Attenuation of initial pilocarpine-induced electrographic seizures by methionine sulfoximine pretreatment tightly correlates with the reduction of extracellular taurine in the hippocampus.

OBJECTIVE: Initiation and development of early seizures by chemical stimuli is associated with brain cell swelling resulting in edema of seizure-vulnerable brain regions. We previously reported that pretreatment with a nonconvulsive dose of glutamine (Gln) synthetase inhibitor methionine sulfoximine (MSO) mitigates the intensity of initial pilocarpine (Pilo)-induced seizures in juvenile rats. We hypothesized that MSO exerts its protective effect by preventing the seizure-initiating and seizure-propagating increase of cell volume. Taurine (Tau) is an osmosensitive amino acid, whose release reflects increased cell volume. Therefore, we tested whether the poststimulus rise of amplitude of Pilo-induced electrographic seizures and their attenuation by MSO are correlated with the release of Tau from seizure-affected hippocampus. METHODS: Lithium-pretreated animals were administered MSO (75 mg/kg ip) 2.5 h before the induction of convulsions by Pilo (40 mg/kg ip). Electroencephalographic (EEG) power was analyzed during 60 min post-Pilo, at 5-min intervals. Extracellular accumulation of Tau (eTau) served as a marker of cell swelling. eTau, extracellular Gln (eGln), and extracellular glutamate (eGlu) were assayed in the microdialysates of the ventral hippocampal CA1 region collected at 15-min intervals during the whole 3.5-h observation period. RESULTS: The first EEG signal became apparent at ~10 min post-Pilo. The EEG amplitude across most frequency bands peaked at ~40 min post-Pilo, and showed strong (r ~ .72-.96) temporal correlation with eTau, but no correlation with eGln or eGlu. MSO pretreatment delayed the first EEG signal in Pilo-treated rats by ~10 min, and depressed the EEG amplitude across most frequency bands, to values that remained strongly correlated with eTau (r > .92) and moderately correlated (r ~ -.59) with eGln, but not with eGlu. SIGNIFICANCE: Strong correlation between attenuation of Pilo-induced seizures and Tau release indicates that the beneficial effect of MSO is due to the prevention of cell volume increase concurrent with the onset of seizures.

Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells.

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.

The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.

TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.

Glutamine: a major player in nitrogen catabolite repression in the yeast Dekkera bruxellensis.

In the present work we studied the expression of genes from nitrogen central metabolism in the yeast Dekkera bruxellensis and under regulation by the Nitrogen Catabolite Repression mechanism (NCR). These analyses could shed some light on the biological mechanisms involved in the adaptation and survival of this yeast in the sugarcane fermentation process for ethanol production. Nitrogen sources (N-sources) in the form of ammonium, nitrate, glutamate or glutamine were investigated with or without the addition of methionine sulfoximine, which inhibits the activity of the enzyme glutamine synthetase and releases cells from NCR. The results showed that glutamine might act as an intracellular sensor for nitrogen availability in D. bruxellensis, by activating NCR. Gene expression analyses indicated the existence of two different GATA-dependent NCR pathways, identified as glutamine-dependent and glutamine-independent mechanisms. Moreover, nitrate is sensed as a non-preferential N-source and releases NCR to its higher level. After grouping genes according to their regulation pattern, we showed that genes for ammonium assimilation represent a regulon with almost constitutive expression, while permease encoding genes are mostly affected by the nitrogen sensor mechanism. On the other hand, nitrate assimilation genes constitute a regulon that is primarily subjected to induction by nitrate and, to a lesser extent, to a repressive mechanism by preferential N-sources. This observation explains our previous reports showing that nitrate is co-consumed with ammonium, a trait that enables D. bruxellensis cells to scavenge limiting N-sources in the industrial substrate and, therefore, to compete with Saccharomyces cerevisiae in this environment.

In Salmonella enterica, the Gcn5-related acetyltransferase MddA (formerly YncA) acetylates methionine sulfoximine and methionine sulfone, blocking their toxic effects.

Protein and small-molecule acylation reactions are widespread in nature. Many of the enzymes catalyzing acylation reactions belong to the Gcn5-related N-acetyltransferase (GNAT; PF00583) family, named after the yeast Gcn5 protein. The genome of Salmonella enterica serovar Typhimurium LT2 encodes 26 GNATs, 11 of which have no known physiological role. Here, we provide in vivo and in vitro evidence for the role of the MddA (methionine derivative detoxifier; formerly YncA) GNAT in the detoxification of oxidized forms of methionine, including methionine sulfoximine (MSX) and methionine sulfone (MSO). MSX and MSO inhibited the growth of an S. enterica ΔmddA strain unless glutamine or methionine was present in the medium. We used an in vitro spectrophotometric assay and mass spectrometry to show that MddA acetylated MSX and MSO. An mddA(+) strain displayed biphasic growth kinetics in the presence of MSX and glutamine. Deletion of two amino acid transporters (GlnHPQ and MetNIQ) in a ΔmddA strain restored growth in the presence of MSX. Notably, MSO was transported by GlnHPQ but not by MetNIQ. In summary, MddA is the mechanism used by S. enterica to respond to oxidized forms of methionine, which MddA detoxifies by acetyl coenzyme A-dependent acetylation.

The two paralogue phoN (phosphinothricin acetyl transferase) genes of Pseudomonas putida encode functionally different proteins.

Phosphinothricin (PPT) is a non-specific inhibitor of glutamine synthetase that has been employed as herbicide for selection of transgenic plants expressing cognate resistance genes. While the soil bacterium Pseudomonas putida KT2440 has been generally considered PPT-sensitive, inspection of its genome sequence reveals the presence of two highly similar open reading frames (PP_1924 and PP_4846) encoding acetylases with a potential to cause tolerance to the herbicide. To explore this possibility, each of these genes (named phoN1 and phoN2) was separately cloned and their activities examined in vivo and in vitro. Genetic and biochemical evidence indicated that phoN1 encodes a bona fide PPT-acetyl transferase, the expression of which suffices to make P. putida tolerant to high concentrations of the herbicide. In contrast, PhoN2 does not act on PPT but displays instead activity against methionine sulfoximine (MetSox), another glutamine synthetase inhibitor. When the geometry of the substrate-binding site of PhoN1 was grafted with the equivalent residues of the predicted PhoN2 structure, the resulting protein increased significantly MetSox resistance of the expression host concomitantly with the loss of activity on PPT. These observations uncover intricate biochemical and genetic interactions among soil microorganisms and how they can be perturbed by exposure to generic herbicides in soil.

Glutamine synthetase plays an important role in ammonium tolerance of Myriophyllum aquaticum.

High-strength ammonium (NH4+), the main characteristic of swine wastewater, poses a significant threat to the rural ecological environment. As a novel phytoremediation technology, Myriophyllum aquaticum wetlands have high tolerance and removal rate of NH4+. Glutamine synthetase (GS), a pivotal enzyme in nitrogen (N) metabolism, is hypothesized to play an important role in the tolerance of M. aquaticum to high NH4+. Herein, the responses of M. aquaticum to GS inhibition by 0.1 mM methionine sulfoximine (MSX) under 15 mM NH4+ were investigated. After 5 days, visible NH4+ toxicity symptoms were observed in MSX-treated plants. Compared with the control, the NH4+ accumulation in the leaves increased by 20.99 times, while that of stems and roots increased by 3.27 times and 47.76 %, suggesting that GS inhibition had a greater impact on the leaves. GS inhibition decreased pigments in the leaves by 8.64 %-41.06 %, triggered oxidative stress, and affected ions concentrations in M. aquaticum. The concentrations of glutamine (Gln) and asparagine decreased by 63.46 %-97.43 % and 12.37 %-76.41 %, respectively, while the concentrations of most other amino acids increased after 5 days of MSX treatment, showing that GS inhibition reprogrammed the amino acids synthesis. A decrease in Gln explains the regulations of N-related genes, including increased expression of AMT in roots and decreased expression of GS, GOGAT, GDH, and AS, which would cause further NH4+ accumulation via promoting NH4+ uptake and decreasing NH4+ assimilation in M. aquaticum. This study revealed for the first time that GS inhibition under high NH4+ condition can lead to phytotoxicity in M. aquaticum due to NH4+ accumulation. The physiological and molecular responses of the leaves, stems, and roots confirmed the importance of GS in the high NH4+ tolerance of M. aquaticum. These findings provide new insights into NH4+ tolerance mechanisms in M. aquaticum and a theoretical foundation for the phytoremediation of high NH4+-loaded swine wastewater.

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies.

To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 µM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 µM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R2 ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 µM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for increasing mAb productivity from GS-CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17-25, 2017.

Phosphoinositide 3-kinase participates in l-methionine sulfoximine-induced cell death via salicylic acid mediated signaling in Nicotiana benthamiana.

Pseudomonas syringae pv. tabaci causes wildfire disease by the action of tabtoxinine-ß-lactam (TßL), a non-specific bacterial toxin. To better understand the molecular mechanisms of wildfire disease and its development, we focused on the phosphoinositide 3-kinase in Nicotiana benthamiana (NbPI3K) and its potential role in the disease outbreak, using l-methionine sulfoximine (MSX) as an easily accessible mimic of the TßL action. The NbPI3K-silenced plants showed accelerated induction of cell death and necrotic lesion formation by MSX, and the expression of hin1, marker gene for the programmed cell death, was strongly induced in the plants. However, the accumulation of ammonium ions, caused by MSX inhibition of glutamine sythetase activity, was not affected by the NbPI3K-silencing. Interestingly, the expression of PR-1a, a marker gene for salicylic acid (SA) innate immunity signaling, and accumulation of SA were both enhanced in the NbPI3K-silenced plants. Accordingly, the acceleration of MSX-induced cell death by NbPI3K-silencing was reduced in NahG plants, and by double silencing of NbPI3K together with the NbICS1 encoding a SA-biosynthetic enzyme. As silencing of NbPI3K accelerated the TßL-induced necrotic lesions, and lesions of wildfire disease caused by P. syringae pv. tabaci, these results suggest that the NbPI3K-related pathway might act as a negative regulator of cell death during development of wildfire disease that involves SA-dependent signaling pathway downstream of TßL action in N. benthamiana.

Development and characterization of a melphalan-resistant human multiple myeloma cell line.

We present data describing a human myeloma cell line (8226/LR-5) selected for resistance to melphalan which exhibits a 7-fold level of resistance to melphalan and is partially cross-resistant to other bifunctional alkylators and X-irradiation. Melphalan resistance is relatively unstable with a decrease in resistance observed within 17 weeks in the absence of drug. The resistance observed in this cell line is not mediated by reduced intracellular melphalan accumulation. DNA interstrand cross-linking at equivalent intracellular drug accumulation is significantly reduced in the resistant subline. Whether this reduction is the result of a decrease in the formation of this lesion or to an increased rate of removal of the lesion remains to be determined. Growth characteristics and cell cycle kinetics, including S phase, were similar between sensitive and resistant cell lines. Intracellular nonprotein thiols were found to be significantly elevated in the resistant 8226/LR-5 cells; as cells revert or lose resistance, intracellular nonprotein sulfhydryl levels decline. Prior treatment of the cells with buthionine sulfoximine significantly reduced nonprotein sulfhydryl levels and enhanced melphalan cytotoxicity in both the sensitive and resistant cell lines. Thiols appear to play a role in mediating melphalan resistance.

Nonenzymatic reductive activation of 7-N-((2-([2-(gamma-L-glutamylamino)ethyl]dithio)ethyl))mitomycin C by thiol molecules: a novel mitomycin C derivative effective on mitomycin C-resistant tumor cells.

7-N-((2-([2-(gamma-L-Glutamylamino)ethyl]dithio)ethyl))mitomycin C (KW-2149) is an analogue of mitomycin C (MMC) and has prominent activities against various tumors. We studied the antitumor effects of KW-2149 in MMC-resistant variants of human colon carcinoma HT-29 (HT-29/MMC) and mouse hepatoma Hepa-I (C4, B13NBii1) cells, which are deficient in DT-diaphorase and cytochrome P450 reductase, respectively. These enzymes mediate the reductive activation of MMC in the cells. Although HT-29/MMC and C4, B13NBii1 cells showed significant resistance to MMC, they showed sensitivity tl KW-2149 comparable to their parental tumors, indicating that DT-diaphorase and cytochrome P450 reductase could not be involved in the activation of KW-2149. In studying the activation mechanism of KW-2149, we found that glutathione (GSH) and cysteine significantly enhanced the cytotoxicity of KW-2149 in HT-29 cells. The DNA adduct of KW-2149 was increased when HT-29 cells or the isolated nuclei of the cells were incubated with KW-2149 in the presence of physiological concentrations of GSH and cysteine. KW-2149 alkylated calf thymus DNA in the presence of GSH and cysteine in vitro. These results indicate that activation of KW-2149 by thiol molecules, unlike MMC, could be an important activation mechanism of KW-2149 to form DNA adduct and to exert its cytotoxicity. This is the reason why KW-2149 is effective against MMC-resistant tumors with deficiencies in the MMC activation enzymes.

Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.

To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500µM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods.

Metabolism and action of amino acid analog anti-cancer agents.

The preclinical pharmacology, antitumor activity and toxicity of seven of the more important amino acid analogs, with antineoplastic activity, is discussed in this review. Three of these compounds are antagonists of L-glutamine: acivicin, DON and azaserine; and two are analogs of L-aspartic acid: PALA and L-alanosine. All five of these antimetabolites interrupt cellular nucleotide synthesis and thereby halt the formation of DNA and/or RNA in the tumor cell. The remaining two compounds, buthionine sulfoximine and difluoromethylornithine, are inhibitors of glutathione and polyamine synthesis, respectively, with limited intrinsic antitumor activity; however, because of their powerful biochemical actions and their low systemic toxicities, they are being evaluated as chemotherapeutic adjuncts to or modulators of other more toxic antineoplastic agents.

Accumulation of aluminum by primary cultured astrocytes from aluminum amino acid complex and its apoptotic effect.

Aluminum salts or doses that are unlikely in the human system have been employed in toxicity studies and much attention had been focused on the secondary target (neurons) of its toxicity rather than the primary target (astroglia). In order to address these issues, we have investigated the uptake and apoptotic effects of aluminum amino acid complex on primary cultured astrocytes because these are fundamental in understanding the mechanism of aluminum neurotoxicity. Aluminum solubilized by various amino acids was differentially internalized by astrocytes (glycine>serine>>glutamine>>glutamate), but aluminum was not internalized from citrate complex following 24 h of exposure. Inhibition of glutamine synthetase, by methionine sulfoximine (MSO), enhanced the uptake of aluminum from various amino acid complexes within 8 h except from glutamine complex. Blockade of selective GLT-1 (EAAT2) and GlyT1, as well as nonspecific transporters, did not inhibit or had no effect on uptake of aluminum in complex with the corresponding amino acids. Ouabain also failed to inhibit uptake of aluminum complexed with glycine. Pulse exposure to aluminum glycinate in the absence or presence of MSO caused apoptosis in over 25% of primary cultured astrocytes, and apoptotic features such as chromatin condensation and fragmentation became evident as early as 3 days of culture in normal medium. Lower doses (as low as 0.0125 mM) also caused apoptosis. The present findings demonstrate that aluminum solubilized by amino acids, particularly glycine, could serve as better candidate for neurotoxicity studies. Citrate may be a chelator of aluminum rather than a candidate for its cellular uptake. Amino acid transporters may not participate in the uptake of aluminum solubilized by their substrates. Another pathway of aluminum internalization may be implicated in addition to passive diffusion but may not require energy in form of Na+/K+-ATPase. Impaired astrocyes' metabolism can aggravate their accumulation of aluminum and aluminum can compromise astrocytes via apoptosis. Thus, loss of astrocytic regulatory and supportive roles in the central nervous system (CNS) may be responsible for neurodegeneration observed in Alzheimer's disease.

Uptake of methionine sulfoximine by some N2 fixing bacteria, and its effect on ammonium transport.

The N2 fixing bacteria Klebsiella pneumoniae, Azospirillum brasilense, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum, but not Azotobacter vinelandii accumulate the glutamine analogue methionine sulfoximine in the cell. In the accumulating cells methionine sulfoximine inhibits ammonium transport. Accumulation and inhibition are prevented by glutamine.

Acute intracarotid glucose injection towards the brain induces specific c-fos activation in hypothalamic nuclei: involvement of astrocytes in cerebral glucose-sensing in rats.

The detection of changes in glucose level constitutes the first step of the control of glucose homeostasis. Glucose sensors are therefore expected to be present in different parts of the body and particularly in the central nervous system. Some studies have already attempted to determine glucose-sensitive cerebral structures either after a glucoprivic stimulus or after prolonged hyperglycaemia. By analogy to beta cells, it was postulated that the glucose sensors in the brain could involve GLUT2, glucokinase and/or ATP-sensitive K(+) channels. Surprisingly, GLUT2 was mainly found in astrocytes. Thus, the aims of the present investigation were to determine, in awake rats: (i) the hypothalamic areas that respond to acute hyperglycaemic condition induced by an intracarotid injection of glucose and (ii) the involvement of astrocytes in glucose-sensing by the use of a glial drug, methionine sulfoximine. Rats were given intracarotid injections of glucose solution to trigger a transient insulin secretion without change in peripheral glycaemia, thus involving only central nervous regulation. Hypothalamic activation was determined by immunodetection of the immediate early gene c-fos protein. Acute glucose injection induces significant activation of arcuate and paraventricular nuclei. This stimulation mainly affects neurones in both nuclei, but also astrocytes in the former as illustrated by double immunohistochemistry (Fos and neuronal nuclei or glial fibrillary acidic protein). After specific impairment of astrocyte metabolism by methionine sulfoximine, cerebral activation disappears in the arcuate nucleus, correlated with the lack of cerebral glucose-induced insulin secretion. Therefore, arcuate and paraventricular hypothalamic nuclei are able to detect acute cerebral hyperglycaemia, leading to a peripheral stimulation of insulin secretion. Arcuate nucleus and more especially astrocytes in this nucleus play a pivotal role in glucose-sensing.

Are neuronal transporters relevant in retinal glutamate homeostasis?

Exposure of isolated retinas to 30 microM D-aspartate, which is a substrate for all high affinity glutamate transporters, for 30 min, resulted in the accumulation of such D-aspartate into Müller glial cells but not glutamatergic neurons as evinced by immunocytochemistry for D-aspartate. Further incubation of such loaded retinas in physiological media, in the absence of D-aspartate, resulted in the slow release of accumulated D-aspartate from the Müller cells and its accumulation into populations of photoreceptors and bipolar cells. This result indicates that after initial transport into Müller cells, reversal of direction of transport of D-aspartate, and thus by inference glutamate, by GLAST, readily occurs. D-aspartate released by Müller cells was strongly accumulated into cone photoreceptors which are known to express GLT-1, and into rod photoreceptors which we demonstrate here to express the retina specific glutamate transporter EAAT5 (excitatory amino transporter 5). Populations of glutamatergic bipolar cells, which express GLT-1 also exhibited avid uptake of D-aspartate. We conclude that the Müller cell glutamate transporter GLAST is responsible for most of the initial glutamate clearance in the retina after its release from neurones. However, some glutamate is also returned from Müller cells, to neurons expressing GLT-1 and EAAT5, albeit at a slow rate. These data suggest that the role of neuronal glutamate transporters in the retina may be to facilitate a slow process of recycling glutamate back from Müller cells to neurons after its initial clearance from perisynaptic regions by GLAST.

Thiol-mediated redox regulation of neutrophil apoptosis.

BACKGROUND: Intracellular glutathione, an endogenous antioxidant, protects cellular function against oxidative stress. Because oxidative stress has been implicated in neutrophil apoptosis, we hypothesized that reduced thiol levels may induce apoptosis through an alteration in cellular redox state. METHODS: Human polymorphonuclear leukocytes (PMNs), were incubated with medium or with increasing concentrations of the reduced glutathione (GSH)-depleting agents diethylmaleate and diamide and buthionine sulfoximine, an inhibitor of GSH synthesis. Apoptosis was assessed by means of flow cytometry with propidium iodide DNA staining and confirmed morphologically. GSH was measured colorimetrically, and tyrosine phosphorylation was assessed by means of immunoblotting. RESULTS: Diethylmaleate and diamide induced a dose-dependent reduction in GSH and a corresponding increase in PMN apoptosis. This effect could be reversed with N-acetylcysteine, suggesting that diethylmaleate induces apoptosis through the depletion of GSH. The antioxidant pyrolidine dithiocarbamate had no effect. Because oxidants can mediate intracellular signaling via tyrosine phosphorylation, we therefore evaluated the effects of the tyrosine kinase inhibition on diethylmaleate-induced PMN apoptosis. Both genistein and herbimycin A reduced diethylmaleate-induced apoptosis and tyrosine phosphorylation. CONCLUSIONS: Sulfhydryl oxidation by diethylmaleate alone induces apoptosis, providing evidence of a redox-sensitive, thiol-mediated pathway of apoptosis. Furthermore, tyrosine phosphorylation appears to play an important role in this process. Because apoptosis is a critical mechanism regulating PMN survival in vivo, manipulation of PMN intracellular thiols may represents a novel therapeutic target for the regulation of cellular function.

Transport into brain of buthionine sulfoximine, an inhibitor of glutathione synthesis, is facilitated by esterification and administration of dimethylsulfoxide.

Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, is poorly transported into the brain of adult mice, and only a slight decrease (approximately 10%) in the level of brain glutathione is found 30-60 min after intraperitoneal administration of BSO. When BSO is given as the ethyl ester, the brain level of BSO increases substantially after 5-15 min, and the glutathione level decreases by about 25% after 30-60 min. When BSO or its ester is given in 15% dimethylsulfoxide solution the brain levels of BSO are increased significantly and the brain glutathione levels are decreased by 20-35%. These observations suggest procedures that may be useful in decreasing the glutathione levels of the brains of adult animals. The finding that administration of BSO ethyl ester led to about a 25% decrease in the brain level of glutathione within 15 min suggests that a fraction of brain glutathione turns over very rapidly and may therefore be of special physiological significance.