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PubMed1997

Regulation of glycogen synthase activity in cultured skeletal muscle cells from subjects with type II diabetes: role of chronic hyperinsulinemia and hyperglycemia.

Human skeletal muscle cultures (HSMCs) from type II diabetic subjects were used to determine whether metabolic abnormalities such as hyperglycemia or hyperinsulinemia contribute to the defective muscle glycogen synthase (GS) activity present in this disorder. Following approximately 6 weeks of growth, diabetic cultures were fused for 4 days in normal, hyperglycemia, or hyperinsulinemia medium. Fusion of diabetic HSMCs in hyperglycemia medium (20 mmol/l vs. 5.5 mmol/l) had no effect on GS fractional velocity (FV) or mRNA levels, but impaired acute insulin-stimulation of glycogen synthesis and GS activity at 0.1 mmol/l glucose-6-phosphate, and reduced GS protein content by approximately 15% (P < 0.05). Fusion of diabetic muscle cultures in hyperinsulinemia medium (30 micromol/l vs. 22 pmol/l) improved basal GS activity, increasing the reduced GS FV by approximately 50% (P < 0.05), and decreasing the elevated Km(0.1) (half-maximal substrate concentration) by approximately 47% (P < 0.05). Hyperinsulinemia also significantly increased (P < 0.05) the reduced GS mRNA and protein levels of diabetic muscle to levels similar to that in nondiabetic subjects. In contrast to the improvements in the basal state, hyperinsulinemia completely abolished acute insulin responsiveness of GS activity and glycogen synthesis in muscle of type II diabetic subjects. The combination of hyperinsulinemia and hyperglycemia produced effects on both basal and insulin-responsive GS FV and mRNA similar to hyperinsulinemia alone, but hyperinsulinemia prevented hyperglycemia's effect of lowering GS protein and glycogen synthesis. We concluded that, in diabetic muscle, hyperinsulinemia may serve to partially compensate for the impaired basal GS activity and for the adverse effects of hyperglycemia on GS protein content, activity, and glycogen formation by both pre- and posttranslational mechanisms. Despite these beneficial effects, hyperinsulinemia also induces severe impairment of insulin-stimulated GS activity and glycogen formation, which may contribute to acquired muscle insulin resistance of type II diabetes.

Adult↗

PubMed1992

Translocation of alpha subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells.

The translocation of the alpha subunits of Gs from the membrane to the cytosol by iloprost, a stable prostacyclin analogue, was studied in mouse mastocytoma P-815 cells. In the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), iloprost stimulated the adenylate cyclase activity, caused the release of both 42- and 45-kDa proteins reactive with the anti Gs alpha carboxyl-terminal antibody, RM/1, from the membrane and attenuated cholera toxin-catalyzed ADP-ribosylation of the 42- and 45-kDa proteins in the membrane. The iloprost-stimulated adenylate cyclase activity and release of Gs alpha from the membrane were markedly suppressed by RM/1. Cholera toxin treatment also stimulated the adenylate cyclase activity and release of Gs alpha from the membrane, and iloprost synergistically potentiated these actions of cholera toxin. In mastocytoma cells, iloprost induced the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol, 45-kDa Gs alpha remaining in the cytosol for a longer time than 42- kDa Gs alpha. Whereas 42-kDa Gs alpha in the cytosol was eluted at the position of Mr = approximately 40,000 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 from a Superose 12 gel filtration column. In contrast, both 42- and 45-kDa Gs alpha released in vitro from the membrane by iloprost plus GTP gamma S were eluted at the position of Mr = approximately 40,000, but only 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 when it was incubated with cytosol. These results taken together demonstrate that iloprost induces the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol and that only the 45-kDa Gs alpha released exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.

Adenosine Diphosphate Ribose↗

PubMed1997

Risk of asymptomatic and symptomatic gallstones in moderately obese women: a longitudinal follow-up study.

Obesity is a rather documented risk factor for the formation of gallstones (GS) in women. The magnitude of the increased risk and the rates of GS occurrence, however, have not been well quantified, except for two studies on the risk of symptomatic stones in obese women. We analyzed the incidence of GS in 157 moderately obese women (body mass index, 31.4 +/- 3.6 kg/ m2) followed up prospectively by ultrasound for 2-6 yr (mean 3.95 yr). Women with morbid obesity (body mass index > 40 kg/m2) were excluded from the study, as well as patients having diseases with lithogenic risk. All the enrolled women had normal cholecystosonogram results at the beginning of the study. Age, family history of GS or obesity, parity, age of obesity onset, hyperlipoproteinemia type, plasma cholesterol (total, HDL, LDL), and triglycerides were assessed. The Student's t, the Mann-Whitney rank sum and the Fisher's exact tests were used, as well as the multiple logistic regression for the multivariate analysis. During the survey, 16 of 157 women (10.2%) developed GS. GS were asymptomatic in 11 persons (68.8%). The cumulative incidence of both asymptomatic and symptomatic GS was 2.6 cases/100 obese women.year. During the follow-up, most of the detected GS were asymptomatic, and this explains the higher GS incidence rate found compared with that previously calculated for symptomatic GS. The following risk factors were associated with GS formation: age (p = 0.002), family history of GS (p = 0.011), early obesity onset (p = 0.003), and hyperlipoproteinemia type IV (p = 0.011). A high risk class might be thus identified among obese women, offering a more realistic approach for the primary prophylaxis of GS.

Adult↗

PubMed1994

Dexamethasone increases jejunal glutamine synthetase expression via translational regulation.

Glutamine provides energy and precursors for nucleotide biosynthesis for the gut mucosa, and it is essential for intestinal metabolism and function. During stress states, glutamine uptake of circulating and luminal glutamine may be diminished, but the ability of the gut mucosa to synthesize glutamine de novo in response to this decreased delivery remains undefined. Since the glucocorticoids play an important role in regulating interorgan glutamine metabolism during catabolic states, we hypothesized that these hormones induce the expression of gut mucosal glutamine synthetase (GS), the enzyme that catalyzes the intracellular biosynthesis of glutamine. Adult rats were treated with dexamethasone (DEX, 0.5 mg/kg intraperitoneally) or saline (controls). At various times after treatment (4, 12, 24, 48, and 72 hours), jejunal mucosal GS-specific activity was assayed, and total RNA was extracted. GS transcripts were detected by Northern blot analysis, using a radiolabeled rat GS cDNA probe. Transcripts were quantitated by phospho-imaging and normalized to beta-actin. An anti-GS polyclonal antibody was used to quantitate GS protein concentrations by Western blot analysis. The relative quantities of GS translated were measured using a cell-free protein-synthesizing system (reticulocyte lysate assay). Data were analyzed using analysis of variance and were considered statistically significant for p < 0.05. DEX increased GS activity by 45% 12 hours after administration. Western blot analysis revealed an increase in the concentration of the GS protein in the jejunum of DEX-treated animals. Northern blot analysis demonstrated no significant change in GS mRNA levels after DEX treatment, indicating the possibility of post-transcriptional regulation. In vitro translational experiments demonstrated that the quantity of GS translated was increased by 25% after the administration of DEX. These data suggest that glucocorticoids may increase jejunal mucosal GS levels by accelerating protein translation. This adaptive response could provide glutamine for the gut mucosa during stress, when exogenous glutamine supplies may be rate limiting.

Animals↗

PubMed1994

Effect of service number on estrus duration in dairy goats.

The object of this research was to study the effect of sterile service number on estrus duration in dairy goats. Twenty-four Nubian goats (20 nulliparous and 4 multiparous) were randomly assigned to 1 of 4 treatment groups (n = 6 animals per group). The following Groups were formed: no service (GS-0); 1 service (GS-1); 2 services (GS-2); 3 services (GS-3). Estrus was synchronized by using fluorogestone acetate intravaginal pessaries (40 mg) over a 12-d period plus 400 IU im pregnant mare serum gonadotropin (PMSG) at pessary removal. Estrus was detected by using a vasectomized buck at 6-h intervals over 5 d after pessary removal (at 0600, 1200, 1800 and 2400 h). In the GS-0 group the teaser was outfitted with an apron and was permitted to mount. In the GS-1, GS-2 and GS-3 groups, the teaser was permitted to mount and service 1, 2 and 3 times, respectively, within the first 12 h after initiation of estrus. The duration of estrus for the 4 groups (GS-0, GS-1, GS-2 and GS-3) was (mean +/- SD) 41.0 +/- 5.9, 24.0 +/- 5.4, 22.0 +/- 4.9 and 22.0 +/- 7.2 h, respectively. These results show differences between the serviced groups and the nonserviced group (P<0.01), but they fail to show differences among the serviced groups (P>0.05). It is concluded that sterile service shortens estrus duration and that service number (1, 2 or 3) does not affect estrus duration.

Journal Article↗

PubMed2006

Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum.

Glutathione (GSH) is synthesized by gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed gamma-GCS-GS catalyzing both gamma-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the gamma-GCS activity, S. agalactiae gamma-GCS-GS had different substrate specificities from those of Escherichia coli gamma-GCS. Furthermore, S. agalactiae gamma-GCS-GS synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-X(aa)-Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding gamma-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae gamma-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed gamma-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-Cys-X(aa). Whereas the substrate specificities of gamma-GCS domain protein and GS domain protein of S. agalactiae gamma-GCS-GS were the same as those of S. agalactiae gamma-GCS-GS.

Clostridium acetobutylicum↗

PubMed2006

Glucosamine sulfate inhibits leukocyte adhesion in response to cytokine stimulation of retinal pigment epithelial cells in vitro.

Glucosamine is an amine-containing sugar that exhibits immunosuppressive effects in vitro and in vivo, although its mechanism of action is unknown. We investigated whether glucosamine sulfate (GS) modulates the proinflammatory cytokine interleukin (IL)-1beta-induced expression and production of intercellular adhesion molecule (ICAM)-1, the mechanism responsible for this effect, and whether GS inhibits leukocyte adhesion to the monolayer of retinal pigment epithelial (RPE) cells stimulated with various cytokines. We used flow cytometry and an ARPE-19 cell model to determine the effect of GS on the production of ICAM-1 in response to IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha plus IL-1beta, TNF-alpha plus IL-6, and TNF-alpha plus interferon (IFN)-gamma. We also used semiquantitative RT-PCR to determine the effect of GS on IL-1beta-induced expression of the ICAM-1 gene, and immunocytochemistry and western blotting to measure the effect of GS on the activation and nuclear translocation of the nuclear factor NF-kappaB and the degradation of cytoplasmic IkappaB. The functionality of GS-modulated ICAM-1 on leukocyte adhesion was demonstrated in an RPE cell-neutrophil adherence assay. IL-1beta increased the expression of ICAM-1 at the mRNA and protein levels in ARPE-19 cells. GS downregulated the production of ICAM-1 induced by IL-1beta, IL-6, TNF-alpha, and IFN-gamma at the protein level in a dose-dependent manner. GS also inhibited the nuclear translocation of NF-kappaB subunit p65 and partially prevented the degradation of cytoplasmic IkappaB in IL-1beta-stimulated ARPE-19 cells. GS significantly decreased the number of neutrophils adhering to the RPE monolayer in response to cytokines IL-1beta, IL-6, TNF-alpha, and IFN-gamma. GS inhibits the expression of the ICAM-1 gene in ARPE-19 cells stimulated with IL-1beta by blocking NF-kappaB subunit p65 translocation and by partially preventing IkappaB degradation. GS also decreases leukocyte adhesion to the monolayer of ARPE-19 cells stimulated with various cytokines by decreasing ICAM-1 production. Our study demonstrates a potentially important property of GS in reducing ICAM-1-mediated inflammatory mechanisms in the eye.

Cell Adhesion↗

PubMed2005

Proton magnetic resonance spectroscopy (1H-MRS) of hippocampus, basal ganglia, and vermis of cerebellum in schizophrenia associated with idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome).

OBJECTIVE: Whether patients with schizophrenia-associated idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome, GS) have specific changes in brain metabolism was examined in this study. METHOD: This study applied proton magnetic resonance spectroscopy (1H-MRS) to the hippocampus, basal ganglia, and vermis of the cerebellum of schizophrenic patients with GS (n=15) or without GS (n=15), all diagnosed according to DSM-IV criteria, and healthy subjects (n=15). RESULTS: In the hippocampus, schizophrenic patients with GS showed a significant decrease of N-acetyl aspartate/creatine-phosphocreatinine (NAA/Cr) and myoinositol/creatine-phosphocreatinine (mI/Cr) ratios compared to healthy subjects and schizophrenic patients without GS, while schizophrenic patients without GS showed only a significant decrease of NAA/Cr compared to healthy subjects. In the basal ganglia, schizophrenic patients with GS showed a significant decrease of ml/Cr compared to schizophrenic patients without GS and healthy subjects, and schizophrenic patients with GS showed a significant decrease of NAA/Cr compared to healthy subjects. In the vermis of the cerebellum, schizophrenic patients with GS showed only a significant decrease of ml/Cr compared to healthy subjects, although schizophrenic patients without GS did not show a significant decrease of ml/Cr compared to healthy subjects. CONCLUSION: The findings suggest that schizophrenia with GS is a more severe sub-type with regard to brain metabolism.

Adult↗

PubMed2004

Choice of vascular surgery as a specialty: survey of vascular surgery residents, general surgery chief residents, and medical students at hospitals with vascular surgery training programs.

PURPOSE: Under the direction of the Association of Program Directors in Vascular Surgery, a survey was mailed to vascular surgery residents (VSRs), general surgery chief residents (GS-CRs), and fourth-year medical students (MSs) to better define reasons why trainees do and do not choose vascular surgery as a career. METHODS: Questionnaires were mailed to all accredited VSR programs and their associated GS programs in the United States and Canada in 2001 (survey 1) and in 2003 (survey 2) and to 2 medical schools with VSR programs in 2001. A total of 197 VSRs, 169 GS-CRs, and 78 MSs responded (overall program response rate of 78% for VSRs, 46% for GSRs, 20% for MSs). A scoring system was assigned, with 1.0 the least important and 5.0 the most important reasons to choose or not choose vascular surgery. RESULTS: Technical aspects, role of mentors, and complex decision making involved in vascular surgery were the most important reasons that VSRs, GS-CRs, and MSs would choose vascular surgery as a specialty (average scores > or =4.0 for VSRs and GS-CRs; > or =3.5 for MSs). Responses of GS-CRs and VSRs did not vary significantly between surveys 1 and 2, except endovascular capabilities of vascular surgeons had a more important role in choosing vascular surgery, and future loss of patients to other interventionalists had a more important role in not choosing this specialty in the more recent survey of GS-CRs and VSRs. MSs identified lifestyle as a surgical resident (4.3) and as a surgeon (4.2) as the most important negative factors. A training paradigm consisting of 4 years general surgery + 2 years vascular surgery with a GS certificate was favored by 64% of GS-CRs and 48% of VSRs, compared with a paradigm of 5 years + 2 years with a general surgery certificate, which was favored by 29% of GS-CRs and 25% of VSRs, or 3 years + 3 years without a general surgery certificate, favored by 7% of GS-CRs and 27% of VSRs. Of note, 86% of MSs favored 3 years general surgery + 3 years vascular surgery or 2 years general surgery + 4 years vascular surgery compared with longer general surgery training periods. CONCLUSION: These findings may help vascular surgery program directors devise strategies to attract future trainees. The importance of mentorship to general surgery junior residents and medical students in choosing vascular surgery cannot be overestimated. Endovascular capabilities of vascular surgeons have an increasingly positive role in career choice by GS-CRs and VSRs, but these residents express increasing concerns about potential loss of patients to other specialists. Lifestyle concerns are the most important reasons why medical students do not choose vascular surgery as a career.

Adult↗

PubMedDate not supplied

Alterations in glutamine synthetase activity in rat skeletal muscle are associated with advanced age.

OBJECTIVE: Glutamine synthetase (GS), a key enzyme in the production of glutamine, is preserved in skeletal muscle during early aging (<24 mo). Because the effects of advanced age on GS are unknown, we investigated the effect of advanced age (>24 mo) on GS activity in skeletal muscle. We hypothesized that advanced age would enhance muscle GS activity. METHODS: Muscle GS activities were assessed in adult (8 mo), mature adult (15 mo), aged (20-22 mo), advanced age (25-27 mo), or very advanced age (29-32 mo) female Wistar rats. Male Wistar (6-27 mo) were used to investigate the effect of gender on this activity. RESULTS: Glutamine synthetase activity remained low and unaltered in rats from 8 to 22 mo of age, as previously demonstrated. In contrast, GS activity was high ( approximately 75% of individual values were higher than the low value mean) in 25-mo to 27-mo-old rats. In very-old-aged rats (29-32 mo), approximately 55% of GS activity data points exhibited low values. Changes in GS protein content paralleled those in GS activities. In male rats, GS activity was also high ( approximately 80% of individual values were higher than the mean value of 6-mo to 19-mo-old rats) at the upper limit of life expectancy (27 mo). CONCLUSION: There is enhanced GS activity in old female and male rats suggesting a greater need for glutamine. In some very old rats, low GS activity may be associated with longevity or reflect a limitation in glutamine production due to extremely advanced age per se.

Adenosine Triphosphate↗

PubMed1999

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes.

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.

Anti-Infective Agents↗

PubMed1999

The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes.

Gramicidin S (GS) is a cyclic decapeptide of primary structure [cyclo-(Val-Orn-Leu-D-Phe-Pro)(2)] secreted by Bacillus brevis. It is a powerful antimicrobial agent with potent cidal action on a wide variety of Gram-negative and Gram-positive bacteria as well as on several pathogenic fungi. Unfortunately, however, GS is rather non-specific in its actions and also exhibits a high hemolytic activity, limiting its use as an antibiotic to topical applications. In a wide variety of environments, the GS molecule exists as a very stable amphiphilic antiparallel beta-sheet structure with a polar and a non-polar surface. Moreover, the large number of structure-activity studies of GS analogs which have been carried out indicate that this 'sidedness' structure is required for its antimicrobial action. In this review, we summarize both published and unpublished biophysical studies of the interactions of GS with lipid bilayer model and with biological membranes. In general, these studies show that GS partitions strongly into liquid-crystalline lipid bilayers in both model and biological membranes, and seems to be located primarily in the glycerol backbone region below the polar headgroups and above the hydrocarbon chains. The presence of GS appears to perturb lipid packing in liquid-crystalline bilayers and GS can induce the formation of inverted cubic phases at lower temperatures in lipids capable of forming such phases at higher temperature in the absence of peptide. The presence of GS at lower concentrations also increases the permeability of model and biological membranes and at higher concentrations causes membrane destabilization. There is good evidence from studies of the interaction of GS with bacterial cells that the destruction of the integrity of the lipid bilayer of the inner membrane is the primary mode of the antimicrobial action of this peptide. The considerable lipid specificity of GS for binding to and destabilization of lipid bilayer model membranes indicates that the design of GS analogs with an improved antimicrobial potency and a markedly decreased toxicity for eukaryotic cell plasma membranes should be possible.

Anti-Bacterial Agents↗

PubMedDate not supplied

Glutamine synthetase in brain: effect of ammonia.

Glutamine synthetase (GS) in brain is located mainly in astrocytes. One of the primary roles of astrocytes is to protect neurons against excitotoxicity by taking up excess ammonia and glutamate and converting it into glutamine via the enzyme GS. Changes in GS expression may reflect changes in astroglial function, which can affect neuronal functions. Hyperammonemia is an important factor responsible of hepatic encephalopathy (HE) and causes astroglial swelling. Hyperammonemia can be experimentally induced and an adaptive astroglial response to high levels of ammonia and glutamate seems to occur in long-term studies. In hyperammonemic states, astroglial cells can experience morphological changes that may alter different astrocyte functions, such as protein synthesis or neurotransmitters uptake. One of the observed changes is the increase in the GS expression in astrocytes located in glutamatergic areas. The induction of GS expression in these specific areas would balance the increased ammonia and glutamate uptake and protect against neuronal degeneration, whereas, decrease of GS expression in non-glutamatergic areas could disrupt the neuron-glial metabolic interactions as a consequence of hyperammonemia. Induction of GS has been described in astrocytes in response to the action of glutamate on active glutamate receptors. The over-stimulation of glutamate receptors may also favour nitric oxide (NO) formation by activation of NO synthase (NOS), and NO has been implicated in the pathogenesis of several CNS diseases. Hyperammonemia could induce the formation of inducible NOS in astroglial cells, with the consequent NO formation, deactivation of GS and dawn-regulation of glutamate uptake. However, in glutamatergic areas, the distribution of both glial glutamate receptors and glial glutamate transporters parallels the GS location, suggesting a functional coupling between glutamate uptake and degradation by glutamate transporters and GS to attenuate brain injury in these areas. In hyperammonemia, the astroglial cells located in proximity to blood-vessels in glutamatergic areas show increased GS protein content in their perivascular processes. Since ammonia freely crosses the blood-brain barrier (BBB) and astrocytes are responsible for maintaining the BBB, the presence of GS in the perivascular processes could produce a rapid glutamine synthesis to be released into blood. It could, therefore, prevent the entry of high amounts of ammonia from circulation to attenuate neurotoxicity. The changes in the distribution of this critical enzyme suggests that the glutamate-glutamine cycle may be differentially impaired in hyperammonemic states.

Amino Acid Transport System X-AG↗

PubMed2003

Characterization and biological activity of gangliosides in buffalo milk.

Gangliosides (GS) were evaluated in Swiss cow's milk (SCM), Italian buffalo milk (IBM) and its serum, Pakistan buffalo colostrum (PBC), Pakistan buffalo mature milk (PBM), and Pakistan buffalo milk from rice-growing areas (PBR). Dairy GS were obtained from the Folch's upper (hydrophilic) and lower (lipophilic) extraction phases, respectively, and determined as lipid-bound sialic acid (LBSA) by colorimetry. Molar ratios of LBSA in the hydro- and lipophilic GS fractions were 52:48 to 79:21. Mature buffalo milk types had 40-100% more LBSA in the lipophilic GS fraction compared to SCM. Liquid PBC was higher in LBSA (24 nmol/g) compared to mature milk types (8-11 nmol/g). Thin-layer chromatography (TLC) and scanning densitometry showed distinct profiles of hydrophilic and lipophilic GS fractions. Lipophilic GS (but importantly not hydrophilic GS) from IBM and its serum decreased prostaglandin series 2 production by 75-80% in cultured human colonic epithelial cells exposed to tumor necrosis factor alpha (TNFalpha). Hydrophilic GD(3) and lipophilic GM(3) selectively bound rotavirus particles prepared from a rhesus strain and its mutant. A GS fraction in IBM showed a GM(1)-specific binding to cholera toxin subunit B (CTB). IBM serum (IBMS) was a rich source of LBSA (420 nmol/g proteins). In summary, improved methodology led to increased LBSA recovery and isolation of additional and bioactive milk GS. Human and Italian buffalo milk had similar CTB binding, and both had increased polysialo-GS compared to cows milk. The toxin binding properties of buffalo milk GS, and the anti-inflammatory activity of the lipophilized GS fraction could be important for developing innovative food applications, as well as the subject of future research.

Animals↗

PubMed2006

Genetic characteristic of high molecular weight glutenin subunits in somatic hybrid wheat lines -- potential application to wheat breeding.

Analysis of 17 derivatives from a somatic fusion between common wheat (Triticum aestivum) and tall wheat grass (Thinopyrum ponticum) showed a diversity of high molecular weight glutenin subunit (HMW-GS) compositions. On the basis of the inheritance of HMW-GS patterns, the derivatives were either (i) bred true over four successive generations, (ii) generated a few novel HMW-GS combinations at each generation, or (iii) showed highly unstable HMW-GS compositions. HMW-GS analysis of F(5) seed and each single seed-generated F(6) progenies further revealed that most of the HMW-GS had genetic stability. The variations of HMW-GS were inferred to occur in early generations and were maintained thereafter. Low molecular weight glutenin subunits (LMW-GS) in hybrid lines with high or low bread-making quality, classified into the first pattern, were compared. The result showed that hybrid lines with the uniform HMW-GS patterns also have identical LMW-GS patterns. The Glu-1 quality score was inferred to be relatively significant to the sodium dodecyl dulfate sedimentation value, as well as to correlate with the gluten exponent and contents of dry gluten and proteins. Sexual hybridization between high-quality somatic hybrid progeny II-12 and Chinese Spring (CS) showed that these high-quality HMW-GS genes could entail progenies. There was not subunit variation in the progenies of II-12 x CS. Therefore, sexual hybridization between somatic hybrid line and cultivars can transfer novel high-quality HMW-GS of somatic hybrids and benefit wheat breeding.

Breeding↗

PubMed2003

Outcome after radical prostatectomy with a pretreatment prostate biopsy Gleason score of >/=8.

OBJECTIVE: To determine the outcome and predictors of recurrence in patients with a pretreatment prostate biopsy Gleason score (GS) of >/= 8 and treated with radical prostatectomy (RP). PATIENTS AND METHODS: We retrospectively reviewed 1048 consecutive patients who underwent RP by one surgeon (M.S.S.); patients who had a pretreatment biopsy GS of >/= 8 were identified. Information was recorded on patient age, initial prostate specific antigen (PSA) level, clinical stage, biopsy GS, pathology GS, extraprostatic extension (EPE), tumour volume, surgical margin status, seminal vesicle invasion (SVI), and lymph node involvement. The results were assessed statistically using the Kaplan-Meier method, univariate log-rank tests and multivariate analysis using Cox's proportional hazards regression. RESULTS: In all, 123 patients met the initial selection criteria; 44 were excluded from further analyses (five salvage RP, 23 < 1 year follow-up and 16 adjuvant treatment). Thus 79 patients were included in the uni- and multivariate analyses; 25 (31%) patients had a GS of </= 7 in the RP specimen and 54 (69%) remained at GS >/= 8. The mean follow-up was 55 months, the age of the patients 63 years and the mean (sd) initial PSA level 13 (12) ng/mL. The overall biochemical failure rate was 38% (41% if the final GS was >/= 8 and 32% if it was </= 7). For those with a GS of >/= 8 in the RP specimen, 20% (11/54) were organ-confined; two patients (2.5%) in this group developed local recurrence. If the final GS was </= 7, 52% (13/25) were organ-confined. In the univariate analysis, significant risk factors for recurrence were PSA >/= 20 ng/mL, EPE, SVI, a positive surgical margin and tumour volume. Cox's proportional regression indicated that a PSA of >/= 20 ng/mL (hazard ratio 7.9, 95% confidence interval 2.6-24.2, P < 0.001), the presence of EPE (4.2, 1.6-10.9, P = 0.004) and a positive surgical margin (3.8, 1.5-9.7, P = 0.005) were significant independent predictors in a multivariate analysis. CONCLUSION: RP is a reasonable treatment option for patients with a prostate biopsy GS of >/=8 and clinical stage T1-2. These patients have a high chance of remaining disease-free if their PSA level is </= 20 ng/mL. Patients with a pretreatment biopsy GS of >/= 8 should be counselled about the potential differences between the biopsy and the RP specimen GS.

Adult↗

PubMed2006

Stomatal conductance is a key parameter to assess limitations to photosynthesis and growth potential in barley genotypes.

Fourteen genotypes of barley were compared for response to salinity by monitoring the parameters gas exchange and chlorophyll fluorescence. We present relationships between stomatal conductance (gs) gas exchange chlorophyll fluorescence parameters and aboveground dry matter (AGDM). We found that genetic variability provided a continuum of data for gs across control and saline conditions. We used this continuum of gs values to test the overall relationships between gs and net photosynthesis (A), leaf internal CO2 concentration (Ci), actual quantum yield of PSII electron transport (PhiPSII), relative electron yield over net CO2 assimilation rate (ETR/A), and AGDM. The relationship between gs and A was highly significant (P < 0.0001) for both control and saline treatments, while correlations between gs and Ci, and Ci and A were significant only under control conditions. Unexpectedly, we found positive correlations between gs and PhiPSII (P < 0.0001) for both conditions. A comparison between relationships of gs and A, and gs and PhiPSII seemed to indicate a possible acclimation to salinity at the chloroplastic level. Finally, the relationships between gs and ETR/A were exceptionally strong for both growing conditions (P < 0.0001) indicating that, as gs values were negatively affected in barley by genetics and salinity as main or interactive effects, there was a progressive increase in photorespiration in barley. Overall, we found that stomatal conductance was a key parameter in the study of barley responses to limiting situations for photosynthesis. We also found a strong relationship between AGDM and gs regardless of growing conditions and genotypes. For breeding evaluations to select barley genotypes for salinity tolerance, it may be possible to replace all measurements of gas exchange and chlorophyll fluorescence by the simple use of a porometer.

Adaptation, Physiological↗

PubMed2000

Enhanced multispecificity of arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2.

Recent investigations have established that Arabidopsis thaliana contains a family of genes encoding ATP-binding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family. So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes. Here we show that Arabidopsis MRP2 (AtMRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast. Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, not trans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each other's transport via distinct but coupled binding sites. The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2. The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione. Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E(2)17betaG uptake by AtMRP2. Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E(2)17betaG uptake, neither competes with DNP-GS for uptake nor is subject to E(2)17betaG-promoted uptake. A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2.

ATP Binding Cassette Transporter, Subfamily B↗