Decreasing glycolysis increases sensitivity to mitochondrial inhibition in primary cultures of renal proximal tubule cells

Summary We have previously shown that shaking the culture plates (SHAKE) of rabbit renal proximal tubule cells (RPTC) to maintain adequate aeration increased aerobic metabolism and decreased the induction of glycolysis compared to RPTC cultured under standard conditions (STILL). However, glycolysis in SHAKE RPTC remained elevated compared to glycolysis in proximal tubules in vivo. In the present study the contribution of culture medium sugar composition and concentration to glycolytic metabolism was assessed in RPTC. SHAKE and STILL RPTC cultured in 5 mM glucose contained lactate levels equivalent to the respective SHAKE and STILL RPTC cultured in standard culture medium which contains 17.5 mM glucose. Similarly, the activity of lactate dehydrogenase was unchanged by lowering the medium glucose concentration. Substituting 5 mM galactose for 5 mM glucose in the culture medium significantly reduced the lactate content of both SHAKE and STILL RPTC but had no effect on lactate dehydrogenase activity. Cell growth was equivalent under all culture conditions. Sensitivity to mitochondrial inhibition was determined for each culture condition by measuring cell death after exposure to the respiratory inhibitor antimycin A. The results showed a hierarchy of sensitivity to antimycin A (5 mM galactose SHAKE >5 mM glucose SHAKE >17.5 mM glucose SHAKE = 17.5 mM glucose STILL), which was generally inversely correlated with the level of glycolysis as measured by lactate content (17.5 mM glucose STILL >17.5 mM glucose SHAKE = 5 mM glucose SHAKE >5 mM galactose SHAKE).     

HPAC,a new human glucocorticoid-sensitive pancreatic ductal adenocarcinoma cell line

Summary A new human pancreatic cancer (HPAC) cell line was established from a nude mouse xenograft (CAP) of a primary human pancreatic ductal adenocarcinoma. In culture, HPAC cells form monolayers of morphologically heterogenous, polar epithelial cells, which synthesize carcinoembryonic antigen, CA 19-9, CA-125, cytokeratins, antigens for DU-PAN-2, HMFG1, and AUA1, but do not express chromogranin A or vimentin indicative of their pancreatic ductal epithelial cell character. In the presence of serum, HPAC cell DNA synthesis was stimulated by insulin, insulin growth factor-I, epidermal growth factor, and TGF-α but inhibited by physiologic concentrations of hydrocortisone and dexamethasone. Dose-dependent inhibition of DNA synthesis was limited to steroids with glucocorticoid activity. The inhibitory effect of dexamethasone was abolished by the glucocorticoid antagonist RU 38486. Binding of [³H]dexamethasone to cytosolic proteins was specific and saturable at 4° C. Scatchard analysis of binding data demonstrated a single class of high-affinity binding sites (K_d=3.8±0.9 nM; B_max=523±128 fmol/mg protein). Western blot analysis revealed a major protein band that migrated at a M_r of 96 kDa. Northern blot analysis identified an mRNA of approximately 7 kilobases which hybridized with a specific glucocorticoid receptor complementary DNA probe (OB7). These findings support a role for glucocorticoids in the regulation of human malignant pancreatic cell function.     

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diiso-propylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.     

INFLUENCE OF NATURAL ULTRAVIOLET RADIATION ON LOTIC PERIPHYTIC DIATOM COMMUNITY GROWTH,BIOMASS ACCRUAL,AND SPECIES COMPOSITION: SHORT-TERM VERSUS LONG-TERM EFFECTS1, 2

Growth rates, accumulation dynamics, and species succession of periphytic diatom communities were examined in the presence and absence of natural ultraviolet (UV) radiation using a series of outdoor, continuous-flow experimental flumes located on the South Thompson River, British Columbia. In a short-term experiment (2–3 wk), log-phase growth rates of naturally seeded diatom communities comprised of Tabellaria fenestrata (Lyngb.) Kütz., T. flocculosa (Roth) Kütz., Fragilaria crotonesis Kitton, and F. vaucheriae (Ehr.) Peter. exposed to 90% ambient photosynthetically active radiation (PAR) + UV were 30–40% lower than growth rates under 90% PAR alone. UV inhibition of growth rate was independent of the degree of P limitation within the range of relative specific growth rates (μ:μ_max-P) of 0.5–1.0. In a long-term trial, inhibition of attached diatom accumulation under 90% PAR + UV during the first 2–3 wk was corroborated. Reduction of full sunlight to 50% PAR + UV prevented the initial inhibition phase. The initial inihibitory effect of 90% PAR + UV on algal accumulation was reversed after 3–4 wk, and by 5 wk total diatom abundance (chlorophyll a, cell numbers and cell biovolumes) in communities exposed to PAR + UV were 2–4-old greater than in communities protected from UV. Under 90% PAR + UV and 50% PAR + UV, a succession to stalked diatom genera (Cymbella and Gomphoneis) occurred. Species succession under UV radiation doubled the mean cell size of the diatom communities. The shift from inhibition to a long-term increase in the autotrophic community under PAR + UV compared to PAR alone provides further evidence against the use of short-term incubation experiments to define the long-term implications of increases in UVB. These results suggest that the ecological effects of present-day levels of UVB and UVB:UVA ratios on autotrophic communities are not well understood and might be mediated through complex trophic level interactions.     

Inhibition of tobacco NADH-hydroxypyruvate reductase by expression of a heterologous antisense RNA derived from a cucumber cDNA: Implications for the mechanism of action of antisense RNAs

Tobacco plants were genetically transformed to generate antisense RNA from a gene construct comprised of a full-length cucumber NADH-dependent hydroxypyruvate reductase (HPR) cDNA placed in reverse orientation between the cauliflower mosaic virus 35S promoter and a nopaline synthase termination/polyadenylation signal sequence. In vivo accumulation of antisense HPR RNA within eight independent transgenic tobacco plants resulted in reductions of up to 50% in both native HPR activity and protein accumulation relative to untransformed tobacco plants (mean transgenote HPR activity=67% wild type, mean transgenote HPR protein=63% wild type). However, in contrast to previous reports describing antisense RNA effects in plants, production of the heterologous HPR antisense RNA did not systematically reduce levels of native tobacco HPR mRNA (mean transgenote HPR mRNA level=135% wild type). Simple regression comparison of the steady-state levels of tobacco HPR mRNA to those of HPR antisense RNA showed a weak positive correlation (r value of 0.548, n=9 ; n is wild type control plus eight independent transformants; significant at 85% confidence level), supporting the conclusion that native mRNA levels were not reduced within antisense plants. Although all transgenic antisense plants examined displayed an apparent reduction in both tobacco HPR protein and enzyme activity, there is no clear correlation between HPR activity and the amount of either sense (r=0.267, n=9) or antisense RNA (r=0.175, n=9). This compares to a weak positive correlation between HPR mRNA levels and the amount of HPR activity observed in wild-type SRI tobacco plants (r=0.603, n=5). The results suggest that in vivo production of this heterologous HPR antisense RNA is inhibitory at the level of HPR-specific translation and produces its effect in a manner not dependent upon, nor resulting in, a reduction in steady-state native HPR mRNA levels. In this context, the observed antisense effect appears to differ mechanistically from most antisense systems described to date.     

Effect of carbon dioxide concentration on the bioleaching of a pyrite-arsenopyrite ore concentrate

The effect of carbon dioxide concentration on the bacterial leaching of a pyrite-arsenopyrite ore concentrate was studied in continuous-flow reactors. Steady-state operation with two feed slurry densities, 6 wt% and 16 wt% solids, were tested for the effect of carbon dioxide concentration. Bacterial growth rates were estimated via the measurement of carbon dioxide consumption rates. Aqueous-phase carbon dioxide concentrations in excess of 10 mg/L were found to be inhibitory to bacterial growth. (c) 1993 John Wiley & Sons, Inc.     

Protein toxicity to aphids: Anin vitro test onAcyrthosiphon pisum

Recent progress in plant transformation for insect resistance has increased the interest in the potential toxicity of proteins towards insect pests. While studies have been targeted to a large array of insect species, phloem-feeding Homoptera have not been investigated yet. The paper describes a routine test for screening toxicity and growth inhibition of purified proteins in artificial diets onAcyrthosiphon pisum (Harris). Twenty-five commercially available proteins of different classes were tested and compared to some non-protein chemicals (an insecticide, an antibiotic …).A. pisum proved to be very sensitive to all proteases tested and to some venoms with general cytolytic properties. A plant lectin, concanavalin A, displayed significant toxicity and growth inhibition, while various proteins such as a soybean proteinase inhibitor, a chitinase, and bovine serum albumin showed measurable impairments of growth only at higher dose (≥250 μg.ml⁻¹). Some proteins were without short-term effect onA. pisum physiology. The influence of these results on aphid-plant interactions are discussed.

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Résumé L'effet de protéines alimentaires sur les insectes phloémophages, dont les pucerons, n'a jamais été étudié. Nous proposons ici un test biologique standardisé sur milieu artificiel permettant d'analyser les effets de différentes classes de protéines sur la physiologie d'A. pisum. La validité de ce test est éprouvée (protocole, reproductibilité) et les différentes données récoltées (mortalité et inhibition de croissance) permettent de définir des paramètres toxicologiques tels que concentration létale 50 ou concentration inhibitrice 50. Cette caractérisation toxicologique a été réalisée sur 25 protéines appartenant à des classes différentes, ainsi que plusieurs substances non protéiques utilisées comme témoin de toxicité (insecticide, antibiotique, inhibiteur de synthèse protéique et glucoside phénolique). Les regroupements de protéines par proximités de profils toxicologiques ont été corrélés aux activités biochimiques des différentes protéines. Les implications de ces résultats sur les interactions plante-puceron sont discutées, ainsi que le potentiel d'une stratégie de création de variétés transgéniques résistantes aux pucerons.

     

Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene. (c) 1993 Wiley & Sons, Inc.