The Trk Potassium Transporter Is Required for RsmB-Mediated Activation of Virulence in the Phytopathogen Pectobacterium wasabiae

Pectobacterium wasabiae (previously known as Erwinia carotovora) is an important plant pathogen that regulates the production of plant cell wall-degrading enzymes through an N-acyl homoserine lactone-based quorum sensing system and through the GacS/GacA two-component system (also known as ExpS/ExpA). At high cell density, activation of GacS/GacA induces the expression of RsmB, a noncoding RNA that is essential for the activation of virulence in this bacterium. A genetic screen to identify regulators of RsmB revealed that mutants defective in components of a putative Trk potassium transporter (trkH and trkA) had decreased rsmB expression. Further analysis of these mutants showed that changes in potassium concentration influenced rsmB expression and consequent tissue damage in potato tubers and that this regulation required an intact Trk system. Regulation of rsmB expression by potassium via the Trk system occurred even in the absence of the GacS/GacA system, demonstrating that these systems act independently and are both required for full activation of RsmB and for the downstream induction of virulence in potato infection assays. Overall, our results identified potassium as an essential environmental factor regulating the Rsm system, and the consequent induction of virulence, in the plant pathogen P. wasabiae.

Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects

IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

Impact of a Potassium-enriched, Chloride-depleted 5% Glucose Solution on Gastrointestinal Function after Major Abdominopelvic Surgery: Results of a Randomized Controlled Trial.

BACKGROUND Gastrointestinal (GI) complications often delay recovery after radical cystectomy with urinary diversion. The authors investigated if perioperative administration of a potassium-enriched, chloride-depleted 5% glucose solution (G5K) accelerates recovery of GI function. METHODS This randomized, parallel-group, single-center double-blind trial included 44 consecutive patients undergoing radical cystectomy and pelvic lymph node dissection with urinary diversion. Patients were randomized to receive either a G5K (G5K group) solution or a Ringer's maleate solution (control group). Fluid management aimed for a zero fluid balance. Primary endpoint was time to first defecation. Secondary endpoints were time to normal GI function, need for electrolyte substitution, and renal dysfunction. RESULTS Time to first defecation was not significantly different between groups (G5K group, 93 h [19 to 168 h] and control group, 120 h [43 to 241 h]); estimator of the group difference, -16 (95% CI, -38 to 6); P = 0.173. Return of normal GI function occurred faster in the G5K group than in the control group (median, 138 h [range, 54 to 262 h] vs. 169 h [108 to 318 h]); estimator of the group difference, -38 (95% CI, -74 to -12); P = 0.004. Potassium and magnesium were less frequently substituted in the G5K group (13.6 vs. 54.5% [P = 0.010] and 18.2 vs. 77.3% [P < 0.001]), respectively. The incidence of renal dysfunction (Risk, Injury, Failure, Loss and End-stage kidney disease stage "risk") at discharge was 9.1% in the G5K group and 4.5% in the control group; P = 1.000. CONCLUSIONS Perioperative administration of a G5K did not enhance first defecation, but may accelerate recovery of normal GI function, and reduces potassium and magnesium substitution after radical cystectomy and urinary diversion.

Organizational maintenance manual : loader-transporter, guided missile, XM501E3 (Hawk guided missile system), part number 11675300, NSN 1450-00-066-8873).

"June 1979."

Thiamine Deficiency Results in Downregulation of the GLAST Glutamate Transporter in Cultured Astrocytes

Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [H-3]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V-max for uptake of [H-3]-D-aspartate, with no change in the K-m value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [H-3]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation. of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder. (C) 2003 Wiley-Liss, Inc.

The mouse sulfate anion transporter gene Sat1 (Slc26a1): Cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation by thyroid hormone

Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.

Secondary prevention of cardiovascular events with long-term pravastatin in patients with diabetes or impaired fasting glucose - Results from the LIPID trial

OBJECTIVE- Diabetes, a major health problem worldwide, increases the risk of cardiovascular disease and its associated mortality. Evidence of the overall benefits of lipid modification in this area is needed. RESEARCH DESIGN AND METHODS- The Long-Term Intervention with Pravastatin in Ischemic Disease (LIPID) trial showed that cholesterol-lowering treatment with pravastatin reduced mortality and coronary heart disease (CHD) events in 9,014 patients aged 31-75 years with CHD and total cholesterol 4.0-7.0 mmol/l. We measured the effects of pravastatin therapy, 40 mg/day over 6.0 years, on the risk of CHD death or nonfatal myocardial infarction and other cardiovascular outcomes in 1,077 LIPID patients with diabetes and 940 patients with impaired fasting glucose (IFG). RESULTS- in patients allocated to placebo, the risk of a major CHD event was 61% higher in patients with diabetes and 23% higher in the IFG group than in patients with normal fasting glucose, and the risk of any cardiovascular event was 37% higher in the diabetic group and 19% higher in the IFG group. Pravastatin therapy reduced the risk of a major CHD event overall from 15.9 to 12.3% (relative risk reduction [RRR] 24%, P < 0.001) and from 23.4 to 19.6% in the diabetic group (19%, P = 0.11); in the diabetic group, the reduction was not significantly different from the reductions in the other groups. Pravastatin reduced the risk of any cardiovascular event from 52.7 to 45.2% (21%, P < 0.008) in patients With diabetes and from 45.7 to 37.1% (26%, P = 0.003) in the IFG group. Pravastatin reduced the risk of stroke from 9.9 to 6.3% in the diabetic group (RRR 39%, Cl 7-61%, P = 0.02) and from 5.4 to 3.4% in the IFG group (RRR 42%, Cl -9 to 69%, P = 0.09). Pravastatin did not reduce the incidence of diabetes. Over 6 years, pravastatin therapy prevented one major,CHD event (CHD death or nonfatal myocardial infarction) in 23 patients with IFG and 18 patients with diabetes. A meta-analysis of other major trials confirmed the high absolute risks of diabetes and IFG and the absolute benefits of statin therapy in these patients. CONCLUSIONS- Cholesterol-lowering treatment with pravastatin therapy prevents cardiovascular events, including stroke, in patients with diabetes or IFG and established CHD.

Glutamate transporter localization does not correspond to the temporary functional recovery and late degeneration after acute ocular ischemia in rats

Purpose: To determine whether the localization of retinal glutamate transporters is affected by retinal ischaemia and whether their ability to transport glutamate decreases with the progression of ischemic retinal and optic nerve degeneration. Methods: Retinal ischemia was induced in rats by acutely increasing the intraocular pressure (IOP, 110 mmHg/60 min). Reperfusion was permitted for periods up to 60 days post-ischemia. Functional evaluation was performed by monitoring the pupil light reflexes (PLRs) and electroretinograms (flash, flicker ERG and oscillatory potentials). Glutamate transporter localization and D-aspartate (glutamate analogue) uptake were assessed by immunohistochemistry. Results: Intense immunoreactivity for the retinal glutamate transporters (GLAST, GLT1, EAAC1 and EAAT5) was observed at all time points after the insult, despite severe retinal degeneration. D-aspartate was also normally accumulated in the ischemic retinas. Ten days post-operatively the PLR ratio (ratio = indirect/direct PLR = 34 +/- 7(.)5%) was significantly less than the pre-operative value (pre-op = 76(.)7 +/- 2 (.)6%, p < 0(.)05). However, 25 and 35 days post-operatively PLR ratios did not differ significantly from pre-operative values (44(.)4 +/- 6(.)9 and 53(.)8 +/- 9(.)6%, p > 0(.)05). Forty-five and 60 days post-operatively the PLR ratio declined again and was significantly lower than the pre-operative value (33(.)8 + 8(.)7 and 26(.)2 + 8(.)9%, p < 0(.)05). Statistical analysis revealed that all tested ERG components had significantly higher values at 32, but not at 42 and 58 days post-operatively when compared to the first time point recorded post-operatively (10 days). Conclusions: While retinal glutamate transport is compromised during an acute ischemic insult, consequent retinal recovery and degeneration are not due to a change in the excitatory amino acid transporter localization or D-aspartate (glutamate analogue) uptake. Rat retina and optic nerve are capable of spontaneous, but temporary, functional recovery after an acute ischemic insult. (C) 2004 Elsevier Ltd. All rights reserved.

Nitric oxide donors inhibit 5-hydroxytryptamine (5-HT) uptake by the human 5-HT transporter (SERT)

1 The aim was to test the hypothesis that nitric oxide ( NO) donor drugs can inhibit the 5-hydroxytryptamine (5-HT) transporter, SERT. 2 The NO donors, MAHMA/NO ( a NONOate; (Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]diazen- 1-ium-1,2-diolate), SIN-1 ( a sydnonimine; 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride), FK409 ( an oxime; (+/-)-(4-ethyl-2E-(hydroxyimino)-5-nitro-3E-hexenamide)) and peroxynitrite, but not Angeli's salt ( source of nitroxyl anion) or sodium nitrite, caused concentration-dependent inhibition of the specific uptake of [H-3]- 5-HT in COS-7 cells expressing human SERT. 3 Superoxide dismutase (150 U ml(-1)) plus catalase ( 1200 U ml(-1)), used to remove superoxide and hence prevent peroxynitrite formation, prevented the inhibitory effect of SIN-1 ( which generates superoxide) but not of MAHMA/NO or FK409. 4 The inhibitory effects of the NO donors were not affected by the free radical scavenger, hydroxocobalamin (1 mM) or the guanylate cyclase inhibitor, ODQ (1H-[ 1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one; 3 muM). 5 L-Cysteine ( 1 mM; source of excess thiol residues) abolished or markedly reduced the inhibitory effects of MAHMA/NO, SIN-1, FK409 and peroxynitrite. 6 It is concluded that inhibition of SERT by the NO donors cannot be attributed exclusively to NO free radical nor to nitroxyl anion. It does not involve guanosine-3',5'-cyclic monophosphate, but may involve nitrosation of cysteine residues on the SERT protein. Peroxynitrite mediates the effect of SIN-1, but not the other drugs. 7 Data in mice with hypoxic pulmonary hypertension suggest that SERT inhibitors may attenuate pulmonary vascular remodelling. Thus, NO donors may be useful in pulmonary hypertension, not only as vasodilators, but also because they inhibit SERT, provided they display this effect in vivo at appropriate doses.

PKC alpha is activated but not required during glucose-induced insulin secretion from rat pancreatic islets

The role of protein kinase C (PKC) in glucose-stimulated insulin secretion (GSIS) is controversial. Using recombinant adenoviruses for overexpression of PKCalpha and PKCdelta, in both wild-type (WT) and kinase-dead (KD) forms, we here demonstrate that activation of these two PKCs is neither necessary nor sufficient for GSIS from batch-incubated, rat pancreatic islets. In contrast, responses to the pharmacologic activator 12-O-tetradecanoylphorbol-13-acetate (TPA) were reciprocally modulated by overexpression of the PKCalphaWT or PKCalphaKD but not the corresponding PKCdelta adenoviruses. The kinetics of the secretory response to glucose (monitored by perifusion) were not altered in either cultured islets overexpressing PKCalphaKD or freshly isolated islets stimulated in the presence of the conventional PKC (cPKC) inhibitor Go6976. However, the latter did inhibit the secretory response to TPA. Using phosphorylation state-specific antisera for consensus PKC phosphorylation sites, we also showed that (compared with TPA) glucose causes only a modest and transient functional activation of PKC (maximal at 2-5 min). However, glucose did promote a prolonged (15 min) phosphorylation of PKC substrates in the presence of the phosphatase inhibitor okadaic acid. Overall, the results demonstrate that glucose does stimulate PKCalphain pancreatic islets but that this makes little overall contribution to GSIS.

The effects of high glucose and atorvastatin on endothelial cell matrix production

Background Statins are known to enhance atherosclerotic plaque stability through influences on extracellular matrix homeostasis. Net matrix production reflects the relative balance of matrix production and degradation through enzymes such as matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of MMP (TIMPs). The effects of statins on endothelial cell production of these parameters following co-exposure with a proatherogenic stimulus such as high glucose are not known. Methods Human endothelial cells were exposed for 72 h to 5 mM> (control) or 25 mM (high) glucose +/- atorvastatin (1 mumol/l). Extracellular matrix homeostasis was assessed by measuring matrix metalloproteinase (MMP)-2 secretion, tissue inhibitor of MMP (TIMP)-1 and -2 secretion and net collagen IV production. Results were expressed as percentage +/- SEM of control values. Results Exposure to high glucose increased cellular collagen IV expression to 190.1 +/- 11.7% (P < 0.0001) of control levels. No change in MMP-2 secretion (111.6 +/- 5.2%; P > 0.05) was observed but both TIMP-1 and TIMP-2 expression were increased to 136.3 +/- 6.4% and 144.0 +/- 27.5%, respectively (both P < 0.05). The presence of atorvastatin in high glucose conditions reduced collagen IV expression to 136.1 +/- 20.6%. This was paralleled by increased secretion of MMP-2 to 145.8 +/- 7.8% (P < 0.01), increased TIMP-2 expression to 208.0 +/- 21.3% (P < 0.005 compared with high glucose) but no change in TIMP-1 expression (155.1 +/- 14.6%) compared with high glucose alone. The presence of atorvastatin in control conditions did not affect levels of collagen IV expression (114.5 +/- 13.2%). Conclusions Endothelial cell exposure to high glucose was associated with a MMP/TIMP profile that increased extracellular matrix production which was attenuated by concurrent exposure to atorvastatin. Consequently, a mechanism by which the atherosclerotic plaque regression that is observed in patients taking these drugs has been demonstrated.

Strength and endurance training lead to different post exercise glucose profiles in diabetic participants using a continuous subcutaneous glucose monitoring system

Background Although both strength training (ST) and endurance training (ET) seem to be beneficial in type 2 diabetes mellitus (T2D), little is known about post-exercise glucose profiles. The objective of the study was to report changes in blood glucose (BG) values after a 4-month ET and ST programme now that a device for continuous glucose monitoring has become available. Materials and methods Fifteen participants, comprising four men age 56.5 +/- 0.9 years and 11 women age 57.4 +/- 0.9 years with T2D, were monitored with the MiniMed (Northridge, CA, USA) continuous glucose monitoring system (CGMS) for 48 h before and after 4 months of ET or ST. The ST consisted of three sets at the beginning, increasing to six sets per week at the end of the training period, including all major muscle groups and ET performed with an intensity of maximal oxygen uptake of 60% and a volume beginning at 15 min and advancing to a maximum of 30 min three times a week. Results A total of 17 549 single BG measurements pretraining (619.7 +/- 39.8) and post-training (550.3 +/- 30.1) were recorded, correlating to an average of 585 +/- 25.3 potential measurements per participant at the beginning and at the end of the study. The change in BG-value between the beginning (132 mg dL(-1)) and the end (118 mg dL(-1)) for all participants was significant (P = 0.028). The improvement in BG-value for the ST programme was significant (P = 0.02) but for the ET no significant change was measured (P = 0.48). Glycaemic control improved in the ST group and the mean BG was reduced by 15.6% (Cl 3-25%). Conclusion In conclusion, the CGMS may be a useful tool in monitoring improvements in glycaemic control after different exercise programmes. Additionally, the CGMS may help to identify asymptomatic hypoglycaemia or hyperglycaemia after training programmes.

Continuous glucose monitoring in diabetic long distance runners

Marathon running is growing in popularity, and many diabetic patients are participating in various marathon races all over the world each year. This study aimed to investigate the prevalence and extent of glycemic excursions (hypo- and hyperglycemic) during a marathon run in patients with well-controlled diabetes mellitus using a continuous glucose monitoring system (CGMS). Five subjects with type 1 and one patient with type 2 diabetes mellitus were monitored with the Medtronic MiniMed CGMS during the 2002 Vienna City Marathon (n = 3) or the Fernwarme run (n = 3) long distance runs of 42.19/15.8 km. All six patients finished their course. The CGSM system was well tolerated in all patients over an average duration of 34 +/- 4.0 hours and it did not limit the patients' activities. The mean running time for the Vienna city marathon was 257 +/- 8 min (247 to 274 min) and for the Fernwarme run 134 +/- 118 min (113 to 150 min). A total of 1470 blood glucose measurements (mean 245 readings per subject) were performed. During and after the marathons frequent hypo and hyperglycemic episodes with and without clinical symptoms were measured. Our data confirm that the CGMS may help to identify asymptomatic hypoglycemia or hyperglycemia during and after a long distance run. The system may also be helpful to improve our understanding about the individual changes of glucose during and after a marathon and may protect hypoglycemic or hyperglycemic periods in future races.

Glucose affects monocarboxylate cotransporter (MCT) 1 expression during mouse preimplantation development

Cleavage-stage embryos have an absolute requirement for pyruvate and lactate, but as the morula compacts, it switches to glucose as the preferred energy source to fuel glycolysis. Substrates such as glucose, amino acids, and lactate are moved into and out of cells by facilitated diffusion. in the case of lactate and pyruvate, this occurs via H+-monocarboxylate cotransporter (MCT) proteins. To clarify the role of MCT in development, transport characteristics for DL-lactate were examined, as were mRNA expression and protein localisation for MCT1 and MCT3, using confocal laser scanning immunofluorescence in freshly collected and cultured embryos. Blastocysts demonstrated significantly higher affinity for DL-lactate than zygotes (K-m 20 +/- 10 vs 87 +/- 35 mmol lactate/l; P = 0.03 by linear regression) but was similar for all stages. For embryos derived in vivo and those cultured with glucose, MCT1 mRNA was present throughout preimplantation development, protein immunoreactivity appearing diffuse throughout the cytoplasm with brightest intensity in the outer cortical region of blastomeres. in expanding blastocysts, MCT1 became more prominent in the cytoplasmic cortex of blastomeres, with brightest intensity in the polar trophectoderm. Without glucose, MCT1 mRNA was not expressed, and immunoreactivity dramatically reduced in intensity as morulae died. MCT3 mRNA and immunoreactivity were not detected in early embryos. The differential expression of MCT1 in the presence or absence of glucose demonstrates that it is important in the critical regulation of pH and monocarboxylate transport during preimplantation development, and implies a role for glucose in the control of MCT1, but not MCT3, expression.

Beneficial associations of physical activity with 2-h but not fasting blood glucose in Australian adults

OBJECTIVE - We examined the associations of physical activity with fasting plasma glucose (FPG) and with 2-h postload plasma glucose (2-h PG) in men and women with low, moderate, and high waist circumference. RESEARCH DESIGN AND METHODS - The Australian Diabetes, Obesity and Lifestyle (AusDiab) study provided data on a population-based cross-sectional sample of 4,108 men and 5,106 women aged >= 25 years without known diabetes or health conditions that could affect physical activity. FPG and 2-h PG were obtained from an oral glucose tolerance test. Self-reported physical activity level was defined according to the current public health guidelines as active (>= 150 min/week across five or more sessions) or inactive (< 150 min/week and/or less than five sessions). Sex-specific quintiles of physical activity time were used to ascertain dose response. RESULTS - Being physically active and total physical activity time were independently and negatively associated with 2-h PG. When physical activity level was considered within each waist circumference category, 2-h PG was significantly lower in active high-waist circumference women (beta-0.30 [95% CI -0.59 to -0.01], P = 0.044) and active low-waist circumference men(beta-0.25 [-0.49 to -0.02],P = 0.036) compared with their inactive counterparts. Considered across physical activity and waist circumference categories, 2-h PG levels were not significantly different between active moderate-waist circumference participants and active low-waist circumference participants. Associations between physical activity and FPG were nonsignificant. CONCLUSIONS - There are important differences between 2-h PG and FPG related to physical activity. It appears that 2-h PG is more sensitive to the beneficial effects of physical activity, and these benefits occur across the waist circumference spectrum.