Epistatic Interaction ofCYP1A1andCOMTPolymorphisms in Cervical Cancer

There is a clear association between the excessive and cumulative exposure to estrogens and the development of cancer in hormone-sensitive tissues, such as the cervix. We studied the association ofCYP1A1 M1(rs4646903) andCOMT(rs4680) polymorphisms in 130 cervical cancer cases (c-cancer) and 179 controls. TheCYP1A1TT genotype was associated with a lower risk for c-cancer (OR = 0.39,p=0.002). The allele C ofCYP1A1was a risk for c-cancer (OR = 2.29,p=0.002). Women withCOMT LLgenotype had a higher risk of developing c-cancer (OR = 4.83,p<0.001). For the interaction of theCYP1A1&COMT, we observed that TC&HL genotypes had a greater risk for c-cancer (OR = 6.07,p=0.006) and TT&HL genotypes had a protection effect (OR = 0.24,p<0.001). TheCYP1A1 TTandCOMT LLgenotypes had higher estradiol levels in c-cancer (p<0.001andp=0.037, resp.). C-cancer is associated with less production of 2-methoxy-estradiol resultant of functional polymorphisms ofCYP1A1andCOMT, separately.CYP1A1andCOMTwork in a metabolic sequence and their interaction could lead to an alternative pathway of estrogen metabolism with production of 16-OH-estrone that is more proliferative.

Sequence Analysis of the GntII (Subsidiary) System for Gluconate Metabolism Reveals a Novel Pathway for l-Idonic Acid Catabolism in Escherichia coli

ABSTRACT The presence of two systems in Escherichia coli for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of l-idonic acid in whichd-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnRoperon, which encodes l-idonate 5-dehydrogenase, 5-keto-d-gluconate 5-reductase, an l-idonate transporter, and an l-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of l-idonate; IdnD catalyzes a reversible oxidation ofl-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to formd-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of d-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, withl-idonate or 5-ketogluconate serving as the true inducer of the pathway. The l-idonate 5-dehydrogenase and 5-keto-d-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of l-idonate andd-gluconate via the intermediate 5-ketogluconate. E. coli K-12 strains are able to utilize l-idonate as the sole carbon and energy source, and as predicted, the ability ofidnD, idnK, idnR, andedd mutants to grow on l-idonate is altered.

Equilibrium enzymes in metabolic pathways

It is commonly believed that certain reactions in a metabolic sequence may be at or close to equilibrium because of the large excess of catalytic capacity compared to the flux through these enzyme loci. Simple algebraic manipulations can show that the equilibrium and steady state conditions are mutually exclusive. However, solution of the complete reaction schemes for model "equilibrium" reactions shows that they can remain far from equilibrium even though the ratio of enzyme flux to steady state flux through the overall pathway is high. These calculations show that a reaction's proximity to equilibrium depends on the overall flux through the enzyme locus as well as on the kinetic parameters of the other enzymes in the pathway. Thus, combinations of kinetic parameters may exist that allow certain reactions to approach equilibrium but these conditions are not universal.Key words: equilibria, theoretical kinetics, metabolic control.

Phosphatidylinositol 3,4,5-trisphosphate is formed from phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets

Platelets accumulate PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to thrombin and thrombin-receptor-directed peptide in a GTP-dependent manner. These phosphoinositides are considered to be mediators of signaling events in a variety of cells. We have examined the metabolic route by which PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are synthesized by briefly (10 min) incubating platelets with high activities of [32P]Pi, followed by 20 or 60 s exposure to thrombin, and analysing the relative radioactivities of the individual phosphate groups in the resulting labelled PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The phosphate group possessing the highest specific activity under such non-equilibrium labelling conditions indicates the last one added in a metabolic sequence. The thrombin-stimulated rate of labelling of PtdIns(3,4)P2 was significantly slower than that of PtdIns(3,4,5)P3. Increased labelled PtdIns3P was not detected within 60 s. The measured relative radioactivities decreased in the order 3 > 5 > 4 >> 1 for PtdIns(3,4,5)P3 and 3 > 4 >> 1 for PtdIns(3,4)P2. On the basis of the results of both rate-of-labelling and specific radioactivity analyses we conclude that PtdIns(3,4,5)Pa is formed by 3-OH phosphorylation of PtdIns(4,5)P2, whereas PtdIns(3,4)P2, may be formed by 3-OH phosphorylation of PtdIns4P and/or dephosphorylation of PtdIns(3,4,5)P3. These findings point to the activation of phosphoinositide 3-kinase as a critical receptor-regulated step in thrombin-stimulated platelets.

Aromatic Degradation in Yeast Rhodotorula rubra

Aromatic degradation by two yeast strains of Rhodotorula (R.) rubra was examined. Separation and identification of phenol and protocatechuic acid (PCA) metabolites were carried out using high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). For HPLC analysis of β -ketoadipic acid, 2,4-dinitrophenylhydrazone derivative was prepared. For GC/MS analysis, metabolites in the cultured broth were extracted with ethyl acetate and trimethylsilylated by N-o-bis(trimethylsilyl)acetoamide. Based on HPLC and GC/MS analyses, phenol metabolites were identified as catechol, eis, cis-muconic acid, muconolactone, β -ketoadipate enol-lactone and β -ketoadipic acid. β -Ketoadipic acid was also found in the PCA metabolites by crude cell-free extract. From the results obtained in this study, a metabolic sequence for aromatic degradation is proposed. Phenol may be hydroxylated to form catechol prior to ring cleavage, and catechol may be further oxidized to eis, cis-muconic acid, muconolactone, β -ketoadipate enol-lactone and β -ketoadipic acid. Catechol branch as well as protocatechuate branch in β -ketoadipate pathway may exist in R. rubra.

Adaptation, constraint, and the function of the gluconeogenic pathway

Gluconeogenesis is responsible for the de novo synthesis of glucose (and glycogen) from precursors including lactate, amino acids, glycerol, and fructose. This metabolic sequence is highly constrained by design features including enzyme composition and tissue localization, but demonstrates a variety of adaptive patterns which are critical to the maintenance of blood glucose levels optimal for animal function. This review identifies the adaptive responses of gluconeogenesis when glucose levels are challenged by changes in diet (both quality and quantity) and in activity level, and by environmental disturbances. Five adaptive patterns are identified: (i) quantitative changes in gluconeogenic enzyme activities and their subcellular distribution; (ii) alterations in tissue demand for glucose; (iii) existence of in situ skeletal muscle lactate cycling; (iv) quantity and type of gluconeogenic precursors; and (v) regulation of gluconeogenesis. The validity of the omnivorous mammalian model in our understanding of gluconeogenesis is discussed.

The stereospecificity of sequential nicotinamide-adenine dinucleotide-dependent oxidoreductases in relation to the evolution of metabolic sequences

The generalization that ‘when a metabolic sequence involves consecutive nicotinamide-adenine dinucleotide-dependent reactions, the dehydrogenases have the same stereospecificity’ was tested and confirmed for three metabolic sequences. (1) NAD+-xylitol (D-xylulose) dehydrogenase and NADP+-xylitol (L-xylulose) dehydrogenase are both B-specific. (2) D-Mannitol 1-phosphate dehydrogenase and D-sorbitol 6-phosphate dehydrogenase are both B-specific. (3) meso Tartrate dehydrogenase and oxaloglycollate reductive decarboxylase are both A-specific. Other dehydrogenases associated with the metabolism of meso-tartrate in Pseudomonas putida, such as hydroxypyruvate reductase and tartronate semialdehyde reductase, were also shown to be A-specific. Malate dehydrogenase from Pseudomonas putida was A-specific, and the proposition is discussed that the common A-stereospecificity among the dehydrogenases involved in meso-tartrate metabolism reflects their origin from malate dehydrogenase.

The metabolic sequence by which some 4,4-dimethyl sterols are converted into cholesterol

Cholest-8(14)-enol is the major radioactive component of the 4-di-demethyl sterol fraction biosynthesized from 4,4-dimethyl[2-3H2]cholest-8(14)-enol by rat liver microsomal fractions, and therefore the first steps in the biosynthesis of cholesterol from the latter compound probably involve removal of the 4-methyl groups. 4,4-Dimethylcholesta-8,14-dienol therefore is not an intermediate in this process, although its presence in the incubation medium at a concentration of 0.146mm almost completely inhibits the demethylation of 4,4-dimethyl[2-3H2]cholest-8(14)-enol. Nor is cholesta-8,14-dienol an intermediate in the conversion of cholest-8(14)-enol into cholest-7-enol and cholesterol. With 4,4-dimethyl[2-3H2]cholesta-8,14-dienol as the cholesterol precursor, 4,4-dimethylcholest-8(9)-enol becomes heavily labelled and there is very little radioactivity associated with cholesta-8,14-dienol.In this case, the most heavily labelled 4-di-demethyl sterols are cholest-7-enol and cholesterol with the former predominating. There is little or no radio-activity associated with cholest-8(14)-enol. A similar labelling pattern amongst the 4-di-demethyl sterols was observed with dihydro[14C]lanosterol as the precursor. The first step therefore in the synthesis of cholesterol from the 4,4-dimethyl[2-3H2]dienol is reduction of the Δ14(15) bond and not removal of the 4α-methyl group. Depending on the nature of the precursor, addition of the soluble fraction of the cell to the microsomal fraction resulted in a two- to four-fold stimulation of 4-di-demethyl sterol biosynthesis from the 4,4-dimethyl sterols studied. Under these conditions, 4,4-dimethylcholesta-8,14-dienol is the most efficient precursor of cholesterol and cholest-7-enol, and dihydrolanosterol is better than 4,4-dimethylcholest-8(14)-enol.