Separation and quantification of d- and l-phosphoserine in rat brain using Nα-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (Marfey's reagent) by high-performance liquid chromatography with ultraviolet detection

d-Serine has recently been described to be present in the brain at high concentrations. However, while prior research has demonstrated that l-phosphoserine is the major precursor of l-serine in the brain, the possible role of d-phosphoserine as the direct precursor of d-serine is unknown. To address this problem, we developed an assay to separate and quantitate d- and l-phosphoserine. A very simple HPLC-UV procedure for the separation and quantification of d- and l-phosphoserine is presented using precolumn derivatization with a chiral reagent, Nα-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (Marfey's reagent), and a conventional C₁₈ reversed-phase column. The procedure is sensitive to 11 pmol on-column and derivatives are stable for at least two weeks at room temperature. Rat brain regions (cortex, hippocampus, striatum, and cerebellum) were analyzed for the presence of d- and l-phosphoserine. It was determined that the brain regions studied contained exclusively l-phosphoserine.     

Metabolism of D-serine in Escherichia coli K-12: mechanism of growth inhibition

Without significant killing, d-serine at concentrations greater than 50 mug/ml inhibits growth in minimal media of mutants of Escherichia coli K-12 unable to form d-serine deaminase. The mutants eventually recover at lower concentrations. There is no evidence of d-serine toxicity in rich media. Toxicity is partially reversed by l-serine. d-Serine does not interfere with l-serine activation, one-carbon metabolism, or (Cronan, personal communication) formation of phosphatidylserine. Pizer (personal communication) finds, however, that it is a powerful feedback inhibitor of the first enzyme of l-serine biosynthesis. In the presence of l-serine, the residual toxicity is largely and noncompetitively over come by pantothenate, indicating that d-serine inhibits growth by affecting two targets: pantothenate biosynthesis and l-serine biosynthesis. l-Serine causes transient growth inhibition in E. coli K-12. Contaminating l-serine in d-serine preparations contributes to the d-serine inhibitory response.     

Author index for volume 88

Age changes in utilization of glucose and galactose were studied in primary cultures of rat hepatocytes. (1) With increasing age of donor from 2 to 10 weeks, the rate of galactose utilization fell while that of glucose utilization increased from a negative value (net production) to a level approximately double that of galactose. Glucose production could account for about 80% of the galactose taken up by cells from 2- to 3-week-old rats. (2) With increasing time of culture there was a fall in galactose utilization and an increase in glucose utilization comparable to the changes with increasing age in vivo. (3) The change in utilization of each sugar was independent of the change in utilization or availability of the other. It is suggested that the switch from galactose to glucose as the preferred substrate in vivo is not determined by dietary availability of these hexoses. (4) The increased utilization of glucose by cells of older animals was associated with a decrease in responsiveness to the glycogenic action of insulin. The change in responsiveness was not due to a decrease in insulin binding capacity of the cells.     

Development of glycogen storage ability under cortisol control in primary cultures of rat fetal hepatocytes

Cortisol has been shown to induce glycogen storage function in primary cultures of fetal hepatocytes. The method we describe provides a homogeneous population of hepatocytes by elimination of hematopoietic cells. Hepatocytes transplanted from 15-day-old fetuses were grown in the absence or presence of cortisol (10^?5M) for periods of up to 4 days. In the presence of cortisol, after a lag period (24 hr), the glycogen content increased sharply, regardless of whether the medium was replaced or not. Incorporation of radioactivity from (U) ¹⁴C-glucose into glycogen paralleled glycogen accumulation, but the specific activity of the stored glycogen was lower than the final specific activity of the glucose in the medium. This result shows that free glucose is a good precursor of glycogen but not the only one. Data from chase and labeling experiments prove that the hormone acts on the synthetic pathway. If cortisol was removed the glycogen content dropped, suggesting that glycogen synthesis depends on the continuous presence of the hormone. The in vitro maturation of hepatocyte can be provoked by the hormone before the normal in vivo maturation stage of the onset of glycogen accumulation. Other studies of the same in vivo phenomenon have demonstrated that accumulation of glycogen in the liver prior to birth is corticosteroid dependent, but only an in vitro study could clearly show that the hormone acts at the cellular level.     

The in vivo Tetrahymena thermophila extracellular glucose drop assay for characterization of mammalian insulin activity

Insulin activity is generally determined by an in vivo rabbit blood glucose drop assay in research and industriel laboratories. The humane experimental techniques imply the use of alternative invertebrate organisms in place of animals, known as replacement rule of the 3Rs. In this study, we report an alternative in vivo extracellular glucose drop assay using unicellular invertebrate Tetrahymena thermophila to replace the use of rabbit and mouse. This assay has four major steps; growing cells, starving cells, treatment of cells and measurement of glucose drop. In this assay, 0.2 mg/ml of human, porcine and bovine insulins dropped extracellular glucose level to 16%, 14% and 12%, respectively in ten minutes. In addition, mammalian insulins respectively increased the cell area about 19%, 15%, and 16% at 6th hour with statistically significant effect on the cell growth, but not in the cell viability. The results showed that the in vivo Tetrahymena thermophila extracellular glucose drop assay could be used as an alternative assay to replace the mouse or the rabbit insulin blood glucose drop assay.     

Circ_0015756 promotes ovarian cancer progression via the miR-145–5p/PSAT1 axis

Circular RNA (circRNA) have been shown to exert vital functions in the pathological progressions of ovarian cancer (OC). Herein, this study aimed to investigate the role and mechanisms of circ_0015756 in OC progression. Levels of circ_0015756, microRNA (miR)? 145–5p and phosphoserine aminotransferase 1 (PSAT1) were detected using quantitative real-time polymerase chain reaction, Western blot or immunohistochemistry assays. Cell proliferation, apoptosis, migration and invasion were determined using cell counting kit-8, 5-Ethynyl-2′-Deoxyuridine (Edu) incorporation, flow cytometry, transwell and Western blot assays. The binding interaction between miR-145–5p and circ_0015756 or PSAT1 was confirmed by bioinformatics prediction and dual-luciferase reporter assay. Tumor formation assay in nude mice was performed to determine the tumor growth in vivo. Circ_0015756 was highly expressed in OC tissues and cells. Knockdown of circ_0015756 suppressed cancer cell growth, migration and invasion in vitro, as well as impeded tumor growth in vivo. In a mechanical study, circ_0015756 directly bound to miR-145–5p, and inhibition of miR-145–5p reversed the effects of circ_0015756 knockdown on OC cells. Moreover, miR-145–5p directly targeted PSAT1, and miR-145–5p weakened OC cell growth, migration and invasion via targeting PSAT1. Importantly, further studies confirmed that circ_0015756 could indirectly regulate PSAT1 expression via sponging miR-145–5p. In all, circ_0015756 accelerated OC tumorigenesis through regulating miR-145–5p/PSAT1 axis, providing a new therapeutic target for OC.     

Ethanol metabolism in guinea pig: Ethanol oxidation and its effect on NADNADH ratios,oxygen consumption,and ketogenesis in isolated hepatocytes of fed and fasted animals

Ethanol metabolism was studied in isolated hepatocytes of fed and fasted guinea pigs. Alcohol dehydrogenase (EC 1.1.1.1) activities of fed or fasted liver cells were 2.04 and 1.88 μmol/g cells/min, respectively. Under a variety of in vitro conditions, alcohol dehydrogenase operates in fed hepatocytes at 34–74% and in fasted liver cells at 23–61% of its maximum velocity, respectively. Hepatocytes of fed animals, incubated in Krebs-Ringer bicarbonate buffer, oxidized ethanol at an average rate of 0.69 μmol/g wet weight cells/min, whereas cells of 48-h fasted animals consumed only 0.44 μmol/g/min under identical conditions. Various substrates and metabolites of intermediary metabolism significantly enhanced ethanol oxidation in fed liver cells. Maximum stimulatory effects were achieved with alanine (+138%) and pyruvate (+102%), followed in decreasing order by propionate, lactate, fructose, dihydroxyacetone, and galactose. In contrast to substrate couples such as lactate/pyruvate and glycerol/dihydroxyacetone, sorbitol with or without fructose significantly inhibited ethanol oxidation. The addition of hydrogen shuttle components such as malate, aspartate, or glutamate to fasted hepatocytes resulted in significantly higher stimulation of ethanol uptake than in fed hepatocytes. Also, the degree of inhibition of shuttle activity by n-butylmalonate was more pronounced in fasted liver cells (77% inhibition) than in fed cells (59% inhibition). These data as well as oxygen kinetic studies in intact guinea pig hepatocytes utilizing uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, dinitrophenol), electron-transport inhibitors (rotenone, antimycin), and malate-aspartate shuttle inhibitors (aminooxyacetate, n-butylmalonate) strongly suggested that the malate-aspartate shuttle is the predominant hydrogen transport system during ethanol oxidation in guinea pig liver.Comparison of the alcohol dehydrogenase-inhibitors 4-methylpyrazole and pyrazole on ethanol oxidation demonstrated that the alcohol dehydrogenase system is quantitatively the most important alcohol-metabolizing pathway in guinea pig liver. Supporting this conclusion, it was found that the H₂O₂-forming substrate glycolate slightly increased ethanol oxidation in liver cells of control animals (+26%), but prior inhibition of catalase by 3-amino-1,2,4-triazole resulted in a significant increase (+25%) instead of a decrease in alcohol oxidation. This finding does not support a quantitatively important role of peroxidatic oxidation of ethanol by catalase in liver.Cytosolic $\text{NAD}\text{NADH}$ ratios were greatly shifted toward reduction during ethanol oxidation. These reductive shifts were even more pronounced when cells were incubated in the presence of fatty acids (octanoate, oleate) plus ethanol. Inhibitor studies with 4-methylpyrazole demonstrated that the decrease of the cytosolic $\text{NAD}\text{NADH}$ ratio during fatty acid oxidation was due to an inhibition of hydrogen transport from cytosol to mitochondria and not the result of transfer of hydrogen, generated by fatty acid oxidation, from mitochondria to cytosol. Lactate plus pyruvate formation was slightly inhibited by ethanol in fed hepatocytes but greatly accelerated in fasted cells; this latter effect was mostly the result of increased lactate formation. Such regulation may represent a hepatic mechanism of alcoholic lactic acidosis as observed in human alcoholics. The ethanol-induced decrease of the mitochondrial $\text{NAD}\text{NADH}$ ratio was prevented by addition of 4-methylpyrazole. Endogenous ketogenesis was greatly increased (+80%) by ethanol in fed liver cells. This effect of ethanol was blunted in the presence of glucose. Propionate, by competing with fatty acid oxidation, was strongly antiketogenic. This effect was alleviated by ethanol. In 48-h fasted hepatocytes, endogenous ketogenesis was enhanced by 84%. Although ethanol did not further stimulate endogenous ketogenesis under these conditions, alcohol significantly increased ketogenesis in the presence of octanoate or oleate. This stimulatory effect of ethanol was almost completely prevented by 4-methylpyrazole. These findings demonstrate that the syndrome of alcoholic ketoacidosis may be due, at least partially, to the additional stimulation of ketogenesis by or from ethanol during fatty acid oxidation in the fasting state.     

Effects of ethanol on gastric mucosal adenosine 3', 5' monophosphate (cAMP)

The effects of ethanol on the gastric mucosal adenosine 3′, 5′-monophosphate (cAMP) system were evaluated. The activity of adenylate cyclase (AC), phosphodiesterase (PDE), and tissue content of cAMP were determined in the presence of ethanol. NaF stimulated AC in rat gastric mucosa was inhibited in vitro and in vivo by 20% ethanol. Basal AC activity was so low (0.05 ± 0.10 pmoles cAMP formed/min/mg protein) that consistent results without NaF could not be obtained. The PDE activity (172 ± 11 pmoles cAMP consumed/min/mg protein) was approximately 350 fold greater than the basal AC activity. All levels of ethanol tested (2.0–20.0%) significantly inhibited (p<0.05) PDE in vitro. Gastric mucosal levels of cAMP are not measurably altered by ethanol in vivo (5–20%).     

Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate

Phosphoserine aminotransferase (PSAT; EC 2.6.1.52), a member of subgroup IV of the aminotransferases, catalyses the conversion of 3-phosphohydroxypyruvate to l-phosphoserine. The crystal structure of PSAT from Escherichia coli has been solved in space group P212121 using MIRAS phases in combination with density modification and was refined to an R-factor of 17.5% (Rfree=20.1 %) at 2.3 A resolution. In addition, the structure of PSAT in complex with alpha-methyl-l-glutamate (AMG) has been refined to an R-factor of 18.5% (Rfree=25.1%) at 2.8 A resolution. Each subunit (361 residues) of the PSAT homodimer is composed of a large pyridoxal-5'-phosphate binding domain (residues 16-268), consisting of a seven-stranded mainly parallel beta-sheet, two additional beta-strands and seven alpha-helices, and a small C-terminal domain, which incorporates a five-stranded beta-sheet and two alpha-helices. A three-dimensional structural comparison to four other vitamin B6-dependent enzymes reveals that three alpha-helices of the large domain, as well as an N-terminal domain (subgroup II) or subdomain (subgroup I) are absent in PSAT. Its only 15 N-terminal residues form a single beta-strand, which participates in the beta-sheet of the C-terminal domain. The cofactor is bound through an aldimine linkage to Lys198 in the active site. In the PSAT-AMG complex Ser9 and Arg335 bind the AMG alpha-carboxylate group while His41, Arg42 and His328 are involved in binding the AMG side-chain. Arg77 binds the AMG side-chain indirectly through a solvent molecule and is expected to position itself during catalysis between the PLP phosphate group and the substrate side-chain. Comparison of the active sites of PSAT and aspartate aminotransferase suggests a similar catalytic mechanism, except for the transaldimination step, since in PSAT the Schiff base is protonated. Correlation of the PSAT crystal structure to a published profile sequence analysis of all subgroup IV members allows active site modelling of nifs and the proposal of a likely molecular reaction mechanism.     

Increased metabolism of retinoic acid after chronic ethanol consumption in rat liver microsomes

In rat liver microsomes, all-trans-[11,12-³H]retinoic acid was found to be metabolized to polar products in the presence of NADPH. One of the metabolites was coeluted with 4-hydroxyretinoic acid on reverse-phase high-pressure liquid chromatography (HPLC). This reaction required oxygen and was inhibited by carbon monoxide as well as aminopyrine, aniline, and ethanol, suggesting the involvement of cytochrome P-450. Isolated rat hepatocytes also metabolized all-trans[³H]retinoic acid to polar compounds, with an elution pattern on HPLC similar to that in microsomal preparations. Microsomal activity was compared in rats pair-fed with diets containing either ethanol or isocaloric carbohydrate for 4–6 weeks. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%, P < 0.01) accompanied by increased microsomal cytochrome P-450 content (34%, P < 0.005). On the other hand, microsomal β-glucuronidation of retinoic acid in the presence of uridine diphosphoglucuronic acid (UDPGA) was not affected by chronic ethanol feeding. The increased hepatic microsomal cytochrome P-450-dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo.     

Neu-Laxova Syndrome Is a Heterogeneous Metabolic Disorder Caused by Defects in Enzymes of the L-Serine Biosynthesis Pathway

Neu-Laxova syndrome (NLS) is a rare autosomal-recessive disorder characterized by a recognizable pattern of severe malformations leading to prenatal or early postnatal lethality. Homozygous mutations in PHGDH, a gene involved in the first and limiting step in L-serine biosynthesis, were recently identified as the cause of the disease in three families. By studying a cohort of 12 unrelated families affected by NLS, we provide evidence that NLS is genetically heterogeneous and can be caused by mutations in all three genes encoding enzymes of the L-serine biosynthesis pathway. Consistent with recently reported findings, we could identify PHGDH missense mutations in three unrelated families of our cohort. Furthermore, we mapped an overlapping homozygous chromosome 9 region containing PSAT1 in four consanguineous families. This gene encodes phosphoserine aminotransferase, the enzyme for the second step in L-serine biosynthesis. We identified six families with three different missense and frameshift PSAT1 mutations fully segregating with the disease. In another family, we discovered a homozygous frameshift mutation in PSPH, the gene encoding phosphoserine phosphatase, which catalyzes the last step of L-serine biosynthesis. Interestingly, all three identified genes have been previously implicated in serine-deficiency disorders, characterized by variable neurological manifestations. Our findings expand our understanding of NLS as a disorder of the L-serine biosynthesis pathway and suggest that NLS represents the severe end of serine-deficiency disorders, demonstrating that certain complex syndromes characterized by early lethality could indeed be the extreme end of the phenotypic spectrum of already known disorders.     

Efficient conversion of glycine to l-serine by a glycine-resistant mutant of a methylotroph using Co2+ as an inhibitor of l-serine degradation

A glycine-resistant mutant, no. 18, which was not lysed by glycine, was obtained from an l-serine-producing mutant, S395 (temperature-sensitive, O-methylserine-resistant), of a facultative methylotroph, Pseudomonas MS31. The mutant stably produced l-serine from glycine. The properties of the enzymes involved in the synthesis and degradation of l-serine were investigated in the wild-type strain MS31 and the l-serine-producing mutants. Mutant derivation had no effect on the activities of methanol dehydrogenase or serine hydroxymethyltransferase, which are involved in l-serine synthesis. On the other hand, the activity of l-serine dehydratase (SDH), which degrades l-serine, was reduced in the mutants. Cobalt (Co²⁺) inhibited SDH activity and its addition (6.5 mM) to the l-serine production culture significantly stimulated l-serine accumulation up to 14.9 mg/ml. The results suggest that blocking of SDH is important for the efficient production of l-serine from glycine by methylotrophs.     

Production of l-serine from glycine by Corynebacterium glycinophilum and properties of serine hydroxymethyltransferase,a key enzyme in l-serine production

Fermentative production of l-serine from glycine by Corynebacterium glycinophilum AJ-3413, an auxotrophic mutant of Leu and Met with increased productivity of l-serine using a one liter jar fermentor was carried out and the properties of serine hydroxymethyltransferase (SHMT), a key enzyme in l-serine synthesis, of the parental strain AJ-3170 were investigated. SHMT was effectively induced by the addition of glycine to the medium at an early stage of cultivation. Under optimal conditions, AJ-3413 produced 16.0 g/l of l-serine from 30 g/l of glycine with a molar yield of 38%. The partially purified SHMT catalyzed the l-allo-threonine degradation in addition to l-serine degradation, but could not catalyze l-threonine degradation. This enzyme showed an absolute tetrahydrofolic acid requirement for l-serine degradation to glycine and formaldehyde, but not for l-allo-threonine degradation. Pyridoxal 5′-phosphate appeared to be required for enzyme activity. The K_m values for glycine and formaldehyde in l-serine synthesis, and for l-serine in l-serine degradation were 1.85, 0.29 and 1.64 mM, respectively.     

Production of l-serine from methanol and glycine by resting cells of a methylotroph under automatically controlled conditions

Serine production from methanol and glycine was tried using frozen-thawed resting cells of a methylotroph, Protomonas extorquens NR-1 under multi-variable controlled conditions. The conditions for l-serine formation were optimized at 30°C. The production of l-serine in 0.4% CaCl₂ solution (initial pH 8.2) was the same as in 0.1 M Tris-HCl buffer (initial pH 8.3). Increasing the initial glycine concentration promoted l-serine formation. A high aeration rate decreased l-serine production. The optimum concentrations of dissolved oxygen and methanol were 0.5 ppm and 10 g/l, respectively. The highest l-serine, 24.9 g/l, was obtained at 24 h from 30.94 gl (as dry weight) resting cells using 100 g/l initial glycine with controlled pH. The relationship between the initial rate of l-serine formation and cell concentration indicated an unusual curve due to the effects of the added NaOH which was used for controlling the pH. In similar experiments without control of pH, a normal profile was observed with respect to the relationship between the initial rate of l-serine formation and cell concentration. The highest l-serine, 54.5 g/l, was obtained at 48 h by 36.4 g/l (as dry weight) resting cells. The yield (mol of l-serine/mol of added substrate) of l-serine from methanol and glycine were 8.3% and 39.3%, respectively. The selectivity of l-serine (mol of l-serine/mol of glycine consumed) was 67.9%. The stoichiometry of the maximum l-serine formation showed that the resting cells carried the highly active methanol dehydrogenase while serine transhydroxymethylase was rather low. Serine glyoxalate aminotransferase was not completely inhibited by the high concentration of glycine (about 68% of synthesized l-serine was detected in the supernatant.     

Influence of Drought Acclimation and CO(2) Enrichment on Osmotic Adjustment and Chlorophyll a Fluorescence of Sunflower during Drought

Osmotic adjustment occurred during drought in expanded leaves of sunflowers (Helianthus annuus var Hysun 30) which had been continuously exposed to 660 microliters CO₂ per liter or had been previously acclimated to drought. The effect was greatest when the treatments were combined and was negligible in nonacclimated plants grown at 340 microliters CO₂ per liter. The concentrations of ethanol soluble sugars and potassium increased during drought but they did not account for the osmotic adjustment. The delay in the decline in conductance and relative water content and in the loss of structural integrity with increasing drought was dependent on the degree of osmotic adjustment. Where it was greatest, conductance fell from 5.8 millimeters per second on the first day of drought to 1.3 millimeters per second on the fourth day and was at approximately the same level on the eighth day. The relative water content remained constant at 85% for three days and fell to 36% on the sixth day. There was no evidence of leaf desiccation even on the eighth day. In contrast, the conductance of leaves showing minimal adjustment fell rapidly after the first day of drought and was negligible after the fourth, at which time the relative water content was 36%. By the sixth day of drought, areas near the margins of the leaves were desiccating and the plants did not recover upon rewatering. Despite the differences in the rate of change of conductance and relative water content during drought, photosynthetic electron transport activity, inferred from measurements of chlorophyll a fluorescence in vivo and PSII activity of isolated thylakoids, remained functional until desiccation occurred.     

The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris

Plant pathogen Xanthomonas campestris pv. campestris produces cis-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of X. campestris pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that X. campestris pv. campestris produces not only DSF but also BDSF (cis-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in Burkholderia cenocepacia to be involved in regulation of motility, biofilm formation, and virulence in B. cenocepacia. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in X. campestris pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in X. campestris pv. campestris. ¹³C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that X. campestris pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.     

Ethanol-Induced Upregulation of 10-Formyltetrahydrofolate Dehydrogenase Helps Relieve Ethanol-Induced Oxidative Stress

Alcoholism induces folate deficiency and increases the risk for embryonic anomalies. However, the interplay between ethanol exposure and embryonic folate status remains unclear. To investigate how ethanol exposure affects embryonic folate status and one-carbon homeostasis, we incubated zebrafish embryos in ethanol and analyzed embryonic folate content and folate enzyme expression. Exposure to 2% ethanol did not change embryonic total folate content but increased the tetrahydrofolate level approximately 1.5-fold. The expression of 10-formyltetrahydrofolate dehydrogenase (FDH), a potential intracellular tetrahydrofolate reservoir, was increased in both mRNA and protein levels. Overexpressing recombinant FDH in embryos alleviated the ethanol-induced oxidative stress in ethanol-exposed embryos. Further characterization of the zebrafish fdh promoter revealed that the −124/+40 promoter fragment was the minimal region required for transactivational activity. The results of site-directed mutagenesis and binding analysis revealed that Sp1 is involved in the basal level of expression of fdh but not in ethanol-induced upregulation of fdh. On the other hand, CEBPα was the protein that mediated the ethanol-induced upregulation of fdh, with an approximately 40-fold increase of fdh promoter activity when overexpressed in vitro. We concluded that upregulation of fdh involving CEBPα helps relieve embryonic oxidative stress induced by ethanol exposure.     

Alcohol Consumption,One-Carbon Metabolites,Liver Cancer and Liver Disease Mortality

Background

Excess alcohol consumption adversely affects one-carbon metabolism and increases the risk of liver disease and liver cancer. Conversely, higher folate levels have been inversely associated with liver damage. The current study investigated the effects of alcohol and one-carbon metabolite intake on liver cancer incidence and liver disease mortality within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study.

Methods

Cox proportional hazards modeling was used to calculate hazard ratios and 95% confidence intervals (CIs) in a population of 27,086 Finnish males with 194 incident liver cancers and 213 liver disease deaths. In a nested case-control subset (95 liver cancers, 103 controls), logistic regression was used to calculate odds ratios and 95% CIs for serum one-carbon metabolites in relation to liver cancer risk.

Results

Daily alcohol consumption of more than 20.44 g was associated with an increased risk of both liver cancer incidence (Hazard Ratio (HR) 1.52, 95%CI 1.06–2.18) and liver disease mortality (HR 6.68, 95%CI 4.16–10.71). These risks were unaffected by one-carbon metabolite intake. Similarly, in the case-control study, none of the serum one-carbon metabolites were associated with liver cancer.

Conclusions

The current study provided no convincing evidence for a protective association of one-carbon metabolite intake or serum level on the risk of liver cancer or liver disease mortality.     

Hypoglycemic activity of alkaloidal fraction of Tinospora cordifolia

The stem of Tinospora cordifolia (TC) is widely used in the therapy of diabetes in traditional folk medicine of India. In the present study, isoquinoline alkaloid rich fraction (AFTC) derived from stem of TC and three alkaloids viz., palmatine, jatrorrhizine and magnoflorine were evaluated for insulin-mimicking and insulin-releasing effect in vitro and in vivo. Their effect on hepatic gluconeogenesis was examined in rat hepatocytes. Insulin releasing effect was detected in vitro using rat pancreatic β-cell line, RINm5F. Furthermore, effects of AFTC and isolated alkaloids on serum glucose and insulin level were studied in fasted and glucose challenged normal rats. AFTC significantly decreased gluconeogenesis in rat hepatocytes as insulin did and it increases insulin secretion in RINm5F cells similar to tolbutamide. In acute 30 min test in vitro, AFTC, palmatine, jatrorrhizine and magnoflorine stimulated insulin secretion from the RINm5F cell line. As in vivo results, administration of AFTC (50, 100, and 200 mg/kg), palmatine, jatrorrhizine and magnoflorine (10, 20 and 40 mg/kg each) orally significantly decreased fasting serum glucose, and suppressed the increase of blood glucose levels after 2 g/kg glucose loading in normal rats. In vivo study further justified their insulin secreting potential by raising the serum insulin level in glucose fed rats. These results demonstrate the alkaloid present in TC contributed for antihyperglycemic activity. AFTC may have hypoglycemic effects via mechanisms of insulin releasing and insulin-mimicking activity and thus improves postprandial hyperglycemia.