Bark extract of Cassia sieberiana DC. (Caesalpiniaceae) displayed good antibacterial activity against MDR gram-negative phenotypes in the presence of phenylalanine-arginine ß-naphthylamide.

BACKGROUND: Multidrug-resistant (MDR) bacteria remain a major cause of morbidity and mortality globally. The present study was designed to investigate the in vitro antibacterial activities of crude methanol extract and constituents isolated by Column Chromatography (CC) from Cassia sieberiana bark (CSB) against ten MDR Gram-negative bacteria, as well as the mechanisms of action of the most active sample. METHODS: The antibacterial activity of the tested samples (extract, the fractions and their compounds isolated by CC and the structures obtained by exploiting 1H and 13C Nuclear magnetic resonance (NMR) spectra) in the presence and absence of an efflux pumps inhibitor, phenylalanine-arginine ß-naphthylamide (PAßN), was evaluated using the micro-dilution method. The effects of the most active sample were evaluated on the cell growth kinetic and on the bacterial H+-ATPase proton pumps. RESULTS: Phytochemical composition of the crude extract showed a rather selective distribution of secondary metabolites (presence of polyphenols, tannins, steroids, triterpenes, flavonoids, alkaloids, saponins and absence of anthocyanins, anthraquinones). The tested samples displayed different antibacterial activities with minimal inhibitory concentrations (MICs) ranging from 64 to 512 µg/mL. Crude extract (CS) and fraction CSc showed the highest inhibitory spectra, both inhibiting all of the studied bacteria except Enterobacter aerogenes EA27 strain. Fraction CSc exerted bactericidal effects on most bacteria meanwhile, crude extract (CS) and sub-fraction CSc2 exerted bacteriostatic effects. Compounds 1 (spectaline) and 2 (iso-6-cassine) inhibited the growth of 70% (Escherichia coli ATCC8739 and AG102, Klebsiella pneumoniae ATCC11296, Enterobacter aerogenes ATCC13048 and EA27, Providencia stuartii ATCC29916, Pseudomonas aeruginosa PA01) and 60% (Escherichia coli ATCC8739, Klebsiella pneumoniae ATCC11296 and KP55, Providencia stuartii ATCC29916, Pseudomonas aeruginosa PA01 and PA124) of bacteria respectively with MICs ranging from 128 to 512 µg/mL. In the presence of PAßN, the activities of crude extract CS, fraction CAc and sub-fraction CSc2 strongly increased on most bacteria strains as their MICs significantly decreased. Sub-fraction CSc2 inhibited the H+-ATPase proton pumps and altered growth kinetic of Escherichia coli ATCC8739. CONCLUSION: The overall results justify the traditional use of C. sieberiana for the treatment of bacterial infections.

YO-001A, a new antifungal agent produced by Streptomyces sp. YO15-A001.

A new antifungal compound YO-001A was found from the culture broth of Streptomyces sp. YO15-A001, which was isolated from a soil sample collected in Toyama Prefecture. YO-001A was identified through morphological changes-based screening of the rice blast fungus, Pyricularia oryzae (P. oryzae). YO-001A is a new 26-membered macrolide of the oligomycin family, which exhibits potent antifungal activity against P. oryzae with an IC50 of 0.012 µM by disrupting mitochondrial respiration via inhibition of the FOF1-ATPase activity.

Biomolecular proteomics discloses ATP synthase as the main target of the natural glycoside deglucoruscin.

Extracts of Ruscus aculeatus are a rich source of bioactive steroidal glycosides, such as ruscogenins which are reported to act against chronic venous disorders. Nowadays, several preparations of its roots, commonly used in traditional medicine, are on the market as food supplements for health care and maintenance. Although spirostanol deglucoruscin is one of the main metabolites in these extracts, literature reports about its pharmacological profile are scarce. In this paper, a multi-disciplinary approach, based on chemical proteomics, molecular modelling and bio-organic assays, has been used to disclose the whole interactome of deglucoruscin and the F0-F1 ATP synthase complex has been found as its main target.

Fungal plasma membrane H⁺-ATPase inhibitory activity of o-hydroxybenzylated flavanones and chalcones from Uvaria chamae P. Beauv.

In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H(+)-ATPase. Ethyl acetate extract of U. chamae was subjected to high-resolution fungal PM H(+)-ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR). This led to identification of a series of uncommon o-hydroxybenzylated flavanones and chalcones, i.e., chamanetin (8), isochamanetin (9), isouvaretin (10), uvaretin (11), dichamanetin (12), and diuvaretin (15). Preparative-scale isolation of the active metabolites allowed determination of IC50 values for inhibition of the PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. These revealed a strong correlation between o-hydroxybenzyl substituents and PM H(+)-ATPase activity, with dichamanetin being the most potent compound, but showing moderate activity in the fungal growth inhibition assays.

High-resolution screening combined with HPLC-HRMS-SPE-NMR for identification of fungal plasma membrane H(+)-ATPase inhibitors from plants.

Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H(+)-ATPase inhibition. This led to identification of 18 extracts showing more than 95% inhibition at a concentration of 7.5 mg/mL and/or a concentration-dependent activity profile. These extracts were selected for semi-high-resolution fungal PM H(+)-ATPase inhibition screening, and, on the basis of these results, Haplocoelum foliolosum (Hiern) Bullock and Sauvagesia erecta L. were selected for investigation by high-resolution fungal PM H(+)-ATPase inhibition screening. Structural analysis performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) led to identification of chebulagic acid (1) and tellimagrandin II (2) from H. foliolosum. Preparative-scale isolation of the two metabolites allowed determination of IC50 values for PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. Chebulagic acid and tellimagrandin II are both potent inhibitors of the PM H(+)-ATPase with inhibitory effect on the growth of S. cerevisiae.

Tonoplast of Beta vulgaris L. contains detergent-resistant membrane microdomains.

The experiments conducted on tonoplast of Beta vulgaris L. roots were performed to identify detergent-resistant lipid-protein microdomains (DRMs, interpreted as lipid rafts).The presence of DRMs can be found when dynamic clustering of sphingolipids, sterols, saturated fatty acids is registered, and the insolubility of these microdomains in nonionic detergents at low temperatures is proven. The elucidation of tonoplast microdomains has been based on results obtained with the aid of high-speed centrifuging in the sucrose gradient. The experiments have shown that tonoplast microdomains are rich in sphingolipids, free sterols and saturated fatty acids (such a lipid content is also typical of lipid-protein microdomains of other membranes), while only few phospholipids are present in tonoplast microdomains. The presence of microdomains has been confirmed by fluorescence and confocal microscopy using filipin and Laurdan as fluorescent probes. The experiments with Laurdan have shown that tonoplast microdomains are characterized by a high order compared to characteristics of the rest of the tonoplast. Thus, the presence of detergent-resistant lipid-protein microdomains in the tonoplast has been demonstrated.

Capsicum annuum L. trypsin inhibitor as a template scaffold for new drug development against pathogenic yeast.

A 6,000 Da peptide, named CaTI, was isolated from Capsicum annuum L. seeds and showed potent inhibitory activity against trypsin and chymotrypsin. The aim of this study was to determine the effect of CaTI on Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Kluyveromyces marxiannus cells. We observed that CaTI inhibited the growth of S. cerevisiae, K. marxiannus as well as C. albicans and induced cellular agglomeration and the release of cytoplasmic content. No effect on growth was observed in C. tropicalis but morphological changes were noted. In the spot assay, different degrees of sensitivity were shown among the strains and concentrations tested. Scanning electron microscopy showed that S. cerevisiae, K. marxiannus and C. albicans, in the presence of CaTI, exhibited morphological alterations, such as the formation of pseudohyphae, cellular aggregates and elongated forms. We also show that CaTI induces the generation of nitric oxide and interferes in a dose-dependent manner with glucose-stimulated acidification of the medium mediated by H(+)-ATPase of S. cerevisiae cells.

Antimicrobial actions of α-mangostin against oral streptococci.

The increasing prevalence of dental caries is making it more of a major world health problem. Caries is the direct result of acid production by cariogenic oral bacteria, especially Streptococcus mutans. New and better antimicrobial agents active against cariogenic bacteria are badly needed, especially natural agents derived directly from plants. We have evaluated the inhibitory actions of α-mangostin, a xanthone purified from ethanolic extracts of the tropical plant Garcinia mangostana L., by repeated silica gel chromatography. α-Mangostin was found to be a potent inhibitor of acid production by S. mutans UA159, active against membrane enzymes, including the F(H+)-ATPase and the phosphoenolpyruvate - sugar phosphotransferase system. α-Mangostin also inhibited the glycolytic enzymes aldolase, glyceraldehyde-3-phosphate dehydrogenase, and lactic dehydrogenase. Glycolysis by intact cells in suspensions or biofilms was inhibited by α-mangostin at concentrations of 12 and 120 µmol·L⁻¹, respectively, in a pH-dependent manner, with greater potency at lower pH values. Other targets for inhibition by α-mangostin included (i) malolactic fermentation, involved in alkali production from malate, and (ii) NADH oxidase, the major respiratory enzyme for S. mutans. The overall conclusion is that α-mangostin is a multitarget inhibitor of mutans streptococci and may be useful as an anticaries agent.

The diarylquinoline TMC207 for multidrug-resistant tuberculosis.

BACKGROUND: The diarylquinoline TMC207 offers a new mechanism of antituberculosis action by inhibiting mycobacterial ATP synthase. TMC207 potently inhibits drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro and shows bactericidal activity in patients who have drug-susceptible pulmonary tuberculosis. METHODS: In the first stage of a two-stage, phase 2, randomized, controlled trial, we randomly assigned 47 patients who had newly diagnosed multidrug-resistant pulmonary tuberculosis to receive either TMC207 (400 mg daily for 2 weeks, followed by 200 mg three times a week for 6 weeks) (23 patients) or placebo (24 patients) in combination with a standard five-drug, second-line antituberculosis regimen. The primary efficacy end point was the conversion of sputum cultures, in liquid broth, from positive to negative. RESULTS: The addition of TMC207 to standard therapy for multidrug-resistant tuberculosis reduced the time to conversion to a negative sputum culture, as compared with placebo (hazard ratio, 11.8; 95% confidence interval, 2.3 to 61.3; P=0.003 by Cox regression analysis) and increased the proportion of patients with conversion of sputum culture (48% vs. 9%). The mean log(10) count of colony-forming units in the sputum declined more rapidly in the TMC207 group than in the placebo group. No significant differences in average plasma TMC207 concentrations were noted between patients with and those without culture conversion. Most adverse events were mild to moderate, and only nausea occurred significantly more frequently among patients in the TMC207 group than among patients in the placebo group (26% vs. 4%, P=0.04). CONCLUSIONS: The clinical activity of TMC207 validates ATP synthase as a viable target for the treatment of tuberculosis. (ClinicalTrials.gov number, NCT00449644.)

The concerted nature between three catalytic subunits driving the F1 rotary motor.

F(1), a rotational molecular motor, shows strong cooperativity during ATP catalysis when driving the rotation of the central gamma subunit surrounded by the alpha(3)beta(3) subunits. To understand how the three catalytic beta subunits cooperate to drive rotation, we made a hybrid F(1) containing one or two mutant beta subunits with altered catalytic kinetics and observed its rotations. Analysis of the asymmetric stepwise rotations elucidated a concerted nature inside the F(1) complex where all three beta subunits participate to rotate the gamma subunit with a 120 degrees phase. In addition, observing hybrid F(1) rotations at various solution conditions, such as ADP, P(i) and the ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) provides additional information for each elementary event. This novel experimental system, which combines single molecule observations and biochemical methods, enables us to dynamically visualize the catalytic coordination inside active enzymes and shed light on how biological machines provide unidirectional functions and rectify information from stochastic reactions.

Effects of hexosamines and omega-3/omega-6 fatty acids on pH regulation by interleukin 1-treated isolated bovine articular chondrocytes.

Previous work has shown that interleukin 1 (IL-1) increases the activity of acid extruders in articular chondrocytes, while the H+-adenosine triphosphatase (ATPase) inhibitor bafilomycin can prevent aggrecanase-mediated cartilage degradation. The H+ transport induced by IL-1 may therefore be required for proteinase activity. In the present study, the effects of hexosamines and fish oils on H+-ATPase activity have been characterised for isolated bovine articular chondrocytes. Cells isolated in the presence of IL-1 were acidified, and the fraction of acid extrusion mediated by Na+-H+ exchange and an H+-ATPase were determined using specific inhibitors. Exposure to IL-1 significantly enhanced both components of acid extrusion. Co-incubation with glucosamine or mannosamine attenuated the H+-ATPase fraction of efflux. The addition of glucosamine at 9 h after exposure to IL-1--when H+-ATPase activation is already apparent--was also able to abolish H+-ATPase activity, implying that hexosamines do not exert effects at the level of protein synthesis. Co-incubation with the glucose transport inhibitor phloretin elicited similar effects to the hexosamines, suggesting that modulation of adenosine triphosphate levels may underlie their effects on H+-ATPase function. The omega-3 fish oil linolenic acid but not the omega-6 fish oil linoleic acid reduced H+-ATPase activity to levels seen in IL-1-untreated cells, although total efflux remained elevated, as a result of an enhanced H+ leak. These observations support a model whereby IL-1 stimulates an H+-ATPase-dependent system, possibly involved in aggrecanase activation, which appears to be one of the target mechanisms interrupted by dietary supplements reported to have symptom-modifying effects on osteoarthritis.

Mitochondrial toxicity and antioxidant activity of a prenylated flavonoid isolated from Dalea elegans.

The prenylated flavanone 2'-4'-dihidroxy-5'-(1" '-dimethylallyl)-6-prenylpinocembrin) (6PP), isolated from the roots of Dalea elegans, shows antimicrobial activity. The aim of this study was to evaluate mitochondrial toxicity and antioxidant properties of 6PP. Addition of micromolar concentrations of 6PP to rat liver mitochondria, stimulated O2 uptake in state 4 and inhibited it in state 3 when malate-glutamate was the respiratory substrate, and inhibited O2 uptake in state 3 when succinate was the substrate. Highest concentration of 6PP also inhibited O2 uptake in state 4 in the latter case; in both conditions, respiratory control index values were decreased. This flavanone collapsed the mitochondrial membrane potential in a concentration-dependent manner. 6PP also inhibited F0F1-ATPase activity in coupled mitochondria and in submitochondrial particles. In the latter, this compound also inhibited NADH oxidase and succinate dehydrogenase activities. HEp-2 cells were incubated for 24 h with 6PP in presence or absence of 0.5% albumin. As measured by reduction of the mitochondrial-related probe MTT, in the albumin-free condition, 6PP was cytotoxic in a concentration-dependent manner; on the other hand, albumin decreased 6PP effect. In addition, in rat liver microsomes 6PP: (1) inhibited the enzymatic lipid peroxidation, (2) exhibited significant scavenging activity, measured by DPPH reduction assay and (3) demonstrated significant antioxidant activity by decreasing the reduction of Mo(VI) to Mo(V). We suggest that 6PP impairs the hepatic energy metabolism by acting as mitochondrial uncoupler and by inhibiting enzymatic activities linked to the respiratory chain. 6PP also exerts both antioxidant and antiradical activities. Due to its cytotoxicity, this molecule, and its future structure developments, can be considered as a potentially promising therapeutic agent, for instance in cancer chemotherapy.

Target identification of drug induced mitochondrial toxicity using immunocapture based OXPHOS activity assays.

Mitochondrial dysfunction has been shown to be a pharmacotoxicological response to a variety of currently-marketed drugs. In order to reduce attrition due to mitochondrial toxicity, high throughput-applicable screens are needed for early stage drug discovery. We describe, here, a set of immunocapture based assays to identify compounds that directly inhibit four of the oxidative phosphorylation (OXPHOS) complexes: I, II, IV, and V. Intra- and inter-assay variation were determined and specificity tested by using classical mitochondrial inhibitors. Twenty drugs, some with known mitochondrial toxicity and others with no known mitochondrial liability, were studied. Direct inhibition of one or more of the OXPHOS complexes was identified for many of the drugs. Novel information was obtained for several drugs including ones with previously unknown effects on oxidative phosphorylation. A major advantage of the immunocapture approach is that it can be used throughout drug screening from early compound evaluation to clinical trials.

Effects of bafilomycin A1, a vacuolar type H+ ATPase inhibitor, on the thermosensitivity of a human pancreatic cancer cell line.

PURPOSE: It has been known that the thermosensitivity of tumour cells can be increased by lowering intra-cellular pH (pHi) by inhibiting pHi control mechanisms. The pHi is partially controlled by transport of H+ from cytoplasm into endocytic and secretary systems in the cells mediated by vacuolar type H+ATPase and also by transport of H+ through plasma membrane. METHODS: This study investigated the effects the bafilomycine A1, an inhibitor of the vacuolar type H+ATPase and the EIPA, an inhibitor of the Na+/H+ exchanger in plasma membrane, on thermosensitivity of AsPC-1 cells, a human pancreatic cancer cell line. It also investigated the effects of combination of bafilomycine A1 and EIPA. RESULTS: The treatment of cancer cells with bafilomycine A1 or EIPA individually slightly lowered pHi of the cells in vitro and increased the thermosensitivity of the cells. CONCLUSION: The combination of these two drugs significantly lowered pHi and increased thermosensitivity of cancer cells in vitro and enhanced the heat-induced the growth delay of AsPC-1 tumours grown s.c in the legs of BALB/cA nude mice.

Low-dose rabeprazole, amoxicillin and metronidazole triple therapy for the treatment of Helicobacter pylori infection in Chinese patients.

BACKGROUND: Rabeprazole in combination with amoxicillin and metronidazole (RAM) has been shown to be an effective second-line treatment of Helicobacter pylori infection. The effects were compared of 7-day low-dose and high dose rabeprazole in RAM for the primary treatment of H. pylori infection in Chinese patients. METHODS: Helicobacter pylori-positive dyspeptic patients were randomized to receive either (i) rabeprazole 10 mg, amoxicillin 1000 mg and metronidazole 400 mg (RAM-10) or (ii) high-dose rabeprazole 20 mg, amoxicillin 1000 mg and metronidazole 400 mg (RAM-20), each given twice daily for 7 days. Helicobacter pylori eradication was confirmed by (13)c-urea breath test 5 weeks after stopping medications. side-effects of treatments were documented. RESULTS: A total of 120 patients were eligible for analysis. By intention-to-treat and per-protocol analysis, the eradication rates were 83% and 86% in the RAM-10 group and 75% and 76% in the RAM-20 group, respectively (P = 0.26 and P = 0.17). Both regimens were well-tolerated and compliance was >98% in both groups. CONCLUSIONS: Low-dose rabeprazole in combination with amoxicillin and metronidazole is an effective, economical and well-tolerated therapy for the treatment of H. pylori infection in Chinese population.

[The role of H+/- ATPase and alternative oxidase in the regulation of intracellular pH at the different stages of development of the tobacco male gametophyte]. / Rol' H+/- ATFazy i al'ternativnoi oksidazy v reguliatsii velichiny vnutrikletochnogo pH na raznykh stadiiakh razvitiia muzhskogo gametofita tabaka.

In order to determine the role of the plasma membrane H(+)-ATPase and alternative oxidase (alternative pathway of respiration) in the regulation of intracellular pH during development of the tobacco male gametophyte, we studied the changes in pH due to the inhibition of these enzymes by orthovadanate and benzhydroxamic acid, respectively. The inhibition of these enzymes decreased the intracellular pH at all three studied stages of the male gametophyte development: middle and late binuclear pollen grains and activated mature pollen grain. The data obtained suggest that H(+)-ATPase and alternative oxidase are involved in the regulation of intracellular pH of the pollen grain during its differentiation and activation that precede germination. At the same time, during the recovery of intracellular pH after its acidification by propionic acid, it was found that other mechanisms, not related to the above mentioned, greatly contribute to the regulation of pH.

A prospective evaluation of esophageal testing and a double-blind, randomized study of omeprazole in a diagnostic and therapeutic algorithm for chronic cough.

OBJECTIVE: Recent studies suggest an association between chronic cough and gastroesophageal reflux. Our study aims were 1) to define the prevalence of acid reflux induced cough in the general community, 2) to examine the ability of esophageal testing to identify gastroesophageal reflux related cough, and 3) to assess the utility of omeprazole in a chronic cough algorithm. METHODS: Patients with chronic cough of unknown etiology, who were mostly from the community, were evaluated. Subjects underwent a chest x-ray, methacholine challenge test, and empiric trial of postnasal drip therapy, and completed daily cough symptom diaries subjectively evaluating cough frequency and severity on a graded scale of 0-4 (combined maximum 8). After excluding other causes of cough, the remaining patients underwent esophageal and pH testing. Those testing positive were randomized to omeprazole 40 mg b.i.d. or placebo for 12 weeks. Follow-up was 1 yr. RESULTS: A total of 71 patients were screened; 48 were excluded. Twenty-three patients were evaluated for gastroesophageal reflux disease; six (26%) were eventually determined to have an acid-related cough. Of these patients, 17 had a positive pH test, six (35%) of whom showed a striking improvement or resolution of their cough during omeprazole treatment which was sustained for up to 1 yr. Six had a negative pH test, none of whom responded to omeprazole therapy. No significant differences were seen between responders (n = 6) and nonresponders (n = 11) for demographic factors, baseline symptom frequency and duration, or physiological parameters (motility/pH). CONCLUSIONS: Acid-related chronic cough was present in 26% (six of 23) of patients evaluated for gastroesophageal reflux disease. Esophageal testing does not reliably identify patients with acid induced chronic cough responsive to proton pump inhibitor therapy. We suggest that the best diagnostic and therapeutic approach, after excluding asthma and postnasal drip syndrome, is empiric treatment for 2 wk with a high dose proton pump inhibitor.

Kup is the major K+ uptake system in Escherichia coli upon hyper-osmotic stress at a low pH.

The K+ uptake was observed in washed cells of Escherichia coli, wild-type, upon hyper-osmotic stress at pH 5.5 when glucose was supplemented. This uptake had apparent a Km of 0.58 mM and Vmax of 0.10 micromol K+/min/mg protein. Such a K+ uptake was investigated using a mutant defective in Kdp and TrkA but with a functional Kup and a mutant defective in Kdp and Kup but having an active TrkA. The K+ uptake to reach the steady state level as well as the initial K+ influx rate in the first mutant were at least 3.5-fold greater than these values with the second mutant and similar to those of the wild-type. Such differences in the K+ uptake activity were correlated with K+ requirements for growth of these mutants. Moreover, the K+ uptake in the wild-type was blocked by a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Valinomycin, arsenate and N,N'-dicyclohexylcarbodiimide were not effective in changing the K+ uptake. It is suggested that Kup is the major K+ uptake system in E. coli upon hyper-osmotic stress at a low pH.

Up-regulation of vascular endothelial growth factor in response to glucose deprivation.

Vascular endothelial growth factor (VEGF), also known as a vascular permeability factor (VPF), is an endothelial specific mitogen and is a potent inducer of angiogenesis. Recently it has been reported that hypoxia induces VEGF mRNA expression in various cells. Since both oxygen and glucose are required for efficient production of energy, we examined the effect of glucose deprivation on VEGF mRNA expression and VEGF protein production in U-937 (a human monocytic cell line) cells. Both the mRNA expression and secretion of VEGF increased after exposure to low glucose. Addition of L-glucose, the L-stereoisomer of D-glucose, did not prevent the up-regulation of VEGF expression. The conditioned medium from glucose-deprived cells, followed by supplementation with glucose, did not up-regulate VEGF mRNA expression in U-937 cells. The low glucose-induced VEGF mRNA expression returned to the control level after supplementation with D-glucose. Furthermore, oligomycin, a mitochondrial ATP synthase inhibitor, increased VEGF protein production. The results suggest that the up-regulation of VEGF mRNA in U-937 cells in response to glucose deprivation is not mediated by autocrine factors from the cells nor is the osmotic change of the medium mediated by the deficiency of glucose metabolism in the cells. Our results also suggest that the intracellular ATP depletion due to glucose deprivation may be one of the causes for increased VEGF mRNA expression. We speculate that local hypoglycemia may act as an essential trigger for angiogenesis through the VEGF gene expression.