Presence Of Glu Research Articles

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H2O2 splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

Biomineralized Cascade Enzyme-Encapsulated ZIF-8 Nanoparticles Combined with Antisense Oligonucleotides for Drug-Resistant Bacteria Treatment.

The unrestrained use of antibiotics accelerates the development of drug-resistant bacteria and leads to an increasing threat to human health. Therefore, there is an urgent need to explore novel and effective strategies for the treatment of bacterial infections. Herein, zeolite imidazole framework-8 (ZIF-8) material was utilized to construct biomineralized nanomaterial (GOx&HRP@ZIF-8/ASO) by encapsulating biological cascade enzymes and combining with antisense oligonucleotides (ASOs), which achieved effective and synergistic antidrug-resistant bacteria therapy. Various in vitro assays confirmed that GOx&HRP@ZIF-8/ASO exhibited excellent antibacterial properties against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) during catalysis of glucose (Glu), especially the minimum inhibitory concentration (MIC) against MRSA was only 16 μg/mL. Compared with simple ZIF-8 (32.85%) and ftsZ ASO (58.65%), GOx&HRP@ZIF-8/ASO+Glu exhibited superb biofilm destruction ability, and the bacteria removal efficiency of the MRSA biofilm could be as high as 88.2%, indicating that the reactive oxygen species (ROS) produced by the cascade enzyme reaction imparted the main synergistic antibacterial capability, and simultaneously, ftsZ ASO significantly enhanced the antibacterial effect by inhibiting the expression of the ftsZ gene. In vivo anti-infection treatment experiments revealed that GOx&HRP@ZIF-8/ASO exhibited the best wound repairing performance and excellent biocompatibility in the presence of Glu. These findings suggested that GOx&HRP@ZIF-8/ASO has favorably realized high-efficiency treatment of MRSA infection and filled the gap in the antibacterial application of biological enzymes.

Detyrosinated α-Tubulin, Vimentin and PD-L1 in Circulating Tumor Cells (CTCs) Isolated from Non-Small Cell Lung Cancer (NSCLC) Patients

Upregulation of Vimentin (VIM), alpha-Tubulin (TUB) and Detyrosinated tubulin (GLU) in circulating tumor cells (CTCs) derived from breast cancer patients is related to poor prognosis. In the current study we evaluated for the first time, these cytoskeletal proteins in sixty Non-Small Cell Lung Cancer (NSCLC) patients’ CTCs (33 treatment-naïve and 27 pre-treated). Samples were isolated using the ISET platform and stained with a pancytokeratin (CK)/CD45/TUB, CK/GLU/VIM and CK/programmed death ligand 1 (PD-L1) combination of antibodies. Subsequently, slides were analyzed using confocal laser scanning microscopy. CTCs were detected in 86.7% of the patients. CTCs with TUB expression were identified in 65.4% (34/52) of the CK (+)-patients. GLU, VIM and PD-L1 were also evaluated. The frequency of the observed phenotypes was as follow: (CK+/GLU−/VIM−): 35.2%, (CK+/GLU+/VIM+): 63.0%, (CK+/GLU+/VIM−): 16.7%, (CK+/GLU−/VIM+): 72.2%, (CK+/PD-L1−): 75% and (CK+/PD-L1+): 55%. The OS was significantly decreased in patients with high GLU (3.8 vs. 7.9 months; p = 0.018) and/or high VIM (3.2 vs. 7.1 months; p = 0.029) expression in their CTCs. PD-L1 was also related to OS (3.4 vs. 7.21 months; p = 0.035). Moreover, TUB-high and TUB-low expression in CTCs inversely influenced patients’ OS as independent prognostic factors (p = 0.041 and p = 0.009). The current study revealed that TUB, GLU, VIM and PD-L1 were overexpressed in CTCs from NSCLC patients. Furthermore, the presence of GLU, VIM-positive and PD-L1 in CTCs is potentially related to patients’ outcomes.

Cd(II) removal from amino acids-bearing wastewater: Critical evaluation and comparison of using magnetic carbon nanotubes vs. magnetite

In the present work, two magnetic adsorbents (magnetic carbon nanotunes “MCNT” and magnetite “Mag”) were tested for cadmium “Cd(II)” removal from water in the presence of various amino acids “AA” (aspartic acid, glutamic acid “Glu”, glycine and cysteine). MCNT was prepared by initially oxidizing carbon nanotubes (CNT) by three different methods: nitric acid method, ammonium persulphate method, Hummer's method. Magnetite was then deposited on the oxidized CNT to produce the corresponding magnetic carbon nanotube adsorbents: NA-MCNT, APS-MCNT, HUM-MCNT, respectively. Adsorption of Cd(II) on various MCNT was investigated in the presence/absence of AA where various factors were studied: pH, oxidation method of CNT, type of AA, magnetite-to-CNT ratio. It was found that the presence of AA significantly reduced the adsorption capacity of MCNT towards Cd(II) at all pHs (reduction occurred from 49.8 mg g−1 to 12.5 mg g−1). Contrary adsorption capacity of Cd(II) on bare magnetite “Mag” was noticeably enhanced in the presence of AA (for Glu, enhancement occurred from 6.9 to 22.3 mg g−1). Due to cheapness of Mag relative to CNT, it was recommended to use Mag rather than MCNT for Cd(II) removal from AA-bearing water. So that the optimum uptake method was proposed using Mag at pH 8 in the presence of Glu at concentration ratio (1:3 Cd(II): Glu). Mag was selective towards Cd(II) even in the presence of co-existing ions. The use of Mag for five cycles decreased adsorption efficiency to 80% which indicated that adsorption system was cost effective. Real water samples gave higher adsorption of Cd(II) in the presence of Glu than in distilled water. As a conclusion, it was recommended to use Mag rather than MCNT for Cd(II) removal in the presence of AA.

An enzyme-activated two-photon ratiometric fluorescent probe with lysosome targetability for imaging β-glucuronidase in colon cancer cells and tissue

Colon cancer is a malignant neoplasm with high mortality that has seriously threatened human life. Accumulating evidence reveals that the β-glucuronidase (GLU), a lysosomal exoglycosidase enzyme, plays important roles in the pathological progression of colon cancer. Unfortunately, understanding the pathological roles of GLU remains a challenge due to the lack of effective detection methods for visualization the fluctuations of GLU in tissues. In this paper, based on hydrolysis function of GLU, an enzyme-activated ratiometric two-photon (TP) fluorescent probe (RN-GLU) was designed. RN-GLU was synthesized by introducing a glucopyranuronic acid methyl ester as the recognition group and 1, 8-naphthalimide as a TP fluorophore. In the presence of GLU, the trigger group was removed made an ICT process occurred induced enhancement of fluorescence ratio (I553 nm/I441 nm, 214-fold). Probe RN-GLU displayed low detection limit (1.2 × 10−2 μg/L) and rapid detection to GLU in vitro through a ratiometric response mode. Meanwhile, RN-GLU exhibited high selectivity for GLU and showed nearly no response to other relevant biological species. The imaging results demonstrated that RN-GLU could be applied for ratiometric monitoring of endogenous GLU levels in HCT116 cells with good lysosome targetable ability. Due to its two-photon excitation, RN-GLU could monitor GLU in colon cancer tumor tissue with good penetration ability (imaging depth of 200 μm). RN-GLU could be developed as a potential method for evaluating and confirming the functions of GLU in colon cancer diagnosis and complex biosystem.

Sulodexide reduces glucose induced senescence in human retinal endothelial cells

Chronic exposure of retinal endothelium cells to hyperglycemia is the leading cause of diabetic retinopathy. We evaluated the effect of high glucose concentration on senescence in human retinal endothelial cells (HREC) and modulation of that effect by Sulodexide. Experiments were performed on HREC undergoing in vitro replicative senescence in standard medium or medium supplemented with glucose 20 mmol/L (GLU) or mannitol 20 mnol/L (MAN). Effect of Sulodexide 0.5 LRU/mL (SUL) on the process of HREC senescence was studied. Glucose 20 mmol/L accelerates senescence of HREC: population doubling time (+ 58%, p < 0.001) β-galactosidase activity (+ 60%, p < 0.002) intracellular oxidative stress (+ 65%, p < 0.01), expression of p53 gene (+ 118%, p < 0.001). Senescent HREC had also reduced transendothelial electrical resistance (TEER) (− 30%, p < 0.001). Mannitol 20 mmol/L used in the same scenario as glucose did not induce HREC senescence. In HREC exposed to GLU and SUL, the senescent changes were smaller. HREC, which became senescent in the presence of GLU, demonstrated higher expression of genes regulating the synthesis of Il6 and VEGF-A, which was reflected by increased secretion of these cytokines (IL6 + 125%, p < 0.001 vs control and VEGF-A + 124% p < 0.001 vs control). These effects were smaller in the presence of SUL, and additionally, an increase of TEER in the senescent HREC was observed. Chronic exposure of HREC to high glucose concentration in medium accelerates their senescence, and that process is reduced when the cells are simultaneously exposed to Sulodexide. Additionally, Sulodexide decreases the secretion of IL6 and VEGF-A from senescent HREC and increases their TEER.

Structural features of the glutamate-binding protein from Corynebacterium glutamicum

L-glutamate (Glu) is the major excitatory transmitter in mammalian brain. Inadequate concentration of Glu in the brain correlates to mood disorder. In industry, Glu is used as a flavour enhancer in food and in foodstuff processing. A high concentration of Glu has several effects on human health such as hypersensitive effects, headache and stomach pain. The presence of Glu in food can be detected by different analytical methods based on chromatography, or capillary electrophoresis or amperometric techniques. We have isolated and characterized a glutamate-binding protein (GluB) from the Gram-positive bacteria Corynebacterium glutamicum. Together with GluC protein, GluD protein and the cytoplasmic protein GluA, GluB permits the transport of Glu in/out of cell.In this study, we have investigated the binding features of GluB as well as the effect of temperature on its structure both in the absence and in the presence of Glu. The results have showed that GluB has a high affinity and selectivity versus Glu (nanomolar range) and the presence of the ligand induces a higher thermal stability of the protein structure.

Electrostatic Tuning of the Ligand Binding Mechanism by Glu27 in Nitrophorin 7

Nitrophorins (NP) 1–7 are NO-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. The isoform NP7 displays peculiar properties, such as an abnormally high isoelectric point, the ability to bind negatively charged membranes, and a strong pH sensitivity of NO affinity. A unique trait of NP7 is the presence of Glu in position 27, which is occupied by Val in other NPs. Glu27 appears to be important for tuning the heme properties, but its influence on the pH-dependent NO release mechanism, which is assisted by a conformational change in the AB loop, remains unexplored. Here, in order to gain insight into the functional role of Glu27, we examine the effect of Glu27 → Val and Glu27 → Gln mutations on the ligand binding kinetics using CO as a model. The results reveal that annihilation of the negative charge of Glu27 upon mutation reduces the pH sensitivity of the ligand binding rate, a process that in turn depends on the ionization of Asp32. We propose that Glu27 exerts a through-space electrostatic action on Asp32, which shifts the pKa of the latter amino acid towards more acidic values thus reducing the pH sensitivity of the transition between open and closed states.

Thermodynamic model for Zr solubility in the presence of gluconic acid and isosaccharinic acid

ABSTRACTZr solubility in the presence of gluconic acid (GLU) and isosaccharinic acid (ISA) was investigated as a function of hydrogen ion concentration (pHc) and the total concentration of GLU or ISA. The dependence of the increase in Zr solubility on the pHc and GLU concentration suggested the existence of Zr(OH)4(GLU)22− in the neutral pH region and Zr(OH)4(GLU)(GLU-H)3− in the alkaline pH region above pHc 10 as the dominant species in the presence of 10−3–10−1 mol/dm3 (M) GLU. In the presence of ISA, the dominant species Zr(OH)4(ISA)22− and Zr(OH)4(ISA)(ISA-H)3− were proposed to occur in the neutral and alkaline pH regions, similar to those found in the presence of GLU. From X-ray diffraction analysis, the solubility-limiting solid phase in the presence of GLU and ISA was considered to be Zr(OH)4(am). The formation constants of the Zr gluconate and isosaccharinate complexes were determined by least-squares fitting analysis of the solubility data, and the obtained values were discussed in comparison with those of tetravalent actinides.

Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells.

Neurotrophin-3 (NT-3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT-3) and the precursor (pro-NT-3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT-3 and pro-NT-3 in this process remains unclear. The current study explores the effects of NT-3 and pro-NT-3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT-3 had no effect on the induced changes in Ca²⁺ homeostasis. In contrast, pro-NT-3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]i ) and Fura-2 fluorescence quenching by Mn²⁺ within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²⁺ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²⁺]i rise was 1.2-fold greater in the presence of pro-NT-3 than in the presence of Glu alone (1.67 ± 0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu-induced changes in Са²⁺ homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²⁺ removal from the cytosol during the DCD latency period.

English

Neuroprotective effects of&nbsp;two medicinally important herbs&nbsp;Withania somnifera&nbsp;(WS) andAsparagus racemosus&nbsp;(AR) were investigated in primary hippocampal neuron cell culture. The root extracts of WS and AR were obtained by Soxhlet extraction using distilled water as solvent. The&nbsp;7 to 8 days old hippocampal cells in culture were treated with100 &micro;M Glutamate (Glu) for 10 min at room temperature in&nbsp;Hank&#39;s balanced saline solution (HBSS). One&nbsp;hour (1 h) after exposure to Glu, cells&nbsp;were treated with root extract of WS, AR or WS + AR [10 &micro;g/ml dissolved in dimethyl sulfoxide (DMSO)], respectively.&nbsp;Results of the study suggested that percent cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay] was more significant in groups treated with Glu + WS and Glu + WS + AR, when compared to group treated with Glu + AR. More than two-fold increase in free Ca2+&nbsp;was observed in culture in presence of Glu, but after treatment with WS, this increase was&nbsp;significantly reduced (P &lt; 0.01). No significant change was observed in presence of AR. A significant decrease in lactic dehydrogenase (LDH) was observed after&nbsp;WS, AR as well as WS&nbsp;+ AR, when compared with Glu treated group. There was significant (P &lt; 0.01) decrease in malondialdehyde (MDA) in&nbsp;presence of WS or AR. Reactive oxygen species (ROS)&nbsp;level&nbsp;was&nbsp;significantly inhibited in both Glu + WS and Glu + AR treated group, compared to Glu and Glu + WS + AR treated group. Significant elevation of&nbsp;reduced glutathione (GSH) and&nbsp;glutathione disulfide (GSSG) was also obtained after WS and AR treatment. A marked increase in&nbsp;nerve growth factor (NGF) and pro-brain derived growth factor (pro-BDNF) expression was observed after AR and WS + AR treatment; whereas no observable change&nbsp;was observed in WS treated group. It was therefore, concluded thatboth plants in combination (WS + AR) have more effective role in neuroprotection. Study also showed neurotrophic factor modulatory activity of these plants. Present study suggest that WS + AR in combination could have alternative therapeutic potency for treatment of diseases associated with neuron cell loss. &nbsp; Key words:&nbsp;Withania somnifera,&nbsp;Asparagus racemosus, hippocampus, glutamate,&nbsp;in vitro, neuroprotection.

Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor

Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNAGln is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNAGln, and an amidotransferase converts Glu-tRNAGln to Gln-tRNAGln. The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNAGln. It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.

The causes for barley root growth retardation in the presence of ammonium and glutamate

In barley (Hordeum vulgare L.) seedlings, the rate of root growth, osmotic pressure (Π), hydraulic conductance (L p), and longitudinal (δl) and transverse (δD/D) extensibility of root cells were measured. The seedlings were grown on Knop solution with nitrate or without nitrate with addition of 5–10 mM NH 4 + or 0.5–1.0 mM glutamate. Root growth retardation on the 1st–4th days of exposure to NH 4 + was determined by a decrease in δl in the zone of elongation, whereas root thickening was evidently related to an increase in Π. Biphasic dynamics of δl in the presence of NH 4 + was imitated by medium acidification near the root surface to pH 3.7, which confirms a conclusion, we have done earlier, about a non-monotonous pH-dependence of longitudinal extensibility. Root growth retardation during the first day of exposure to Glu was also determined by a decrease in the δl, which was, however, accompanied by an increase in the δD/D and L p. Fast Glu-induced changes of measured root parameters were imitated by root exposure to oryzalin, ionomycin, and inhibitors of the H+-pump. It was supposed that a decrease in δl in the presence of NH 4 + and Glu was related to cortical microtubule disorganization with the involvement of cytosolic calcium Ca cyt 2+ . A decrease in the δD/D and L p in the presence of NH 4 + was related to apoplast acidification and a high activity of the plasmalemmal H+-pump. An increase in the δD/D and L p in the presence of Glu indicates the inhibition of the plasmalemmal H+-pump. On the 2nd–4th days of exposure to Glu, root growth ceased, as distinct from treatment with NH 4 + . This complete root growth inhibition by Glu was possibly related to a rapid uptake of Ca2+ through Glu-sensitive Ca2+-channels, Ca2+-dependent inhibition of the plasmalemmal H+-pump, and a decrease in mitotic activity.

Role of Ca 2+/H + Exchange and Disturbance of Oxidative Phosphorilation in Glutamate-Induced Delayed Calcium Deregulation in Cultured Neurons

Prolonged application of toxic doses of glutamate (Glu) to neurons, resulting in their death, induces a biphasic Ca2+ response, of which the second phase termed delayed calcium deregulation (DCD) is not fully understood. Using fluorescence microscopy, we monitored cytosolic Ca2+ concentration ([Ca2+]i), mitochondrial membrane potential (Vm) and pH (pHm), cytosolic pH (pHc) and NAD(P)H in cultured rat hippocampal and cerebellar granule neurons. Glu (0.1mM+Gly-Mg) caused a rapid drop in pHc and a smaller decrease in pHm. Oligomycin (Oligo), applied in the presence of Glu prior to DCD, increased Vm and NAD(P)H but did not affect the pH gradient between mitochondria and cytosol. When cyanide was applied in the presence of Glu and Oligo, we observed a rapid increase in [Ca2+]i, drop in Vm, and simultaneous changes in pHm and pHc in opposite directions. Similar changes in [Ca2+]i, Vm, and pH were observed during DCD, except of NAD(P)H became insensitive to Oligo indicating to oxidative phosophorylation uncoupling. PLA2 inhibition increased the period of DCD onset indicating to free fatty acids as possible uncoupling agents. We suppose that DCD is due in part to Ca2+ release from mitochondria and can be formally classified as Ca2+/H+ exchange. Such exchange acts as a positive feedback loop because H+ influx to mitochondria promotes dissociation of Ca2+-phosphate complexes and leads to further Ca2+ release into cytosol. Oxidative phosophorylation uncoupling results in ATP deficiency for ion pumps making worse Ca2+ extrusion and acting as aggravating factor. Supported by RFBR.

Glutamate Inhibits Chondral Mineralization through Apoptotic Cell Death Mediated by Retrograde Operation of the Cystine/Glutamate Antiporter

Although we have previously demonstrated the functional significance of excitatory amino acid transporters as well as glutamate (Glu) receptors (GluRs) expressed by chondrocytes, little attention has been paid to the possible expression of the cystine/Glu antiporter responsible for the bi-directional transmembrane transport of Glu in chondrocytes to date. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, the chondral mineralization was significantly decreased in the presence of Glu at a high concentration. Apoptotic cells were detected within the late proliferating and prehypertrophic chondrocytic layers in metatarsals cultured in the presence of Glu. A group III metabotropic GluR (mGluR) antagonist partially, but significantly, prevented the inhibition of mineralization by Glu in metatarsals without affecting the number of apoptotic cells. Both decreased mineralization and apoptosis by Glu were significantly prevented by the addition of the cystine/Glu antiporter inhibitor homocysteic acid, as well as reduced glutathione (GSH) and cystine. Expression of mRNA for xCT and 4F2hc subunits, which are components of the cystine/Glu antiporter, was seen in both cultured mouse metatarsals and rat costal chondrocytes. In chondrocytes cultured with Glu, a significant decrease was seen in intracellular GSH levels, together with increases in the number of apoptotic cells and the level of intracellular reactive oxygen species. These results suggest that Glu could regulate chondrogenic differentiation toward mineralization through a mechanism associated with apoptosis mediated by the depletion of intracellular GSH after the retrograde operation of the cystine/Glu antiporter, in addition to the activation of group III mGluR, in chondrocytes.

Differential inactivation of glucose- and glutamate dependent acid resistance of Escherichia coli TMW 2.497 by high-pressure treatments

The inactivation by 200–400 MPa and post-pressure survival at acid conditions of E. coli TMW 2.497 was characterized by the measurement of intracellular pH (pH in), viable cell counts, glutamate (Glu) and arginine (Arg) consumption, and the influence of mild adaptation to mild acid stress prior to pressure treatment. Glutamate and arginine did not affect viable cell counts or the pH in during pressure application but improved the ability to maintain a high pH in after pressure treatment. In pH 4.0 buffer without arg and glu, a 3 log reduction of cell counts occurred after 24 h of incubation, whereas little or no loss of viability was observed after 24 h incubation in the presence of glu and arg. During post-pressure incubation at pH 4.0, 10 mM glutamate were metabolized but only 2 mM arginine were used, indicating that glutamate rather than arginine was responsible for the protective effect on pH in and survival. In conclusion, the pressure induced, irreversible loss of the transmembrane ΔpH correlates to cell death and glu stabilizes the pH in of E. coli during post-pressure incubation.

Analysis of Glutamine Accumulation in Rat Brain Mitochondria in the Presence of a Glutamine Uptake Inhibitor, Histidine, Reveals Glutamine Pools With a Distinct Access to Deamidation

Rat cerebral nonsynaptic mitochondria were incubated in medium containing 2 mM glutamine (Gln) or 2 mM glutamate (Glu), in the presence of a Gln uptake inhibitor histidine (His) as well as other basic amino acids, lysine and arginine (Lys, Arg) not inhibiting Gln uptake. Subsequently, the mitochondrial contents of Glu and Gln were determined by HPLC. Incubation in the presence of Glu alone increased the Glu content from approximately 3.5 to 15 nmol/mg protein, without affecting the Gln content. On the other hand, incubation with Gln increased the content of Gln from approximately 1.5 to approximately 12 nmol/mg, and that of Glu to 10 nmol/mg. As expected, addition of His did not alter the Glu and Gln content resulting from incubation with Glu. However, His significantly decreased to almost the preincubation level the content of Glu in mitochondria incubated with Gln, without affecting the content of Gln. No other amino acid had any effect on these parameters. The results point to the existence of distinct Gln pools, one of which is accessible to external Gln via a His-sensitive transporter and is accessible for deamidation in the mitochondria.

A Search for Hyperglycosylation Signals in Yeast Glycoproteins

N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13-14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200-300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of alpha-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection.

Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases.

The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

Nitric oxide-mediated Ca ++-influx in neuronal nuclei and cortical synaptosomes of normoxic and hypoxic newborn piglets

Previous studies have shown that hypoxia results in the generation of nitric oxide (NO) free radicals in the cerebral cortex of newborn animals. The present study tested the hypothesis that NO increases Ca ++-influx in neuronal nuclei as well as N-methyl- d aspartate (NMDA) receptor-mediated Ca ++-influx in cortical synaptosomes of newborn piglets. Studies were performed in five normoxic (Nx) and 6 hypoxic (Hx) newborn piglets. Cerebral tissue hypoxia was documented by determining the levels of ATP and phosphocreatine (PCr). 45Ca ++ -influx was determined in the presence of sodium nitroprusside (SNP, 10 μM), a NO donor, and peroxynitrite (10 μM). In the Hx group, ATP levels decreased to 1.40±0.69 vs 4.27±0.80 μmoles/g brain in the Nx group ( P<0.05). Similarly, PCr levels decreased to 0.91±0.57 vs 3.40±0.99 μmoles/g brain ( P<0.001). Nuclear 45Ca ++-influx increased from 3.57±1.46 pmoles/mg protein in Nx nuclei to 8.64±3.50 in Hx nuclei ( P<0.05). SNP increased neuronal nuclear Ca ++ influx in the Nx from 3.57±1.46 to 5.47±2.52 pmoles/mg protein ( P<0.05) but did not affect Ca ++ influx in the Hx group (8.64±3.50 vs. 10.17±4.00 pmoles/mg protein). The level of Ca ++ influx in the presence of SNP in Nx nuclei was similar to that seen in Hx nuclei alone. Peroxynitrite did not affect nuclear Ca ++-influx in either Nx or Hx group. Synaptosomal Ca ++-influx in the presence of glu+gly was 40±11 pmoles/mg protein in the Nx group and 80±16 pmoles/mg protein in the Hx group ( P<0.05). Both SNP and peroxynitrite increased Ca ++ influx in Nx and Hx synaptosomes. These results show that hypoxia results in increased nuclear and synaptosomal Ca ++-influx. Further, the data demonstrate that NO increases intranuclear as well as intrasynaptosomal Ca ++-influx and suggest that during hypoxia, the increase in intranuclear and intraynaptosomal Ca ++ is NO-mediated. We propose that NO-mediated modification (by nitrosylation/nitration) of nuclear membrane high affinity Ca ++-ATPase and neuronal membrane NMDA receptor, resulting in increased intranuclear and intracellular Ca ++ influx, are potential NO-mediated mechanisms of Hx neuronal injury.