Lysophosphatidic Acid Induces a Sustained Elevation of Neuronal Intracellular Calcium

Abstract: Lysophosphatidic acid (LPA) is a lipid biomediator enriched in the brain. A novel LPA-induced response in rat hippocampal neurons is described herein, namely, a rapid and sustained elevation in the concentration of free intracellular calcium ([Ca²⁺]_i). This increase is specific, in that the related lipids phosphatidic acid and lysophosphatidylcholine did not induce an alteration in [Ca²⁺]_i. Moreover, consistent with a receptor-mediated process, there was no further increase in [Ca²⁺]_i after a second addition of LPA. The LPA-induced increase in [Ca²⁺]_i required extracellular calcium. However, studies with Cd²⁺, Ni²⁺, and nifedipine and nystatin-perforated patch clamp analyses did not indicate involvement of voltage-gated calcium channels in the LPA-induced response. In contrast, glutamate appears to have a significant role in the LPA-induced increase in [Ca²⁺]_i, because this increase was inhibited by NMDA receptor antagonists and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonists. Thus, LPA treatment may result in an increased extracellular glutamate concentration that could stimulate AMPA/kainate receptors and thereby alleviate the Mg²⁺ block of the NMDA receptors and lead to glutamate stimulation of an influx of calcium via NMDA receptors.     

Manipulation of Intracellular Calcium in NCB-20 Cells

A number of lines of evidence indicate that the Ca2+ and cyclic AMP signalling systems interact in NCB-20 cells. However, to date, the regulation of [Ca2+]i homeostasis has not been studied in this cell line. The present study aimed to clarify our understanding of [Ca2+]i homeostasis in these cells and to evaluate tools that manipulate [Ca2+]i, independently of protein kinase C effects. Bradykinin, by a B2-receptor, elevated [Ca2+]i by a pertussis-toxin-insensitive mechanism. The BK-stimulated [Ca2+]i rise originated from intracellular sources, without a contribution from Ca2+ entry mechanisms. The effect of BK was precluded by pretreatment with thapsigargin and ionomycin--compounds that elevated [Ca2+]i independent of phospholipase C activation. Both compounds, however, exerted effects in addition to stimulating release of Ca2+ from BK-sensitive stores; the BK-sensitive Ca2+ pool was a subset of the thapsigargin-sensitive pool; ionomycin strongly stimulates Ca2+ entry. Activation of protein kinases A and C attenuated the duration of the BK-induced rise in [Ca2+]i, without affecting the peak [Ca2+]i, suggesting interference with the BK response at a step downstream of the activation of phospholipase C. Application of these approaches should enhance the delineation of the consequences of Ca2+ mobilization on cyclic AMP accumulation.     

Expression of antigenically distinct fimbriae with hemagglutination and HeLa cell adherence properties by an enteroaggregative Escherichia coli strain belonging to the enteropathogenic serogroup

Abstract An enteroaggregative Escherichia coli (EAggEC) strain (DS92), isolated from a case of infantile diarrhea, was shown to express mannose-resistant hemagglutination and HeLa cell adhering properties when grown at 37°C but not at 28°C. Cellular adherence properties of DS92, which belonged to enteropathogeci serogroup 0125, were shown to correlate well with the expression of fimbriae that were encoded by a 112 kb plasmid. The fimbriae of the EAggEC strain DS92 were composed of 20 kDa subunit proteins and were serologically distinct from fimbrial or non-fimbrial cell surface antigen(s) of other diarrheagenic E. coli strains including the reference EAggEC strain 17-2. Interestingly, the 20-kDa fimbrial protein was found to be antigenically related to 18- and 14.5-kDa cell surface proteins of two other locally isolated EAggEC strains belonging to the enteropathogenic serogroup 086.     

Regulation of TRPV5 Single-Channel Activity by Intracellular pH

The transient receptor potential channel TRPV5 contributes to the apical entry pathway for transcellular calcium reabsorption in the kidney. Acid load causes hypercalciuria in animals and humans. We have previously reported that intracellular protons directly inhibit TRPV5. Here, we examined the effects of intracellular pH on single-channel activity of TRPV5. We found that TRPV5 channels exhibit full and subconductance open states in excised inside–out patches of Chinese hamster ovary cells. The slope conductance values (Na⁺ as a charge carrier, between −25 and −75 mV) for full and subconductance opening at intracellular pH 7.4 were 59 ± 6 and 29 ± 3 pS, respectively. Intracellular acidification caused a small decrease in single-channel conductance. Importantly, intracellular acidification decreased open probability for the full and subconductance states and increased probability for closing. To investigate how intracellular protons decrease open probability of the channel, we proposed a simple three-state model for open–subconductance–closed state transition and examined the effects of acidification on the respective forward and reverse rate constants. We found that intracellular acidification decreases opening of TRPV5 predominantly by promoting a transition from the subconductance to the closed state. Thus, intracellular acidification directly inhibits TRPV5 by causing a conformational change(s) leading to a decrease of open probability of TRPV5 as well as of the single-channel conductance. Seung-Kuy Cha and Wasey Jabbar contributed equally to this work.     

Ca2+ signaling, intracellular pH and cell volume in cell proliferation

Mitogens control progression through the cell cycle in non-transformed cells by complex cascades of intracellular messengers, such as Ca²⁺ and protons, and by cell volume changes. Intracellular Ca²⁺ and proton concentrations are critical for linking external stimuli to proliferation, motility, apoptosis and differentiation. This review summarizes the role in cell proliferation of calcium release from intracellular stores and the Ca²⁺ entry through plasma membrane Ca²⁺ channels. In addition, the impact of intracellular pH and cell volume on cell proliferation is discussed.     

Biosynthesis of avermectins and lipids in Streptomyces avermitilis

Abstract The gene coding for a β- d -xylosidase (E.C. 3.2.1.37) of the thermophile Caldocellum saccharolyticum was isolated previously as part of a gene cluster which has been cloned in Escherichia coli . The enzyme characteristics were determined in E. coli using toluenized cell extracts. The pH optimum was 6.5 and temperature optimum 70°C. The enzyme was stable at 60°C and the half life at 80°C was 45 minutes. The temperature optimum and the temperature stability exceed those reported for other bacterial or fungal β- d -xylosidases. The enzyme showed no other detectable xylanolytic or cellulolytic enzyme activity.     

Intracellular pH and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy

Photometric fluorescence microscopy has been used to measure intracellular pH (pHi) and free calcium concentrations [( Ca]i) in individual mouse thymocytes and 2H3 rat basophil leukaemic cells containing indicators for pH (quene 1) or calcium (quin 2). The pHi and [Ca]i measurements in individual 2H3 cells and mouse thymocytes and their responses to various stimuli were consistent with the corresponding data obtained from suspensions of these cells measured in a spectrofluorimeter. Photometric fluorescence microscopy of these indicators in individual cells provides a sensitive and fast method of following pHi and [Ca]i responses in individual cells.     

应用激光共聚焦扫描显微镜技术检测缺氧人肺微血管内皮细胞内钙离子浓度动态变化

目的：探讨应用激光共聚焦扫描显微镜（LSCM）技术检测缺氧状态的人肺微血管内皮细胞（HPMVEC）内钙离子（Ca2＋）浓度动态变化的价值。方法：HPMVEC常规培养,按观察时间点不同分为5个缺氧培养组（1h hyp组、2h hyp组、4h hyp组、6h hyp组和8h hyp组）以及1个对照组（0h con组）共6个组,每组设8个复孔,应用LSCM技术测定缺氧后HPMVEC内Ca2＋浓度水平及随时间推移的变化。结果：LSCM技术显示HPMVEC内Ca2＋的荧光强度1h hyp组与0h con组比较、2h hyp组与1h hyp组比较、4h hyp组与2h hyp组比较、6h hyp组与4h hyp组比较、8h hyp组与6h hyp组比较有显著差异（P〈0.05）。线性回归分析结果显示Ca2＋荧光强度与缺氧时间成正相关（r=0.969,P〈0.01）。结论：HPMVEC内Ca2＋浓度随缺氧时间增长而增高;LSCM在动态检测缺氧状态下HPMVEC内的Ca2＋浓度变化中具有明显优势。     

钙离子浓度动态变化

目的:探讨应用激光共聚焦扫描显微镜(LSCM)技术检测缺氧状态的人肺微血管内皮细胞(HPMVEC)内钙离子(Ca2+)浓度动态变化的价值。方法:HPMVEC常规培养,按观察时间点不同分为5个缺氧培养组(1h hyp组、2h hyp组、4h hyp组、6h hyp组和8h hyp组)以及1个对照组(0h con组)共6个组,每组设8个复孔,应用LSCM技术测定缺氧后HPMVEC内Ca2+浓度水平及随时间推移的变化。结果:LSCM技术显示HPMVEC内Ca2+的荧光强度1h hyp组与0h con组比较、2h hyp组与1h hyp组比较、4h hyp组与2h hyp组比较、6h hyp组与4h hyp组比较、8h hyp组与6h hyp组比较有显著差异(P<0.05)。线性回归分析结果显示Ca2+荧光强度与缺氧时间成正相关(r=0.969,P<0.01)。结论:HPMVEC内Ca2+浓度随缺氧时间增长而增高;LSCM在动态检测缺氧状态下HPMVEC内的Ca2+浓度变化中具有明显优势。     

构建一种检测水中As3+的敏感型大肠杆菌

为构建一种敏感性强、特异性好的检测砷离子的大肠杆菌荧光报告菌株,本研究利用基因敲除技术,敲除了大肠杆菌中负责向胞外转运砷离子的ars B基因,构建对As~(3+)敏感的菌株;利用egfp基因作为报告基因,构建融合检测载体p ET28b-Pars-ars R-egfp。然后将检测载体p ET28b-Pars-ars R-egfp转化至ars B基因敲除菌中完成敏感型As~(3+)检测菌株的构建。接着对此微生物传感器进行了检测条件的优化,以及线性检测范围、最低检出限、特异性等性能的确定。研究结果表明,与利用野生大肠杆菌作为检测宿主相比,此敏感型砷检测菌株对检测As~(3+)的灵敏度有显著提高,最适检测As~(3+)浓度范围为0.013-42.71μmol/L,最低检出下限为5.13 nmol/L。因此,本研究利用基因敲除技术对大肠杆菌进行改造,成功地提高了砷检测微生物传感器的灵敏度,为重金属微生物传感器的优化研究工作提供了有用方案。     

Modulation of lectinophagocytosis of Escherichia coli by variation of pH and temperature

The effect of variation of pH and temperature on the lectinophagocytosis of enteropathogenic Escherichia coli by polymorphonuclear leukocytes and macrophages elicited by thioglycolate medium was evaluated. The phagocytosis of enteropathogenic E. coli is dependent on pH, being maximal at pH 7.0 and reduced at pH 5.5 or 6.0. Mannan and mannose (as representative sugars that bind to phagocyte lectin receptors), are recognized by mannose receptors and reduced the phagocytic index at pH 7.0 (from 41.6 +/- 8.5 to 17.0 +/- 6.1) and at pH 6.0 (from 24.1 +/- 5.1 to 14.5 +/- 5.0), suggesting that mannose receptors, despite their reduced affinity for ligand at pH 6.0, also participate in phagocytosis of enteropathogenic E. coli. The inhibition of phagocytosis by anti-substance A antibody was also examined at pH 7.0 and at pH 6.0, decreasing (from 41.6 +/- 8.5 to 21.1 +/- 3.4) and (from 24.1 +/- 5.1 to 12.0 +/- 3.5), respectively. This antibody reduced the phagocytosis of enteropathogenic E. coli in phagocytic assays at 37 or 41 degrees C. These results suggest that the acidic pH decreased the affinity of mannose receptors to ligands on the surface of E. coli and also affected the binding of lectin from E. coli to N-acetylgalactosamine on phagocytes.     

The Effects of GPX-1 Knockout on Membrane Transport and Intracellular Homeostasis in the Lens

Glutathione peroxidase-1 (GPX-1) is an enzyme that protects the lens against H₂O₂-mediated oxidative damage. The purpose of the present study was to determine the effects of GPX-1 knockout (KO) on lens transport and intracellular homeostasis. To investigate these lenses we used (1) whole lens impedance studies to measure membrane conductance, resting voltage and fiber cell gap junction coupling conductance; (2) osmotic swelling of fiber cell membrane vesicles to determine water permeability; and (3) injection of Fura2 and Na⁺-binding benzofuran isophthalate (SBFI) into fiber cells to measure [Ca²⁺] _i and [Na⁺] _i , respectively, in intact lenses. These approaches were used to compare wild-type (WT) and GPX-1 KO lenses from mice around 2 months of age. There were no significant differences in clarity, size, resting voltage, membrane conductance or fiber cell membrane water permeability between WT and GPX-1 KO lenses. However, in GPX-1 KO lenses, coupling conductance was 72% of normal in the outer shell of differentiating fibers and 45% of normal in the inner core of mature fibers. Quantitative Western blots showed that GPX-1 KO lenses had about 50% as much labeled Cx46 and Cx50 protein as WT, whereas they had equivalent labeled AQP0 protein as WT. Both Ca²⁺ and Na⁺ accumulated significantly in the core of GPX-1 KO lenses. In summary, the major effect on lens transport of GPX-1 KO was a reduction in gap junction coupling conductance. This reduction affected the lens normal circulation by causing [Na⁺] _i and [Ca²⁺] _i to increase, which could increase cataract susceptibility in GPX-1 KO lenses.     

促甲状腺激素对甲状腺细胞胞内游离钙和钙调蛋白的影响

本文研究TSH和forskolin对原代培养的猪甲状腺细胞[Ca~(2+)]_i和钙调蛋白的影响。结果表明,TSH可引起甲状腺细胞[Ca~(2+)]_1急性升高。此反应是剂量依赖关系,而与细胞外钙的存在与否无关。其反应性在细胞单层高于细胞是液,近汇合细胞单层高于汇合细胞单层。TSH作用3天,可使甲状腺细胞的钙调蛋白含量增高,此作用与TSH对甲状腺细胞数的影响无关。Forskolin对甲状腺细胞的[Ca~(2+)]_i和钙调蛋白均无明显的影响。     

Effects of sub-minimal inhibitory concentrations of EDTA on growth of Escherichia coli and the release of lipopolysaccharide

Abstract Release of lipopolysaccharide from E. coli was studied in the presence of sub-minimal inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). In untreated cells no release was detected with 50 mM Mg²⁺ in the medium, but a steady release of over 50% of the synthesized lipopolysaccharide was observed with 0.1 mM Mg²⁺. EDTA at MIC/8 led to a 2- to 3-fold higher release, presumably by an adjustment of the concentration of unchelated Mg²⁺ to a value still sustaining normal growth but giving rise to a highly unstable outer membrane. No structural difference was observed between cell-bound and released lipopolysaccharide.     

Early Events in Free Radical-Mediated Damage of Isolated Nerve Terminals: Effects of Peroxides on Membrane Potential and Intracellular Na+ and Ca2+ Concentrations

Abstract: The effects of peroxides were investigated on the membrane potential, intracellular Na⁺ ([Na⁺]_i) and intracellular Ca²⁺ ([Ca²⁺]_i) concentrations, and basal glutamate release of synaptosomes. Both H₂O₂ and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na⁺]_i over an incubation period of 15 min. A steady rise of the [Ca²⁺]_i due to peroxides was also observed that was external Ca²⁺ dependent and detected only at an inwardly directed Ca²⁺ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H₂O₂. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.     

Regulation of Intracellular Calcium in Cerebellar Granule Neurons: Effects of Depolarization and of Glutamatergic and Cholinergic Stimulation

The regulation of the cytosolic free Ca2+ concentration ([Ca2+]i) was investigated by microfluorimetry in single cerebellar granule neurons exposed to various treatments (high K+, glutamate, or acetylcholine) and drugs. The responses to the treatments developed asynchronously during cell culture, with high K+ and glutamate reaching their maxima at 6 and 7 days in vitro and acetylcholine at 9 days in vitro. The biphasic [Ca2+]i transients induced by high K+ (an initial peak, followed by a plateau 30-40% of the peak, both sustained by dihydropyridine-sensitive voltage-gated Ca2+ channels) were dissipated by washing with fresh medium or, more rapidly, by addition of excess EGTA (t1/2 = 11 +/- 2 and 3 +/- 0.6 s, respectively). Compared to those induced by high K+, the [Ca2+]i transients induced by glutamate administered in Mg2(+)-free medium were much more variable. An initial peak, sustained by voltage-gated Ca2+ channels, was visible in only approximately 50% of the cells and disappeared when multiple glutamate pulses were administered. In the rest of the population, the transients were monophasic, with persistent plateaus sustained only in part (30-40%) by voltage-gated Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular distribution of acridine derivatives in platelets and their suitability for cytoplasmic pH measurements

The site and mechanism of accumulation of acridine derivatives into platelets and their isolated organelles were investigated. In addition, their suitability as indicators of cytoplasmic pH was analysed. Direct microscopic observation showed that quinacrine and 9-aminoacridine are concentrated inside organelles in platelets. Using fractionation studies, the acridine derivatives were found to accumulate particularly in dense and α-granules. Uptake into these organelles is driven by a pH differential across their membrane (acidic inside). Because of their cellular distribution, acridine derivatives were found to be poor indicators of cytoplasmic pH. In contrast, a poorly permeant dicarboxylated fluorescein derivative, generated in situ by cytosolic enzymes, is shown to be a more reliable probe of intracellular pH. The results are compared with previous reports of the use of 9-aminoacridine as a cytoplasmic pH probe in platelets and of quinacrine as a selective dense-granule marker.