GABase and glutaminase inhibitory activities of herbal extracts and acylated flavonol monoglycosides isolated from the leaves of Laurus nobilis L.

This study was to compare GABase [a mixture of γ-aminobutyric acid (GABA) aminotransferase and succinic semialdehyde dehydrogenase] and glutaminase inhibitory activities of 20 herbal extracts and investigate the isolation, structural elucidation and those inhibitory activities of three acylated flavonol monoglycosides from the selected extract of Laurus nobilis L. (laurel). On the basis of the NMR spectroscopic data and the ESI MS spectra together with the comparison with the literature values, three compounds were identified as kaempferol-3-O-(4″-E-p-coumaroyl)-α-l-rhamnopyranoside (1), kaempferol-3-O-(3″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (2) and kaempferol-3-O-(2″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (3), respectively. The IC50 values of GABase inhibitory activity of 1-3 and p-hydroxybenzaldehyde (HBA) as control were 0.24 mM, 0.14 mM, 0.12 mM and 0.43 mM, respectively. Additionally, the IC50 values of glutaminase inhibitory activity of 1-3 and 6-diazo-5-oxo-l-norleucine (DON) as control were 0.34 mM, 0.13 mM, 0.14 mM and 0.33 mM, respectively. The results suggest that the extract from laurel shows the strongest biological activities among 20 herbal extracts and three acylated flavonol monoglycosides may serve as potential lead compounds for the prevention and treatment of neurodegenerative and lifestyle-related diseases by targeting GABase and glutaminase. This is the first report on GABase and glutaminase inhibitory activities of 1-3.

Studies on the potential link between antidepressant effect of Xiaoyao San and its pharmacological activity of hepatoprotection based on multi-platform metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM) theory, depression is considered to be "liver qi stagnation", and relieving "liver qi stagnation" is regarded as an effective method for treating depression. Xiaoyao San (XYS) is a well-known TCM formula for the treatment of depression by relieving "liver qi stagnation". This formula consists of Radix Paeoniae Alba (Paeonia lactiflora Pall.), Radix Bupleuri (Bupleurum chinense DC.), Poria (Poria cocos (Schw.) Wolf), Rhizoma Atractylodis Macrocephalae (Atractylodes macrocephala Koidz.), Radix Angelicae Sinensis (Angelica sinensis (Oliv.) Diels), Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch.), Rhizoma Zingiberis Recens (Zingiber officinale Roscoe) and Herba Menthae Haplocalycis (Mentha haplocalyx Briq.). AIM OF THE STUDY: Several studies have suggested that depression is associated with liver injury. XYS was a well-known TCM formula for the treatment of depression and liver stagnancy. However, it was still unknown whether the antidepressant effect of XYS is related to the pharmacological activity of hepatoprotection. The aim of this study was to elucidate the potential link between the antidepressant and hepatoprotective effect of XYS. MATERIALS AND METHODS: A depression rat model was established by the CUMS (chronic unpredictable mild stress) procedure. The antidepressant effect of XYS was assessed by the behavioral indicators, and the hepatoprotective effect of XYS was evaluated through biochemical assays. 1H-NMR and LC/MS-based liver metabolomics were performed to discover key metabolic pathways involved in the antidepressant and hepatoprotective effects of XYS. Further, the key pathway was validated using commercial kits. RESULTS: The results demonstrated that XYS pretreatment could significantly improve the depressive symptom induced by CUMS. More importantly, the results demonstrated that liver injury was observed in the CUMS model rats, and XYS had a hepatoprotective effect by reducing the activities of AST and ALT in serum, increasing the levels of SOD and GSH-Px and reducing the contents of MDA, IL-6, and IL-1ß in the liver. In addition, the NMR and LC/MS-based metabolomics results indicated that XYS improved 23 of the 35 perturbed potential liver biomarkers that were induced by CUMS. Among them, 9 biomarkers were significantly correlated with both depression and liver pathology, according to Pearson correlation analysis. Metabolic pathway analyses of these 9 biomarkers showed that glutamine and glutamate metabolism were the most important metabolic pathways. Furthermore, to verify glutamine and glutamate metabolism, the levels of glutamine and glutamate, and the activity of glutamine synthetase (GS) and glutaminase (GLS) were quantitatively determined in the liver by commercial kits, and these results were consistent with the metabolomics results. CONCLUSIONS: XYS could significantly improve the depressive and liver injury symptoms induced by CUMS. The metabolomics results indicate that the regulation of glutamine and glutamate metabolism to maintain the balance of ammonia and promote energy metabolism is a potential junction between the antidepressant and hepatoprotective effects of XYS.

Increased Glutaminolysis Marks Active Scarring in Nonalcoholic Steatohepatitis Progression.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) occurs in the context of aberrant metabolism. Glutaminolysis is required for metabolic reprograming of hepatic stellate cells (HSCs) and liver fibrogenesis in mice. However, it is unclear how changes in HSC glutamine metabolism contribute to net changes in hepatic glutaminolytic activity during fibrosis progression, or whether this could be used to track fibrogenic activity in NASH. We postulated that increased HSC glutaminolysis marks active scarring in NASH. METHODS: Glutaminolysis was assessed in mouse NASH fibrosis models and in NASH patients. Serum and liver levels of glutamine and glutamate and hepatic expression of glutamine transporter/metabolic enzymes were correlated with each other and with fibrosis severity. Glutaminolysis was disrupted in HSCs to examine if this directly influenced fibrogenesis. 18F-fluoroglutamine positron emission tomography was used to determine how liver glutamine assimilation tracked with hepatic fibrogenic activity in situ. RESULTS: The serum glutamate/glutamine ratio increased and correlated with its hepatic ratio, myofibroblast content, and fibrosis severity. Healthy livers almost exclusively expressed liver-type glutaminase (Gls2); Gls2 protein localized in zone 1 hepatocytes, whereas glutamine synthase was restricted to zone 3 hepatocytes. In fibrotic livers, Gls2 levels reduced and glutamine synthase zonality was lost, but both Slc1a5 (glutamine transporter) and kidney-type Gls1 were up-regulated; Gls1 protein was restricted to stromal cells and accumulated in fibrotic septa. Hepatocytes did not compensate for decreased Gls2 by inducing Gls1. Limiting glutamine or directly inhibiting GLS1 inhibited growth and fibrogenic activity in cultured human HSCs. Compared with healthy livers, fibrotic livers were 18F-fluoroglutamine-avid by positron emission tomography, suggesting that glutamine-addicted myofibroblasts drive increased hepatic utilization of glutamine as fibrosis progresses. CONCLUSIONS: Glutaminolysis is a potential diagnostic marker and therapeutic target during NASH fibrosis progression.

Acetone immersion enhanced MALDI-MS imaging of small molecule metabolites in biological tissues.

Profiling the endogenous tissue metabolites with spatial features is significant for our understanding of molecular histology, and provides an insightful way to uncover the complex associations between tissue metabolic response and external stimuli. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an effective molecular imaging technology to illustrate the spatial locations of molecules in tissue. However, due to the limited sensitivity and the presence of multiple matrix-related ions, it is still challenging to globally image the small molecule metabolites (SMMs) using MALDI, especially for those low-content functional ones. Here, a simple acetone washing method was developed to improve the sensitivity of MALDI-MS for imaging SMMs. After immersing in acetone and shaken for 15 min, key functional SMMs were well-visualized with significantly enhanced ion intensities. In addition to lipids, more than 160 SMM ions, including polyamines, cholines, carnitines, amino acids, nitrogenous bases, nucleosides, carbohydrates, organic acids, vitamins were imaged. The acetone washes-based MALDI-MSI was then applied to profile the metabolic alternations that occurred in osteosarcoma, and the abnormally altered SMMs and lipids were clearly visualized. Moreover, with the protection of acetone against tissue antigenicity, we successfully characterized the expression of three metabolites-related enzymes, fatty acid synthase (FASN), glutaminase (GLS), and cytosolic phospholipase A2 (cPLA2) in osteosarcoma. The spatially-resolved metabolite and corresponding enzyme information reveals what occured in osteosarcoma at the molecular level, providing new insights into the understanding of tumour metabolic reprogramming.

Glutaminase activity in GLS1 Het mouse brain compared to putative pharmacological inhibition by ebselen using ex vivo MRS.

Glutaminase mediates the recycling of neurotransmitter glutamate, supporting most excitatory neurotransmission in the mammalian central nervous system. A constitutive heterozygous reduction in GLS1 engenders in mice a model of schizophrenia resilience and associated increases in Gln, reductions in Glu and activity-dependent attenuation of excitatory synaptic transmission. Hippocampal brain slices from GLS1 heterozygous mice metabolize less Gln to Glu. Whether glutaminase activity is diminished in the intact brain in GLS1 heterozygous mice has not been assessed, nor the regional impact. Moreover, it is not known whether pharmacological inhibition would mimic the genetic reduction. We addressed this using magnetic resonance spectroscopy to assess amino acid content and 13C-acetate loading to assess glutaminase activity, in multiple brain regions. Glutaminase activity was reduced significantly in the hippocampus of GLS1 heterozygous mice, while acute treatment with the putative glutaminase inhibitor ebselen did not impact glutaminase activity, but did significantly increase GABA. This approach identifies a molecular imaging strategy for testing target engagement by comparing genetic and pharmacological inhibition, across brain regions.

Expression of glutamine metabolism-related proteins in Hürthle cell neoplasm of thyroid: Comparison with follicular neoplasm.

PURPOSE: We evaluated the expression of glutaminolysis-related proteins in Hurthle cell neoplasms (HCN) and follicular neoplasms (FN) of the thyroid, and investigated its clinical implication. METHODS: Tissue microarrays were constructed from 264 FNs (112 follicular carcinomas [FCs] and 152 follicular adenomas [FAs]) and 108 HCNs (27 Hurthle cell carcinomas [HCCs] and 81 Hurthle cell adenomas [HCAs]. The immunohistochemical staining result of 3 glutaminolysis-related proteins (Glutaminase 1 [GLS1], glutaminate dehydrogenase [GDH] and alanine- serine, cysteine-preferring transporter 2 [ASCT2]) was analyzed. RESULTS: GLS1 and GDH showed significantly higher expression rates in HCN compared to FN (P<0.001). More HCN cases showed co-positivity of multiple glutaminolysis-related proteins than those of FN cases (P<0.001). In silico analysis, both GLUD1 and GLUD2 showed higher expression rate in HCA compared to FA (P=0.027 and P=0.018, respectively). SLC1A5 expression was highest in HCA, followed by FC and FA (HCA vs FC, P=0.023; FC vs FA, P=0.002). CONCLUSION: FN and HCN exhibit a different expression pattern for glutaminolysis-related proteins, and GLS1 and GDH have higher expression rates in HCN and FN.

Glutaminase expression is a poor prognostic factor in node-positive triple-negative breast cancer patients with a high level of tumor-infiltrating lymphocytes.

Glutamine metabolism is emerging as one aspect of dysregulated metabolism of tumors. Triple-negative breast cancer (TNBC) cells are glutamine dependent, whereas luminal-type cells tend to be glutamine independent. Therefore, TNBC patients might benefit from therapies targeting glutamine metabolism. To investigate the clinical significance of glutamine metabolism, we examined expression and prognostic significance of glutaminase in tumor cells and tumor-infiltrating lymphocytes (TILs) in TNBC. We retrieved 658 surgically resected TNBCs and analyzed glutaminase expression in tumor cells and TILs by immunohistochemical staining. Glutaminase expression was observed in 237 cases (36.0%) in tumor cells and 104 cases (15.5%) in TILs. Although glutaminase expression in tumor cells was significantly associated with a low level of TILs (p = 0.018), glutaminase expression in TILs was significantly higher in cases with a high level of TILs (p = 0.031). Glutaminase expression in tumor cells was significantly associated with poor disease-free survival in patients with lymph node metastasis and high levels of TILs (p = 0.020). In addition, it was an independent poor prognostic factor (hazard ratio = 10.643, 95% confidence interval = 1.999-56.668; p = 0.006). Glutaminase expression in tumor cells was observed in a subset of TNBC patients. It was significantly associated with a low level of TILs and poor disease-free survival in TNBCs presenting with lymph node metastasis and high levels of TILs.

Aplicación de nuevos criterios diagnósticos de enfermedad celiaca en Castilla-La Mancha / Application of new criteria for diagnosis of celiac disease in Castilla-La Mancha

Objetivo: Estudiar la aplicación de los nuevos criterios diagnósticos de enfermedad celiaca en Castilla-La Mancha, así como las características de los pacientes. Material y métodos: Estudio descriptivo transversal, con recogida prospectiva de datos en 7 hospitales de pacientes diagnosticados entre el 1 de julio de 2011 y el 31 de diciembre de 2012. Se analizó la frecuencia con la que se reúnen requisitos para el diagnóstico sin biopsia intestinal mediante la aplicación de los nuevos criterios y el seguimiento de los mismos. Resultados: Se incluyeron 158 pacientes (un 63,3% mujeres) con una media de edad de 5,5 años. La presencia de síntomas compatibles fue detectada en el 54,4% de los pacientes. El hallazgo analítico más frecuente fue la ferropenia (29,7%), y se encontró anemia en el 8,9% e hipertransaminasemia en el 5,7%. El 79,1% presentaba en el momento del diagnóstico títulos de anticuerpos antitransglutaminasa más de 10 veces superiores al valor de corte de normalidad, con positividad comprobada de anticuerpos antiendomisio en el 49,6% de ellos. El 39,2% de los pacientes cumplían requisitos para ser diagnosticados mediante la aplicación de los nuevos criterios. Se realizó biopsia intestinal en el 32,2% por diferentes motivos, y todos estos casos presentaban una lesión de grado 3 según la clasificación de Marsh. Conclusiones: La introducción de los nuevos criterios diagnósticos para la enfermedad celiaca podría suponer en nuestro medio una reducción del 40% de los procedimientos endoscó- picos en estos pacientes. La variabilidad interprovincial en el acceso a determinadas técnicas no permite su aplicación de manera homogénea en nuestra comunidad, y actualmente se tiene que recurrir al estudio histológico en muchos casos (AU)

Objective: To study the use of the new diagnostic criteria for celiac disease in the Castilla-La Mancha region, and the characteristics of these patients. Material and methods: Prospective data were collected in 7 hospitals within a descriptive transversal study that included patients diagnosed between July 2011 and the end of December 2012. The frequency with which they met requirements for diagnosis without intestinal biopsy based on the new criteria and the monitoring thereof were analyzed. Results: A total of 158 patients (63.3% female), with a median age of 5.5 years, were included in the study. In 54.4% of these patients celiac disease related symptoms were detected. The most frequent laboratory findings were ferropenia (29.7%), anemia (8.9%) and hypertransaminasemia (5.7%). At diagnosis, 79.1% of the patients had transglutaminase antibody titers 10 times above the upper limit of the normal range, with proven positive endomysial antibodies in 49.6% of them. Of the total patients, 39.2% fulfilled the new diagnostic criteria and 32.2% undergone an intestinal biopsy for different reasons. In all these cases, grade 3 lesions according to Marsh classification for celiac disease were found. Conclusions: The introduction of the new diagnostic criteria for celiac disease could lead to a 40% reduction in endoscopic procedures for these patients in our region. The interprovince variability in the access to certain techniques, does not allow the global use of these new criteria in the studied region where histology continues to be the main available option (AU)

Isolation and identification of actinomycetes for production of novel extracellular glutaminase free L-asparaginase.

Over the recent years glutaminase free L-asparaginase has gained more importance due to better therapeutic properties for treatment of acute lymphoblastic leukemia. Actinomycetes are known for L-asparaginase activity. In the current study, 80 actinomycetes were isolated from various soil habitats by serial dilution technique. Presence of L-asparaginase was investigated in a total of 240 actinomycetes by tubed agar method using modified M-9 medium. A total of 165 actinomycetes were found positive for L-asparaginase activity. Among these, 57 actinomycetes producing larger zones of L-asparagine hydrolysis were further screened for their capacity to produce glutaminase-free L-asparaginase. Four L-glutaminase-free actinomycetes were found to be potential L-asparaginase producers. These actinomycetes were identified as Streptomyces cyaneus (SAP 1287, CFS 1560), S. exfoliates (CFS 1557) and S. phaeochromogenes (GS 1573) on the basis of morphological and biochemical identification studies. Maximum L-asparaginase activity (19.2 Uml(-1)) was observed in culture filtrate of S. phaeochromogenes under submerged fermentation. Results indicate that S. phaeochromogenes could be a potential source of glutaminase free L-asparaginase for commercial purpose. To the best of our knowledge, this is the first report on production of glutaminase free L-asparaginase from S. cyaneus, S. exfoliatus and S. phaeochromogenes.

Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition.

Extracellular shed vesicles (ESVs) facilitate a unique mode of cell-cell communication wherein vesicle uptake can induce a change in the recipient cell's state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

[Glutaminase in the cerebellum of patients with Alzheimer's disease: a postmortem brain study].

Phosphate activated glutaminase (PAG) was quantified in human cerebellar cortex extracts in 13 patients with Alzheimer's disease (AD) and 13 controls by Western immunoblotting using antibody to C-terminus of PAG kidney isoform. The majority of samples from the AD group contained less amount of PAG in comparison with control samples. Although medians in these groups were slightly different (21 and 28 arbitrary units in AD patients and controls, respectively), the Mann Whitney U-test demonstrated a significant between-group difference (U= 28.5, Z= -2.87, p=0.004). Since the both groups were matched for gender, age and postmortem interval, the difference in the PAG level was probably due to the presence of AD. The alteration in the PAG level observed in the cerebellum of patients with AD results in the disturbance of probably not only glutamate metabolism but also many other pathways involving PAG and leads to crucial consequences, particularly, to neurodegeneration.

Salt-tolerant and thermostable glutaminases of cryptococcus species form a new glutaminase family.

Genes encoding salt-tolerant and thermostable glutaminases were isolated from Cryptococcus species. The glutaminase gene, CngahA, from C. nodaensis NISL-3771 was 2,052 bp in length and encoded a 684-amino acid protein. The gene, CagahA, from C. albidus ATCC20293 was 2,100 bp in length and encoded a 700-amino acid protein. These glutaminases showed 44% identity. By searches on public databases, we found that these glutaminases are not similar to any other characterized glutaminases, but are similar to certain hypothetical proteins. On searching the conserved domain with the basic local alignment search tool (BLAST), it was found that they have the amidase domain and are members of the amidase signature superfamily. They were expressed in Saccharomyces cerevisiae, and their activity was detected on the cell surface. This study revealed that they are a new type of glutaminase with the amidase signature sequence, and that they form a new glutaminase family.

Prolonged ingestion of prehydrolyzed whey protein induces little or no change in digestive enzymes, but decreases glutaminase activity in exercising rats.

Because consumption of whey protein hydrolysates is on the increase, the possibility that prolonged ingestion of whey protein hydrolysates affect the digestive system of mammals has prompted us to evaluate the enzymatic activities of pepsin, leucine-aminopeptidase, chymotrypsin, trypsin, and glutaminase in male Wistar rats fed diets containing either a commercial whey isolate or a whey protein hydrolysate with medium degree of hydrolysis and to compare the results with those produced by physical training (sedentary, sedentary-exhausted, trained, and trained-exhausted) in the treadmill for 4 weeks. The enzymatic activities were determined by classical procedures in all groups. No effect due to the form of the whey protein in the diet was seen in the activities of pepsin, trypsin, chymotrypsin, and leucine-aminopeptidase. Training tended to increase the activity of glutaminase, but exhaustion promoted a decrease in the trained animals, and consumption of the hydrolysate decreased it even further. The results are consistent with the conclusion that chronic consumption of a whey protein hydrolysate brings little or no modification of the proteolytic digestive system and that the lowering of glutaminase activity may be associated with an antistress effect, counteracting the effect induced by training in the rat.

Development of medium for enhanced production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428.

Glutaminase-free L-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of L-asparaginase using the Plackett-Burman experimental design. The medium components, viz., glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O, were screened based on their high confidence levels (P<0.04). The optimum levels of glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O were found to be 2.076, 5.202, 1.773, and 0.373 g L(-1), respectively, using the central composite experimental design. The maximum specific activity of L-asparaginase in the optimized medium was 27.88 U mg(-1) of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.

13C MR spectroscopy measurements of glutaminase activity in human hepatocellular carcinoma cells using hyperpolarized 13C-labeled glutamine.

Dynamic nuclear polarization (DNP) is an emerging technique for increasing the sensitivity of (13)C MR spectroscopy (MRS). [5-(13)C(1)]Glutamine was hyperpolarized using this technique by up to 5%, representing a 6000-fold increase in sensitivity. The conversion of hyperpolarized glutamine to glutamate by mitochondrial glutaminase was demonstrated using (13)C-MRS measurements in cultured human hepatoma cells (HepG2). These results represent the first step in developing an imaging technique for detecting glutamine metabolism in vivo. Furthermore, since glutamine utilization has been correlated with cell proliferation, the study suggests a new technique for detecting changes in tumor cell proliferation.

Production of glutaminase (E.C.3.2.1.5) from Zygosaccharomyces rouxii: statistical optimization using response surface methodology.

A face centered central composite design was employed to investigate the interactive effects of four variables, viz. concentrations of sucrose, yeast extract, sodium chloride, and glutamine, identified earlier by one-factor-at-a-time approach, on glutaminase production by Zygosaccharomyces rouxii. A significant influence of yeast extract on glutaminase production was noted. Response surface methodology (RSM) showed that a medium containing (g/l) sucrose, 17.8; yeast extract, 48.0; glutamine, 5.0 and sodium chloride, 55.6 to be optimum for the production of glutaminase. This medium was projected to produce, theoretically, an enzyme activity of 149.98 U/l and a specific activity of 0.488 U/mg protein. The applied methodology was validated using this optimized media and enzyme activity 155.89+/-1.68 U/l and specific activity of 0.468+/-0.088 U/mg protein was obtained. Further, this optimization strategy combined with an increase in inoculum enhanced the enzyme activity and specific activity by 2.94 and 3.58 fold, respectively, as compared to the unoptimized media.

L-asparaginase and L-glutaminase activities in submerged rice soil amended with municipal solid waste compost and decomposed cow manure.

The field study was conducted to evaluate the effect of municipal solid waste compost (MSWC) as a soil amendment on L-asparaginase (LA) and L-glutaminase (LG) activities. Experiments were conducted during the wet seasons of 1997, 1998 and 1999 on rice grown under a submerged condition, at the Agriculture Experimental Farm, Calcutta University at Baruipur, West Bengal, India. The treatments consisted of control, no input; MSWC, at 60 Kg N ha(- 1); well-decomposed cow manure (DCM), at 60 Kg N ha(- 1); MSWC (30 Kg N ha(- 1)) + Urea (U) (30 Kg N ha(- 1)); DCM (30 Kg N ha(- 1)) + U (30 Kg N ha(- 1)) and Fertilizer, (at 60:30:30 NPK kg ha(- 1)) through urea, single superphosphate and muriate of potash respectively). LA and LG activities alone and their ratio with organic-C (ratio index value, RIV), straw and grain yield were higher in DCM than MSWC-treated soils, due to higher amount of biogenic organic materials like water-soluble organic carbon, carbohydrate and mineralizable nitrogen in the former. The studied parameters were higher when urea was integrated with DCM or MSWC, compared to their single applications. The heavy metals in MSWC did not detrimentally influence the above-measured activities of soil. In the event of long term MSWC application, changes in soil quality parameters should be monitored regularly, since heavy metals once entering into soil persist over a long period.

VGluT1- and GAD-immunoreactive terminals in synaptic contact with PAG-immunopositive neurons in principal sensory trigeminal nucleus of rat.

AIM: To trace the origin of abundant vesicular glutamate transporter 1-like immunoreactive (VGluT1-LI) axon terminals in the dorsal division of the principal sensory trigeminal nucleus (Vpd) and the relationships between VGluT1-LI, as well as the glutamic acid decarboxylase (GAD)-LI axon terminals, and phosphate- activated glutaminase (PAG)-LI thalamic projecting neurons in the Vpd. METHODS: Following unilateral trigeminal rhizotomy, triple-immunofluorescence histochemistry for VGluT1, GAD and PAG and the immunogold-silver method for VGluT1 or GAD, combined with the immunoperoxidase method for PAG were performed, respectively. RESULTS: After unilateral trigeminal rhizotomy, the density of VGluT1-like immunoreactivity (IR) in the Vpd on the lesion side was reduced compared to its contralateral counterpart. Under the confocal laser-scanning microscope, the VGluT1-LI or GAD-LI axon terminals were observed to be in close apposition to the PAG-LI thalamic projecting neuronal profiles, and further electron microscope immunocytochemistry confirmed that VGluT1- and GAD-LI axon terminals made asymmetrical and symmetrical synapses upon the PAG-LI neuronal structures. CONCLUSION: The present results suggest that the VGluT1-LI axon terminals, which mainly arise from the primary afferents of the trigeminal ganglion, along with the PAG-LI neuronal profiles, form the key synaptic connection involved in sensory signaling.

Distribution and morphological characterization of phosphate-activated glutaminase-immunoreactive neurons in cat visual cortex.

Phosphate-activated glutaminase (PAG) is the major enzyme involved in the synthesis of the excitatory neurotransmitter glutamate in cortical neurons of the mammalian cerebral cortex. In this study, the distribution and morphology of glutamatergic neurons in cat visual cortex was monitored through immunocytochemistry for PAG. We first determined the specificity of the anti-rat brain PAG polyclonal antibody for cat brain PAG. We then examined the laminar expression profile and the phenotype of PAG-immunopositive neurons in area 17 and 18 of cat visual cortex. Neuronal cell bodies with moderate to intense PAG immunoreactivity were distributed throughout cortical layers II-VI and near the border with the white matter of both visual areas. The largest and most intensely labeled cells were mainly restricted to cortical layers III and V. Careful examination of the typology of PAG-immunoreactive cells based on the size and shape of the cell body together with the dendritic pattern indicated that the vast majority of these cells were pyramidal neurons. However, PAG immunoreactivity was also observed in a paucity of non-pyramidal neurons in cortical layers IV and VI of both visual areas. To further characterize the PAG-immunopositive neuronal population we performed double-stainings between PAG and three calcium-binding proteins, parvalbumin, calbindin and calretinin, to determine whether GABAergic non-pyramidal cells can express PAG, and neurofilament protein, a marker for a subset of pyramidal neurons in mammalian neocortex. We here present PAG as a neurochemical marker to map excitatory cortical neurons that use the amino acid glutamate as their neurotransmitter in cat visual cortex.

Glutamate transport, glutamine synthetase and phosphate-activated glutaminase in rat CNS white matter. A quantitative study.

Glutamatergic signal transduction occurs in CNS white matter, but quantitative data on glutamate uptake and metabolism are lacking. We report that the level of the astrocytic glutamate transporter GLT in rat fimbria and corpus callosum was approximately 35% of that in parietal cortex; uptake of [3H]glutamate was 24 and 43%, respectively, of the cortical value. In fimbria and corpus callosum levels of synaptic proteins, synapsin I and synaptophysin were 15-20% of those in cortex; the activities of glutamine synthetase and phosphate-activated glutaminase, enzymes involved in metabolism of transmitter glutamate, were 11-25% of cortical values, and activities of aspartate and alanine aminotransferases were 50-70% of cortical values. The glutamate level in fimbria and corpus callosum was 5-6 nmol/mg tissue, half the cortical value. These data suggest a certain capacity for glutamatergic neurotransmission. In optic and trigeminal nerves, [3H]glutamate uptake was < 10% of the cortical uptake. Formation of [14C]glutamate from [U-14C]glucose in fimbria and corpus callosum of awake rats was 30% of cortical values, in optic nerve it was 13%, illustrating extensive glutamate metabolism in white matter in vivo. Glutamate transporters in brain white matter may be important both physiologically and during energy failure when reversal of glutamate uptake may contribute to excitotoxicity.