Ferredoxin-dependent glutamate synthase: involvement in ammonium assimilation in Haloferax mediterranei

Glutamate synthase (GOGAT) is one of the two important enzymes involved in the ammonium assimilation pathway glutamine synthetase (GS)/GOGAT, which enables Hfx. mediterranei to thrive in media with low ammonium concentration or containing just nitrate as single nitrogen source. The gene coding for this enzyme, gltS, has been sequenced, analysed and compared with other GOGATs from different organisms from the three domains of life. According to its amino acid sequence, Hfx. mediterranei GOGAT displays high homology with those from other archaeal halophilic organisms and with the bacterial alpha-like subunit. Hfx. mediterranei GOGAT and GS expression was induced under conditions of ammonium restriction. The GOGAT protein was found to be a monomer with a molecular mass of 163.78 kDa, which is consistent with that estimated by gel filtration, 198 ± 30 kDa. The enzyme is highly ferredoxin dependent: activity was only observed with one of the two different 2Fe–2S ferredoxins chromatographically isolated from Hfx. mediterranei. The enzyme also displayed typical halophilic behaviour, being fully stable, and producing maximal activity, at salt concentrations from 3 to 4 M NaCl, pH 7.5 and a temperature of 50 °C.

NADH-Glutamate Synthase in Alfalfa Root Nodules. Genetic Regulation and Cellular Expression1

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric β-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer

BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced/absent in human NSCLC protein samples (P <.0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P =.004) and in male patients (P <.05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P <.001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. © 2011 American Cancer Society.

Thymidylate synthase expression and outcome of patients receiving pemetrexed for advanced nonsquamous non-small-cell lung cancer in a prospective blinded assessment phase II clinical trial

INTRODUCTION In retrospective analyses of patients with nonsquamous non-small-cell lung cancer treated with pemetrexed, low thymidylate synthase (TS) expression is associated with better clinical outcomes. This phase II study explored this association prospectively at the protein and mRNA-expression level. METHODS Treatment-naive patients with nonsquamous non-small-cell lung cancer (stage IIIB/IV) had four cycles of first-line chemotherapy with pemetrexed/cisplatin. Nonprogressing patients continued on pemetrexed maintenance until progression or maximum tolerability. TS expression (nucleus/cytoplasm/total) was assessed in diagnostic tissue samples by immunohistochemistry (IHC; H-scores), and quantitative reverse-transcriptase polymerase chain reaction. Cox regression was used to assess the association between H-scores and progression-free/overall survival (PFS/OS) distribution estimated by the Kaplan-Meier method. Maximal χ analysis identified optimal cutpoints between low TS- and high TS-expression groups, yielding maximal associations with PFS/OS. RESULTS The study enrolled 70 patients; of these 43 (61.4%) started maintenance treatment. In 60 patients with valid H-scores, median (m) PFS was 5.5 (95% confidence interval [CI], 3.9-6.9) months, mOS was 9.6 (95% CI, 7.3-15.7) months. Higher nuclear TS expression was significantly associated with shorter PFS and OS (primary analysis IHC, PFS: p < 0.0001; hazard ratio per 1-unit increase: 1.015; 95%CI, 1.008-1.021). At the optimal cutpoint of nuclear H-score (70), mPFS in the low TS- versus high TS-expression groups was 7.1 (5.7-8.3) versus 2.6 (1.3-4.1) months (p = 0.0015; hazard ratio = 0.28; 95%CI, 0.16-0.52; n = 40/20). Trends were similar for cytoplasm H-scores, quantitative reverse-transcriptase polymerase chain reaction and other clinical endpoints (OS, response, and disease control). CONCLUSIONS The primary endpoint was met; low TS expression was associated with longer PFS. Further randomized studies are needed to explore nuclear TS IHC expression as a potential biomarker of clinical outcomes for pemetrexed treatment in larger patient cohorts. © 2013 by the International Association for the Study of Lung Cancer.

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods TXS and VEGF metabolite levels were measured in NSCLC serum samples (n=46) by EIA. TXB2 levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results Serum TXB2 levels were correlated with VEGF (p<0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p<0.0001) and microvessel density (CD-31; p<0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p<0.0001) and female patients (p<0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p<0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.

Regulation of lipocalin prostaglandin-D synthase expression by interleukin-1β in human chondrocytes

L'arthrose est une maladie multifactorielle complexe. Parmi les facteurs impliqués dans sa pathogénie, les certains prostaglandines exercent un rôle inflammatoire et d’autres un rôle protecteur. La prostaglandine D2 (PGD2) est bien connue comme une PG anti-inflammatoire, qui est régulée par l’enzyme «Lipocalin prostaglandine D-synthase». Avec l’inflammation de l'arthrose, les chondrocytes essaient de protéger le cartilage en activant certaines voies de récupération dont l'induction du gène L-PGDS. Dans cette étude, nous étudions la voie de signalisation impliquée dans la régulation de l'expression du (L-PGDS) sur les chondrocytes traités avec différents médiateurs inflammatoires. Le but de projet: Nous souhaitons étudier la régulation de la L-PGDS dans le but de concevoir des approches thérapeutiques qui peuvent activer la voie intrinsèque anti-inflammatoire. Méthode et conclusions: In vivo, l'arthrose a été suivie en fonction de l’âge chez la souris ou chirurgicalement suivant une intervention au niveau des genoux de souris. Nous avons confirmé les niveaux d’expression de L-PGDS histologiquement et par immunohistochimie. In vitro, dans les chondrocytes humains qui ont été traités avec différents médiateurs de l'inflammation, nous avons observé une augmentation de l’expression de la L-PGDS dose et temps dépendante. Nous avons montré, in vivo et in vitro que l’inflammation induit une sécrétion chondrocytaire de la L-PGDS dans le milieu extracellulaire. Enfin, nous avons observé la production de différentes isoformes de la L-PGDS en réponse à l'inflammation.

Regulation of GABA(A) and glutamate receptor expression, synaptic facilitation and long-term potentiation in the hippocampus of prion mutant mice

Background: Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP(sc)) of the natural cellular prion protein (PrP(c)) encoded by the Prnp gene. Although several roles have been attributed to PrP(c), its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP(c) studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Principal Findings: Here we explore the role of PrP(c) expression in neurotransmission and neural excitability using wild-type, Prnp -/- and PrP(c)-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp -/- mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp -/- and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA(A) and AMPA-kainate receptors are co-regulated in both Prnp -/- and Tg20 mice. Conclusions/Significance: Present results demonstrate that PrP(c) is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABA(A) and AMPA-Kainate neurotransmission. New PrP(c) functions have recently been described, which point to PrP(c) as a target for putative therapies in Alzheimer's disease. However, our results indicate that a "gain of function" strategy in Alzheimer's disease, or a "loss of function" in prionopathies, may impair PrP(c) function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

Trigeminal nitric oxide synthase expression correlates with new bone formation during distraction osteogenesis

Nitric oxide synthase (NOS) has been reported to be involved with both bone healing and bone metabolism. The aim of this study was to test the null hypothesis that there is no correlation between new bone formation during mandibular distraction osteogenesis and NOS expression in the trigeminal ganglion of rats. Newly formed tissue during distraction osteogenesis and trigeminal NOS expression measured by the NADPH-diaphorase (NADPH-d) reaction were evaluated in 72 male Wistar rats by histomorphometric and histochemical methods. In animals submitted to 0.5 mm/day distraction osteogenesis, the percentage of bone tissue was higher in the basal area of the mandibles compared with the center and significantly increased through the experimental periods (P < 0.05). At the sixth postoperative week, the difference in bone formation between the continuous and acute distraction osteogenesis groups was the highest. Significant correlation between new bone formation by distraction osteogenesis and NADPH-d-reactive neurons was found, varying according to neuronal cell size (r = -0.6, P = 0.005, small cells strongly stained; r = 0.5, P = 0.018, large cells moderately stained). The results suggest that NOS may play a role in the bone healing process via neurogenic pathways, and the phenomenon seems to be neuronal cell morphotype-dependent. Further studies are now warranted to investigate the mechanistic link between the expression of trigeminal NOS and mandibular new bone formation by distraction osteogenesis.

Preliminary report: Leucine supplementation enhances glutamate dehydrogenase expression and restores glucose-induced insulin secretion in protein-malnourished rats

Low-protein diet impairs insulin secretion in response to nutrients and may induce several metabolic disorders including diabetes, obesity, and cardiovascular disease. In the present study, the influence of leucine supplementation on glutamate dehydrogenase (GDH) expression and glucose-induced insulin secretion (GIIS) was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal-protein diet (17%) without or with leucine supplementation or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine (1.5%) was supplied in the drinking water. Western blotting analysis revealed reduced GIN! expression in LP, whereas LPL displayed improved GDH expression, similar to control. The GHS and leucinc-induced insulin release were also enhanced in LPL compared with LP and similar to those observed in rats fed a normal-protein diet without leucine supplementation. In addition, GDH allosteric activators produced an increased insulin secretion in LPL. These findings indicate that leucine supplementation was able to increase GDH expression leading to Cl IS restoration, probably by improved leucine metabolic pathways. (C) 2010 Elsevier Inc. All rights reserved.

Ebselen and cytokine-induced nitric oxide synthase expression in insulin-producing cells

Interleukin-1 (IL-1) may be a mediator of β-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor κB (NF-κB), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H2O2, have been proposed as second messengers for NF-κB activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1β, H2O2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 μM) prevented the increase in nitrite production by rat islets exposed to IL-1β for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H2O2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1β for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1β, tumor necrosis factor-α and interferon-γ). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-β but failed to block IL-1β-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1β-induced NF-κB activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic β-cells, an effect not associated with inhibition of NF-κB activation.

NADH-Glutamate Synthase in Alfalfa Root Nodules. Immunocytochemical Localization1

In root nodules of alfalfa (Medicago sativa L.), N2 is reduced to NH4+ in the bacteroid by the nitrogenase enzyme and then released into the plant cytosol. The NH4+ is then assimilated by the combined action of glutamine synthetase (EC 6.3.1.2) and NADH-dependent Glu synthase (NADH-GOGAT; EC 1.4.1.14) into glutamine and Glu. The alfalfa nodule NADH-GOGAT protein has a 101-amino acid presequence, but the subcellular location of the protein is unknown. Using immunocytochemical localization, we determined first that the NADH-GOGAT protein is found throughout the infected cell region of both 19- and 33-d-old nodules. Second, in alfalfa root nodules NADH-GOGAT is localized predominantly to the amyloplast of infected cells. This finding, together with earlier localization and fractionation studies, indicates that in alfalfa the infected cells are the main location for the initial assimilation of fixed N2.

Quantitative Intercellular Localization of NADH-Dependent Glutamate Synthase Protein in Different Types of Root Cells in Rice Plants1

The quantitative analysis with immunogold-electron microscopy using a single-affinity-purified anti-NADH-glutamate synthase (GOGAT) immunoglobulin G (IgG) as the primary antibody showed that the NADH-GOGAT protein was present in various forms of plastids in the cells of the epidermis and exodermis, in the cortex parenchyma, and in the vascular parenchyma of root tips (<10 mm) of rice (Oryza sativa) seedlings supplied with 1 mm NH4+ for 24 h. The values of the mean immunolabeling density of plastids were almost equal among these different cell types in the roots. However, the number of plastids per individual cell type was not identical, and some parts of the cells in the epidermis and exodermis contained large numbers of plastids that were heavily immunolabeled. Although there was an indication of labeling in the mitochondria using the single-affinity-purified anti-NADH-GOGAT IgG, this was not confirmed when a twice-affinity-purified IgG was used, indicating an exclusively plastidial location of the NADH-GOGAT protein in rice roots. These results, together with previous work from our laboratory (K. Ishiyama, T. Hayakawa, and T. Yamaya [1998] Planta 204: 288–294), suggest that the assimilation of exogeneously supplied NH4+ ions is primarily via the cytosolic glutamine synthetase/plastidial NADH-GOGAT cycle in specific regions of the epidermis and exodermis in rice roots. We also discuss the role of the NADH-GOGAT protein in vascular parenchyma cells.