Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in <Emphasis Type="Italic">Taxus chinensis</Emphasis> cultures

The roles of glucose-6-phosphate dehydrogenase (G6PDH) in paclitaxel production were investigated in cell suspension cultures of Taxus chinensis. In the normal cultures, the trend of G6PDH activity was similar to that of cell growth. Addition of glutamate increased G6PDH activity, while dehydroepiandrosterone (DHEA) decreased G6PDH activity. In elicitor-treated cultures, cell growth was depressed, while G6PDH activity and taxol production were enhanced compared with the control. Glutamate recovered the depression of cell growth, and resulted in further increase in G6PDH activity and taxol production. Contrarily, DHEA exacerbated the depression of cell growth, and decreased G6PDH activity and taxol production induced by fungal elicítor. The results indicated that G6PDH played a critic role of taxol production by affecting cell viability.     

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.     

Effects of dehydroepiandrosterone on obesity and glucose-6-phosphate dehydrogenase activity in the lethal yellow mouse (strain 129/Sv-Ay/Aw)

We investigated the anti-obesity effects of the adrenal androgen, dehydroepiandrosterone (DHEA), on genetically predisposed obese lethal yellow mice (Ay/Aw). Secondly, we tested the hypothesis that DHEA promotes its anti-obesity effects by decreasing the activity of glucose-6-phosphate dehydrogenase (G6PDH). We subjected four genotype-sex combinations of yellow and agouti (control) mice to four dietary treatments and determined weight changes, food consumption, and G6PDH activity. Although G6PDH activities of yellow mice were considerably decreased in the 0.4% DHEA treatment group, they were elevated in the 0.0 and 0.1% DHEA treatment groups. In contrast, G6PDH activities of DHEA-treated control agouti mice remained relatively constant. These studies confirm that DHEA prevents the Ay gene from promoting excess fat deposition via some mechanism(s) other than reduced dietary intake. However, the overall absence of agreement between weight change (gain or loss) and G6PDH activity suggests that the anti-obesity activity of DHEA is not mediated via G6PDH. Since yellow obese (Ay/Aw) mice were found to be more susceptible to DHEA's effects than their agouti (Aw/Aw) littermates, Ay appears to induce an altered metabolism in Ay/Aw mice which is more susceptible to the effects of DHEA than the normal metabolism of Aw/Aw mice.     

Inhibition of Trypanosoma brucei glucose-6-phosphate dehydrogenase by human steroids and their effects on the viability of cultured parasites

Dehydroepiandrosterone (DHEA) is known as an intermediate in the synthesis of mammalian steroids and a potent uncompetitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH), but not the enzyme from plants and lower eukaryotes. G6PDH catalyzes the first step of the pentose-phosphate pathway supplying cells with ribose 5-phosphate, a precursor of nucleic acid synthesis, and NADPH for biosynthetic processes and protection against oxidative stress. In this paper we demonstrate that also G6PDH of the protozoan parasite Trypanosoma brucei is uncompetitively inhibited by DHEA and epiandrosterone (EA), with K_i values in the lower micromolar range. A viability assay confirmed the toxic effect of both steroids on cultured T. brucei bloodstream form cells. Additionally, RNAi mediated reduction of the G6PDH level in T. brucei bloodstream forms validated this enzyme as a drug target against Human African Trypanosomiasis. Together these findings show that inhibition of G6PDH by DHEA derivatives may lead to the development of a new class of anti-trypanosomatid compounds.     

Anti-proliferative action of endogenous dehydroepiandrosterone metabolites on human cancer cell lines

Dehydroepiandrosterone (DHEA) is a naturally occurring steroid synthesized in the adrenal cortex, gonads, brain, and gastrointestinal tract, and it is known to have chemopreventive and anti-proliferative actions on tumors. These effects are considered to be induced by the inhibition of glucose-6-phosphate dehydrogenase (G6PD) and/or HMG-CoA reductase (HMGR) activities. The present study was undertaken to investigate whether endogenous DHEA metabolites, i.e. DHEA-sulfate, 7-oxygenated DHEA derivatives, androsterone, epiandrosterone, and etiocholanolone, have anti-proliferative effects on cancer cells and to clarify which enzyme, G6PD or HMGR, is responsible for growth inhibition. Growth of Hep G2, Caco-2, and HT-29 cells, evaluated by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays, was time- and dose-dependently inhibited by addition of all DHEA-related steroids we tested. In particular, the growth inhibition due to etiocholanolone was considerably greater than that caused by DHEA in all cell lines. The suppression of growth of the incubated steroids was not correlated with the inhibition of G6PD (r=-0.031, n=9, NS) or HMGR (r=0.219, n=9, NS) activities. The addition of deoxyribonucleosides or mevalonolactone to the medium did not overcome the inhibition of growth induced by DHEA or etiocholanolone, while growth suppression by DHEA was partially prevented by the addition of ribonucleosides. These results demonstrate that endogenous DHEA metabolites also have an anti-proliferative action that is not induced by inhibiting G6PD or HMGR activity alone. These non-androgenic DHEA metabolites may serve as chemopreventive or anti-proliferative therapies.     

Cyclic strain modulates resistance to oxidant stress by increasing G6PDH expression in smooth muscle cells

Vascular smooth muscle cells (VSMC) may be subjected to mechanical forces, such as cyclic strain, that promote the formation of reactive oxygen species (ROS). We hypothesized that VSMC modulate this adverse milieu by increasing the expression of glucose-6-phosphate dehydrogenase (G6PDH) to maintain or restore intracellular glutathione (GSH) levels. Cyclic strain increased superoxide formation, which resulted in diminished GSH because of an increase in oxidized glutathione formation; there was also an increase in glutathione peroxidase and glutathione reductase activities. G6PDH activity and protein expression were enhanced concomitant with decreases in GSH levels and remained elevated until intracellular GSH levels were restored. To confirm the role of G6PDH in repleting GSH stores, we inhibited G6PDH activity with DHEA or inhibited enzyme expression with an antisense oligodeoxynucleotide. Diminished G6PDH activity or expression was associated with persistently depleted GSH levels and inhibition of the cyclic strain-mediated increase in glutathione reductase activity. These observations demonstrate that cyclic strain promotes oxidant stress in VSMC, which, in turn, induces G6PDH expression. When G6PDH is inhibited, GSH levels are not restored because of impaired glutathione reductase activity. These data suggest that G6PDH is a critical determinant of the response to oxidant stress in VSMC.     

Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis

In green tissues of plants under illumination, photosynthesis is the primary source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is utilized in reductive reactions such as carbon fixation and nitrogen assimilation. In non-photosynthetic tissues or under non-photosynthetic conditions, the oxidative pentose phosphate pathway contributes to basic metabolism as one of the major sources of NADPH. The first and committed reaction is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). We characterized the six members of the G6PDH gene family in Arabidopsis. Transit peptide analysis predicted two cytosolic and four plastidic isoforms. Five of the six genes encode active G6PDHs. The recombinant isoforms showed differences in substrate requirements and sensitivities to feedback inhibition. Plastidic isoforms were redox sensitive. One cytosolic isoform was insensitive to redox changes, while the other was inactivated by oxidation. The respective genes had distinct expression patterns that did not correlate with the activity of the proteins, implying a regulatory mechanism beyond the control of mRNA abundance. Two cytosolic and one plastidic isoform were detected in vivo using zymograms, and the respective genes were identified using T-DNA insertion lines. The activity of a plastidic isoform was detected in all tissues including photosynthetic tissues despite its sensitivity to reduction observed in vitro. Genomic data, gene expression, and in vivo enzyme activity data were integrated with in vitro biochemical data to propose in vivo roles for individual G6PDH isoforms in Arabidopsis.     

16-Bromoepiandrosterone,an activator of the mammalian immune system,inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the pentose-phosphate pathway which supplies cells with ribose 5-phosphate (R5P) and NADPH. R5P is the precursor for the biosynthesis of nucleotides while NADPH is the cofactor of several dehydrogenases acting in a broad range of biosynthetic processes and in the maintenance of the cellular redox state. RNA interference-mediated reduction of G6PDH levels in bloodstream-form Trypanosoma brucei validated this enzyme as a drug target against Human African Trypanosomiasis. Dehydroepiandrosterone (DHEA), a human steroidal pro-hormone and its derivative 16α-bromoepiandrosterone (16BrEA) are uncompetitive inhibitors of mammalian G6PDH. Such steroids are also known to enhance the immune response in a broad range of animal infection models. It is noteworthy that the administration of DHEA to rats infected by Trypanosoma cruzi, the causative agent of Human American Trypanosomiasis (also known as Chagas’ disease), reduces blood parasite levels at both acute and chronic infection stages. In the present work, we investigated the in vitro effect of DHEA derivatives on the proliferation of T. cruzi epimastigotes and their inhibitory effect on a recombinant form of the parasite’s G6PDH (TcG6PDH). Our results show that DHEA and its derivative epiandrosterone (EA) are uncompetitive inhibitors of TcG6PDH, with K_i values of 21.5 ± 0.5 and 4.8 ± 0.3 μM, respectively. Results from quantitative inhibition assays indicate 16BrEA as a potent inhibitor of TcG6PDH with an IC₅₀ of 86 ± 8 nM and those from in vitro cell viability assays confirm its toxicity for T. cruzi epimastigotes, with a LD₅₀ of 12 ± 8 μM. In summary, we demonstrated that, in addition to host immune response enhancement, 16BrEA has a direct effect on parasite viability, most likely as a consequence of TcG6PDH inhibition.     

DHEA inhibits cell growth and induces apoptosis in BV-2 cells and the effects are inversely associated with glucose concentration in the medium

Dehydroepiandrosterone (DHEA), a major steroid secreted by the adrenal gland which decreases with age after adolescence, is available as a nutritional supplement. DHEA is known to have antiproliferative effects but the mechanism is unclear. In this study using BV-2 cells, a murine microglial cell line, we investigated the effect of DHEA on cell viability and the interaction between DHEA and glucose concentrations in the medium. We showed that DHEA inhibited cell viability and G6PD activity in a dose-dependent manner and that the effect of DHEA on cell viability was inversely associated with glucose concentrations in the medium, i.e. lowered glucose strongly enhanced the inhibition of cell viability by DHEA. DHEA inhibited cell growth by causing cell cycle arrest primarily in the G0--G1 phase, and the effect was more pronounced at zero glucose (no glucose added, G0) than high glucose (4.5 mg/ml of the medium, G4.5). Glucose deprivation also enhanced apoptosis induced by DHEA. At G4.5, DHEA did not induce formation of DNA ladder until it reached 200 microM. However, at G0, 100 microM DHEA was able to induce apoptosis, as evidenced by the formation of DNA ladder, elevation of histone-associated DNA fragmentation and increase in cells positively stained with annexin V-FITC and annexin V-FITC/propidium iodide. The interactions between DHEA and glucose support the contention that DHEA exerts its antiproliferative effects through alteration of glucose metabolism, possibly by inhibition of G6PD activity leading to decreased supply of ribose-5-phosphate for synthesis of DNA and RNA. Although DHEA is only antiproliferative at pharmacological levels, our results indicate that its antiproliferative effect can be enhanced by limiting the supply of glucose such as by energy restriction. In addition, the present study shows that glucose concentration is an important factor to consider when studying the antiproliferative and toxicological effects of DHEA.     

Evidence for the involvement of the fatty acid and peroxisomal β-oxidation pathways in the inhibition by dehydroepiandrosterone (DHEA) and induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benz(a)anthracene (BA) of cytochrome P4501B1 (CYP1Bl) in mouse embryo fibroblasts (C3H10T1/2 cells)

Treatment of intact C3H10T1/2 cells or microsomes therefrom with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzanthracene (BA) enhanced CYP1B1 activity and CYP1B1 expression as revealed by elevations of CYP1B1-catalyzed DMBA metabolism, CYP1B1 apoprotein level and CYP1B1 gene expression. One hundred µM DHEA caused an 80-90% inhibition of cellular DMBA metabolism without inflicting cell death. Cytosolic glucose-6-phosphate dehydrogenase (G6PDH) was also inhibited in DHEA-treated cells, presumably due to the inhibition of NADP reduction. In contrast, neither DMBA metabolism nor CYP1B1 apoprotein was inhibited by DHEA in the microsomes isolated from these cells. DHEA (100 M), TCDD (10 nM) and BA (10 M) stimulated the activities and increased the apoprotein levels of two peroxisomal enzymes, namely, acyl CoA oxidase (ACOX) and acyl CoA hydrolase (ACH₂) and also induced the expression of CYP1B1 and ACOX genes. Cytosolic fatty acyl-CoA -oxidation was also stimulated by DHEA, TCDD and BA. In corroboratory experiments, it was found that concomittant with the stimulation of the activity of a key enzyme regulator of fatty acid homeostasis, namely, glycerol-3-phosphate dehydrogenase (G3PDH), these agents enhanced arachidonic acid (AA) metabolism as judged by the release of [³H] from AA into the culture medium. Collectively, these data suggest that DHEA mediates the regulation of CYP1B1 and inhibits BA and TCDD-induced CYP1B1-catalyzed carcinogen (DMBA) activation in 10T1/2 cells through metabolic interactions that involve the activation of the peroxisomal and fatty acid -oxidation signaling pathways. These results also present evidence for the first time, for the possible peroxisomal effects of TCDD and BA which are similar to those of DHEA in this mouse embryo fibroblast cell line.     

Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal alpha-glucosidase

The levels of functional mRNA encoding glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) were examined in hepatocytes from fasted and fasted/carbohydrate-refed rats and in hepatocytes inoculated into primary culture. Functional G6PDH mRNA was assessed in a cell-free protein synthesis system in vitro. We observed that hepatocytes from fasted/carbohydrate-refed rats had a 12-fold higher level of mRNA than did hepatocytes from fasted rats. The possibility that the adrenal glucocorticoids and insulin were responsible for the increase in G6PDH mRNA in refed rats was examined by studying the effect of insulin and the synthetic glucocorticoid, dexamethasone, on the level of functional G6PDH mRNA in primary cultures of rat hepatocytes maintained in a chemically defined medium. Hepatocytes from fasted rats were inoculated into primary culture and maintained for 48 h either in the absence of hormones or in the presence of insulin alone, dexamethasone alone or both hormones together. We observed that dexamethasone alone caused a fourfold increase in G6PDH mRNA while insulin caused about a twofold increase. Both hormones together elicited an increase that was additive. A comparison of functional G6PDH mRNA levels with the effect of the hormones on G6PDH activity and relative rate of enzyme synthesis suggests that the glucocorticoid elevates the level of G6PDH mRNA within the cell without causing a concommitant increase in the rate of synthesis of the enzyme or the level of G6PDH activity. The results obtained with the primary cultures of hepatocytes indicate that insulin and the glucocorticoids are probably involved with the regulation of hepatic G6PDH mRNA. However, involvement of other hormones, such as thyroid hormone, seems likely since the induced levels of G6PDH mRNA in hepatocytes in culture was one-third of that observed in refed rats.     

Altered activity of the P2 isoform of plastidic glucose 6-phosphate dehydrogenase in tobacco (Nicotiana tabacum cv. Samsun) causes changes in carbohydrate metabolism and response to oxidative stress in leaves

Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.     

Engineered sorbitol accumulation induces dwarfism in Japanese persimmon

A cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), which is a key enzyme in sorbitol biosynthesis in Rosaceae, was introduced into the Japanese persimmon (Diospyros kaki) to increase the environmental stress tolerance. Resultant transformants exhibited salt-tolerance with dwarfing phenotypes. Therefore, we studied two transgenic lines to understand the physiological mechanism of this dwarfism: lines PS1 and PS6 accumulated high and moderate levels of sorbitol, respectively. The average length of shoots was significantly shorter as compared with the wild-type in line PS1, while no such decrease was observed in line PS6. The myo-inositol and glucose 6-phosphate (G6P) contents were measured in the transgenic lines because previous work with tobacco transformed with S6PDH had suggested that growth inhibition was due to depletion of these metabolites. Although the myo-inositol content was decreased in PS1 plants, the decrease was much smaller than that observed in transgenic tobacco that accumulates sorbitol. The G6P contents were the same in PS1 plants and phenotypically normal PS6 plants. The level of indole-3-acetic acid (IAA), which affects stem elongation, in line PS1 was similar to the levels in the other lines. A decrease in gibberellin (GA) content generally induces dwarfism in plants. However, GA was not decreased in PS1 plants compared with wild-type or control plants. Therefore, we focused on sorbitol accumulation as the most remarkable feature of PS1 plants. As one possibility, the observed growth inhibition was likely caused by an osmotic imbalance between the cytosol and vacuole.     

Importance of glucose-6-phosphate dehydrogenase activity in cell death

The intracellular redox potential plays an important role incell survival. The principal intracellular reductant NADPH is mainlyproduced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by6-phosphogluconate dehydrogenase. Considering the importance of NADPH,we hypothesized that G6PDH plays a critical role in cell death. Ourresults show that 1) G6PDHinhibitors potentiatedH₂O₂-inducedcell death; 2) overexpression ofG6PDH increased resistance toH₂O₂-induced cell death; 3) serum deprivation, astimulator of cell death, was associated with decreased G6PDH activityand resulted in elevated reactive oxygen species (ROS);4) additions of substrates for G6PDHto serum-deprived cells almost completely abrogated the serumdeprivation-induced rise in ROS; 5)consequences of G6PDH inhibition included a significant increase inapoptosis, loss of protein thiols, and degradation of G6PDH; and6) G6PDH inhibition caused changesin mitogen-activated protein kinase phosphorylation that were similarto the changes seen withH₂O₂.We conclude that G6PDH plays a critical role in cell death by affectingthe redox potential.     

Comparative C19-radiosteroid metabolism by MA 160 and HeLa cell lines

Summary Since the designation of the human MA 160 line as prostatic epithelial cells has been questioned and the possibility of HeLa cross contamination raised, this comparative study of C₁₉-radiosteroid transformation in MA 160 and HeLa monolayer cultures was done to determine whether these cells possess the distinguishing features of reductive and oxidative androgen metabolism expected in male and female genital organs, respectively. We compared the radiometabolite patterns produced by incubating [¹⁴C]testosterone (300nM) and [³H]testosterone (3nm) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) with cultures of prostatic MA 160 and HeLa Parent, TCRC-1, TCRC-2 and ATC 229 cells. C₁₉-Radiosteroid metabolite patterns from MA 160 cell incubations also were compared with patterns generated by MA 196 fibroblasts from abdomnal skin of the same donor. MA 160 cells metabolized radiotestosterone predominantly to 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol. The diol epimers were the principal metabolites of 5α-dihydrotestosterone radiosubstrate. In contrast, radiotestosterone metabolism by MA 196 and HeLa Parent, TCRC-1 and TCRC-2 cells was overwhelmingly to the 17-oxosteroids 4-androstene-3,17-dione and androsterone. Another pathway was operative in HeLa 229 and, to a minor extent, in TCRC-1, which converted radiotestosterone to 4-androstene-3α,17β-diol and 5α-androstane-3α,17β-dol, with little formation of 5α-dihydrotestosterone. MA 160 cells thus metabolize radiotestosterone preponderantly to 5α-reduced 17β-hydroxysteroids as expected for prostatic epithelial cells, whereas HeLa cells show heterogeneity in metabolizing the labeled hormone by the alternative 17-oxosteroid and Δ⁴ pathways. This work was supported by Public Health Service Research Grants CA 13417 and CA 12924 from the National Cancer Institute, AM 11011 from the National Institute of Arthritis, Metabolism and Digestive Diseases, and by appropriations of the Commonwealth of Massachusetts, Item No. 4532-9003-01.     

Effect of phenolic compounds on glucose-6-phosphate dehydrogenase isoenzymes

Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.     

Monoclonal antibodies to glucose-6-phosphate dehydrogenase (G6PDH) form cyclic 1:1 complexes with G6PDH and act as regulatory subunits

A number of IgG monoclonal antibodies against L. mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) have been prepared. Four of the antibodies form 1:1 enzyme-antibody complexes which are stabilized in the presence of glucose-6-phosphate (G6P) and have greatly reduced enzyme activity. In the absence of G6P, the 1:1 complexes convert gradually to a more active multimeric form. Reduction of the IgG inter-heavy chain disulfides partially relieves inhibition and removes the G6P requirement for stability. F(ab')2 fragments of one of the antibodies behave similarly to the intact IgG. Reduction of the disulfides in the G6PDH-F(ab')2 complex leads to complete recovery of activity. The activity of complexes of G6PDH with reduced antibodies or Fab with digoxin bound to the antibody or Fab sulfhydryl groups can be modulated with antibodies to digoxin. The anti-G6PDH antibodies bridge two identical epitopes of this two subunit enzyme and simulate the function of regulatory subunits in which anti-digoxin acts as an activator. The system can be used to provide a sensitive homogeneous immunoassay for digoxin.     

Glucose-6P dehydrogenase in Chlorella sorokiniana (211/8k): an enzyme with unusual characteristics

In Chlorella sorokiniana (211/8k), glucose-6 phosphate dehydrogenase (G6PDH—EC 1.1.1.49) activity is similar in both N-starved cells and nitrate-grown algae when expressed on a PCV basis. A single G6PDH isoform was purified from Chlorella cells grown under different nutrient conditions; the presence of a single G6PDH was confirmed by native gels stained for enzyme activity and by Western blots. The algal G6PDH is recognised only by antibodies raised against higher plants plastidic protein, but not by chloroplastic and cytosolic isoform-specific antisera. Purified G6PDH showed kinetic parameters similar to plastidic isoforms of higher plants, suggesting a different biochemical structure which would confer peculiar regulative properties to the algal G6PDH with respect to higher plants enzymes. The most remarkable property of algal G6PDH is represented by the response to NADPH inhibition. The algal enzyme is less sensitive to NADPH effects compared to higher plants G6PDH: Ki_NADPH is 103 μM for G6PDH from nitrogen-starved C. sorokiniana, similarly to root plastidic P2-G6PDH. In nitrate-grown C. sorokiniana the Ki_NADPH decreased to 48 μM, whereas other kinetic parameters remained unchanged. These results will allow further investigations in order to rule out possible modifications of the enzyme, and/or the expression of a different G6PDH isoform during nitrate assimilation.     

Glucocorticoid receptors and cell cycle progression in human melanoma cell lines

Proliferation of six established human melanoma cell lines was inhibited after treatment for 1 h with a high dose of glucocorticoid. Four of the lines with the capacity of colony formation were used to quantify final plating efficiency. Specific glucocorticoid binding sites in these cell lines ranged from 51,000 to 170,000 sites per cell as measured with a whole-cell assay. Growth inhibition was completely reversible in one cell line, irreversible in another, and partially reversible in two lines. Receptor content per cell correlated with the reduction in final plating efficiency of glucocorticoid-treated cells, suggesting a receptor-mediated event. A more than 90% growth inhibition and a 40% reduction in cell survival in the most sensitive cell line, M-5A, was accompanied by a dual blockage in G1 and G2/M phase that lasted till at least 96 h after treatment with 2.5 microM dexamethasone for 1 h. Evidence is presented of a real arrest of M-5A cells in G1 phase and a markedly retarded progression through G2; the blockage of G1-S transition was immediate and complete. Accumulation of G1 cells was observed in two other cell lines but was inconsistent in the fourth line studied by flow cytometry; in none of the three cell lines was G2/M accumulation observed. Stimulated melanogenesis after glucocorticoid treatment of M-5A and NKI-26 cells suggested differentiation of the cells during glucocorticoid-induced arrest.     

Dehydroepiandrosterone improves hepatic antioxidant reserve and stimulates Akt signaling in young and old rats

This study examined, in the liver of young and old (3- and 24-month-old, respectively) healthy Wistar rats, the in vivo effect of dehydroepiandrosterone (DHEA) (10mg/kg body weight) administered subcutaneously for 5 weeks. Reduced (GSH) and oxidized (GSSG) glutathione levels, glucose-6-phosphate dehydrogenase (G6PDH), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities, hydrogen peroxide concentration, GST and p-Akt/Akt immunocontent ratio were assessed in hepatic tissue. DHEA treatment significantly increased total glutathione content (17%) and GSH (22%) in 3- and 24-month-old treated groups when compared to control groups. The aging factor increased G6PDH (51%) and GPx (22%) activities as well as the hydrogen peroxide concentration (33%), independently of treatment. DHEA treatment increased p-Akt (54%) and p-Akt/Akt ratio (36%) immunocontents in both treated groups. Increased serum levels of alanine aminotransferase (ALT) in aged rats were reduced by DHEA treatment (34%).