Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

Multiple ecto-nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression

The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surface-located enzymes (ecto-nucleotidases) of which several families with partially overlapping substrate specificities exist. In order to identify ecto-nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface-located ATPase and ADPase activity with apparent K(m)-values of 283 microM and 243 microM, respectively. Using PCR we identified the mRNA of all members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto-nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue-non-specific alkaline phosphatase and ecto-5'-nucleotidase. The surface-located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto-nucleoside triphosphate diphosphohydrolase family. Activity of ecto-5'-nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent. For a detailed analysis of the cellular distribution of ecto-nucleotidases single and double transfections of PC12 cells were performed, followed by fluorescence analysis. Ecto-nucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto-nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells.     

Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography

We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feedstock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography.The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration.     

Regio-selective preparation of vinyl adenosine ester catalyzed by alkaline protease

The transesterification of divinyladipate with adenosine in DMF containing 20% (v/v) DMSO was catalyzed by Streptomyces sp. alkaline protease and esterification occurred exclusively at the 3-position of hydroxyl group of ribofuranose in adenosine to give 3-O-vinyladipoyl adenosine without other products.     

Silicon bioavailability studies in young rapidly growing rats and turkeys fed semipurified diets

Two experiments were conducted using completely randomized designs to study the bioavailability of Si from three sources to growing rats and turkeys fed semipurified diets. The basal diets were dextrose-egg albumin for rats and dextrose-casein for turkeys. The Si sources were tetraethylorthosilicate (TES), sodium silicate (NaSil), and sodium zeolite A (NaZA). Rats and turkeys were supplemented at 500 and 270 ppm Si, respectively, from each source. A control group of unsupplemented rats and turkeys was included in each experiment. In general, irrespective of Si source, Si supplementation slowed (p < 0.05 orp < 0.01) growth rates in both rats and turkeys. Although dietary Si supplementation reduced (p < 0.05) plasma Mg levels and liver Zn concentrations in rats, it increased (p < 0.05) plasma P and reduced (p < 0.05) plasma Cu levels in turkeys. Rats on TES had significantly slower (p < 0.05 orp < 0.01) growth rates (5–10%) than those on NaSil or NaZA. In rats, NaZA and TES reduced (p < 0.05) hemoglobin concentrations and plasma Zn, respectively. However, plasma Mg levels were higher (p < 0.05) in TES than NaSil-or NaZA-fed rats. The source of the dietary Si did not affect (p < 0.05) the organ weights of rats and their mineral concentrations. Turkeys on TES diets grew at a significantly faster (p < 0.05) rate (15%) than those on NaSil or NaZA diets during the first 2 wk of experimentation. However, after 4 wk, there were no significant (p > 0.05) differences in growth between the Si sources. In turkeys, NaZA increased (p < 0.05) hematocrit levels and plasma Mg levels. Turkeys on NaZA diets had larger (p < 0.05) hearts and livers than those on NaSil but not TES. Liver Mn content was higher (p < 0.05) in turkeys on NaSil than TES or NaZA. Heart Zn was lower (p < 0.05) in turkeys on NaSil than TES, but not NaZA.     

Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin-based ELISA

AIMS: Different indicator enzymes and fluorogenic or chromogenic substrates were compared as detector systems in a novel polymyxin-based enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157 lipopolysaccharide (LPS) antigens. METHODS AND RESULTS: An ELISA system was developed using polymyxin immobilized in the wells of a microtitre plate as a high-affinity adsorbent for E. coli O157 LPS antigens, which were immunoenzymatically detected using anti-E. coli O157 antibody-enzyme conjugates. With peroxidase as the indicator enzyme the fluorogenic substrates Amplex Red and QuantaBlu produced only slight improvement in the performance characteristics of the polymyxin-ELISA compared with the use of the chromogenic substrate tetramethylbenzidine (TMB). On the other hand, with alkaline phosphatase as the indicator enzyme a pronounced improvement in assay performance was noted using the fluorogenic substrate Attophos compared with the chromogenic substrate p-nitrophenylphosphate. CONCLUSIONS: The detection system exhibiting the best characteristics with respect to cost, ease of use and overall performance in the detection of E. coli O157 in enrichment cultures from a variety of solid foods was based on the use of peroxidase as the indicator enzyme with the chromogenic substrate TMB. SIGNIFICANCE AND IMPACT OF THE STUDY: The polymyxin-ELISA provides a rapid, simple and inexpensive assay system for the detection of E. coli O157 in foods.     

A novel alkaline α-galactosidase gene is involved in rice leaf senescence

We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the structure and function of an SAG-Osh69 encoding alkaline α-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H₂O₂ and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal α-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards α-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence. The nucleotide sequence data reported will appear in the GenBank Nucleotide Sequence Database under the accession number AF251068.     

Design, synthesis and anti-microbial activity of 1H-pyrazole carboxylates

In a SAR study, we have synthesized a few 1H-pyrazole carboxylate related microbicides using Vilsmeier reagent. The anti-microbial screening results of 1H-pyrazole-3-carboxylate are reported here for the first time. The effect of 1H-pyrazole carboxylates on the mycelial growth of plant pathogenic fungi is revealed. The first X-ray structure in the family of microbicidal 1H-pyrazole-4-carboxylates is presented.     

Recombinant alkaline serine protease II degrades scrapie isoform of prion protein

Summary An efficient Escherichia coli expression system for the production of mature-type alkaline serine protease II (mASP II) has been constructed. Complementary deoxyribonucleic acid-encoding mASP II was inserted into the inducible bacterial expression vector pGE-30. After introduction into E, coli, the plasmid was expressed by isopropyl-1-thio-β-d-galactopyranoside, and the recombinant product was purified using a Ni-nitrilotriacetic acid column The purified product had the expected NH₂-terminal sequence and showed a scrapie isoform of prion protein-degrading activity using hamster scrapie 263K prions as a substrate.