar rats underwent a 60% small bowel resection and jugular vein catheterization and were randomized into two groups. The control group (n = 10) received a standard intravenous nutritional solution and the study group (n = 10) received a similar solution but enriched with alanylglutamine dipeptide. After 7 days blood was taken for amino acid analysis, and bowel was harvested to determine mucosal morphology and expression of mucosal cell glutaminase and IGF-I mRNA. Mesentery lymphnodes were cultured to determine the presence of bacteria and thus access bacteria translocation. Serum glutamine concentration and mucosal architecture were maintained in the study group compared to the controls. Seventy percent of lymphnodes were cultured positive in control vs. only 20% in the study group (P

Development and distribution of PAG-immunoreactive neurons in the central pathway of trigeminal proprioception of the rat brainstem*

Objective: To investigate the development and distribution of phosphate-activated glutaminase like immunoreactive (PAG-LI) neurons in the central pathway of trigeminal proprioception of the rat brainstem. Methods: The immunohistochemitry techniques were used. Results: (1) At embryonic day 17 (E17), PAG-LI neurons were initially observed in the mesencephalic trigeminal nucleus (Vme). All PAG-LI neurons were large round neurons with moderate immunostaining. The immunoreactivity grew intense and attained adult-like pattern at P10. (2) Not until postnatal day 10 (P10) did a few PAG-LI neurons appear in the area ven-tral to the motor trigeminal nucleus (AVM) and area dorsal to the superior olivery nucleus (ADO), and not until P12 in the dorsomedial part of the subnucleus oralis of the spinal trigeminal nucleus (Vodm) and dorso-medial part of the principal sensory trigeminal nucleus (Vpdm). As development proceeded, more and more neurons in them were immunostained, and some PAG-LI neurons were detected in the lateral reticular forma-tion adjacent to the Vodm(LRF)and the caudolateral part of the supratrigeminal nucleus (Vsup-CL) at P21. Conclusion: In the central pathway of trigeminal proprioception of the rat brainstem, PAG-LI neurons ap-peared during two stages: The first stage from E17 to P10, PAG-LI neurons appeared in the Vme and reached adult-like pattern; the second stage from P10 to P21, PAG-LI neurons appeared in the Vodm, LRF, Vpdm, Vsup-CL, ADO, AVM and gradually reached adult-like pattern. This might be relative to the estab-lishment of jaw movement patterns.

Anaplerosis in cancer: Another step beyond the warburg effect

Biosynthesis is up-regulated in tumors and thus the demand for anabolic intermediates is increased. The metabolic routes providing the building blocks for macromolecules are thus a very attractive target as they are not normally up-regulated in a normal quiescent cell. Some routes for glycolysis-derived intermediates production have been identified, but these do not constitute the whole pool of biosynthetic molecules in the cell, as many of these derive from mitochondria in the Krebs cycle. Indeed, this metabolic pathway is considered a “biosynthetic hub” from which anabolism is fed. If a metabolite efflux is indeed occurring, anaplerotic reactions must keep a steady supply of substrates. In spite of this obvious relevance of anaplerosis, it has been poorly characterized in the malignant cell context. Glutaminolysis and and pyruvate carboxylation are two pathways that function in an anaplerotic fashion. In spite of the increasing evidence implicating these two processes in cancer metabolism their role as intermediate providers is overlooked. In this review we analyze the implications of an active anaplerosis in cancer and we discuss experimental evidence showing the relevance of these metabolic routes in tumor physiology.

PLASTIC SURGERY AND BURNS

The amount and constitution of protein, itsultraviolet photo-absorption ability, andesterase in homogenates of human cadavericand guinea pig skin (each consisted of newborn,fresh adult and stored adult skin) were assayedby using ultraviolet spectrophotometer,polyacrylamide gel electrophoresis and sodiumdodecyl sulphate polyacrylamide gel electro-

PRIMARY CLONING AND EXPRESSION OF A GENE ENCODING THE ANTILEUKEMIA AGENT(ASPARAGINASE)

Microbial asparaginase has been shown to have antitumor activity for lymphosarcoma, and carcinosarcoma, and is particularly effective in the treatment for acute lumphocytic leukemia. Since the 1960s, asparaginase purified from E. coli has been used clinically and found to be a very effective antileukemia agent but with certain side effects. In recent years, asparaginases from other bacteria, such as