Co-expression of mCysLT1 receptors and IK channels in Xenopus laevis oocytes elicits LTD4-stimulated IK current, independent of an increase in [Ca2+]i

Addition of LTD4 (10 nM) to Xenopus laevis oocytes expressing the mCysLT1 receptor together with hBK or hIK channels resulted in the activation of both channels secondary to an LTD4-induced increase in [Ca2+]i. In addition, the hIK channel is activated by low concentrations of LTD4 (<0.1 nM), which did not result in any increase in [Ca2+]i. Even though activation of hIK by low concentrations of LTD4 was independent of an increase in [Ca2+]i, a certain "permissive" level of [Ca2+]i was required for its activation, since buffering of intracellular Ca2+ by EGTA completely abolished the response to LTD4. Neither hTBAK1 nor hTASK2 was activated following stimulations with LTD4 (0.1 and 100 nM).     

Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin

The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P₂] in this process. In Ehrlich-Lettre ascites (ELA) cells, a current with biophysical and pharmacological properties characteristic of VRAC was activated by hypotonic swelling. A 44% increase in cellular cholesterol content had no detectable effects on F-actin organization or VRAC activity. A 47% reduction in cellular cholesterol content increased cortical and stress fiber-associated F-actin content in swollen cells. Cholesterol depletion increased VRAC activation rate and maximal current after a modest (15%), but not after a severe (36%) reduction in extracellular osmolarity. The cholesterol depletion-induced increase in maximal VRAC current was prevented by F-actin disruption using latrunculin B (LB), while the current activation rate was unaffected by LB, but dependent on Rho kinase. Rho activity was decreased by 20% in modestly, and 50% in severely swollen cells. In modestly swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P₂ analogue or a PtdIns(4,5)P₂-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P₂. It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part by preventing the hypotonicity-induced decrease in Rho activity and eliciting actin polymerization. cell swelling; kinase; phospholipid phosphatidylinositol-(4,5)-bisphosphate; cytoskeleton     

Gastrointestinal nematode infection is associated with variation in innate immune responsiveness

Ex vivo monocyte cytokine responses (IL-1beta, TNF-alpha, IL-12p70, IL-10, TGF-beta) to bacterial TLR2 and TLR4 ligands were quantified in 47 gastrointestinal (GI) nematode-exposed children in Pemba Island, Tanzania. Worminess (estimated by faecal egg counts (FEC)) had a positive relationship with pro-inflammatory TNF-alpha and IL-1beta responsiveness to the TLR ligands. In particular, there was a strong significant relationship with TNF-alpha response to TLR4 ligand (LPS). There were no significant associations between regulatory responses (IL-10, TGF-beta) and worminess. These results are consistent with the possibility that GI nematodes modulate innate responses and may indicate a potential mechanism for interactions between GI nematodiasis and important bystander pathogens.     

Membranes as possible pacemakers of metabolism.

Basal metabolic rate (BMR) varies dramatically among vertebrate species, both (i) being several fold higher in the endothermic mammals and birds compared to the ectothermic reptiles, amphibians and fish, and (ii) being much greater, on a body mass basis, in small vertebrates compared to large vertebrates. These differences in whole animal BMR are also manifest at the cellular level with substantial contributions to basal metabolic activity from the maintenance of various trans-membrane gradients. The percentage contribution of various processes to basal metabolism is remarkably consistent between different vertebrates and when BMR varies, the components of metabolic activity vary in relative unison. Membrane composition also varies between vertebrates and the degree of polyunsaturation of membrane phospholipids is correlated with cellular metabolic activity. In general, the tissue phospholipids and thus membrane bilayers of endotherms are more polyunsaturated than those from similar-sized ectotherms. In mammals membrane polyunsaturation is allometrically related to body mass. We suggest that membranes can act as pacemakers for overall metabolic activity. We propose that such membrane polyunsaturation increases the molecular activity of many membrane-bound proteins and consequently some specific membrane leak-pump cycles and cellular metabolic activity. We hypothesize a possible mechanistic basis for this effect that is based on a greater transfer of energy during intermolecular collisions of membrane proteins with the unsaturated two carbon units (C=C) of polyunsaturates compared to the single carbon units of saturated acyl chains, as well as the more even distribution of such units throughout the depth of the bilayer when membranes contain polyunsaturated acyl chains compared to monounsaturated ones. The proposed pacemaker role of differences in membrane bilayer composition is briefly discussed with respect to the brain (and sensory cells), evolution of mammalian endothermic metabolism, and its clinical implications for humans.     

Predictors of Weight Loss in Adults with Topiramate‐Treated Epilepsy

Objective: We examined predictors of weight loss with topiramate, an anticonvulsant associated with weight loss in adults. Research Methods and Procedures: In this uncontrolled, prospective clinical trial, topiramate was added to existing anticonvulsants in adults (40 to 110 kg) with partial‐onset seizures. Primary measurements were change from baseline weight after 3 months and 1 year in patients completing 1 year of topiramate treatment (N = 38). Physiological and metabolic measures were analyzed for correlation with weight loss during topiramate treatment. Results: In patients who completed 1 year of topiramate treatment, baseline weight was reduced in 82% at 3 months and in 86% at 1 year. Mean body weight was reduced 3.0 kg (3.9% of baseline) at 3 months and 5.9 kg (7.3%) at 1 year. In obese patients [body mass index (BMI) ≥ 30 kg/m²], mean weight loss was 4.2 kg (4.3%) at 3 months and 10.9 kg (11.0%) at 1 year. Weight loss was primarily caused by reduction in body fat mass. For all patients, weight loss at 3 months correlated most strongly with reduced caloric intake (p = 0.02). At 1 year, caloric intake had returned to baseline levels; weight loss correlated most strongly with higher baseline BMI (p = 0.0007). Discussion: Our results suggest that weight loss occurs in most adults treated with topiramate and is sustained for at least 1 year. Reduced caloric intake may account, in part, for weight loss during early treatment. The pattern of weight loss differs according to baseline BMI, with obese patients experiencing greater weight loss during continued therapy.     

Studies on methods for determining varietal utilization of nutrients

Summary In pot experiments with a phosphorus-deficient soil and four barley varieties, Kenia, Maja, Bonus, and Pallas, receiving increasing amounts of phosphorus, it was found that the order of susceptibility to phosphorus deficiency of the varieties was the same for grain as for grain + straw, when the ranking was based on the percentage decrease in dry matter yield as a result of phosphorus deficiency based on the maximum obtained dry matter yield. Estimations of the maximum phosphorus utilization quotients of dry matter yield of grain + straw graduated the susceptibility to phosphorus deficiency of the varieties in the same order.     

Telomere protection by mammalian Pot1 requires interaction with Tpp1

The shelterin complex at mammalian telomeres contains the single-stranded DNA-binding protein Pot1, which regulates telomere length and protects chromosome ends. Pot1 binds Tpp1, the shelterin component that connects Pot1 to the duplex telomeric DNA-binding proteins Trf1 and Trf2. Control of telomere length requires that Pot1 binds Tpp1 as well as the single-stranded telomeric DNA, but it is not known whether the protective function of Pot1 depends on Tpp1. Alternatively, Pot1 might function similarly to the Pot1-like proteins of budding and fission yeast, which have no known Tpp1-like connection to the duplex telomeric DNA. Using mutant mouse cells with diminished Tpp1 levels, RNA interference directed to mouse Tpp1 and Pot1, and complementation of mouse Pot1 knockout cells with human and mouse Pot1 variants, we show here that Tpp1 is required for the protective function of mammalian Pot1 proteins.     

Uptake of l-Glutamate into Rat Brain Synaptic Vesicles: Effect of Inhibitors that Bind Specifically to the Glutamate Transporter

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron-donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl⁻. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A₁, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.