Search

Hydatid cyst is a zoonotic infestation caused by Echinococcus granulosus, and it is known that some parasites found in humans cause cancer in humans or some may have a protective effect against cancer. Cancer is one of the most serious health problems of today and it has been shown in some studies that parasites such as Echinococcus granulosus can have an inhibitory effect. The aim of this study was determined as whether Echinococcus granulosus has an inhibitory effect on exocrine pancreatic cancer with the help of the azaserine-rat model used in different cancer studies.  Material and Methods: During experimental process a total of 45 male Wistar rats used, 14-day-old male Wistar rats were divided into groups according to the experimental protocol, administered azaserine injection protocol or kept as a control group without azaserine injection. Animals are grouped as Group 1, Control Group (group not treated with Azaserine and not injected with protoscolex.) (E-A-) (n=7); Group 2, Group injected with (IP) Azaserine only (30mg/kg) (E-A+)  (n=8);Group 3, Group injected (IP) with protoscolex suspension of 1 cc only (E+A-) (n=15);Group 4, Group injected both Azaserine (IP) and protoscolex suspension (IP) (E+A+) (n=15). Atypical Acinar Cell Foci (AACF) load in the exocrine pancreas of each rat was measured quantitatively with the help of a video image analyzer and the AACF load was calculated with the help of a mathematical model. Results: Findings showed that the Atypical Acinar Cell Foci (AACF) burden was statistically significantly lower in the Azaserine+ protoscolex (Azaserine-injected-protoscolex-implanted) rat group compared to the other groups, suggesting that Echinococcosis in the azaserine-rat model could inhibit the development of precursor foci of neoplastic changes in the exocrine pancreas. Conclusion: The most significant aspect of our study is that it contributes new insights into the controversy that Echinococcosis suppresses pancreatic cancer.

Objectives: High-fat diet has been considered a risk factor for the development of pancreatic cancer. It is also shown to significantly impact composition and dysbiosis of gut microbiota in both humans and animals. However, there is little information on the effect of high-fat diet on the development of pancreatic cancer or upon the gut microbiota of patients with pancreatic cancer in humans or animal models., Methods: In this study, the effect of high-fat diet on cancer pathology and the gut microbiota was investigated by a carcinogen-induced pancreatic cancer mouse model., Results: Compared with carcinogen alone, mice with high-fat diet and carcinogen showed more obvious pathological changes in pancreatic tissue; increased levels of proinflammatory cytokine tumor necrosis factor-α, interleukin-6, interleukin-10, and carbohydrate antigen 242; and increased expression of cancer-associated biomarkers mucin-4 and claudin-4 in pancreatic tissue. Moreover, there is a significant change in the gut microbiota between the carcinogen group and the carcinogen with high-fat diet group. We identified that Johnsonella ignava especially existed in the carcinogen with high-fat diet group, which may contribute to pancreatic cancer development., Conclusions: Our results revealed that high-fat diet changed the composition of the gut microbiota and was involved in carcinogen-induced pancreatic cancer progression., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Trypanosoma cruzi is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, T. cruzi passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to T. cruzi survival. Recently, we described that T. cruzi can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for T. cruzi survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.

The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that negatively regulates glucose uptake into cells. However, little information is available regarding the function of TXNIP in bovine oocytes. Accordingly, the present study was performed to investigate the influence of TXNIP on glucose metabolism in bovine oocytes during in vitro maturation. Pharmacological inhibition of TXNIP by azaserine enhanced glucose uptake and imparted a specific metabolic effect on glycolysis and pentose phosphate pathway (PPP). RNA interference (RNAi) was adopted to further determine the biological significance of TXNIP in regulating glucose metabolism. The maturation rate and the developmental competence of TXNIP siRNA-treated oocytes were significantly improved. Knockdown of TXNIP in bovine oocytes significantly increased glycolysis by increasing the activities of phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase; pyruvate and lactate production; and intracellular ATP level, as well as mitochondrial activity. Furthermore, glucose metabolism through PPP was also enhanced by TXNIP depletion, as TXNIP siRNA treatment promoted glucose-6-phosphate dehydrogenase (G6PDH) activity and NADPH content, and helped maintain a high level of glutathione and a low level of reactive oxygen species within the oocytes. Further studies revealed that inhibition of TXNIP resulted increases in glucose transporter 1 (GLUT1) expression, as well as PFK1 platelet isoform ( PFKP ) and G6PDH mRNA levels. These results reveal that TXNIP depletion promotes oocyte maturation by enhancing both glycolysis and the PPP. During in vitro maturation of bovine oocytes, TXNIP serves as a key regulator of glucose uptake by controlling GLUT1 expression.

Cells under stress generate reactive oxygen species (ROS) in excess, which causes mitochondrial dysfunction and stimulates the apoptotic cascade. However, mild stress or pre-conditioning lead to the evasion of apoptosis by activating mitogenic signaling, including the signaling of inhibitors of apoptosis proteins (IAPs), or by inactivating certain apoptotic molecules. The silkworm (Bombyx mori) is an important economic insect which serves as a model organism in biological research. Bombyx mori apoptotic protease inducing factor (BmApaf1), a death-related ced-3/Nedd2-like protein (BmDredd), and BmSurvivin-2 (BmSvv2) are known to play significant roles in metamorphosis. Azaserine is an analogue of glutamine and irreversibly inhibits glutamine-utilizing enzymes and cysteine-glutamate transporter genes EAAT2. In the present study, we experimentally demonstrated stress induced by azaserine along with the capacity of antioxidants to modulate apoptotic/anti-apoptotic gene expression in determining the fate of the larvae. We observed higher larval survival with higher azaserine dosages and attributed this to the quantum of ROS generated and AOEs response, which favoured the BmSvv2 expression. Meanwhile higher levels of ROS with concomitant changes in AOEs were found to be responsible for BmApaf1 and BmDredd expression, which reflected a higher mortality rate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Artemisinin and its analogues (ARTs) are currently the most effective anti-malarial drugs, but the precise mechanism of action is still highly controversial. Effects of ARTs on Plasmodium genes expression are studied in our Lab. The overexpression of an interesting amidotransferase, NADH-dependent glutamate synthase (NADH-GltS) was found in treated by dihydroartemisinin (DHA). The increased expression occurred not only from global transcriptomics analysis on the human malaria parasite Plasmodium falciparum ( P. falciparum ) 3D7 and gene expression screening on all of iron-sulphur cluster proteins from P . f . 3D7 in vitro but also from Plasmodium berghei ( P. berghei ) ANKA in mice. Influence of DHA on NADH-GltS was specifically at trophozoite stage of P. falciparum and in a dose-dependent manner below the effective doses. L-glutamine (Gln) and L-glutamate (Glu) are the substrate and product of NADH-GltS respectively. Azaserine (Aza) is specific inhibitor for NADH-GltS. Experimental data showed that Glu levels were significantly decreasing with DHA dose increasing but NADH-GltS enzyme activities were still remained at higher levels in parasites, and appropriate amount of exogenous Glu could significantly reduce anti-malarial action of DHA but excessive amount lost the above effect. Aza alone could inhibit proliferation of P. falciparum and had an additive effect in combination with DHA. Those results could suggest that: Glutamate depletion is one of the anti-malarial actions of DHA; overexpression of NADH-GltS would be a feedback pattern of parasite itself due to glutamate depletion, but not a direct action of DHA; the "feedback pattern" is one of protective strategies of Plasmodium to interfere with the anti-malarial actions of DHA; and specific inhibitor for NADH-GltS as a new type of anti-malarial agents or new partner in ACT might provide a potential.

The structures and amounts of glycosaminoglycan (GAG) produced by cells have attracted much interest because GAG biosynthesis activity can change in cellular processes such as disease and differentiation. β-Xylosides, also called saccharide primers, have been used as artificial acceptors not only to generate GAG oligosaccharides in cells and tissues but also to investigate their biosynthetic pathways. Various analytical methods have been applied to confirm the structure and amounts of GAG oligosaccharides elongated using saccharide primers, yet sample preparation processes such as solid-phase extraction in analysis can cause experimental error and disrupt accurate comparative quantification of glycosylated products. In this study, we developed a new quantification method using a deuterium-labeled saccharide primer. The "heavy" and "light" primers were chemically synthesized, and priming abilities were confirmed by liquid chromatography-tandem mass spectrometry. Relative peak areas of light/heavy products showed good linearity and were well correlated with the theoretical amounts of glycosylated products. Then, as a validation study, we carried out a biosynthesis inhibition assay using known GAG biosynthesis inhibitors. According to the relative quantification using saccharide primers, differences in the mode-of-action among the four GAG biosynthesis inhibitors were dependent on the GAG biosynthetic pathway. Our results indicate that the method will likely forge a new path for comparative glycosaminoglycomics using cultured cells and tissues.

Sirtuin 3 (SIRT3) is a NAD + -dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet (HFD)-induced metabolic disorders. Here, we performed a small-molecule screen and identified an unexpected metabolic vulnerability associated with SIRT3 loss. Azaserine, a glutamine analog, was the top compound that inhibited growth and proliferation of cells lacking SIRT3. Using stable isotope tracing of glutamine, we observed its increased incorporation into de novo nucleotide synthesis in SIRT3 knockout (KO) cells. Furthermore, we found that SIRT3 KO cells upregulated the diversion of glutamine into de novo nucleotide synthesis through hyperactive mTORC1 signaling. Overexpression of SIRT3 suppressed mTORC1 and growth in vivo in a xenograft tumor model of breast cancer. Thus, we have uncovered a metabolic vulnerability of cells with SIRT3 loss by using an unbiased small-molecule screen., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without affecting downstream targets previously suggested to be involved, such as p70 ribosomal S6 protein kinase, calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal-regulated kinases. Instead, cardiomyocyte hypertrophy is accompanied by increased protein O-GlcNAcylation, which is reversed by AMPK activation. Decreasing O-GlcNAcylation by inhibitors of the glutamine:fructose-6-phosphate aminotransferase (GFAT), blocks cardiomyocyte hypertrophy, mimicking AMPK activation. Conversely, O-GlcNAcylation-inducing agents counteract the anti-hypertrophic effect of AMPK. In vivo, AMPK activation prevents myocardial hypertrophy and the concomitant rise of O-GlcNAcylation in wild-type but not in AMPKα2-deficient mice. Treatment of wild-type mice with O-GlcNAcylation-inducing agents reverses AMPK action. Finally, we demonstrate that AMPK inhibits O-GlcNAcylation by mainly controlling GFAT phosphorylation, thereby reducing O-GlcNAcylation of proteins such as troponin T. We conclude that AMPK activation prevents cardiac hypertrophy predominantly by inhibiting O-GlcNAcylation.

The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational protein modification that adds GlcNAc to nuclear and cytoplasmic proteins. Increased GlcNAc has been linked to regulatory factors involved in cancer cell growth and survival. However, the biological significance of GlcNAc in diffuse large B-cell lymphoma (DLBCL) is not well defined. This study is the first to show that both the substrate and the endpoint O-GlcNAc transferase (OGT) enzyme of the HBP were highly expressed in DLBCL cell lines and in patient tumors compared with normal B-lymphocytes. Notably, high OGT mRNA levels were associated with poor survival of DLBCL patients. Targeting OGT via small interference RNA in DLBCL cells inhibited activation of GlcNAc, nuclear factor kappa B (NF-κB), and nuclear factor of activated T-cells 1 (NFATc1), as well as cell growth. Depleting both glucose and glutamine in DLBCL cells or treating them with an HBP inhibitor (azaserine) diminished O-GlcNAc protein substrate, inhibited constitutive NF-κB and NFATc1 activation, and induced G0/G1 cell-cycle arrest and apoptosis. Replenishing glucose-and glutamine-deprived DLBCL cells with a synthetic glucose analog (ethylenedicysteine-N-acetylglucosamine [ECG]) reversed these phenotypes. Finally, we showed in both in vitro and in vivo murine models that DLBCL cells easily take up radiolabeled technetium-99m-ECG conjugate. These findings suggest that targeting the HBP has therapeutic relevance for DLBCL and underscores the imaging potential of the glucosamine analog ECG in DLBCL.

Humans are exposed to N-nitroso compounds through environmental exposure and endogenous metabolism. Some N-nitroso compounds can be metabolically activated to yield diazoacetate, which is known to induce DNA carboxymethylation. DNA lesion measurement remains one of the core tasks in toxicology and in evaluating human health risks associated with carcinogen exposure. In this study, we developed a highly sensitive nanoflow liquid chromatography-nanoelectrospray ionization-multistage tandem mass spectrometry (nLC-nESI-MS(3)) method for the simultaneous quantification of O(6)-carboxymethyl-2'-deoxyguanosine (O(6)-CMdG), O(6)-methyl-2'-deoxyguanosine (O(6)-MedG), and N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA). We were able to measure the levels of these three lesions with the use of low-microgram quantities of DNA from cultured human skin fibroblasts and human colorectal carcinoma cells treated with azaserine, a DNA carboxymethylating agent. Our results revealed that the levels of O(6)-CMdG and O(6)-MedG increased when the dose of azaserine was increased from 0 to 450 μM. We, however, did not observe an apparent dose-dependent induction of N(6)-CMdA, suggesting the presence of repair mechanism(s) for the rapid clearance of this lesion in cells. This is the first report about the application of nLC-nESI-MS(3) technique for the simultaneous quantification of O(6)-CMdG, O(6)-MedG, and N(6)-CMdA. The method reported here will be useful for future investigations about the repair of the carboxymethylated DNA lesions and about the implications of these lesions in carcinogenesis.

Cancer was hypothesized to be driven by cancer stem cells (CSCs), but the metabolic determinants of CSC-like phenotype still remain elusive. Here, we present that hexosamine biosynthetic pathway (HBP) at least in part rescues cancer cell fate with inactivation of glycolysis. Firstly, metabolomic analysis profiled cellular metabolome in CSCs of hepatocellular carcinoma using CD133 cell-surface marker. The metabolic signatures of CD133-positive subpopulation compared to CD133-negative cells highlighted HBP as one of the distinct metabolic pathways, prompting us to uncover the role of HBP in maintenance of CSC-like phenotype. To address this, CSC-like phenotypes and cell survival were investigated in cancer cells under low glucose conditions. As a result, HBP inhibitor azaserine reduced CD133-positive subpopulation and CD133 expression under high glucose condition. Furthermore, treatment of N-Acetylglucosamine in part restores CD133-positive subpopulation when either 2.5 mM glucose in culture media or glycolytic inhibitor 2-deoxy-D-glucose in HCC cell lines was applied, enhancing CD133 expression as well as promoting cancer cell survival. Together, HBP might be a key metabolic determinant in the functions of hepatic CSC marker CD133.

Background: We previously demonstrated that hyperglycemia could suppress apolipoprotein M (apoM) synthesis both in vivo and in vitro; however, the mechanism of hyperglycemia-induced downregulation of apoM expression is unknown yet., Methods: In the present study we further examined if hexosamine pathway, one of the most important pathways of glucose turnover, being involved in modulating apoM expression in the hyperglycemia condition. We examined the effect of glucosamine, a prominent component of hexosamine pathway and intracellular mediator of insulin resistance, on apoM expression in HepG2 cells and in rat's models. In the present study we also determined apolipoprotein A1 (apoA1) as a control gene., Results: Our results demonstrated that glucosamine could even up-regulate both apoM and apoA1 expressions in HepG2 cell cultures. The glucosamine induced upregulation of apoM expression could be blocked by addition of azaserine, an inhibitor of hexosamine pathway. Moreover, intravenous infusion of glucosamine could enhance hepatic apoM expression in rats, although serum apoM levels were not significantly influences., Conclusions: It is concluded that both exogenous and endogenous glucosamine were essential for the over-expression of apoM, which may suggest that the increased intracellular content of glucosamine does not be responsible for the depressed apoM expression at hyperglycemia condition.

Objectives: Through the hexosamine biosynthetic pathway (HBP) proteins are modified by O-linked-β-N-acetylglucosamine (O-GlcNAc), which acts as a stress sensor. Augmentation of O-GlcNAc confers cardioprotection against ischemia- reperfusion injury, but its role in ischemic preconditioning (IPC) is unknown. Azaserine and alloxan are unspecific blockers of the HBP and have been used to block the cardioprotective effects of O-GlcNAc. We hypothesized that IPC reduces infarct size and increases O-GlcNAc levels in hearts subjected to ischemia-reperfusion injury, and that these effects could be blocked by azaserine and alloxan., Design: Isolated rat hearts subjected to 40 min global ischemia and 120 min reperfusion were randomized to control, IPC, IPC + azaserine or alloxan, or control + azaserine or alloxan. The effects on infarct size, hemodynamic recovery, myocardial O-GlcNAc levels, and HBP enzyme activities were determined., Results: IPC reduced infarct size, increased O-GlcNAc levels, O-GlcNAc-transferase levels, and O-GlcNAc-transferase activity. Azaserine and alloxan did not block the effect of IPC on O-GlcNAc levels and O-GlcNAc-transferase activity., Conclusions: IPC increased O-GlcNAc levels though increased O-GlcNAc-transferase expression and activity. Azaserine and alloxan failed to block these effects presumably due to poor specificity and sensitivity of the blockers, and IPC-mediated cardioprotection may therefore still be dependent on O-GlcNAc.

Early hyperglycemic insult can lead to permanent, cumulative damage that might be one of the earliest causes for a pre-diabetic situation. Despite this, the early phases of hyperglycemic exposure have been poorly studied. We have previously demonstrated that mitochondrial injury takes place early on upon hyperglycemic exposure. In this work, we demonstrate that just 1 h of hyperglycemic exposure is sufficient to induce increased mitochondrial membrane potential and generation. This is accompanied (and probably caused) by a decrease in the cells' NAD(+)/NADH ratio. Furthermore, we show that the modulation of the activity of parallel pathways to glycolysis can alter the effects of hyperglycemic exposure. Activation of the pentose phosphate pathway leads to diminished effects of glucose on the above parameters, either by removing glucose from glycolysis or by NADPH generation. We also demonstrate that the hexosamine pathway inhibition also leads to a decreased effect of excess glucose. So, this work demonstrates the need for increased focus of study on the reductive status of the cell as one of the most important hallmarks of initial hyperglycemic damage.

We investigated whether the acrylamide formed during cooking carbohydrate-rich foods at high temperatures causes neoplastic changes in rat pancreas. Azaserine, which is an amino acid derivative that has the ability to initiate neoplastic changes in rat pancreas, was injected into 14-day-old male rats once a week for three weeks. Acrylamide was given to both azaserine-injected and non-injected rats at doses of 5 and 10 mg/kg/day in drinking water for 16 weeks after which tissue slides were prepared from the pancreata. Pancreas weights and body weights of rats treated with azaserine and acrylamide together increased significantly compared to the other groups. Moreover, the size, average diameter and volume of atypical acinar cell foci that developed in the pancreata of rats treated with azaserine and acrylamide together increased significantly compared to rats treated with either azaserine or acrylamide alone and control groups. Atypical acinar cell adenoma or adenocarcinoma was not observed in the pancreata of rats in any group.

The short half-life protooncogene β-catenin acquires a remarkable stability in a large subset of cancers, mainly from mutations affecting its proteasomal degradation. In this sense, colorectal cancers (CRC) form a group of pathologies in which early steps of development are characterized by an aberrant expression of β-catenin and an uncontrolled proliferation of epithelial cells. Diet has long been described as an influence in the emergence of CRC, but the molecular events that link metabolic disorders and CRC remain elusive. Part of the explanation may reside in hexosamine biosynthetic pathway (HBP) flux. We found that fasted mice being force-fed with glucose or glucosamine leads to an increase of β-catenin and O-GlcNAcylation levels in the colon. MCF7 cells possessing intact Wnt/β-catenin signaling heavily expressed β-catenin when cultured in high glucose; this was reversed by the HBP inhibitor azaserine. HBP inhibition also decreased the expression of β-catenin in HT29 and, to a lesser extent, HCT116 cells. The same observation was made with regard to the transcriptional activity of β-catenin in HEK293 cells. Inhibition of HBP also blocked the glucose-mediated proliferation capacity of MCF7 cells, demonstrating that glucose affects both β-catenin expression and cell proliferation through the HBP. The ultimate element conducting these events is the dynamic posttranslational modification O-GlcNAcylation, which is intimately linked to HBP; the modulation of its level affected the expression of β-catenin and cell proliferation. In accordance with our findings, we propose that metabolic disorders correlate to CRC via an upregulation of HBP that reverberates on high O-GlcNAcylation levels including modification of β-catenin.

CTP synthase is compartmentalized within a subcellular structure, termed the cytoophidium, in a range of organisms including bacteria, yeast, fruit fly and rat. Here we show that CTP synthase is also compartmentalized into cytoophidia in human cells. Surprisingly, the occurrence of cytoophidia in human cells increases upon treatment with a glutamine analog 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of glutamine-dependent enzymes including CTP synthase. Experiments in flies confirmed that DON globally promotes cytoophidium assembly. Clonal analysis via CTP synthase RNA interference in somatic cells indicates that CTP synthase expression level is critical for the formation of cytoophidia. Moreover, DON facilitates cytoophidium assembly even when CTP synthase level is low. A second glutamine analog azaserine also promotes cytoophidum formation. Our data demonstrate that glutamine analogs serve as useful tools in the study of cytoophidia., (Copyright © 2011. Published by Elsevier Ltd.)

Glutamate synthase (E.C. 1.4.1.14) (GOGAT) activity was not detectable in L3 Haemonchus contortus, but was present in L3 Teladorsagia circumcincta and adult worms of both species. GOGAT activity was inhibited by 80% by azaserine. Activity (nmol min(-1) mg(-1) protein) was 33-59 in adult H. contortus, 51-91 in adult T. circumcincta and 24-41 in L3 T. circumcincta, probably depending on exposure to ammonia, as incubation with 1mM NH(4)Cl doubled GOGAT activity. The pH optimum was 7.5 in both species. Either NAD or NADP acted as co-factor. The mean apparent K(m) for 2-oxoglutarate was 0.7 (0.5-0.9) mM and for glutamine was 1.0 (0.5-1.7) mM for different homogenates. There was no detectable activity in whole parasite homogenates of glutamate decarboxylase (E.C. 4.1.1.15) or succinic semialdehyde dehydrogenase (E.C. 1.2.1.24), the first and third enzymes of the GABA shunt, respectively, suggesting that the GABA shunt is not important in general metabolism in these species., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aim: To study the phenomenon that human erythroid leukemia K-562 cells are more sensitive to cytotoxic effect of antimetabolites when cultured in a serum-free medium than in a conventional medium containing fetal calf serum (FCS)., Methods: Cytotoxic effects of methotrexate, azaserine and 5-fluorouracil were estimated by accessing the lactate dehydrogenase (LDH) activity of viable tumor cells. Proteins of FCS were separated using two-dimensional electrophoresis followed by mass spectrometry analysis., Results: Addition of 10% FCS attenuated anti-tumor activity of methotrexate and azaserine against K-562 cells compared with serum-free medium. Such an activity of FCS was different for each serum lot. Comparison of the proteins in active serum lot with those in not active one using two-dimensional electrophoresis showed that in the active serum there were proteins 150 kDa, which were absent in the not active serum lot. Mass spectrometry indicated that all those proteins had the amino acid sequence of albumin. Sera of one healthy volunteer and two patients with thyroid cancer also attenuated the activity of the agent., Conclusion: Several lots of FCS and human serum demonstrated the ability to attenuate the cytotoxic effect of methotrexate in vitro, possibly due to the formation of albumin dimers/MTX complexes.

Objective: To further study the safety and effect of the umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) on apoptosis of human cardiac myocyte (HCM)., Methods: The UCB was collected at the time of delivery with informed consent obtained from 10 donors. The UCB-derived MSCs was treated with 5-azaserine (5-AZA), and further introduced differentiation into cardiomyocytes. The telomerase activity, G-banding patterns of chromosomal karyotypes, tumor formation in nude mice, reverse transcription polymerase chain reaction (RT-PCR), and the inhibited apoptosis of UCB-derived MSCs were further investigated. This study was carried out in the laboratory of Beijing Shijitan Hospital, Beijing, China and Inheritance Research Section of Chinese Medical Institute, Beijing, China from July 2005 to December 2007., Results: The MSCs-derived from UCB were differentiated into cardiomyocytes in vitro, possessed telomerase activity after 5-AZA induction, and no abnormal chromosomal karyotypes were observed. Expression of p53, cyclinA, cdk2, -actin, C-fos, h-TERT and c-myc were similar in MSCs before and after 5-AZA treatment. There was no tumor formation injected into nude mice. The UCB-derived MSCs significantly inhibited apoptosis of human cardiomyocytes., Conclusion: Umbilical cord blood-derived MSCs are safe and effective source of cell-transplantation treatment, and can inhibit the apoptosis of human cardiomyocytes in co-cultured.

Hexosamine biosynthetic pathway (HBP) accounts for some cardiovascular adverse effects of hyperglycemia. We investigated whether the HBP inhibitor azaserine protects against hyperglycemia-induced endothelial damage dependently of HBP. Human endothelial cells isolated from umbilical veins were exposed either to a high (30.5 mmol/l) or low concentration of glucose (5.5 mmol/l) for 4 days, followed by a stimulation with TNF-alpha (1 ng/ml, 24 h). The blockade of the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase inhibited HBP flux and oxidative stress (generation of superoxide and peroxynitrite) under the hyperglycemic condition and prevented the synergistic stimulation of VCAM-1 and ICAM-1 expression by hyperglycemia and TNF-alpha. In the cells cultured under a low-glucose condition when no increased HBP flux occurred, azaserine enhanced the manganese-superoxide dismutase (MnSOD) protein level and also inhibited the oxidative stress and the expression of VCAM-1 and ICAM-1 in response to TNF-alpha. Moreover, the polyphenol resveratrol inhibited the oxidative stress and adhesion molecule expression and did not decrease the HBP flux under the hyperglycemia condition. In addition, in isolated rat aortas exposed to hyperglycemic buffer for 5 h when no significant HBP flux occurred, azaserine upregulated the MnSOD protein level and prevented decreased endothelium-dependent relaxations to acetylcholine. In conclusion, hyperglycemia independently increases oxidative stress and HBP flux, amplifies endothelial inflammation, and impairs endothelial function mainly through oxidative stress and not the HBP pathway. Azaserine protects against hyperglycemic endothelial damage through its antioxidant effect independently of inhibiting HBP pathway.

Petal pigmentation is the most important aspect in natural flower coloration. In the present study, the inhibition of petal pigmentation by exogenous ammonium was investigated. Ray floret petals detached from inflorescences of Gerbera hybrida (Shenzhen No. 5) were cultured in vitro on media supplied with different forms of nitrogen and its assimilated compounds. The expression of a set of genes involved in anthocyanin biosynthesis and regulation was determined by Northern blotting assay. It was found that ammonium (NH4+), not nitrate (NO3-), in millimolar concentrations inhibited anthocyanin accumulation. The expressions of Gerbera chalcone synthase 1 (GCHS1), Gerbera chalcone synthase 2 (GCHS2) and Gerbera dihydroflavonol-4-reductase (GDFR) decreased, while six other related genes showed no significant changes after NH4+ treatment. Further studies on NH4+ function indicated that glutamine (Gln) acted as a downstream factor of NH4+ to suppress petal pigmentation. Both exogenous Gln and NH4+ were found to inhibit anthocyanin accumulation in the petals, and the application of Gln was also found to inhibit the expressions of GCHS1, GCHS2 and GDFR. The application of NH4+ also resulted in an increase in the activity of Gerbera glutamine synthetase (EC 6.3.1.2) along with a rapid increase of Gln content. When methionine sulfoximine, an inhibitor of glutamine synthetase (GS), was added, it was found to block the NH4+-induced inhibition of pigmentation. From these experiments, we conclude that the NH4+-induced suppression of petal pigmentation is not because of NH4+ toxicity, and the inhibition of pigmentation caused by the addition of exogenous NH4+ is the result of its assimilation into Gln.

Purines are synthesized de novo in 10 chemical steps that are catalyzed by six enzymes in eukaryotes. Studies in vitro have provided little evidence of anticipated protein-protein interactions that would enable substrate channeling and regulation of the metabolic flux. We applied fluorescence microscopy to HeLa cells and discovered that all six enzymes colocalize to form clusters in the cellular cytoplasm. The association and dissociation of these enzyme clusters can be regulated dynamically, by either changing the purine levels of or adding exogenous agents to the culture media. Collectively, the data provide strong evidence for the formation of a multi-enzyme complex, the "purinosome," to carry out de novo purine biosynthesis in cells.

Although mouse oocytes and cleavage-stage embryos are unable to utilize glucose as a metabolic fuel, they have a specific requirement for a short exposure to glucose prior to compaction. The reason for this requirement has been unclear. In this study we confirm that cleavage-stage exposure to glucose is required for blastocyst formation and show that the absence of glucose between 18-64 h after hCG causes an irreversible decrease in cellular proliferation and an increase in apoptosis. More importantly, this glucose signals to activate expression of Slc2a3 transcript and SLC2A3 protein, a facilitative glucose transporter (previously known as GLUT3) associated with developmental competence and increased glucose uptake used to fuel blastocyst formation. Glucosamine could substitute for glucose in these roles, suggesting that hexosamine biosynthesis may be a nutrient-sensing mechanism involved in metabolic differentiation. Inhibition of the rate-limiting enzyme in this pathway, glutamine-fructose-6-phosphate amidotransferase (GFPT), inhibited expression of the SLC2A3 transporter protein and blastocyst formation. Glucosamine, a substrate that enters this pathway downstream of GFPT, was able to overcome this inhibition and support SLC2A3 expression. These data suggest that early embryos rely on hexosamine biosynthesis as a glucose-sensing pathway to initiate metabolic differentiation.

N-methyl-N'-nitro-N-nitrosoguanidine (NH)-induced mutagenesis with subsequent selection for resistance to toxic amino acid analogues (azaserine, m-fluoro-DL-phenylalanine, and 6-diazo-5-oxo-L-norleucine) was applied to Pseudomonas aurantiaca B-162. The resulting strains produced phenazine antibiotics three times more efficiently than the wild type strain and ten times more efficiently than the known pseudomonad strains. Overproduction of phenazine antibiotics was shown to result either from deregulation of 3-deoxi-D-arabinohepulosonate-7-phosphate synthase (DAHP synthase), the key enzyme of the aromatic pathway (removal of inhibition by phenylalanine, tyrosine, and phenazine), or overproduction of N-hexanoyl homoserine lactone, the regulatory molecule of positive control of cellular metabolism (QS system).

We herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis, specifically a more efficient transformation and a gene expression system. We evaluated several parameters that influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions and host cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensis mixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78+/-9 transformants/co-cultivation (5+/-1 transformants/10(6) target cells). P. brasiliensis GFP-expressing isolates were also constructed by insertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopy and flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differences in fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random single gene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use in targeted mutagenesis and linking mutations to phenotypes.

Deposition of islet amyloid polypeptide (IAPP) as amyloid in the pancreatic islet occurs in approximately 90% of individuals with Type 2 diabetes and is associated with decreased islet beta-cell mass and function. Human IAPP (hIAPP), but not rodent IAPP, is amyloidogenic and toxic to islet beta-cells. In addition to IAPP, islet amyloid deposits contain other components, including heparan sulfate proteoglycans (HSPGs). The small molecule 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha-D-xylo-hexopyranose (WAS-406) inhibits HSPG synthesis in hepatocytes and blocks systemic amyloid A deposition in vivo. To determine whether WAS-406 inhibits localized amyloid formation in the islet, we incubated hIAPP transgenic mouse islets for up to 7 days in 16.7 mM glucose (conditions that result in amyloid deposition) plus increasing concentrations of the inhibitor. WAS-406 at doses of 0, 10, 100, and 1,000 microM resulted in a dose-dependent decrease in amyloid deposition (% islet area occupied by amyloid: 0.66 +/- 0.14%, 0.10 +/- 0.06%, 0.09 +/- 0.07%, and 0.004 +/- 0.003%, P < 0.001) and an increase in beta-cell area in hIAPP transgenic islets (55.0 +/- 2.6 vs. 60.6 +/- 2.2% islet area for 0 vs. 100 microM inhibitor, P = 0.05). Glycosaminoglycan, including heparan sulfate, synthesis was inhibited in both hIAPP transgenic and nontransgenic islets (the latter is a control that does not develop amyloid), while O-linked protein glycosylation was also decreased, and WAS-406 treatment tended to decrease islet viability in nontransgenic islets. Azaserine, an inhibitor of the rate-limiting step of the hexosamine biosynthesis pathway, replicated the effects of WAS-406, resulting in reduction of O-linked protein glycosylation and glycosaminoglycan synthesis and inhibition of islet amyloid formation. In summary, interventions that decrease both glycosaminoglycan synthesis and O-linked protein glycosylation are effective in reducing islet amyloid formation, but their utility as pharmacological agents may be limited due to adverse effects on the islet.

It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identified. Glutamine:fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP), and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5 mM glucose, and global, no-flow ischemia was induced for 20 min followed by 60 min of reperfusion. Thirty-minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6+/-5.7% vs. 59.4+/-6.1%; p<0.05) and decreased cardiac troponin I release (25.4+/-3.0 vs. 4.7+/-1.9 ng/ml; p<0.05) during reperfusion. This protection was associated with a significant increase in the levels of protein O-GlcNAc and ATP. Pre-treatment with 80 muM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in protein O-GlcNAc. O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc, and inhibition of OGT with 5 mM alloxan also reversed the protection associated with glutamine. These data support the hypothesis that in the ex vivo perfused heart glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels.

Changes in glucose levels are known to directly alter gene expression. A number of previous studies have found that these effects are in part mediated by modulating the levels and the activity of transcription factors. We have investigated an alternative mechanism by which glucose might regulate gene expression by modulating levels of a transcriptional repressor. We have focused on Id2, which is a protein that indirectly regulates gene expression by sequestering certain transcription factors and preventing them from forming functional dimers. Id2 targets include the class A basic helix-loop-helix transcription factors and the sterol regulatory element-binding protein (SREBP)-1. We demonstrate that increases in glucose levels cause a rapid increase in levels of Id2 in J774.2 macrophages, and a number of lines of evidence indicate that this is via the hexosamine pathway because 1) the effect of glucose requires glutamine; 2) the effect of glucose is mimicked by low levels of glucosamine; 3) the effect of glucose is inhibited by azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT); and 4) adenoviral mediated overexpression of GFAT increases levels of Id2. We go on to show that increases in Id2 can have functional effects on metabolic genes, because Id2 blocked the SREBP-1-induced induction of hormone-sensitive lipase (HSL) promoter activity, whereas Id2 alone does not modulate activity of the HSL promoter. In summary, these studies define a new mechanism by which glucose uses the hexosamine pathway to regulate gene expression by increasing levels of a transcriptional repressor.

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.

In humans, initial events of pancreatic carcinogenesis remain unknown, and the question of whether this cancer, which has a ductal phenotype, exclusively arises from duct cells has been raised. Previous studies have demonstrated that transgenic expression of the CCK2 receptor in acinar cells of ElasCCK2 mice plays a role in the development of pancreatic neoplasia. The aim of our study was to examine initial steps of carcinogenesis in ElasCCK2 mice, adding a supplementary defect by using a chemical carcinogen, azaserine. Results of posttreatment sequential immunohistochemical examinations and quantifications demonstrate that mice responded to azaserine. Transition of acinar cells into duct-like cells expressing Pdx1 and gastrin, as well as proliferation of acinar cells, were transiently observed in both transgenic and control mice. The carcinogen also induced formation of preneoplastic lesions, adenomas, exhibiting properties of autonomous growth. Importantly, expression of the CCK2 receptor increased the susceptibility of pancreas to azaserine. Indeed, treated ElasCCK2 mice exhibited larger areas of pancreatic acinar-ductal transition, increased cellular proliferation as well as larger adenomas areas vs. control mice. These amplified responses may be related to auto/paracrine stimulation of CCK2 receptor by gastrin expressed in newly formed duct-like cells. Our results demonstrate that activation of CCK2 receptor and azaserine result in cumulative effects to favor the emergence of a risk situation that is a potential site for initiation of carcinogenesis., ((c) 2005 Wiley-Liss, Inc.)

Aim: To set up an IR-HIRc cell model for screening the inhibitor of GFAT (glutamine: fructose-6-phosphate amidotransferase) , the key enzyme in the hexosamine biosynthesis pathway (HBP)., Methods: For GFAT activity assay, the GDH method was improved by adjusting the value of pH in the reaction system and the concentrations of the reactants. The sensitivity to insulin in the cells was estimated by the measurement of insulin-induced glucose-uptake. The IR-HIRc model was set up by the stimulation of long-action insulin for 36 h. The IR-HIRc model and GDH method was used for screening GFAT inhibitor., Results: With the administration of 25 nmol x L(-1) long-action insulin in HIRe cells for 36 hours, the GFAT activity increased by 47% and the insulin-induced glucose-uptake decreased by 21%. Azaserine, a GFAT inhibitor, inhibited GFAT activity significantly in a dose-dependent manner in IR-HIRc model., Conclusion: With the stimulation of 25 nmol x L(-1) long-action insulin for 36 h, excess hexosamine flux and insulin resistant in IR-HIRc cell model was set up, which can be used for screening

We reported previously that insulin inhibits the stimulatory effect of high glucose on the expression of angiotensinogen (ANG) gene in both rat immortalized renal proximal tubular cells (IRPTCs) and non-diabetic rat renal proximal tubular cells (RPTCs), but has no effect in diabetic rat RPTCs. In the present study we investigated whether hyperglycaemia-induced resistance to the insulin-induced inhibition of expression of the ANG gene is mediated via the generation of reactive oxygen species (ROS) in RPTCs. Rat IRPTCs were cultured for 2 weeks in high-glucose (25 mM) or normal-glucose (5 mM) medium plus angiotensin II (Ang II) with or without a superoxide scavenger (tiron), or inhibitors of: NADPH oxidase (diphenylene iodinium, DPI), Ang II type 1 and 2 receptors (losartan and PD123319), angiotensin-converting enzyme (perindopril), protein kinase C (GF 109203X), or glutamine:fructose-6-phosphate amino-transferase (azaserine). Cellular generation of ROS, and ANG and renin mRNA levels were assessed by lucigenin assay and specific reverse transcriptase-PCR respectively. Phosphorylation of p44/42 mitogen-activated protein kinase (p44/42 MAPK) was evaluated by western blotting. Prolonged exposure of IRPTCs to high concentrations of glucose or Ang II evoked generation of ROS and resistance to the insulin-induced inhibition of expression of the ANG gene and of p44/42 MAPK phosphorylation. Co-incubation of IRPTCs with tiron, DPI, losartan, PD123319, perindopril, GF 109203X or azaserine prevented ROS generation, restoring the inhibitory action of insulin on ANG gene expression and on p44/42 MAPK phosphorylation. In conclusion, our studies demonstrate that blockade of both ROS generation and activation of the intrarenal renin-angiotensin system improves the inhibitory action of insulin on ANG gene expression in IRPTCs in conditions of high glucose.

In vivo (15)N nuclear magnetic resonance (NMR) as well as (15)N solid-state magic angle spinning (MAS) NMR spectroscopy were used to investigate nitrogen metabolism in cultured white spruce (Picea glauca) buds. Long-term as well as short-term experiments were carried out involving the use of inhibitors of the nitrogen pathways such as methionine sulfoximine (MSO), azaserine (AZA) and aminooxyacetate (AOA). Both in vivo and solid-state NMR showed that when MSO blocked glutamine synthetase (GS) no NH(4)(+) is incorporated. When glutamate synthase (GOGAT) is blocked by AZA there is some incorporation into glutamine (Gln), but very little into alpha-amino groups (glutamate, Glu). The transamination inhibitor AOA does not affect the metabolism of (15)NH(4)(+) into Gln and Glu, but blocks the production of arginine (Arg), as would be expected. Proline (Pro) and gamma-aminobutyric acid (GABA), which are produced directly from Glu without a transamination step, were not affected. The solid-state NMR experiments showed that protein synthesis occurred. Collectively, our results show that NH(4)(+) can only be assimilated through the GS/GOGAT pathway in P. glauca buds.

The growth of Escherichia coli is inhibited by an antibiotic compound, azaserine (O-diazoacetyl-L-serine). Previous studies revealed the biochemical properties of azaserine, which involves inhibition of various enzymatic reactions as well as introduction of DNA breakage. However, genetically, nothing has been elucidated except that all the azaserine-resistant strains isolated so far carry lesions in the aroP gene as a primary determinant. Here, we demonstrate that, in addition to AroP, the LIV-I/LS system, an ATP-binding cassette type transporter, is involved in azaserine sensitivity of E. coli, by genetic analysis and transport studies, in which Ki value for azaserine was determined to be approximately 10(-3) M.

The present study investigated whether activation of the hexosamine biosynthesis pathway might mediate at least in part the high glucose effect on angiotensinogen (ANG) gene expression and immortalized renal proximal tubular cell (IRPTC) hypertrophy. IRPTC were cultured in monolayer. ANG, renin, and beta-actin mRNA expression were determined by specific RT-PCR assays. Phosphorylation of p38 MAPK, activating transcription factor-2 (ATF-2), and cAMP-responsive element-binding protein (CREB) was determined by Western blot analysis. Cell hypertrophy was assessed by flow cytometry, intracellular p27kip1 protein levels, and [3H]leucine incorporation into proteins. Glucosamine stimulated ANG and renin mRNA expression and enhanced p38 MAPK, ATF-2, and CREB phosphorylation in normal glucose (5 mm) medium. Azaserine and 6-diazo-5-oxo-l-norleucine (inhibitors of glutamine: fructose-6-phosphate amino transferase enzyme) blocked the stimulatory effect of high glucose, but not that of glucosamine, on ANG gene expression in IRPTCs. SB 203580 (a specific p38 MAPK inhibitor) attenuated glucosamine action on ANG gene expression as well as p38 MAPK and ATF-2 phosphorylation, but not that of CREB. GF 109203X and calphostin C (inhibitors of protein kinase C) blocked the effect of glucosamine on ANG gene expression and CREB phosphorylation, but had no impact on p38 MAPK and ATF-2 phosphorylation. Finally, both glucosamine and high glucose induced IRPTC hypertrophy. The hypertrophic effect of glucosamine was blocked in the presence of GF 109203X, but not azaserine and SB 203580. In contrast, the hypertrophic effect of high glucose was blocked in the presence of azaserine and GF 109203X, but not SB203580. Our studies demonstrate that the stimulatory effect of high glucose on ANG gene expression and IRPTC hypertrophy may be mediated at least in part via activation of hexosamine biosynthesis pathway signaling.

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.

The hexosamine biosynthesis pathway plays a role in the modification of cellular proteins via the provision of substrate for addition of O-linked N-acetylglucosamine (GlcNAc). The relative importance of the GlcNAc modification of proteins to insulin secretion from pancreatic beta-cells has not been investigated and so remains unclear. In the present study, we show that inhibition of the hexosamine biosynthesis pathway decreases insulin secretion from mouse islets in response to a number of secretagogues, including glucose. This impairment in beta-cell function could not be attributed to reduced islet insulin content, altered ATP levels, or cell death and was restored with the addition of N-acetylglucosamine, a substrate that enters the pathway below the point of inhibition. Western blot analysis revealed that decreased islet protein glycosylation paralleled the decrease in insulin secretion following inhibition of the pathway. In conclusion, the data suggest a role for the hexosamine biosynthesis pathway in regulating the secretion of insulin by altering protein glycosylation. This finding may have implications for the development of type 2 diabetes, as chronic increase in flux through the hexosamine biosynthesis pathway may lead to the deterioration of beta-cell function via abnormal protein glycosylation.

Although an accumulating amount of research clearly indicates that plants are capable of taking up exogenous amino acids, the actual importance of such organic N sources for plant N nutrition is under debate. In this study, we show that amino acid uptake by Scots pine (Pinus sylvestris L.) is significantly decreased by elevated internal NH(4)(+) levels, while it increases following exposure to exogenous amino acids. Furthermore, amino acid uptake is larger in N-deficient plants than in plants grown with a large access of N. The regulatory pattern of amino acid uptake shows important similarities to the regulation of NO(3)(-) and NH(4)(+) transport as well as to the regulation of yeast amino acid transporters. In addition, our data suggest that uptake may be regulated by factors not originating from N metabolism. The up-regulation of uptake in response to N deficiency suggests that amino acid uptake may be a significant contributor to the N economy of P. sylvestris.

The production of toxins A and B by Clostridium difficile was greatly enhanced under biotin-limited conditions, in which a 140-kDa protein was expressed strongly. Gene cloning revealed that this protein was a homologue of formylglycinamidine ribonucleotide synthetase (FGAM synthetase, EC 6.3.5.3), which is known as PurL in Escherichia coli and catalyses the fourth step of the de novo purine biosynthesis pathway. This enzyme consisted of a single polypeptide, although FGAM synthetases of gram-positive bacteria usually consist of two subunits. Inhibition of the enzymic activity of C. difficile PurL by O-diazoacetyl-L-serine (azaserine) resulted in enhanced toxin B production even in biotin-sufficient conditions. In contrast, blockade of the preceding step of the PurL catalysing step by sulfamethoxazole inhibited toxin B production almost completely. These results suggest that accumulation of formylglycinamide ribonucleotide (FGAR), a substrate of FGAM synthetase, enhances toxin production by C difficile and depletion of FGAR reduces toxin production.

In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mm glucose for 24 h attenuated the ability of 10 nm insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mm glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased approximately 2- and 15-fold after a 24-h incubation in 20 mm glucose and 1.5 mm glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mm glucose, but not that of 1.5 mm glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, glucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes.

Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cells with high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long period of high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosamine biosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culture media enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF beta-receptor was increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF beta-receptor association with PLC gamma was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, and MAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF beta-receptor association with the p85 regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P<0.01) and 36% (P<0.01), respectively. The intracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors. Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitory effect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantly inhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involved in the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway.

In vivo studies suggest that sterol regulatory element-binding protein (SREBP)-1 plays a key role in the up-regulation of lipogenic genes in the livers of animals that have consumed excess amounts of carbohydrates. In light of this, we sought to use an established mouse hepatocyte cell line, H2-35, to further define the mechanism by which glucose regulates nuclear SREBP-1 levels. First, we show that these cells transcribe high levels of SREBP-1c that are increased 4-fold upon differentiation from a prehepatocyte to a hepatocyte phenotype, making them an ideal cell culture model for the study of SREBP-1c induction. Second, we demonstrate that the presence of precursor and mature forms of SREBP-1 protein are positively regulated by medium glucose concentrations ranging from 5. 5 to 25 mm and are also regulated by insulin, with the amount of insulin in the fetal bovine serum being sufficient for maximal stimulation of SREBP-1 expression. Third, we show that the increase in SREBP-1 protein is due to an increase in SREBP-1 mRNA. Reporter gene analysis of the SREBP-1c promoter demonstrated a glucose-dependent induction of transcription. In contrast, expression of a fixed amount of the precursor form of SREBP-1c protein showed that glucose does not influence its cleavage. Fourth, we demonstrate that the glucose induction of SREBP could not be reproduced by fructose, xylose, or galactose nor by glucose analogs 2-deoxy glucose and 3-O-methyl glucopyranose. These data provide strong evidence for the induction of SREBP-1c mRNA by glucose leading to increased mature protein in the nucleus, thus providing a potential mechanism for the up-regulation of lipogenic genes by glucose in vivo.

To better understand the molecular mechanisms for hyperglycemia-induced proatherogenic changes in endothelial cells, the effect of high glucose on activation of members of the mitogen-activated protein kinase (MAPK) family, including c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)-1, -2, and -5, and p38 kinase, was examined in bovine pulmonary artery endothelial cells (PAEC). Glucose, fructose, and raffinose induced a concentration-dependent decrease in PAEC growth. Addition of 25 mM glucose, fructose, or raffinose to normal growth medium stimulated an approximately twofold increase in JNK1 activity that was maximal after 24 h, whereas only glucose markedly increased ERK5 activity. Neither ERK1/2 nor p38 kinase activity was increased by glucose, fructose, or raffinose. The antioxidant N-acetylcysteine partially abrogated the glucose-induced increase in ERK5 activity but had no effect on the increase in JNK1 activity. In contrast, azaserine, which prevents increased flux through the hexosamine pathway, decreased glucose-induced JNK1 activity but had no effect on fructose- or raffinose-induced JNK1 activity. Consistent with this finding, glucosamine stimulated a 2.4-fold increase in JNK1 activity and reproduced the inhibitory effect of glucose on PAEC growth. In summary, glucose activates different members of the MAPK family in PAEC via distinct mechanisms. Moreover, the correlation between the ability of different sugars to activate JNK1 and inhibit cell growth suggests that activation of this signaling pathway may contribute to the growth inhibitory effect of glucose in endothelial cells.

Heritable gene silencing is an important mechanism of tumor suppressor gene inactivation in a variety of human cancers. In the present study, we show that methylation-associated silencing of the autosomal adenine phosphoribosyltransferase (Aprt) locus occurs in primary mouse kidney cells. Aprt-deficient cells were isolated from mice that were heterozygous for Aprt, i.e., they contained one wild-type Aprt allele and one targeted allele bearing an insertion of the bacterial neo gene. Although silencing of the wild-type allele alone was sufficient for the cells to become completely Aprt-deficient, biallelic methylation of the promoter region was found to occur. Moreover, despite the absence of selective pressure against the targeted allele, phenotypic silencing of the inserted neo gene accompanied silencing of the wild-type Aprt allele. A potential role for allelic homology in these events is discussed.

L-Glutamine (the most abundant free amino acid in plasma and the body) is a potent inhibitor of endothelial NO synthesis. However, little is known about glutamine metabolism in endothelial cells (EC). As an initial step toward understanding the role of glutamine in endothelial physiology, the present study was conducted to quantify glutamine catabolism in microvascular, aortic and venous EC. For metabolic studies, EC were incubated for 1 h in Krebs bicarbonate buffer containing 5 mM glucose and 0.5-4 mM L-[U-(14)C]-glutamine. For enzymological studies, cell extracts and mitochondrial fractions were prepared to determine the activities of glutamine-degrading enzymes. Our results reveal extensive hydrolysis of glutamine to glutamate and ammonia in a concentration-dependent manner via phosphate-dependent glutaminase in all EC studied. In addition, both metabolic and enzymological evidence indicate a novel pathway for endothelial synthesis of ornithine from glutamine via pyrroline-5-carboxylate synthase. This new knowledge of glutamine metabolism may pave a new path for understanding the physiological role of glutamine in vascular function.

The majority of human pancreatic adenocarcinomas display a ductal phenotype; experimental studies indicate that tumors with this phenotype can arise from both acinar and ductal cells. In normal pancreas acinar cells, the pancreas transcription factor 1 transcriptional complex is required for gene expression. Pancreas transcription factor 1 is a heterooligomer of pancreas-specific (p48) and ubiquitous (p75/E2A and p64/HEB) basic helix-loop-helix proteins. We have examined the role of p48 in the phenotype of azaserine-induced rat DSL6 tumors and cancers of the human exocrine pancreas. Serially transplanted acinar DSL6 tumors express p48 whereas DSL6-derived cell lines, and the tumors induced by them, display a ductal phenotype and lack p48. In human pancreas cancer cell lines and tissues, p48 is present in acinar tumors but not in ductal tumors. Transfection of ductal pancreas cancers with p48 cDNA did not activate the expression of amylase nor a reporter gene under the control of the rat elastase promoter. In some cell lines, p48 was detected in the nucleus whereas in others it was cytoplasmic, as in one human acinar tumor. Together with prior work, our findings indicate that p48 is associated with the acinar phenotype of exocrine pancreas cancers and it is necessary, but not sufficient, for the expression of the acinar phenotype.

Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48h to anaerobic conditions. Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA, inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8h of anaerobic treatment and, by immunoprecipitation of 35S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during the treatment. These findings indicate that the glutamine synthetase/glutamate synthase cycle play an important role in anaerobic amino acid accumulation.

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.