Impact of specific inhibitors on metallo-ß-carbapenemases detected in Escherichia coli and Klebsiella pneumoniae isolates.

Carbapenems are widely regarded as the drugs of choice for the treatment of severe infections caused by extended-spectrum beta lactamases producing Enterobacteriaceae. The emergence of carbapenem-resistant organisms is worrisome due to the limited treatment options. Detection of carbapenemase-producing bacteria is critical for the choice of appropriate therapy. However, Inhibition of carbapenemases is an alternative approach to combat resistance to carbapenms. In this study, Escherichia coli and Klebsiella pneumoniae carbapenem resistant isolates were recovered from 300 clinical isolates. They were subjected phenotypically for detection of class B metallo-carbapenemase (MBL) producers (by carbapenem disks with or without EDTA), and were subjected for confirmation genotypically by PCR. In addition, the synergistic activities of MBL-inhibitors in combination with carbapenems were elucidated. Two E. coli and 15 K. pneumoniae isolates were carbapenem resistant. The genes encoding blaNDM-1 carbapenemase were detected in 16/17 isolates solely, or collaboratively with either blaVIM, or blaIMP or both in all carbapenem resistant isolates, by PCR method. The VIM-carbapenemase was encoded by one isolate. In pre-clinical trials for development of MBL-specific inhibitors, Sub-inhibitory concentrations of citric acid, malic acid, ascorbic acid and ciprofloxacin in combination with imipenem or meropenem exerted synergistic activities against metallo-carbapenemases. Their activities are probably attributed to the chelation of zinc ions in the active site of carbapenemase. Conclusively, these promising combined therapies might represent a new strategy for combating such serious infections caused by metallo-B-carbapenemase producers of K. pneumoniae and E. coli isolates.

Opposite effects of vitamin C and vitamin E on the antifungal activity of honokiol.

The aim of the present study was to evaluate the effects of two well-known natural antioxidants vitamin C (VC) and vitamin E (VE) on the antifungal activity of honokiol against Candida albicans. The broth microdilution method was employed to test the antifungal activities of honokiol with or without antioxidants in the medium against C. albicans strain. Intracellular reactive oxygen species (ROS) and lipid peroxidation were determined by fluorescence staining assay. Mitochondrial dysfunction was assessed by detecting the mitochondrial DNA and the mitochondrial membrane potential. We observed that VC could significantly potentiate the antifungal activities of honokiol while VE reduced the effectiveness of honokiol against C. albicans. In addition, VC accelerated honokiol-induced mitochondrial dysfunction and inhibited glycolysis leading to a decrease in cellular ATP. However, VE could protect against mitochondrial membrane lipid peroxidation and rescue mitochondrial function after honokiol treatment. Our research provides new insight into the understanding of the action mechanism of honokiol and VC combination against C. albicans.

Ascorbic acid inhibits visceral obesity and nonalcoholic fatty liver disease by activating peroxisome proliferator-activated receptor α in high-fat-diet-fed C57BL/6J mice.

BACKGROUND/OBJECTIVES: Ascorbic acid is a known cofactor in the biosynthesis of carnitine, a molecule that has an obligatory role in fatty acid oxidation. Our previous studies have demonstrated that obesity is regulated effectively through peroxisome proliferator-activated receptor α (PPARα)-mediated fatty acid ß-oxidation. Thus, this study aimed to determine whether ascorbic acid can inhibit obesity and nonalcoholic fatty liver disease (NAFLD) in part through the actions of PPARα. DESIGN: After C57BL/6J mice received a low-fat diet (LFD, 10% kcal fat), a high-fat diet (HFD, 45% kcal fat), or the same HFD supplemented with ascorbic acid (1% w/w) (HFD-AA) for 15 weeks, variables and determinants of visceral obesity and NAFLD were examined using metabolic measurements, histology, and gene expression. RESULTS: Compared to HFD-fed obese mice, administration of HFD-AA to obese mice reduced body weight gain, visceral adipose tissue mass, and visceral adipocyte size without affecting food consumption profiles. Concomitantly, circulating ascorbic acid concentrations were significantly higher in HFD-AA mice than in HFD mice. Ascorbic acid supplementation increased the mRNA levels of PPARα and its target enzymes involved in fatty acid ß-oxidation in visceral adipose tissues. Consistent with the effects of ascorbic acid on visceral obesity, ascorbic acid not only inhibited hepatic steatosis but also increased the mRNA levels of PPARα-dependent fatty acid ß-oxidation genes in livers. Similarly, hepatic inflammation, fibrosis, and apoptosis were also decreased during ascorbic acid-induced inhibition of visceral obesity. In addition, serum levels of alanine aminotransferase, aspartate aminotransferase, total cholesterol, and LDL cholesterol were lower in HFD-AA-fed mice than in those of HFD-fed mice. CONCLUSIONS: These results suggest that ascorbic acid seems to suppress HFD-induced visceral obesity and NAFLD in part through the activation of PPARα.

Potential pro-oxidative effects of single dose of mephedrone in vital organs of mice.

BACKGROUND: Mephedrone is a recreationally used synthetic cathinone, relatively new abusive substances with molecular structure similar to amphetamine. As there is still lack of scientific data regarding mechanisms of action as well as metabolism of mephedrone, especially in aspects other than neurotoxicity, addiction or behavioral changes, therefore we aimed, for the first time, to investigate potential pro-oxidative actions of a single dose of mephedrone in organs other than brain and its structures, i.e. in liver, kidneys, heart and spleen of Swiss mice. METHODS: The following biomarkers of oxidative stress were measured: concentration of ascorbic acid (AA) and malondialdehyde (MDA) as well as total antioxidant capacity (TAC) of the tissues homogenates. RESULTS: Our study revealed that mephedrone intoxication induces oxidative stress by reducing concentration of AA and TAC and increasing concentration of MDA in these organs. CONCLUSIONS: Such occurred state of antioxidant-oxidant imbalance may be etiopathological factor of a number of severe diseases within cardiovascular, digestive as well as immunological systems.

Mannich-Benzimidazole Derivatives as Antioxidant and Anticholinesterase Inhibitors: Synthesis, Biological Evaluations, and Molecular Docking Study.

A series of Mannich bases of benzimidazole derivatives having a phenolic group were designed to assess their anticholinesterase and antioxidant activities. The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities were evaluated in vitro by using Ellman's method. According to the activity results, all of the compounds exhibited moderate to good AChE inhibitory activity (except for 2a), with IC50 values ranging from 0.93 to 10.85 µM, and generally displayed moderate BuChE inhibitory activity. Also, most of the compounds were selective against BuChE. Compound 4b was the most active molecule on the AChE enzyme and also selective. In addition, we investigated the antioxidant effects of the synthesized compounds against FeCl2 /ascorbic acid-induced oxidative stress in the rat brain in vitro, and the activity results showed that most of the compounds are effective as radical scavengers. Molecular docking studies and molecular dynamics simulations were also carried out.

U937 cell apoptosis induced by arsenite is prevented by low concentrations of mitochondrial ascorbic acid with hardly any effect mediated by the cytosolic fraction of the vitamin.

Arsenite directly triggers cytochrome c and Smac/DIABLO release in mitochondria isolated from U937 cells. These effects were not observed in mitochondria pre-exposed for 15 min to 10 µM L-ascorbic acid (AA). In other experiments, intact cells treated for 24-72 h with arsenite were found to die by apoptosis through a mechanism involving mitochondrial permeability transition. Pre-exposure (15 min) to low micromolar concentrations of AA and dehydroascorbic acid (DHA), resulting in identical cytosolic levels of the vitamin, had a diverse impact on cell survival, as cytoprotection was only observed after treatment with AA. Also the mitochondrial accumulation of the vitamin was restricted to AA exposure. An additional indication linking cytoprotection to the mitochondrial fraction of the vitamin was obtained in experiments measuring susceptibility to arsenite in parallel with loss of mitochondrial and cytosolic AA at different times after vitamin exposure. Finally, we took advantage of our recent findings that DHA potently inhibits AA transport to demonstrate that DHA abolishes all the protective effects of AA, under the same conditions in which the mitochondrial accumulation of the vitamin is prevented without affecting the overall cellular accumulation of the vitamin.

Ascorbic acid enhances adipogenesis of 3T3-L1 murine preadipocyte through differential expression of collagens.

BACKGROUND: Adipogenesis from preadipocytes into mature adipocyte is precisely coordinated by transcription factors such as CCAAT-enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor γ (PPARγ), cytokines, and hormones, which is accompanied by extracellular matrix remodeling. Besides anti-oxidant activity, ascorbic acid (ASC) is participating in collagen biosynthesis and increase production and processing of collagens. Moreover, several studies demonstrated that ASC enhanced differentiation from preadipocytes into mature adipocytes. METHODS: The adipogenic effect of ascorbic acid was evaluated in chemical induced 3T3-L1 by Oil Red O staining. This effect was elucidated by immunoblotting which detected the expression level of collagens and transcription factors in adipogenesis. The immunocytochemical determination of type I collagen was performed in 3T3-L1 adipocyte to show the change of extracellular matrix during adipogenesis. RESULTS: In this study, Oil Red O staining in 3T3-L1 preadipocytes was increased dose-dependently by addition of ASC. These ASC-treated adipocytes increased collagen processing of α1(I) and α1(V) and expressed α1(VI) and α2(VI) collagens differentially. ASC also stimulated expression of C/EBPα and PPARγ, which is preceded by collagen enhancement. In addition, inhibition of ASC activity by ethyl-3,4-dihydroxybenzoate showed reduction of lipid accumulation by removal of large lipid droplets, not by inhibition of lipid production. This observation went with loss of α1(I) deposition on adipocyte surface, increase of α1(V) and α2(VI) collagens and decrease of C/EBPs. CONCLUSION: Our findings imply that various actions of ASC on adipogenesis through differential collagen expression may provide diverse applications of ASC to adipose tissue technology.

Dose-dependent effects of nicotine on proliferation and differentiation of human bone marrow stromal cells and the antagonistic action of vitamin C.

A range of biological and molecular effects caused by nicotine are considered to effect bone metabolism. Vitamin C functions as a biological antioxidant. This study was to evaluate the in vitro effects of nicotine on human bone marrow stromal cells and whether Vitamin C supplementation show the antagonism action to high concentration nicotine. We used CCK-8, alkaline phosphatase (ALP) activity assay, Von Kossa staining, real-time polymerase chain reaction and Western Blot to evaluate the proliferation and osteogenic differentiation. The results indicated that the proliferation of BMSCs increased at the concentration of 50, 100 ng/ml, got inhibited at 1,000 ng/ml. When Vitamin C was added, the OD for proliferation increased. For ALP staining, we found that BMSCs treated with 50 and 100 ng/ml nicotine showed a higher activity compared with the control, and decreased at the 1,000 ng/ml. Bone morphogenetic protein-2 (BMP-2) expression and the calcium depositions decreased at 100 and 1,000 ng/ml nicotine, while the addition of Vitamin C reversed the down regulation. By real-time PCR, we detected that the mRNA expression of collagen type I (COL-I) and ALP were also increased in 50 and 100 ng/ml nicotine groups (P < 0.05), while reduced at 1,000 ng/ml (P < 0.05). When it came to osteocalcin (OCN), the changes were similar. Taken all together, it is found that nicotine has a two-phase effect on human BMSCs, showing that low level of nicotine could promote the proliferation and osteogenic differentiation while the high level display the opposite effect. Vitamin C could antagonize the inhibitory effect of higher concentration of nicotine partly.

[Mouse melanoma cell line B16F10-derived conditioned medium inhibits sodium L-ascorbate-induced B16F10 cell apoptosis].

OBJECTIVE: To investigate the effect of mouse melanoma cell line B16F10-derived conditioned medium on the apoptosis of B16F10 cells. METHODS: B16F10 cells were cultured in high-glucose DMEM in the presence of 10% fetal bovine serum, and upon cell confluence, the growth medium was replaced with serum-free high-glucose DMEM. After 8 h, the medium was collected and infiltrated to serve as the conditioned medium. B16F10 cells cultured in normal growth medium or the conditioned medium were exposed to 10 mmol/L sodium L-ascorbate, and the cell apoptosis was analyzed. The ingredients in the conditioned medium with relative molecular mass less or more than 5 000 were extracted to assess their effect on sodium L-ascorbate-induced cell apoptosis. RESULTS: The conditioned medium for B16F10 cells significantly inhibited cell apoptosis induced by sodium L-ascorbate, and the effective ingredients in the medium showed a relative molecular mass below 5,000. CONCLUSION: Mouse melanoma cell line B16F10-derived conditioned medium can suppress sodium L-ascorbate- induced apoptosis of B16F10 cells, and the ingredients with relative molecular mass less than 5 000 are responsible for this effect.

Ciprofloxacin-induced antibacterial activity is reversed by vitamin E and vitamin C.

In the present study, we investigated the possible involvement of oxidative stress in ciprofloxacin-induced cytotoxicity against several reference bacteria including Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). Oxidative stress was assessed by measurement of hydrogen peroxide generation using a FACScan flow cytometer. The antibacterial activity of ciprofloxacin was assessed using the disk diffusion method and by measuring the minimum inhibitory concentration (MIC). Ciprofloxacin induced a dose-dependent antibacterial activity against all bacteria where the highest tested concentration was 100 ug/ml. Results revealed that E. coli cells were highly sensitive to ciprofloxacin (MIC = 0.21 µg/mL ± 0.087), P. aeruginosa and S. aureus cells were intermediately sensitive (MIC = 5.40 µg/mL ± 0.14; MIC = 3.42 µg/mL ± 0.377, respectively), and MRSA cells were highly resistant (MIC = 16.76 µg/mL ± 2.1). Pretreatment of E. coli cells with either vitamin E or vitamin C has significantly protected cells against ciprofloxacin-induced cytotoxicity. These results indicate the possible antagonistic properties for vitamins C or E when they are used concurrently with ciprofloxacin.

Up-regulation of GLT1 reverses the deficit in cortically evoked striatal ascorbate efflux in the R6/2 mouse model of Huntington's disease.

A corticostriatal-dependent deficit in the release of ascorbate (AA), an antioxidant vitamin and neuromodulator, occurs concurrently in striatum with dysfunctional GLT1-dependent uptake of glutamate in the R6/2 mouse model of Huntington's disease (HD), an autosomal dominant condition characterized by overt corticostriatal dysfunction. To determine if deficient striatal AA release into extracellular fluid is related to altered GLT1 activity in HD, symptomatic R6/2 mice between 6 and 9 weeks of age and age-matched wild-type (WT) mice received single daily injections of 200 mg/kg ceftriaxone, a ß-lactam antibiotic that elevates the functional expression of GLT1, or saline vehicle for five consecutive days. On the following day, in vivo voltammetry was coupled with corticostriatal afferent stimulation to monitor evoked release of AA into striatum. In saline-treated mice, we found a marked decrease in evoked extracellular AA in striatum of R6/2 relative to WT. Ceftriaxone, in contrast, restored striatal AA in R6/2 mice to WT levels. In addition, intra-striatal infusion of either the GLT1 inhibitor dihydrokainic acid or dl-threo-beta-benzyloxyaspartate blocked evoked striatal AA release. Collectively, our results provide compelling evidence for a link between GLT1 activation and release of AA into the striatal extracellular fluid, and suggest that dysfunction of this system is a key component of HD pathophysiology.

Copper-induced synthesis of ascorbate, glutathione and phytochelatins in the marine alga Ulva compressa (Chlorophyta).

In order to analyze the synthesis of antioxidant and heavy metal-chelating compounds in response to copper stress, the marine alga Ulva compressa (Chlorophyta) was exposed to 10 µM copper for 7 days and treated with inhibitors of ASC synthesis, lycorine, and GSH synthesis, buthionine sulfoximine (BSO). The levels of ascorbate, in its reduced (ASC) and oxidized (DHA) forms, glutathione, in its reduced (GSH) and oxidized (GSSG) forms, and phytochelatins (PCs) were determined as well as activities of enzymes involved in ASC synthesis, L-galactose dehydrogenase (GDH) and L-galactono 1,4 lactone dehydrogenase (GLDH), and in GSH synthesis, γ-glutamylcysteine synthase (γ-GCS) and glutathione synthase (GS). The level of ASC rapidly decreased to reach a minimum at day 1 that remained low until day 7, DHA decreased until day 1 but slowly increased up to day 7 and its accumulation was inhibited by lycorine. In addition, GSH level increased to reach a maximal level at day 5 and GSSG increased up to day 7 and their accumulation was inhibited by BSO. Activities of GDH and GLDH increased until day 7 and GLDH was inhibited by lycorine. Moreover, activities of γ-GCS and GS increased until day 7 and γ-GCS was inhibited by BSO. Furthermore, PC2, PC3 and PC4, increased until day 7 and their accumulation was inhibited by BSO. Thus, copper induced the synthesis of ascorbate, glutathione and PCs in U. compressa suggesting that these compounds are involved in copper tolerance. Interestingly, U. compressa is, until now, the only ulvophyte showing ASC, GSH and PCs synthesis in response to copper excess.

Antioxidant properties of argpyrimidine.

Argpyrimidine, the product of non-enzymatic protein glycation by methylglyoxal, has been implicated in the pathophysiology of diabetes mellitus and neurodegenerative diseases. Chemically, argpyrimidine is a substituted pyrimidinol with structural features common to known antioxidants. The objective of this study was to investigate the antioxidant properties of argpyrimidine. Argpyrimidine was synthesized by mixing L-arginine with 3-acetoxypentane-2,4-dione under acidic conditions and purified by chromatography. Argpyrimidine inhibited lipid peroxidation of rat brain homogenates catalyzed by hydroxyl radicals, metal ions, and autooxidation in a concentration- and time-dependent manner. In addition, argpyrimidine scavenged superoxide anion, 1,1-diphenyl 2-picryl-hydrazyl-stable free radical, intracellular-hydrogen peroxide, and inhibited free-radical-mediated nicking of plasmid-DNA. Taken together, our data suggest that argpyrimidine has antioxidant properties and may therefore have biological relevance in pathophysiologies associated with diabetes mellitus and neurodegenerative diseases.

Development of a new class of potential antiatherosclerosis agents: NO-donor antioxidants.

A new class of NO-donor phenol derivatives is described. The products were obtained by joining appropriate phenols with either nitrooxy or 3-phenylsulfonylfuroxan-4-yloxy moieties. All the compounds proved to inhibit the ferrous salt/ascorbate induced lipidic peroxidation of membrane lipids of rat hepatocytes. They were also capable of dilating rat aorta strips precontracted with phenylephrine.

Functional expression of sodium-dependent vitamin C transporter 2 in human endothelial cells.

Since oxidative stress plays an important role in dysregulation of the microcirculation as well as the pathogenesis of atherosclerosis, therapeutic intervention with antioxidants has been speculated to prevent cardiovascular diseases. Ascorbic acid (AA) has been reported to improve endothelial function; however, its intracellular metabolic pathway has not been fully determined. Sodium-dependent vitamin C transporter (SVCT) types 1 and 2 were recently cloned. In the present study, we investigated whether SVCT-2 is functionally expressed in vascular endothelial cells and, if so, what factors modulate its activity. The uptake of AA into human umbilical vein endothelial cells (HUVECs) was examined by incubation with radiolabeled AA (14C-AA). AA was transported into HUVECs in a dose- and time-dependent manner. Replacement of sodium chloride with choline chloride in the medium suppressed the uptake of AA. RT-PCR revealed that HUVECs expressed SVCT-2 mRNA, but not SVCT-1. Transfection of HUVECs with the antisense oligonucleotide of SVCT-2 significantly suppressed the uptake of AA. Furthermore, tumor necrosis factor-alpha and interleukin-1beta inhibited the transport activity of AA. Thus, SVCT-2 is functionally expressed in human endothelial cells, and its activity is negatively regulated by inflammatory cytokines. Our findings might provide a new insight into understanding the treatment of cardiovascular diseases with AA.

Inhibition of stimulated ascorbic acid and luteinizing hormone-releasing hormone release by nitric oxide synthase or guanyl cyclase inhibitors.

Ascorbic acid (AA), an antioxidant, is present in high concentrations in the hypothalamus. Previously, we have shown that AA inhibited stimulated release of luteinizing hormone-releasing hormone (LHRH) from medial basal hypothalami in vitro. We have also demonstrated that cell membrane depolarization by high [K(+)] media-induced AA release that is blocked by N(G)-mono-methyl-L-arginine, a competitive inhibitor of nitric oxide synthase (NOS), indicating that the release process is mediated by NO. The release of LHRH is also mediated by NO. We hypothesized that AA is a co-transmitter released with classical transmitters from synaptic vesicles that acts to reduce chemically the NO formed, thereby providing feed-forward inhibitory control over LHRH release. Because NO acts by activating guanylyl cyclase (GC) resulting in production of cGMP, in the present investigation we studied the effects of an NOS inhibitor LY 83583 and GC inhibitor, O.D.Q. to further characterize the role of NO in high [K(+)]-induced AA and LHRH release. Medial basal hypothalami were incubated in 0.5 ml of Krebs-Ringer Bicarbonate buffer or medium containing increased potassium [K(+) = 56 mM] for 1 hr or combinations of high [K(+)] + LY 83583 or O.D.Q. for 1 hr. AA and LHRH released into the incubation medium were measured by high-pressure liquid chromatography and radioimmunoassay, respectively. Cell membrane depolarization with high [K(+)] produced a significant increase in both AA and LHRH release. A combination of high [K(+)] + LY 83583 or high [K(+)] + O.D.Q. decreased basal AA and completely blocked high [K(+)]-induced AA and LHRH release. As in the case of high [K(+)], LHRH release induced by the excitatory amino acid N-methyl-D-aspartic acid (NMDA) was blocked by both the inhibitors. NMDA alone failed to alter AA release, but the combined presence of NMDA and the inhibitors totally blocked AA release. Because LY 83583 and O.D.Q. were shown to inhibit NOS and soluble GC, respectively, the data demonstrate that basal and high [K(+)]-induced AA and high [K(+)] and NMDA-stimulated LHRH release were mediated by NO by its activation of GC and consequent generation of cGMP.

Vitamin C activity in guinea pigs of 6-O-acyl-2-O-alpha-D-glucopyranosyl-L- ascorbic acids with a branched-acyl chain.

A series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain (6-bAcyl-AA-2G) were recently developed in our laboratory as stable and lipophilic ascorbate derivatives. In this study, the bioavailability of 6-bAcyl-AA-2G was investigated in guinea pigs. Various tissue homogenates from guinea pigs hydrolyzed 6-bAcyl-AA-2G to give ascorbic acid (AA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), and 6-O-acyl AA. The releasing pattern of the three hydrolysates suggested that 6-bAcyl-AA-2G was hydrolyzed via 6-O-acyl AA to AA as a main pathway and via AA-2G to AA as a minor pathway. The former pathway seems to be of advantage, because 6-O-acyl AA, as well as AA, can have vitamin C activity. In addition, we found that a derivative with an acyl chain of C(12), 6-bDode-AA-2G, had a pronounced therapeutic effect in scorbutic guinea pigs by its repeated oral administrations. These results indicate that 6-bAcyl-AA-2G is a readily available source of AA in vivo, and may be a promising antioxidant for skin care and treatment of diseases associated with oxidative stress.

The natural antioxidant otobaphenol delays the permeability transition of mitochondria and induces their aggregation.

The lignan otobaphenol, (8R,8'R,7R)-4'-hydroxy-5'-methoxy-3,4-methylenedioxy-2',7,8,8'-neolignan, extracted from Virola Aff. Pavonis leaves, completely inhibits at a concentration of 2.5 micro M the Fe(3+)-ascorbate-induced lipoperoxidation of rat liver mitochondria that was determined by oxygen consumption and accumulation of thiobarbituric acid-reactive species. At 25 micro M, it delays the mitochondrial permeability transition induced by tert-butyl hydroperoxide or Ca(2+), substantially inhibits the state 3 respiration, does not affect the state 4 respiration and the ADP/O ratio (with succinate), diminishes the rate of Ca(2+) uptake by mitochondria, and delays the ruthenium red-insensitive uncoupler-induced release of the loaded Ca(2+). Dose-dependent delaying of the calcium-induced swelling of mitochondria in the presence of otobaphenol nonlinearly correlates with its 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity. At 75 micro M and higher, this lignan causes mitochondrial aggregation and is able to aggregate itself, without mitochondria. The formed aggregates of otobaphenol do not cause an aggregation of subsequently added mitochondria. Thus, otobaphenol seems to be a promising target to prevent the oxidative stress death of cells.

Reversal of P-glycoprotein expressed in Escherichia coli leaky mutant by ascorbic acid.

It has been reported that functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in E. coli is useful for screening P-gp substrates and inhibitors. In the present study, we have constructed by nitrosoguanidine and UV mutagenesis 28 leaky mutants of E. coli UT5600. These mutants are significantly susceptible to the toxic effect of known P-gp substrates and lipophilic cancer drugs. Mouse mdr1 was functionally expressed in the most permeable E. coli mutant (UTP17). Expression of P-gp in this mutant confers cross-resistance to mitomycin C, tegafur, daunorubicin, rhodamine 6G, tetraphenylphosphonium bromide and ciprofloxacin. To examine the reversal of P-gp expressed in this heterologous system, UTP17 cells expressing mouse mdr1 or lac permease as negative control were treated with various concentrations of mitomycin C with or without ascorbic acid. We found that ascorbic acid abrogated P-gp mediated multidrug resistance, suggesting that ascorbic acid might be used in combination with anticancer drugs to reduce emergence of multidrug resistance. We also demonstrated that tomato lectin antagonized the inhibitory action of ascorbic acid. This study provide a heterologous system for mdr1 expression in E. coli leaky mutant that can be used as a system for the screening of P-gp inducers and inhibitors, since it is quick and simple.

Inhibition of ascorbic acid-induced modifications in lens proteins by peptides.

The effects of three dipeptides L-phenylalanyl-glybine, glycyl-L-phenylalanine,and aspartame (L-aspartyl-L-phenylalanine, methyl ester) as inhibitors of the ascorbic acid-induced modifications in lens proteins were studied. Their efficiency was compared to that of two known inhibitors--aminoguanidine and carnosine. The tested dipeptides diminished protein carbonyl content by 32-58% and most moderated the formation of chromophores, as measured by the absorbency at 325 nm of the glycated proteins. The appearance of non-tryptophan fluorescence (excitation 340 nm/emission 410 nm) was observed for proteins glycated with ascorbic acid. All of the dipeptides examined, as well as aminoguanidine, decreased this glycation-related fluorescence. The potential inhibitors prevented the intensive formation of very high molecular weight aggregates. A competitive mechanism of their inhibitory effect was proposed, based on the reactivity of individual substances toward ascorbic acid. These findings indicate that they have a potential for use as alternatives for aminoguanidine as an anti-glycation agent.