Cytosolic pH and the inflammatory microenvironment modulate cell death in human neutrophils after phagocytosis.
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Following phagocytosis in vivo, acidification of extracellular pH (pH(o)) and intracellular metabolic acid generation contribute to cytosolic proton loading in neutrophils. Cytosolic pH (pH(i)) affects neutrophil function, although its regulation is incompletely understood. Its effect on mechanisms of neutrophil death is also uncertain. Thus, we investigated pH(i) regulation in Escherichia coli-exposed neutrophils, at various pathogen-to-phagocyte ratios (0:1-50:1), under conditions simulating the inflammatory milieu in vivo and correlated pH(i) changes with mechanisms of neutrophil death. Following phagocytosis, proton extrusion was dominated early by passive proton conductance channels, and later by Na(+)/H(+) exchange (NHE). H(+)-translocating adenosine triphosphatase (V-ATPase) pH(i) regulation was evident primarily at lower bacterial densities. At physiologic pH(o), lower pathogen-to-phagocyte ratios alkalinized pH(i) and inhibited apoptosis, whereas higher ratios acidified pH(i) (despite proton extrusive mechanisms) and promoted apoptosis. Necrosis was induced by high-density bacterial exposure at reduced pH(o). Following phagocytosis, targeted inhibition of NHEs, proton conductance channels, or V-ATPases (amiloride, ZnCl(2), or bafilomycin, respectively) moderately hyperacidified pH(i) and accelerated apoptosis. However, in combination they profoundly acidified pH(i) and induced necrosis. Proinflammatory mediators in vivo might affect both pH(i) regulation and cell death, so we tested the effects of bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF) and healthy subjects. Only CF BAL fluid alkalinized pH(i) and suppressed apoptosis at physiologic pH(o), but failed to prevent necrosis following phagocytosis at low pH(o). Thus, a precarious balance between cytosolic proton loading and extrusion after phagocytosis dictates the mode of neutrophil cell death. pH(i)/pH(o) might be therapeutically targeted to limit neutrophil necrosis and protect host tissues during necrotizing infections. (+info)
Supragingival calculus: formation and control.
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Dental calculus is composed of inorganic components and organic matrix. Brushite, dicalcium phosphate dihydrate, octacalcium phosphate, hydroxyapatite, and whitlockite form the mineral part of dental calculus. Salivary proteins selectively adsorb on the tooth surface to form an acquired pellicle. It is followed by the adherence of various oral micro-organisms. Fimbriae, flagella, and some other surface proteins are essential for microbial adherence. Microbial co-aggregation and co-adhesion enable some micro-organisms, which are incapable of adhering, to adhere to the pellicle-coated tooth surface. Once organisms attach to the tooth surface, new genes could be expressed so that mature dental plaque can form and biofilm bacteria assume increased resistance to antimicrobial agents. Supersaturation of saliva and plaque fluid with respect to calcium phosphates is the driving force for plaque mineralization. Both salivary flow rate and plaque pH appear to influence the saturation degree of calcium phosphates. Acidic phospholipids and specific proteolipids present in cell membranes play a key role in microbial mineralization. The roles of crystal growth inhibitors, promoters, and organic acids in calculus formation are discussed. Application of biofilm culture systems in plaque mineralization is concisely reviewed. Anti-calculus agents used--centering on triclosan plus polyvinyl methyl ether/maleic acid copolymer, pyrophosphate plus polyvinyl methyl ether/maleic acid copolymer, and zinc ion-in commercial dentifrices are also discussed in this paper. (+info)
A novel procedure for simple and efficient genotyping of single nucleotide polymorphisms by using the Zn2+-cyclen complex.
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The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilized in the study of various genetic determinants. Here, we introduce a simple, rapid, low-cost and accurate procedure for the detection of SNPs by polyacrylamide gel electrophoresis (PAGE) with a novel additive, the Zn2+- cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane). The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE, which is due to Zn2+-cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. Various nucleotide substitutions (e.g. AT to GC) in DNA fragments (up to 150 bp) can be visualized with ethidium bromide staining. Furthermore, heteroduplex and homoduplex DNAs are clearly separated as different bands in the gel. We demonstrate the analysis of single- and multiple-nucleotide substitutions in a voltage-dependent sodium channel gene by using this novel procedure (Zn2+-cyclen-PAGE). (+info)
Inhibition of aquaporin-mediated CO2 diffusion and voltage-gated H+ channels by zinc does not alter rabbit lung CO2 and NO excretion.
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Aquaporins (AQs) increase cell membrane CO(2) diffusivity, and it has been proposed that they may serve as transmembrane channels for CO(2) and other small gas molecules. In addition, it has been hypothesized that voltage-gated H(+) channels located on the apical membrane of the alveolar epithelium contribute to CO(2) elimination by the lung. To test whether these membrane proteins contribute to CO(2) elimination in vivo, we measured CO(2) exchange in buffer- and blood-perfused rabbit lungs before and after addition of 0.5 mM ZnCl(2), an inhibitor of both AQ-mediated CO(2) diffusion and voltage-gated H(+) channels. For comparison, red cell and lung carbonic anhydrases (CAs) were inhibited by 0.1 mM methazolamide. ZnCl(2) had no effect on CO(2) exchange when inspired CO(2) was altered between 2% and 5% in 5-min intervals. Pulmonary vascular and airway resistances were not altered by ZnCl(2). In contrast, methazolamide inhibited CO(2) exchange by 30% in buffer-perfused lungs and by 65% in blood-perfused lungs. Exhaled NO concentrations were unaffected by ZnCl(2) or by CA inhibition. Lung capillary gas exchange modelling shows that under normal resting conditions it would be necessary to reduce the alveolar-capillary membrane CO(2) diffusion capacity by >90% to lower CO(2) elimination by 10%. Therefore we conclude that red cell and lung AQs and voltage-gated H(+) channels in the alveolar epithelium contribute minimally to normal physiological CO(2) elimination. (+info)
Forms of zinc accumulated in the hyperaccumulator Arabidopsis halleri.
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The chemical forms of zinc (Zn) in the Zn-tolerant and hyperaccumulator Arabidopsis halleri and in the non-tolerant and nonaccumulator Arabidopsis lyrata subsp. petraea were determined at the molecular level by combining chemical analyses, extended x-ray absorption spectroscopy (EXAFS), synchrotron-based x-ray microfluorescence, and muEXAFS. Plants were grown in hydroponics with various Zn concentrations, and A. halleri specimens growing naturally in a contaminated site were also collected. Zn speciation in A. halleri was independent of the origin of the plants (contaminated or non-contaminated) and Zn exposure. In aerial parts, Zn was predominantly octahedrally coordinated and complexed to malate. A secondary organic species was identified in the bases of the trichomes, which contained elevated Zn concentrations, and in which Zn was tetrahedrally coordinated and complexed to carboxyl and/or hydroxyl functional groups. This species was detected thanks to the good resolution and sensitivity of synchrotron-based x-ray microfluorescence and muEXAFS. In the roots of A. halleri grown in hydroponics, Zn phosphate was the only species detected, and is believed to result from chemical precipitation on the root surface. In the roots of A. halleri grown on the contaminated soil, Zn was distributed in Zn malate, Zn citrate, and Zn phosphate. Zn phosphate was present in both the roots and aerial part of A. lyrata subsp. petraea. This study illustrates the complementarity of bulk and spatially resolved techniques, allowing the identification of: (a) the predominant chemical forms of the metal, and (b) the minor forms present in particular cells, both types of information being essential for a better understanding of the bioaccumulation processes. (+info)
Anti-diabetes effect of Zn(II)/carnitine complex by oral administration.
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A novel bis(L-carnitinato)Zn(II) complex, Zn(car)(2)Cl(2), was prepared, and its insulinomimetic and antidiabetic activities were examined. The complex showed a tendency to lower the high blood glucose levels of KK-A(y) mice with type 2 diabetes mellitus when given by oral administration at a dose of 20 mg Zn/kg body weight for 16 d. In addition, the complex improved glucose tolerance ability when examined by the oral glucose tolerance test (1 g glucose/kg body weight). (+info)
Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress.
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Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels. (+info)
Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.).
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Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazur (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca(2+) ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn(2+) ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far. (+info)