Sequential Statistical Improvement of the Liquid Cultivation of Helicobacter pylori

Background: Colonization of the gastric mucosa by Helicobacter pylori is one of the most important causes of acute and chronic gastric pathologies in humans. Achieving the growth of H. pylori in liquid media is of great importance in the development of clinical studies. In this study, we developed a sequential optimization strategy based on statistical models to improve the conditions of liquid culture of H. pylori. Materials and Methods: Four statistical models were sequentially used. First, a Box‐Behnken design was used to select the best process conditions (shaking speed, inoculum concentration, and final volume of culture). Secondly, a general factorial design was used to evaluate the influence of adding gel blocks or gel beads (shape and composition). Then a D‐optimal reduce design was carried out to allow the selection of the most influential factors in increasing the cell concentration (culture media components). Finally, another Box‐Behnken design was used to optimize the concentration of the culture media components previously selected. Results: After 12 hours of liquid culture a concentration of 25 × 10⁸ cells per mL (9.4 log₁₀ cells per mL) of H. pylori was obtained, compared with a predicted 32 × 10⁸ (9.5 log₁₀ cells per mL), which means between 1 and 5 log₁₀ units higher than some previous reports. Conclusions: The sequential statistical approach increased the planktonic H. pylori cell culture. The final culture media and conditions were: Brain Heart Infusion, blood agarose (1.5% w/v), lamb’s blood (3.18% v/v), DENT (0.11% v/v), and Vitox (0.52% v/v) at 60 rpm and 37 °C with filtered CO₂ (5% v/v) bubbled directly into the culture media in a final volume of 76.22 mL.     

Effects of gamma irradiation on the plasma membrane of suspension-cultured apple cells. Rapid irreversible inhibition of H+-ATPase activity

The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells ( Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H⁺-ATPase activity. Plasma membrane-enriched vesicles were prepared and the H⁺-ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H⁺-ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2m M dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H⁺-ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH.
This rapid irreversible inhibition of the plasma membrane H⁺-ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material.     

Salinity tolerance of Kosteletzkya virginica. II. Root growth, lipid content, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicot halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m ³ NaCl, and the effects of external salinity on root growth, ion and water levels, and lipid content were examined in successive harvests. Root growth paralleled shoot growth trends, with some enhancement observed at 85 mol m ³ NaCl and a reduction noted at the higher salinities. Root Na⁺ content increased with increasing external NaCl, but remained constant with time for each treatment. K⁺ content, although lower in salt-grown plants after 14 d salinization, subsequently increased to levels comparable to unsalinized plants. A strong K⁺ affinity was reflected in the increased K⁺/Na⁺ selectivity of salt-grown plants and by their low Na⁺/K⁺ ratios. Cl levels rose in salinized plants and values were double or more those for Na⁺, indicating the possibility of a sodium-excluding mechanism in roots. Root phospholipids and sterols, principal membrane constituents, were maintained or elevated and the free sterol/phospholipids ratio increased in salinized K. virginica plants, suggesting retention of overall membrane structure and decreased permeability. This response, considered in light of root calcium maintenance and high potassium levels, suggests that salinity-induced changes in membrane lipid composition may be important in preventing K⁺ leakage from cells.     

转铁蛋白在低钾刺激Madin Darby狗肾细胞钠-钾ATP酶中的作用

体外低钾培养肾细胞能刺激细胞膜钠-钾ATP酶。本研究利用Madin Darby狗肾细胞能在无血清培养液中健康生存48h这一特征,研究体外低钾刺激细胞膜钠-钾ATP酶所依赖的血清中的活性因子,观察了表皮生长因子(EGF)、胰岛素样生长因子(IGF1)、前列腺素1(PGE1)和转铁蛋白(tranderrin)在这一过程中的作用。结果表明,在无血清培养液中低钾并不能刺激细胞膜钠—钾ATP酶,而添加转铁蛋白可模拟血清的作用。转铁蛋白能剂量依赖性地增加ouabain结合位点,对细胞膜钠-钾ATP酶作用呈良好的时间效应关系。在低钾无血清培养液中,细胞膜钠-钾ATP酶α1亚基启动子活性增强,α1与β1亚基蛋白质表达的增加依赖于转铁蛋白的存在。进一步研究结果表明,低钾在转铁蛋白的无血清培养液环境中能增加细胞对铁的摄取(^59Fe),该作用可被铁螯合剂(deferoxamine,DFO;35 μmol/L)所阻断。DFO也可阻断转铁蛋白依赖性低钾刺激细胞膜钠-钾ATP酶数目的增多,α1亚基启动子活性增强,α1与β1亚基蛋白质表达增加。以上结果表明,低钾对细胞膜钠-钾ATP酶活性的刺激作用依赖于转铁蛋白所调节的铁的摄取。     

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.     

活细胞内5-羟色胺可见荧光的多光子激发成像

应用多光子激发激光扫描显微镜对5-羟色胺（5-hydroxytryptamine, 5-HT）孵育的大鼠粘膜型肥大细胞进行自发荧光成像,首次观察到了活细胞内5-HT相关的可见荧光,并对其产生机理进行了初步探讨.实现了对活细胞内5-HT空间分布的高分辨成像,为研究活组织或细胞内5-HT的空间分布和含量与细胞功能状态的关系提供了新的实验方法.     

[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5-Hydroxytryptamine and 5-Hydroxytryptamine Uptake Inhibitors

Abstract: The nature of [³H]imipramine binding to human platelets was investigated. Desipramine and 5-hydroxytryptamine (5-HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [³H]imipramine binding was investigated in saturation experiments. Increases in K d without changes in B _max were noted with the addition of 5-HT, desipramine, norzimeldine, or 5-methoxytryptoline. Reductions in B _max without alterations in K _D were obtained when citalopram or clomipramine was added. It is concluded that the [³H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5-HT uptake. In addition, [³H]imipramine and other 5-HT uptake inhibitors have bonds to other parts of the 5-HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.     

Breaking the HxNy outbreak

The latest emergence of influenza A (H1N1) virus outbreak demonstrated how swiftly a new strain of flu can evolve and spread around the globe. The A/H1N1 flu has been spreading at unprecedented speed, and further spread within the countries being affected and to other adjacent or far way countries is considered inevitable due to the rapid emigration of infected individuals across the world. In this bioinformation, we discuss the mechanism of evolution of a new HxNy strain and the essential criteria for potentially breaking the outbreak of these extremely harmful and rapidly evolving viral strains in the near future by taking the recent H1N1 pandemic as a classical paradigm.