Glutamine Synthetase Expression Research Articles

Small loci of astroglial glutamine synthetase deficiency in the postnatal brain cause epileptic seizures and impaired functional connectivity.

The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain. We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GSflox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS. The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity. These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels.

Glutamine synthetase regulation by dexamethasone, RU486, and compound A in astrocytes derived from aged mouse cerebral hemispheres is mediated via glucocorticoid receptor.

Glucocorticoids (GCs) regulate astrocyte function, while glutamine synthetase (GS), an enzyme highly expressed in astrocytes, is one of the most remarkable GCs-induced genes. GCs mediate their effects through their cognate glucocorticoid receptor (GRα and GRβ isoforms); however, the mechanism via which these isoforms regulate GS activity in astrocytes remains unknown. We used dexamethasone (DEX), a classical GRα/GRβ agonist, RU486, which is a specific GRβ ligand, and Compound A, a known "dissociated" ligand, to delineate the mechanism via which GR modulates GS activity. Aged Mouse Cerebral Hemisphere astrocytes were treated with DEX (1μM), RU486 (1nM-1μM) or compound A (10μM), alone or in combination with DEX. GS activity and expression, GR isoforms (mRNA and protein levels), and GRα subcellular trafficking were measured. DEX increased GS activity in parallel with GRα nuclear translocation. RU486 increased GS activity in absence of GRα nuclear translocation implicating thus a role of GRβ-mediated mechanism compound A had no effect on GS activity implicating a GRα-GRE-mediated mechanism. None of the compounds affected whole-cell GRα protein content. DEX reduced GRα and GRβ mRNA levels, while RU486 increased GRβ gene expression. We provide evidence that GS activity, in astrocytes, is regulated via GRα- and GRβ-mediated pathways with important implications in pathological conditions in which astrocytes are involved.

Exercise increases striatal Glu reuptake and improves motor dysfunction in 6-OHDA-induced Parkinson’s disease rats

The purpose of this study is to clarify that exercise may improve the motor dysfunction of Parkinson's disease (PD) model rats by increasing the reuptake of glutamate (Glu) in the striatum. The neurotoxin 6-hydroxydopamine (6-OHDA) was injected into the medial forebrain bundle (MFB) of the rats' right brain to establish PD model rats with unilateral injury, and the sham operation group was given the same dose of normal saline at the same site as the control group. The reliability of the model was evaluated by apomorphine (APO)-induced rotation test combined with tyrosine hydroxylase (TH) immunohistochemical staining in the substantia nigra and striatum. The exercise group started treadmill training intervention (11m/min, 30min/day, 5d/week, and 4weeks in total) 1 week after the operation. The balance bar test, suspension test, and the tail-lifting handstand test were used to evaluate exercise performance of rats; RT-PCR and western blotting were used to detect protein and mRNA expression of glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) in the striatum; and isotope labeling was used to detect the ability of Glu reuptake in the striatum. (1) Compared with PD group, the number of TH immunoreactive cells in the substantia nigra and the content of TH immunoreactive fibers in the striatum did not change significantly in PD + Ex group. (2) Compared with PD group, the latency and total time of crossing the balance beam were significantly shorter (P < 0.01), the retention time of two forepaws on the metal wire was significantly longer (P < 0.01), the maximum lifting of head and trunk was significantly increased (P < 0.01) in PD + Ex group. (3) Compared with PD group, the ability of Glu reuptake in the striatum was significantly increased (P < 0.05), the expression levels of GLT-1 and GS mRNA in the striatum were significantly increased (P < 0.05), the protein expression of GLT-1 and GS in the striatum was significantly upregulated (P < 0.05) in PD + Ex group. Exercise intervention can significantly improve the motor dysfunction of PD model rats, increase the ability of striatal Glu reuptake significantly, and upregulate the expression levels of GLT-1 and GS protein and GS mRNA significantly. Exercise intervention may increase the protein expression level of GLT-1 and increase the reuptake ability of Glu in the striatum, thereby reducing the excitotoxic effect of excessive Glu on the postsynaptic membrane, and ultimately alleviate the motor dysfunction in PD model rats.

"Glu/GABA-Gln" metabolic loop abnormalities in iminodipropionitrile (IDPN)-induced dyskinetic syndrome.

Iminodipropionitrile (IDPN)-induced dyskinetic syndrome is characterized by abnormal repetitive involuntary movements with abnormalities in the neuro-transmission. This study explored the mechanism of glutamate (Glu)/γ-aminobutyric acid (GABA)-glutamine (Gln) metabolic circuit in rat dyskinetic syndrome and the possible regulation mechanism of "tiapride (Tia)." Male Wistar rats were assigned to the control group, dyskinetic syndrome group, and Tia group. Dyskinetic syndrome was induced by injecting with 3,3'-iminodipropionitrile for 7days. Tia group was treated with tiapride, while the control and dyskinetic syndrome groups were gavaged with saline. Eventually the Glu, GABA, and Gln concentrations in striatum were detected using UPLC-3QMS, additionally another amino acid neurotransmitters (aspartate, glycine) were also detected. Expressions of glutamine synthetase (GS), glutamate transporter (EAAT2), glutamate decarboxylase (GAD65/67), and γ-aminobutyric acid transporter protein (GAD-T) were observed using Western blot and real-time polymerase chain reaction. The behavior test scores of dyskinetic syndrome group were increased compared with the control group. Tia group decreased the behavior test scores compared with dyskinetic syndrome group. For amino acid neuro-transmission, dyskinetic syndrome group increased Glu level (p < 0.01), decreased GABA level (p < 0.01), increased Glu/GABA ratio (p < 0.01), and decreased Asp level (p < 0.01) compared with control group. Tia group decreased Glu level (p < 0.01), increased GABA level (p < 0.01), decreased Glu/GABA ratio (p < 0.01), and increased Asp level (p < 0.05) compared with dyskinetic syndrome group. For Glu/GABA-Gln circuit, the protein and mRNA expression of GS and EAAT2 in dyskinetic syndrome group were decreased (p < 0.05). Tia group increased protein and mRNA expression level of GS (p < 0.05) and EAAT2 (p < 0.01). The rat dyskinetic syndrome has Glu/GABA-Gln abnormalities. "Tiapride" upregulated the protein expression of GS and EAAT2, reduce Glu levels, increase γ-GABA levels, and eventually improve amino acid neurotransmitter imbalance.

Evidence for glutamine synthetase function in mouse spinal cord oligodendrocytes.

Glutamine synthetase (GS) is a key enzyme that metabolizes glutamate into glutamine. While GS is highly enriched in astrocytes, expression in other glial lineages has been noted. Using a combination of reporter mice and cell type-specific markers, we show that GS is expressed in myelinating oligodendrocytes (OL) but not oligodendrocyte progenitor cells of the mouse and human ventral spinal cord. To investigate the role of GS in mature OL, we used a conditional knockout (cKO) approach to selectively delete GS-encoding gene (Glul) in OL, which caused a significant decrease in glutamine levels on mouse spinal cord extracts. GS cKO mice (CNP-cre+ :Glulfl/fl ) showed no differences in motor neuron numbers, size or axon density; OL differentiation and myelination in the ventral spinal cord was normal up to 6months of age. Interestingly, GS cKO mice showed a transient and specific decrease in peak force while locomotion and motor coordination remained unaffected. Last, GS expression in OL was increased in chronic pathological conditions in both mouse and humans. We found a disease-stage dependent increase of OL expressing GS in the ventral spinal cord of SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Moreover, we showed that GLUL transcripts levels were increased in OL in leukocortical tissue from multiple sclerosis but not control patients. These findings provide evidence towards OL-encoded GS function in spinal cord sensorimotor axis, which is dysregulated in chronic neurological diseases.

Protective Effect of Raf-1 Kinase Inhibitory Protein on Diabetic Retinal Neurodegeneration through P38-MAPK Pathway

ABSTRACT Purpose This study aimed to investigate the effect of Raf-1 kinase inhibitory protein (RKIP) on diabetic retinal neurodegeneration in streptozotocin-treated rat model and high glucose-treated rat Müller cells. Methods Control and streptozotocin-treated rats were intravitreally injected with saline, RKIP gene overexpression lentivirus (oeRKIP) or negative control lentivirus (RKIP-vector). Normal or high glucose-treated Müller cells were transfected with saline, RKIP gene overexpression lentivirus or negative control lentivirus. Western blotting and immunofluorescence assay were utilized to evaluate the function of RKIP on the expression of RKIP, p38 mitogen-activated protein kinase (p38-MAPK), glutamate/aspartate transporter (GLAST), glutamine synthetase (GS), glial fibrillar acidic protein (GFAP) and cysteine-aspartic acid protease-3 (caspase-3). A glutamate assay kit was adopted to detect glutamate level in retina samples. Apoptosis of Müller cells was determined by Annexin-V/PI staining and flow cytometry. Results High glucose-treated Müller cells exhibited promoted apoptosis, while RKIP overexpression in high glucose-treated Müller cells down-regulated the enhanced apoptosis. Compared with rats injected with saline, streptozotocin-treated hyperglycemic rats displayed enhancement in the immunoreactivities of p38-MAPK and GFAP as well as in the protein expression of p38-MAPK and caspase-3. Strikingly, intravitreal injection of RKIP gene overexpression lentivirus in the hyperglycemic rats reversed the augmented immunoreactivities and protein expression mentioned above. Meanwhile, RKIP overexpression in the hyperglycemic rats improved the immunoreactivities and protein expression of RKIP, GS and GLAST. Besides, RKIP down-regulated the increased level of retinal glutamate in the hyperglycemic rats. Conclusions Intravitreal injection of RKIP gene overexpression lentivirus functioned in preventing diabetic retinal neurodegeneration in a rat model of diabetes presumably by inhibiting p38-MAPK pathway.

N-acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.

Modulation of Astrocytic Glutamine Synthetase by Endocannabinoid 2-Arachidonoylglycerol in JNK-Independent Pathway.

Background and Objective: The glutamine synthetase (GS), an astrocyte-specific enzyme, plays an important role in neuroprotection through the glutamate/glutamine shuttle and can be modulated by endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) through extracellular signal-regulated protein kinase ½ (ERK1/2) and p38 signaling pathways. However, the role of c-Jun N-terminal kinase (JNK) signaling pathway in the modulation of GS in astrocytes by 2-AG is not clear.Materials and Methods: The expression of GS and JNK in astrocytes following the exposure to lipopolysaccharide (LPS) was examined with Western blotting and immunochemistry.Results: The results revealed that short-term exposure to LPS activated GS and increased phosphorylation of JNK in astrocytes in a time-dependent manner. Treatment with 2-AG reversed the changes in GS but had no effect on the activation of JNK.Conclusions: These findings suggest that the activation of JNK induced by LPS is not involved in the modulation of astrocytic GS by 2-AG.

Improvement of the efficiency and quality in developing a new CHO host cell line.

Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high-producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi-allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS-knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS-knockout hosts with desired growth and recombinant protein expression characteristics.

Dysembryoplastic neuroepithelial tumors: A single-institutional series with special reference to glutamine synthetase expression

Dysembryoplastic neuroepithelial tumors (DNT) is a benign (World Health Organisation, WHO, grade I) glioneuronal tumor and it represent one of the most frequent neoplasm in patient affected by seizures. The epileptic neuronal activity can be determined by abnormal synchronization, excessive glutamate excitation and\\or inadequate GABA inhibition. Increasing evidence suggests that the astrocytes might be involved in this process even if neurons play a relevant role. In particular astrocytes promote the clearance of glutamate, a potent excitatory neurotransmitter of the central nervous system. Indeed, elevated concentrations of extracellular glutamate may determine iper-excitability and seizures as well as other neurological disorders. So, astrocytes, converting glutamate into glutamine via the enzyme glutamine synthetase (GS), could play a protective anti-seizures role. In the present study, we analyzed the immunohistochemical expression of GS in 20 DNTs specimens documenting a constant immunoistochemical expression of GS in astrocytes of the lesional tissue and of the cerebral cortex.

Expression dynamics of genes encoding nitrate and ammonium assimilation enzymes in rice genotypes exposed to reproductive stage salinity stress

Understanding the reproductive stage salinity stress tolerance is a key target for breeding stress tolerant rice genotypes. Nitrate and ammonium are equally important nitrogen forms utilized by rice. We evaluated nitrate and ammonium assimilation during reproductive stage in control and salinity (10dSm−1 using NaCl) stressed rice plants. Osmotic stress tolerant rice genotype Shabhagidhan (SD) and high yielding yet osmotic and salinity stress sensitive genotype Pusa sugandh-5 (PS5) were evaluated. Salinity stress was given to plants during panicle emergence and flag leaves was collected after 1d, 3d 5d, 7d, 9d,12d and 15d after anthesis. Reproductive stage salinity stress resulted in decrease of membrane stability, relative water content and osmotic potential of rice plants. Reproductive stage salinity stress decreased the expression of nitrate reductase (OsNIA), nitrite reductase (OsNiR), Glutamine synthetase (OsGLN1.1, OsGLN1.2, OsGLN2) and glutamate synthase/GOGAT (OsFd-GOGAT, OsNADH-GOGAT) in flag leaves. In response to stress, SD showed better stress tolerance than PS5 in terms of higher yield stability. Variety SD showed higher leaf nitrate and ammonium content and maintained comparatively higher nitrate and ammonia metabolism enzyme activity than PS5. Salinity stress upregulated the activity of glutamate dehydrogenase enzyme and indirectly contributed to the higher proline content and maintenance of favourable osmotic potential in SD. Expression of GS2 which has role in photo respiratory ammonia assimilation was upregulated by salinity stress in PS5 in comparison to SD. Rice genotype showing better induction of nitrogen assimilatory genes will be more tolerant to reproductive stage salinity stress.

Pharmacological Vitamin C Treatment Impedes the Growth of Endogenous Glutamine-Dependent Cancers by Targeting Glutamine Synthetase.

Purpose: Glutamine synthetase (GS) is the only currently known enzyme responsible for synthesizing endogenous glutamine (Gln). GS exerts a critical role in the oncogenesis of endogenous Gln-dependent cancers, making it an attractive target for anti-tumor therapies. A mixed-function oxidation system consisting of vitamin C (VC), oxygen, and trace metals can oxidize GS and promote its degradation. The current study aims to explore the effect of pharmacological VC treatment on GS. Methods: Endogenous Gln-dependent cancer lines (breast cancer MCF7 and prostate cancer PC3) were selected to establish chronic Gln-deprived MCF7 and PC3 cell models. The expression of GS in parental and chronic Gln-deprived tumor cells exposed to VC treatment and control was determined by Western blot analysis. The anti-cancer effects of VC on parental and chronic Gln-deprived tumor cells were assessed by CCK-8 and annexin V-FITC/PI FACS assays. In addition, changes in cellular reactive oxygen species (ROS), glutathione (GSH) levels and NADPH/NADP + ratio were analyzed to explore the underlying mechanisms. Moreover, BALB/c nude mice xenografting with parental and chronic Gln-deprived prostate cancer cells were constructed to evaluate the in vivo therapeutic effect of VC. Finally, tumor 13N-ammonia uptake in mice bearing prostate cancer xenografts was analyzed following treatment with VC and the expression of GS in xenografts were detected by immunohistochemistry. Results: Cells overexpressing GS were obtained by chronic Gln deprivation. We found that the cytotoxic effect of VC on cancer cells was positively correlated with the expression of GS. Additionally, VC treatment led to a significant increase in ROS production, as well as GSH depletion and NADPH/NADP + reduction. These changes could be reversed by the antioxidant N-acetyl-L-cysteine (NAC). Furthermore, pharmacological VC treatment exhibited a more significant therapeutic effect on xenografts of prostate cancer cells overexpressing GS, that could be well monitored by 13N-ammonia PET/CT imaging. Conclusion: Our findings indicate that VC can kill cancer cells by targeting glutamine synthetase to induce oxidative stress. VC could be used as an anti-cancer treatment for endogenous glutamine-dependent cancers.

Dysregulation of Ambient Glutamate and Glutamate Receptors in Epilepsy: An Astrocytic Perspective.

Given the important functions that glutamate serves in excitatory neurotransmission, understanding the regulation of glutamate in physiological and pathological states is critical to devising novel therapies to treat epilepsy. Exclusive expression of pyruvate carboxylase and glutamine synthetase in astrocytes positions astrocytes as essential regulators of glutamate in the central nervous system (CNS). Additionally, astrocytes can significantly alter the volume of the extracellular space (ECS) in the CNS due to their expression of the bi-directional water channel, aquaporin-4, which are enriched at perivascular endfeet. Rapid ECS shrinkage has been observed following epileptiform activity and can inherently concentrate ions and neurotransmitters including glutamate. This review highlights our emerging knowledge on the various potential contributions of astrocytes to epilepsy, particularly supporting the notion that astrocytes may be involved in seizure initiation via failure of homeostatic responses that lead to increased ambient glutamate. We also review the mechanisms whereby ambient glutamate can influence neuronal excitability, including via generation of the glutamate receptor subunit GluN2B-mediated slow inward currents, as well as indirectly affect neuronal excitability via actions on metabotropic glutamate receptors that can potentiate GluN2B currents and influence neuronal glutamate release probabilities. Additionally, we discuss evidence for upregulation of System , a cystine/glutamate antiporter expressed on astrocytes, in epileptic tissue and changes in expression patterns of glutamate receptors.

Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells

BackgroundCisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance.MethodsWe evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.ResultsThe results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2′-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance.ConclusionsThe results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

Angiocentric glioma-associated seizures: The possible role of EATT2, pyruvate carboxylase and glutamine synthetase

PurposeOur purpose was to better understand the pathogenesis of seizures associated with angiocentric glioma. Angiocentric glioma is an indolent and rare low-grade glioma. Its typical clinical presentation is with epileptic seizures. The pathogenesis of tumor-associated seizures is poorly understood. Among the possible pathomechanisms, the increased neurotoxic concentrations of the glutamate has been proposed. Glutamate transporters, pyruvate carboxylase and glutamine synthetase are involved in maintaining the physiological concentration of glutamate in the inter synaptic spaces. MethodsWe evaluated the immunohistochemical expression of EAAT2 (the most important glutamate transporter), pyruvate carboxylase and glutamine synthetase in 17 angiocentric gliomas. ResultsEAAT2 was never expressed (0%) in the neoplastic cells in none of the cases studied. Pyruvate carboxylase was expressed in the cytoplasm of the neoplastic cells in 16/17 cases (94 %). Glutamine synthetase was expressed in the cytoplasm of the neoplastic cells in 15/17 cases (88 %). ConclusionThe net result of this enzymatic expression, in particular considering the loss of EAAT2, could be an increased glutamate concentration in the synaptic clef, which might increase local network excitability initially involving intratumoral neurons. The observation that the angiocentric glioma-associated epilepsy might be at least in part related to EAAT2 deficiency opens up interesting therapeutic perspectives.

Predictive Patterns of Glutamine Synthetase Immunohistochemical Staining in CTNNB1-mutated Hepatocellular Adenomas.

Some hepatocellular adenoma (HCA) subtypes are characterized by different CTNNB1 mutations, leading to different beta-catenin activation levels, hence variable immunostaining patterns of glutamine synthetase (GS) expression, and different risks of malignant transformation. In a retrospective multicentric study of 63 resected inflammatory (n=33) and noninflammatory (n=30) molecularly confirmed CTNNB1-mutated b-(I)HCA, we investigated the predictive potential of 3 known GS patterns as markers for CTNNB1 exon 3, 7/8 mutations. Pattern 1 (diffuse homogenous) allowed recognition of 17/21 exon 3 non-S45 mutated b-(I)HCA. Pattern 2 (diffuse heterogenous) identified all b-(I)HCA harboring exon 3 S45 mutation (20/20). Pattern 3 (focal patchy) distinguished 12/22 b-(I)HCA with exon 7/8 mutations. In exon 3 S45 and 7/8 mutations, both b-HCA and b-IHCA showed a GS+/CD34- rim with diffuse CD34 positivity in the center of the lesion. Interobserver reproducibility was excellent for exon 3 mutations. Comparative analysis of GS patterns with molecular data showed 83% and 80% sensitivity (b-HCA/b-IHCA) and 100% specificity for exon 3 non-S45. For exon 3 S45, sensitivity was 100% for b-(I)HCA, and specificity 93% and 92% (b-HCA/b-IHCA). For exon 7/8, sensitivity was 55% for both subtypes and specificity 100% and 96% (b-HCA/b-IHCA). Preliminary data from 16 preoperative needle biopsies from the same patients suggest that this panel may also be applicable to small samples. In surgically resected HCA, 2 distinct GS patterns can reliably predict CTNNB1 exon 3 mutations, which are relevant because of the higher risk for malignant transformation. The third pattern, although specific, was less sensitive for the identification of exon 7/8 mutation, but the GS+/CD34- rim is a valuable aid to indicate either an exon 3 S45 or exon 7/8 mutation.

Mechanical Brain Injury Increases Cells' Production of Cystathionine β-Synthase and Glutamine Synthetase, but Reduces Pax2 Expression in the Telencephalon of Juvenile Chum Salmon, Oncorhynchus keta.

The considerable post-traumatic brain recovery in fishes makes them a useful model for studying the mechanisms that provide reparative neurogenesis, which is poorly represented in mammals. After a mechanical injury to the telencephalon in adult fish, lost neurons are actively replaced due to the proliferative activity of neuroepithelial cells and radial glia in the neurogenic periventricular zone. However, it is not enough clear which signaling mechanisms are involved in the activation of adult neural stem cells (aNSC) after the injury (reactive proliferation) and in the production of new neurons (regenerative neurogenesis) from progenitor cells (NPC). In juvenile Pacific salmon, the predominant type of NSCs in the telencephalon are neuroepithelial cells corresponding to embryonic NSCs. Expression of glutamine synthetase (GS), a NSC molecular marker, was detected in the neuroepithelial cells of the pallium and subpallium of juvenile chum salmon, Oncorhynchus keta. At 3 days after a traumatic brain injury (TBI) in juvenile chum salmon, the GS expression was detected in the radial glia corresponding to aNSC in the pallium and subpallium. The maximum density of distribution of GS+ radial glia was found in the dorsal pallial region. Hydrogen sulfide (H2S) is a proneurogenic factor that reduces oxidative stress and excitotoxicity effects, along with the increased GS production in the brain cells of juvenile chum salmon. In the fish brain, H2S producing by cystathionine β-synthase in neurogenic zones may be involved in maintaining the microenvironment that provides optimal conditions for the functioning of neurogenic niches during constitutive neurogenesis. After injury, H2S can determine cell survivability, providing a neuroprotective effect in the area of injury and reducing the process of glutamate excitotoxicity, acting as a signaling molecule involved in changing the neurogenic environment, which leads to the reactivation of neurogenic niches and cell regeneration programs. The results of studies on the control of the expression of regulatory Sonic Hedgehog genes (Shh) and the transcription factors Paired Box2 (Pax2) regulated by them are still insufficient. A comparative analysis of Pax2 expression in the telencephalon of intact chum salmon showed the presence of constitutive patterns of Pax2 expression in neurogenic areas and non-neurogenic parenchymal zones of the pallium and subpallium. After mechanical injury, the patterns of Pax2 expression changed, and the amount of Pax2+ decreased (p < 0.05) in lateral (Dl), medial (Dm) zones of the pallium, and the lateral zone (Vl) of the subpallium compared to the control. We believe that the decrease in the expression of Pax2 may be caused by the inhibitory effect of the Pax6 transcription factor, whose expression in the juvenile salmon brain increases upon injury.

Inoculation with Clariodeoglomus etunicatum improves leaf food quality of tea exposed to P stress

The present study aimed to evaluate the effect of an arbuscular mycorrhizal fungus (AMF), Clariodeoglomus etunicatum, on leaf food quality and relevant gene expression levels of tea (Camellia sinensis cv. ‘Fuding Dabaicha’) seedlings exposed to 0.5 μM P (P0.5) and 50 μM P (P50) levels. Twenty-four weeks later, the seedlings recorded higher root mycorrhizal fungal colonization in P50 than in P0.5. AMF-inoculated tea plants represented significantly higher leaf fructose and glucose contents and lower sucrose content than non-inoculated plants, irrespective of substate P levels. AMF treatment also increased total amino acids content in P0.5 and P50, accompanied with higher expression of glutamate dehydrogenase (CsGDH) and lower expression of glutamine synthetase (CsGS) and glutamine oxoglutarate aminotransferase (CsGOGAT). The total flavonoid content was higher in mycorrhizal versus non-mycorrhizal plants under P0.5 and P50, together with induced expression of phenylalanine ammonia-lyase (CsPAL) and cinnamic acid 4-hydroxylase (CsC4H). Mycorrhizal fungal inoculation improved catechins content, which is due to the up-regulated expression of flavanone 3-hydroxylase (CsF3H), flavonoid 3'-hydroxylase (CsF3'H), dihydroflavonol 4-reductase (CsDFR), leucoanthocyanidin reductase (CsLAR), anthocyanidin reductase (CsANR), and chalcone isomerase (CsCHI) under P0.5. However, under P50, the gene involved in catechins synthesis was not affected or down-regulated by mycorrhization, implying a complex mechanism (e.g. nutrient improvement). AMF also inhibited the tea caffeine synthase 1 (CsTCS1) expression regardless of P levels. Therefore, the results of this study concluded that inoculation with C. etunicatum improves leaf food quality of tea exposed to P stress, but the improved mechanisms were different between P0.5 and P50.

Rhophilin-2 Upregulates Glutamine Synthetase by Stabilizing c-Myc Protein and Confers Resistance to Glutamine Deprivation in Lung Cancer.

IntroductionRHPN2, a member of rhophilin family of rho-binding proteins, regulates actin cytoskeleton and vesicular trafficking, and promotes mesenchymal transformation in cancer. We have found that RHPN2 was significantly mutated in lung adenocarcinoma (LUAD). However, the role of RHPN2 in lung cancer is not fully understood.MethodsIn the present study, we investigated the expression of RHPN2 in 125 patients with LUAD by qRT-PCR and correlated its expression with clinical characteristics. The effects of RHPN2 on the proliferation and invasion of lung cancer cells were determined by CCK-8 and in vitro transwell assays, clonogenic assay, and xenograft mouse model. The RhoA pull down assay and Western blotting were performed to elucidate the mechanism of RNPN2 in tumorigenesis of lung cancer.ResultsRHPN2 was overexpressed in tumors from LUAD, and high levels of RHPN2 were associated with poor prognosis of LUAD patients. RHPN2 was required for proliferation and invasion of lung cancer cells. Intriguingly, overexpression of RHPN2 conferred the resistance to glutamine depletion in lung cancer cells. Mechanistic studies revealed that ectopic overexpression of RHPN2 promoted the stability of c-Myc protein via phosphorylation at Ser62 and increased c-Myc target glutamine synthetase (GS). Analysis of GS expression in clinical sample showed that the expression of GS was elevated in tumor cells. Kaplan-Meier analysis revealed that high levels of GS were significantly associated with worse overall survival time of the patients with LUAD.ConclusionsTaken together, this study suggested that RHPN2 was involved in tumorigenesis of lung cancer via modulating c-Myc stability and the expression of its target GS in lung adenocarcinoma, which links RHPN2 and glutamine metabolism.

Effect of a mix of oligogalacturonides on symbiotic nitrogen fixation in common bean

The objective of this study was to evaluate the effect of a mix of pectic oligosaccharides in the common bean-rhizobia symbiosis. Cuba Cueto-25-9-N bean seeds were inoculated with Rhizobium tropici CIAT 899 and treated with a mix of oligogalacturonides, either by application to the seed (10 mg L-1, 1 ml per seed) at sowing and inoculation or by foliar spray (5 or 100 mg L-1, 1.5 ml per plant) to plants with two trefoils. Plant growth, nodulation, nitrogenase activity, and the gene expression of glutamine synthetase and glutamate synthase in nodules were evaluated in inoculated plants and oligogalacturonide-treated inoculated plants at 18 d post-inoculation. The oligogalacturonide-treated plants showed increases in shoot and root growth and number of nodules. Regarding nodule function, the oligogalacturonides increased the nitrogenase activity and the expression level of the gene coding for NADH-dependent glutamate synthase. The positive effects of oligogalacturonides resulted in higher effectiveness of the symbiotic nitrogen fixation. The application of oligogalacturonides can offer an alternative to increasing the symbiotic nitrogen fixation in common bean plants.