Effects Of Metabolic Inhibitors Research Articles

Effect of Metabolic Inhibitors on the Size and Structure of Halos Induced by the Powdery Mildew Pathogen Blumeria graminis f. sp. tritici on Wheat Leaves

Effect of Metabolic Inhibitors on the Size and Structure of Halos Induced by the Powdery Mildew Pathogen Blumeria graminis f. sp. tritici on Wheat Leaves

The immunometabolic ecosystem in cancer.

Our increased understanding of how key metabolic pathways are activated and regulated in malignant cells has identified metabolic vulnerabilities of cancers. Translating this insight to the clinics, however, has proved challenging. Roadblocks limiting efficacy of drugs targeting cancer metabolism may lie in the nature of the metabolic ecosystem of tumors. The exchange of metabolites and growth factors between cancer cells and nonmalignant tumor-resident cells is essential for tumor growth and evolution, as well as the development of an immunosuppressive microenvironment. In this Review, we will examine the metabolic interplay between tumor-resident cells and how targeted inhibition of specific metabolic enzymes in malignant cells could elicit pro-tumorigenic effects in non-transformed tumor-resident cells and inhibit the function of tumor-specific T cells. To improve the efficacy of metabolism-targeted anticancer strategies, a holistic approach that considers the effect of metabolic inhibitors on major tumor-resident cell populations is needed.

Targeting the monocarboxylate transporter MCT2 and lactate dehydrogenase A LDHA in cancer cells with FX-11 and AR-C155858 inhibitors.

In 1930, Otto Warburg reported that "aerobic glycolysis" is the intrinsic property of all tumor cells' fermentation of glucose to L-Lactate by lactate dehydrogenase A (LDHA) activity. This only produces per mole of glucose two moles of adenosine triphosphate (ATP), compared with 32 moles of ATP in a normal cell. Thus, tumor cells have to uptake 30 folds more glucose, the resulting accumulated lactate are then transported by a monocarboxylate transporter (MCT) with the participation of a CD147 molecule. Inhibition of MCT1 by RNA interference (RNAi) disrupted the unique metabolism of the tumor and caused tumor cell death. However, the effectiveness of the strategies depends on the targeted delivery of the therapeutics. In this study, a synergistic approach was used to target LDHA and MCT1 with small molecule inhibitors FX11 and AR-C155858, respectively. Cell cytotoxicity assays (AlamarBlue assay), and Mitochondria Membrane Potential (JC-1) dye assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. To achieve this aim, the following objectives were proposed: the effect of metabolic inhibitors on tumor glycolytic metabolite environment, and the efficacy of metabolite inhibitors on human breast and colorectal cancer cells in vitro. Then, gene expression analysis was performed using Qiagen RT2 Profiler PCR array for apoptosis. All these assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. Normal human fibroblasts were used as control cells under normal and hypoxic culture conditions. In this study, the use of FX-11 inhibitors under normoxia or hypoxia in two or more cancer and normal cell lines has a direct effect on LDHA, whereby it inhibits its production, and this reduces the growth and cell proliferation of tumors. One of the more significant findings to emerge from this study is that using AR-C155858 inhibitor alone has increased the cell proliferation and showed no significant changes compared with the control. The other major finding was that combination of the two inhibitors, FX-11 and AR-C155858, under normoxia or hypoxia in two different cell lines MCF-7 and HCT-116 measured a decrease in the cells proliferative and red/green ratio. We successfully demonstrated that a combination of MCT1 inhibitor and LDHA inhibitor led to better outcomes. Indeed, this makes LDHA an ideal metabolic therapeutic target.

Evaluation of extracellular electron transfer in Pseudomonas aeruginosa by co-expression of intermediate genes in NAD synthetase production pathway.

Pseudomonas aeruginosa (PA) is an electrogenic bacterium, in which extracellular electron transfer (EET) is mediated by microbially-produced phenazines, especially pyocyanin. Increasing EET rate in electrogenic bacteria is key for the development of biosensors and bioelectrofermentation processes. In this work, the production of pyocyanin, Nicotinamide Adenine Dinucleotide (NAD) and NAD synthetase by the electrogenic strain PA-A4 is determined using a Microbial Fuel Cell (MFC). Effects of metabolic inhibition and enhancement of pyocyanin and NAD synthetase on NAD/NADH levels and electrogenicity was demonstrated by short chronoamperometry measurements (0-48h). Combined overexpression of two intermediate NAD synthetase production genes-nicotinic acid mononucleotide adenyltransferase (nadD) and quinolic acid phosphoribosyltransferase (nadC) genes, which are distant on the PA genomic map, enabled co-transcription and increased NAD synthetase activity. The resulting PA-A4 nadD + nadC shows increases in pyocyanin concentration, NAD synthetase activity, NAD/NADH levels, and MFC potential, all significantly higher than its wild type. Extracellular respiratory mechanisms in PA are linked with NAD metabolism, and targeted increased yield of NAD could directly lead to enhanced EET. A previous attempt at enhancing NAD synthetase for electrogenicity by targeting the terminal NAD synthetase gene (nadE) in standard P. aeruginosa PA01 had earlier been reported. Our work however, poses another route to electrogenicity enhancement in PA using; a combination of nadD and nadC. Further experiments are needed to understand specific intracellular mechanisms governing how over-expression of nadD and nadC induced activity of NadE protein. These findings significantly advance the knowledge of the versatility of NAD biosynthetic genes in PA electrogenicity.

Metformin and 2-Deoxyglucose Collaboratively Suppress Human CD4+ T Cell Effector Functions and Activation-Induced Metabolic Reprogramming.

Metabolic reprogramming plays a central role in T cell activation and differentiation, and the inhibition of key metabolic pathways in activated T cells represents a logical approach for the development of new therapeutic agents for treating autoimmune diseases. The widely prescribed antidiabetic drug metformin and the glycolytic inhibitor 2-deoxyglucose (2-DG) have been used to study the inhibition of oxidative phosphorylation and glycolysis, respectively, in murine immune cells. Published studies have demonstrated that combination treatment with metformin and 2-DG was efficacious in dampening mouse T cell activation-induced effector processes, relative to treatments with either metformin or 2-DG alone. In this study, we report that metformin + 2-DG treatment more potently suppressed IFN-γ production and cell proliferation in activated primary human CD4+ T cells than either metformin or 2-DG treatment alone. The effects of metformin + 2-DG on human T cells were accompanied by significant remodeling of activation-induced metabolic transcriptional programs, in part because of suppression of key transcriptional regulators MYC and HIF-1A. Accordingly, metformin + 2-DG treatment significantly suppressed MYC-dependent metabolic genes and processes, but this effect was found to be independent of mTORC1 signaling. These findings reveal significant insights into the effects of metabolic inhibition by metformin + 2-DG treatment on primary human T cells and provide a basis for future work aimed at developing new combination therapy regimens that target multiple pathways within the metabolic networks of activated human T cells.

Metabolic heterogeneity and plasticity of glioma stem cells in a mouse glioblastoma model.

Glioblastomas have been shown to rely on glycolysis as an energy source. However, recent evidence suggests that at least a subset of glioma cells with stem cell-like properties can thrive on oxidative phosphorylation. It remains unclear whether both metabolic phenotypes support tumor propagation, if they are independent, and how stable they are. The present study investigated these questions with the use of isogenic murine glioma stem cells (GSCs). GSCs were established from tumors formed by Ink4a/Arf-null, H-RasV12-expressing glioma-initiating cells that differed in extracellular acidification potential. Metabolic characteristics of GSCs were determined by measurement of glucose, oxygen, and glutamine uptake, ATP content, and lactate production. Effects of metabolic inhibitors and changes in oxygen or nutrient availability on lactate production and tumorsphere growth were also determined. GSCs were found either to consume more glucose and produce more lactate or to consume more oxygen and maintain a higher ATP content depending on the metabolic characteristics of the tumor cells of origin. The latter, mitochondrial-type GSCs increased lactate production after treatment with the oxidative phosphorylation inhibitor oligomycin or phenformin. Exposure to hypoxia also increased lactate production and expression of glycolysis-related enzymes and metabolites in mitochondrial-type GSCs in a reversible manner. Both glycolytic and mitochondrial-type energy production can sustain tumor propagation by isogenic GSCs. Whereas both phenotypes can be independent and stable, cells that rely on oxidative phosphorylation can also switch to a more glycolytic phenotype in response to metabolic stress, suggesting that plasticity is a further characteristic of GSC metabolism.

Abstract 5422: Synergistic effects of metabolic inhibitors on radiochemosensitized spheroid prostate cancer cells

Abstract One of the ways by which prostate cancer cells survive or become resistant to conventional therapies is through the ability to reprogram their metabolic pathways. Thus, justifying the need for studies to elucidate the efficacy of metabolic inhibitors in prostate cancer intervention. The purpose of this study was to investigate the synergistic effects of two metabolic pathway inhibitors - 3-bromopyruvate (3-BrPA) and genistein - on androgen sensitive (LNCaP) and androgen insensitive (DU145) spheroid prostate cancer cells separately primed or irradiated with very low doses of x-ray radiation (VLDR) and low frequency weak magnetic field radiation (MFR). Methods: Both PCa cells were cultured until they form spheroids and then irradiated with either VLDR (20mGy/hr) or MFR (10mT; 50Hz/hr) in separate groups. Cells were seeded and treated with varying doses of 3-BrPA and genistein in single or combination regimen. Cell viability, metabolic activity and cytotoxicity or LDH release levels were measured using MTT, Alamar blue and LDH assays respectively. NBT assay was used to determine treatment-induced ROS levels. mitochondrial membrane potential was determined by Rh123 and ethidium bromide fluorescent staining. Modes of Cell death were determined by fluorescent staining with acridine orange and ethidium bromide dyes, and by morphological identification of apoptotic and autophagic PCa cells. CompuSyn program was used to determine the time- and dose-dependent synergism between combination regimens. Results: Our results show that VLDR and weak MFR moderately inhibit cell proliferation in both PCa cell lines. However, LNCaP PCa cells were found to be more responsive to both VLDR or weak MFR compared to DU-145 PCa cells. Both VLDR and MFR suppresses ATP generation and increases ROS generation time-dependently in both cell lines. The most common cell death event found in MFR-exposed cells was a mixture of mitotic catastrophe and autophagy, while autophagic and slight apoptotic responses were observed in VLDR-exposed cells. 3-BrPA triggers more autophagic response in irradiated DU145 cells compared more apoptotic response in LNCaP cells. In contrast, genistein induced cell growth inhibition in both cell lines, but more effect was observed in LNCaP cells with dose-dependent apoptotic response. Combination of 3-BrPA and genistein forced PCa cells to undergo reprogrammed cell death, decreased the mitochondrial membrane potential and LDH levels significantly in a dose and time-dependently. Conclusion: The combination treatments show synergistic and significant inhibition of the glycolytic and mitochondrial metabolic pathways resulting in induction of apoptosis in radiochemosensitized PCa cells. Priming of cells with VLDR or MFR also enhanced the therapeutic effects of both metabolic inhibitors. Hence, reinforcing the potential benefit of metabolic inhibitors in prostate cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Saheed O. Oseni, Rolando Branly, Mirjana Pavlovic, James Kumi-Diaka. Synergistic effects of metabolic inhibitors on radiochemosensitized spheroid prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5422. doi:10.1158/1538-7445.AM2017-5422

禾本科作物に対するケイ素の栄養学的意義(第3報) : 水稲のケイ素吸収に対する各種代謝阻害剤の影響

禾本科作物に対するケイ素の栄養学的意義(第3報) : 水稲のケイ素吸収に対する各種代謝阻害剤の影響

Effect of Metabolic Inhibitors on the Hepatic Disposition of 5-Fluorouracil after Application to the Rat Liver Surface.

We evaluated the effects of 5-fluorouracil (5-FU) metabolic inhibitors, gimeracil or uridine, on the hepatic disposition of 5-FU after application to the liver surface in rats, aiming to enhance the availability of 5-FU in the liver. 5-FU solution with or without metabolic inhibitors was applied to the rat liver surface using a cylindrical diffusion cell. The liver, blood and the remaining solution in the diffusion cell were collected at specified times, and assayed for 5-FU content. 5-FU absorption properties were not altered by addition of gimeracil and uridine. The 5-FU concentration in the diffusion cell attachment site of the rat liver (site 1) at 0.1-0.4 M ratios of gimeracil to 5-FU was significantly higher than that of the control. On the contrary, the addition of uridine did not increase the 5-FU concentration at site 1. At a 0.1 M ratio of gimeracil to 5-FU, the maximum 5-FU plasma concentration was the lowest, and the area under the 5-FU concentration-time curve at site 1 was 3.4 times greater than that of the control. We demonstrated that applying 5-FU with gimeracil to the rat liver surface could increase the availability of 5-FU in the liver.

Metabolism of excised embryos of Lupinus luteus L. I. Effect of metabolic inhibitors and growth substances on the water uptake

Metabolism of excised embryos of Lupinus luteus L. I. Effect of metabolic inhibitors and growth substances on the water uptake

Mechanisms of Repair of Low Water Activity and pH-Injured Z ygosaccharomyces rouxii YSa40 in Glycerol and Sucrose/CPB Liquid Holding System

This study demonstrated injury of a 2-day-old stressed Zygosaccharomyces rouxii YSa40 (Xerotolerant yeast) cells in 0.64 water activity (aw)/pH 3.5 glycerol (GLY)/CPB stress system. The effects of metabolic inhibitors on repair of the low aw injury were also investigated by resuscitating the low aw injured cells over a time period of 24 h in a suitably designed 0.87 aw/pH 3.5 GLY and sucrose (SUC)/citrate phosphate buffer (CPB) liquid holding (LH) system. On use of appropriate inhibitory concentrations of metabolic inhibitors, repair processes in low aw-injured populations involved cell membrane and cell wall synthesis, protein and DNA synthesis and adenosine triphosphate (ATP) from glycolysis, but without oxidative phosphorylation. During 24 h of LH period, ATP was vital to maintain the cell viability of the surviving population on 0.94 aw/pH 3.5 sugars/basal medium agar (lactic acid); while allowing its injured population to repair at low aw injury in the LH system. This oxidative phosphorylation pathway can provide insight into various mechanisms of repair in low aw-injured yeast cells. Practical Applications Results generated in this study are expected to benefit food industries where problems related to recovery of injured microbes are involved, especially in Intermediate Moisture Food products.

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy.

Effect of 5-fluoro-2′-deoxyuridine on toxin production and cell cycle regulation in marine dinoflagellate, Alexandrium tamarense

The effect of metabolic inhibitor, 5-fluoro-2′-deoxyuridine (FUdR) on toxin production and the cell cycle of marine dinoflagellate, Alexandrium tamarense, was investigated. Compared to untreated cells, FUdR at 3μM (p<0.05) to 300μM (p<0.01) inhibited the cell proliferation and toxin production in a dose-dependent manner for A. tamarense cultured in modified T1 medium. FUdR at 203μM resulted in cell cycle arrest at the S phase at day 4 and toxigenesis was inhibited after day 2. The toxin profiles of the FUdR-treated cultures were similar to those of the control culture. These results suggest that FUdR inhibits saxitoxin (STX) biosynthesis in the early stage of the pathway. This report is the first to demonstrate the inhibition of toxin production in A. tamarense by a nucleoside analog.

Abstract C152: Characterizing rhabdomyosarcoma and osteosarcoma cellular metabolism.

Abstract Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. A renewed interest in the metabolic properties of cancer cells, as well as the recognition of altered cellular metabolism as a critical hallmark of cancer, has led to an exploration of targeting metabolic dependencies of cancer cells as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human rhabdomyosarcoma and osteosarcoma cell lines in order to identify potential differences in sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Glycolytic and mitochondrial bioenergetic profiles were generated using Seahorse extracellular flux analysis to measure extracellular acidification rates and oxygen consumption rates, and significant differences in glycolytic and respiratory bioenergetics between rhabdomyosarcoma and osteosarcoma cell lines were identified. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely-used diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells versus embryonal rhabdomyosarcoma, osteosarcoma, and normal cells, whereas inhibition of mitochondrial respiration by oligomycin or metformin did not significantly affect viability. Combined inhibition of glycolysis and respiration decreased cell growth more effectively than either treatment alone in all sarcoma lines tested. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels in all sarcoma lines tested, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C152. Citation Format: Sameer Issaq, Beverly Teicher, Anne Monks. Characterizing rhabdomyosarcoma and osteosarcoma cellular metabolism. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C152.

Effect of Metabolic Inhibitors on Growth and Production of L-glutamic Acid by the Mutant Micrococcus glutamicus AB100

Effect of Metabolic Inhibitors on Growth and Production of L-glutamic Acid by the Mutant Micrococcus glutamicus AB100

Effect of metabolic inhibitors on growth and carotenoid production in Dunaliella bardawil

Dunaliella bardawil, a green alga accumulates high amount of β-carotene under stress conditions. This organism has been exploited for β-carotene at industrial scale. In the present work, various metabolic inhibitors like diphenylamine (DPA), nicotine, basta, glyphosate, DCMU [3-(3',4'-dichlophenyl)-1,1-dimethylurea] and caffeine were used in autotrophic medium, to understand their influence on carotenoid biosynthesis. The results indicated that these metabolic inhibitors influenced the production of carotenoids like wise, DPA and basta increased the contents of β-carotene (1.7 fold), glyphosate and DCMU for lutein (2.4 and 2 fold) caffeine for biomass yields (1.1 fold), while nicotine decreased the biomass yield (3.6 fold), β-carotene (2 fold) and lutein (10.5 fold).

Effects of metabolic inhibitors and incubation temperature on the saturable uptake of propranolol by isolated rat lung tissue

The effects of several metabolic inhibitors (50 microM) on the initial uptake rate of propranolol (0.5 to 500 micrograms mL-1) by the minced lungs (0.4 g) isolated from 7-week-old rats has been investigated in oxygenated, pH 7.4 Krebs-Ringer bicarbonate buffer solution (20 mL) containing 3% BSA at 37 degrees C for 5 min. The effect of the incubation temperature was also examined. Metabolism of propranolol was almost insignificant (i.e. less than 1.3% of the initial load). The overall initial uptake rate was considered to be a combination of apparent linear transport and saturable processes. For the control uptake rate, the linear transport rate constant was 1.26 +/- 0.16 g-1 mL-1 min-1, while Vmax and Km' of the capacity limited uptake process were estimated as 0.727 +/- 0.074 mg g-1 min-1 and 24.8 +/- 2.71 micrograms mL-1, respectively. No metabolic inhibitor tested had an effect on the linear transport rate of propranolol but 2,4-dinitrophenol and potassium cyanide inhibited saturable uptake rate (i.e. Vmax) of propranolol significantly (P less than 0.01) while ouabain, phloridine and iodoacetic acid did not do so significantly. Reduction of the incubation temperature to 15 degrees C decreased and at 25 degrees C tended to decrease, both linear transport and saturable uptake rates.

The biosynthesis and accumulation of β-carotene in Dunaliella salina proceed via the glyceraldehyde 3-phosphate/pyruvate pathway

In Dunaliella salina, we studied the early steps in the isoprenoid pathway for the biosynthesis of carotenoids and β-carotene and the effects of metabolic inhibitors. When D. salina was grown under carotenogenic and non-carotenogenic conditions, mevinolin did not inhibit growth or the accumulation of carotenoids, β-carotene or chlorophyll. In contrast, fosmidomycin progressively inhibited cell growth and the biosynthesis of carotenoids, β-carotene and chlorophyll. In this work, we reported for the first time that in D. salina, β-carotene biosynthesis does not proceed via the classical acetate/mevalonate pathway but via the novel glyceraldehyde 3-phosphate/pyruvate pathway. This favours the yield of C5 isoprenoid units for synthesis of isopentenyl diphosphate, the precursor in the biosynthesis of C20 compounds, including geranylgeranyl diphosphate. Consequently, this pathway promotes carotenogenesis and the biosynthesis of C40 β-carotene in D. salina.

Metabolic inhibition increases activity of connexin-32 hemichannels permeable to Ca2+ in transfected HeLa cells

Numerous cell types express functional connexin (Cx) hemichannels (HCs), and membrane depolarization and/or exposure to a divalent cation-free bathing solution (DCFS) have been shown to promote HC opening. However, little is known about conditions that can promote HC opening in the absence of strong depolarization and when extracellular divalent cation concentrations remain at physiological levels. Here the effects of metabolic inhibition (MI), an in vitro model of ischemia, on the activity of mouse Cx32 HCs were examined. In HeLa cells stably transfected with mouse Cx32 (HeLa-Cx32), MI induced an increase in cellular permeability to ethidium (Etd). The increase in Etd uptake was directly related to an increase in levels of Cx32 HCs present at the cell surface. Moreover, MI increased membrane currents in HeLa-Cx32 cells. Underlying these currents were channels exhibiting a unitary conductance of approximately 90 pS, consistent with Cx32 HCs. These currents and Etd uptake were blocked by HC inhibitors. The increase in Cx32 HC activity was preceded by a rapid reduction in mitochondrial membrane potential and a rise in free intracellular Ca(2+) concentration ([Ca(2+)](i)). The increase in free [Ca(2+)](i) was prevented by HC blockade or exposure to extracellular DCFS and was virtually absent in parental HeLa cells. Moreover, inhibition of Cx32 HCs expressed by HeLa cells in low-confluence cultures drastically reduced cell death induced by oxygen-glucose deprivation, which is a more physiological model of ischemia-reperfusion. Thus HC blockade could reduce the increase in free [Ca(2+)](i) and cell death induced by ischemia-like conditions in cells expressing Cx32 HCs.

Effects of metabolic inhibitors on activity of Cry1Ab toxin to inhibit growth of Ephestia kuehniella larvae

Although the toxins of Bacillus thuringiensis Berliner (Bt) are frequently used to control lepidopteran pests, the tolerance or resistance of some lepidopteran moth strains may limit Bt applications. In this study, insecticidal cocktails consisting of sublethal doses of Cry1Ab toxin and additive compounds were tested for their suppressive effect on larval relative growth rate (RGR) in Ephestia kuehniella Zeller under laboratory conditions. In the first step, the suppressive effect of diflubenzuron, soybean trypsin inhibitor (STI) and chitinase on RGR was confirmed. In the second step, these compounds were incorporated into a mixture of crushed kernels of Bt maize hybrid MON 810-YieldGard and its isoline with concentrations of Cry1Ab toxin ranging from 0.011 to 0.091 microg g(-1) diet. An additive effect on the suppression of larval RGR in E. kuehniella was found in a combination of diflubenzuron, STI and STI + chitinase as secondary compounds in insecticidal cocktails. Chitinase showed no additive effect on RGR. The highest suppression level was found in cocktails with STI + chitinase as a secondary compound. It is hypothesized that the protease inhibitor (STI) protects both chitinase and Cry1Ab proteins from endogenous proteases in the larval midgut and prolongs their insecticidal activities. The possible application of insecticidal cocktails in the control of E. kuehniella is discussed.