Different Concentrations Of Substrate Research Articles

Persimmon leaf (Diospyros kaki), a potent -glucosidase inhibitor and antioxidant: Alleviation of postprandial hyperglycemia in normal and diabetic rats

This research aims to investigate the α-glucosidase (baker’s yeast and rat intestinal acetone powder) inhibitory potential of Diospyros kaki Thunb. (Ebenaceae) leaf extract (DKLE) along with its antioxidant activity against metal-catalysed protein oxidation by pro-oxidant model (Fe2+/ascorbate) in rat liver homogenates, in vitro. DKLE evidenced the outstanding inhibitory effect with IC50 values of 1.22 and 32 µg/ml for baker’s yeast and rat intestinal α-glucosidase enzymes. The inhibition mode of this extract was determined by measuring enzyme activity in different concentrations of substrate for Lineweaver-Burk plot analysis. Its inhibition was found to be noncompetitive. The addition of Fe2+/ascorbate to the liver homogenate significantly increased the extent of lipid peroxidation which was significantly lowered with the extract treatment. Furthermore, DKLE ability to alleviate postprandial hyperglycemia in normal and streptozotocin (STZ)-induced diabetic rats was scrutinized. Oral administration of this leaf extract (250 to 1000 mg/kg body wt.) considerably decreased blood glucose in normal and diabetic rats after glucose and maltose loading in a dose-dependent manner. These results propose that DKLE might show an anti-diabetic effect by suppressing carbohydrate and glucose absorption from the intestine and can reduce the postprandial blood glucose rise. Key words: Diospyros kaki, α-glucosidase, antioxidant, diabetic rats, postprandial hyperglycemia, streptozotocin.

Improved specific productivity in cephalexin synthesis by immobilized PGA in silica magnetic micro‐particles

There is a marked trend in pharmaceutical industry towards the replacement of classical organic methods by "green" alternatives that minimize or eliminate the generation of waste and avoid, where possible, the use of toxic and/or hazardous reagents and solvents. In this work the kinetically controlled synthesis of cephalexin by soluble and penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties was performed in aqueous media with PGME and 7-ADCA as substrates, at different concentrations of substrate, temperature, pH, enzyme to substrate ratio and acyl donor to nucleophile ratio. Excess acyl donor had a strong effect on cephalexin productivity. A PGME/7-ADCA ratio of 3 was considered optimum. A maximum specific productivity of 5.9 mmol h(-1), gbiocatalyst(-1) at 160 mM 7-ADCA, 480 mM PGME and low enzyme to substrate ratio at 32.5 U mmol(-1) 7-ADCA was obtained with immobilized PGA in full aqueous medium, suggesting that diffusional limitations were minimized when compared with other commercial biocatalysts. A half-life of 133 h for the immobilized biocatalyst was estimated during cephalexin synthesis in the presence of 100 mM 7-ADCA and 300 mM PGME, in 50 mM Tris/HCl at pH 7.2 and 14°C. These results compare quite favorably with those previously reported for the kinetically controlled synthesis of cephalexin.

Nutritional care: from the dark ages to the renaissance, to the age of enlightenment

Nutritional care: from the dark ages to the renaissance, to the age of enlightenment

Inhibitory effect of methanol extract of Rosa damascena Mill. flowers on α-glucosidase activity and postprandial hyperglycemia in normal and diabetic rats

The effect of a methanol extract of Rosa damascena Mill. flowers was studied, in comparison to the alpha-glucosidase inhibitor acarbose, in normal and diabetic rats. The inhibition mode of this extract was examined by measuring enzyme activity in different concentrations of substrate for Lineweaver-Burk plot analysis. The results show that Rosa damascena extract has an intensive inhibitory effect on alpha-glucosidase. Its inhibition was found to be noncompetitive. Oral administration of this plant extract (100 to 1000 mg/kg body wt.) significantly decreased blood glucose after maltose loading in normal and diabetic rats in a dose-dependent manner. These results suggest that Rosa damascena might exert an anti-diabetic effect by suppressing carbohydrate absorption from the intestine and can reduce the postprandial glucose level.

Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

The Suggested Physiologic Aryl Hydrocarbon Receptor Activator and Cytochrome P4501 Substrate 6-Formylindolo[3,2-b]carbazole Is Present in Humans

Dioxins and other polycyclic aromatic compounds formed during the combustion of waste and fossil fuels represent a risk to human health, as well as to the well being of our environment. Compounds of this nature exert carcinogenic and endocrine-disrupting effects in experimental animals by binding to the orphan aryl hydrocarbon receptor (AhR). Understanding the mechanism of action of these pollutants, as well as the physiological role(s) of the AhR, requires identification of the endogenous ligand(s) of this receptor. We reported earlier that activation of AhR by ultraviolet radiation is mediated by the chromophoric amino acid tryptophan (Trp), and we suggested that a new class of compounds derived from Trp, in particular 6-formylindolo[3,2-b]carbazole (FICZ), acts as natural high affinity ligands for this receptor. Here we describe seven new FICZ-derived indolo[3,2-b]carbazole-6-carboxylic acid metabolites and two sulfoconjugates, and we demonstrate the following. (i) FICZ is formed efficiently by photolysis of Trp upon exposure to visible light. (ii) FICZ is an exceptionally good substrate for cytochromes P450 (CYP) 1A1, 1A2, and 1B1, and its hydroxylated metabolites are remarkably good substrates for the sulfotransferases (SULTs) 1A1, 1A2, 1B1, and 1E1. Finally, (iii) sulfoconjugates of phenolic metabolites of FICZ are present in human urine. Our findings indicate that formylindolo[3,2-b]carbazols are the most potent naturally occurring activators of the AhR signaling pathway and may be the key substrates of the CYP1 and SULT1 families of enzymes. These conclusions contradict the widespread view that xenobiotic compounds are the major AhR ligands and CYP1 substrates.

Biotransformation of Vinclozolin in Rat Precision-Cut Liver Slices: Comparison with in Vivo Metabolic Pattern

Vinclozolin is a dicarboxymide fungicide that presents antiandrogenic properties through its two hydrolysis products M1 and M2, which bind to the androgen receptor. Because of the lack of data on the biotransformation of vinclozolin, its metabolism was investigated in vitro in precision-cut rat liver slices and in vivo in male rat using [ (14)C]-vinclozolin. Incubations were performed using different concentrations of substrate, and the kinetics of formation of the major metabolites were studied. Three male Wistar rats were fed by gavage with [ (14)C]-VZ. Urine was collected for 24 h and analyzed by radio-HPLC for metabolic profiling. Metabolite identification was carried out on a LCQ ion trap mass spectrometer. In rat liver slices and in vivo, the major primary metabolite has been identified as 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (M5) and was mainly present as glucuronoconjugates. M5 is produced by dihydroxylation of the vinyl group of M2. Other metabolites have been identified as 3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione (M4), a dihydroxylated metabolite of vinclozolin, which undergoes further conjugation to glucuronic acid, and 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3,4-dihydroxy-butanoic acid (M6), a dihydroxylated metabolite of M1.

Functional Coupling between the Kv1.1 Channel and Aldoketoreductase Kvβ1

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.

The impact of Cytochrome P450‐2D6 genotype on the use and interpretation of therapeutic drug monitoring in long‐stay patients treated with antidepressant and antipsychotic drugs in daily psychiatric practice

This retrospective follow-up study investigates whether cytochrome P450-2D6 (CYP2D6) genotype explains variability in plasma concentrations of psychotropic drugs in daily psychiatric practice. The study population consisted of 62 hospitalised psychiatric patients genotyped for CYP2D6. Primary endpoint was the normalised plasma concentration ratio which was defined as the [measured concentration]/[mean therapeutic concentration] allowing comparison of plasma concentrations of different substrates. Secondary endpoint was a plasma concentration above the therapeutic range. The determinant was CYP2D6 genotype classified as ultrarapid metaboliser (UM), extensive metaboliser (EM), intermediate metaboliser (IM), or poor metaboliser (PM). The relation between CYP2D6 genotype and the normalised plasma concentration ratio was assessed with a linear mixed-effects model after adjustment for the Prescribed Daily Dose (PDD). The risk of having a plasma concentration above the therapeutic range was assessed with a logistic mixed-effects model. For antidepressants, CYP2D6 genotype PM (1.68 (95%CI: 1.01-2.28)) and IM (1.09 (95%CI: 0.77-1.29)) were associated with higher normalised plasma concentration ratios of antidepressants compared to EMs (0.56 (95%CI: 0.26-0.74)). In addition, the risk of a plasma concentration above the therapeutic range was increased for PMs (OR 33.1 (95%CI: 2.0-544.6)) and IMs (OR 8.2 (95%CI: 1.1-60.3)) relative to EMs using antidepressants. CYP2D6 genotype could not clearly explain variability in plasma concentrations of antipsychotics possibly due to a low frequency of therapeutic drug monitoring (TDM) in antipsychotics primarily metabolised by CYP2D6 in daily psychiatric practice. CYP2D6 genotype contributes to clinically relevant variability in plasma concentrations of antidepressants but probably not antipsychotics in daily clinical practice.

Kinetic model for whey protein hydrolysis by alcalase multipoint-immobilized on agarose gel particles

Partial hydrolysis of whey proteins by enzymes immobilized on an inert support can either change or evidence functional properties of the produced peptides, thereby increasing their applications. The hydrolysis of sweet cheese whey proteins by alcalase, which is multipoint-immobilized on agarose gel, is studied here. A Michaelis-Menten model that takes into account competitive inhibition by the product was fitted to experimental data. The influence of pH on the kinetic parameters in the range 6.0 to 11.0 was assessed, at 50oC. Initial reaction-rate assays in a pHstat at different concentrations of substrate were used to estimate kinetic and Michaelis-Menten parameters, k and KM. Experimental data from long-term batch assays were used to quantify the inhibition parameter, KI. The fitting of the model to the experimental data was accurate in the entire pH range.

Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors.

This paper is a review of the authors' publications concerning the development of biosensors based on enzyme field-effect transistors (ENFETs) for direct substrates or inhibitors analysis. Such biosensors were designed by using immobilised enzymes and ion-selective field-effect transistors (ISFETs). Highly specific, sensitive, simple, fast and cheap determination of different substances renders them as promising tools in medicine, biotechnology, environmental control, agriculture and the food industry. The biosensors based on ENFETs and direct enzyme analysis for determination of concentrations of different substrates (glucose, urea, penicillin, formaldehyde, creatinine, etc.) have been developed and their laboratory prototypes were fabricated. Improvement of the analytical characteristics of such biosensors may be achieved by using a differential mode of measurement, working solutions with different buffer concentrations and specific agents, negatively or positively charged additional membranes, or genetically modified enzymes. These approaches allow one to decrease the effect of the buffer capacity influence on the sensor response in an aim to increase the sensitivity of the biosensors and to extend their dynamic ranges. Biosensors for the determination of concentrations of different toxic substances (organophosphorous pesticides, heavy metal ions, hypochlorite, glycoalkaloids, etc.) were designed on the basis of reversible and/or irreversible enzyme inhibition effect(s). The conception of an enzymatic multibiosensor for the determination of different toxic substances based on the enzyme inhibition effect is also described. We will discuss the respective advantages and disadvantages of biosensors based on the ENFETs developed and also demonstrate their practical application.

Thermal oscillations in the bromate – oxalic acid – MnSO4– H2SO4– acetone system: A calorimetric study

The oscillatory reaction of potassium bromate, oxalic acid, and MnSO4in the presence of acetone in aq H2SO4solutions has been studied calorimetrically at different concentrations of substrates. Acetone and oxalic acid have been found to accelerate the reaction by increasing the oscillation frequency. On the other hand, MnSO4has a retarding effect; the frequency of oscillation decreases systematically with increasing concentration of MnSO4. Potassium bromate shows mixed behavior in this respect. The total heat evolved during the reaction per mol of oxalic acid consumed has been evaluated, which can be used to calculate the concentrations of the various reactants at different stages of the reaction. The effects of different electrolytes (KCl, NaCl, BaCl2, CaCl2, SrCl2, NaBr, NaI, KNO3, NaNO3, and Ca(NO3)2) and solvents (dimethylformamide, 1,4-dioxane, acetonitrile, and tetrahydrofuran) on the extents of oscillation and the enthalpy change for the process have been also examined.Key words: Belousov–Zhabotinsky reaction, oscillatory reaction, oxalic acid, calorimetry, acetone.

Molecular Cloning and Characterization of the Gene Coding for Azoreductase from Bacillus sp. OY1-2 Isolated from Soil

Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp. OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH. A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp. OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp. OY1-2. In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli.

Inhibition and activation of enzymes. The effect of a modifier on the reaction rate and on kinetic parameters.

A combined analysis of enzyme inhibition and activation is presented, based on a rapid equilibrium model assumption in which one molecule of enzyme binds one molecule of substrate (S) and/or one molecule of a modifier X. The modifier acts as activator (essential or non-essential), as inhibitor (total or partial), or has no effect on the reaction rate (v), depending on the values of the equilibrium constants, the rate constants of the limiting velocity steps, and the concentration of substrate ([S]). Different possibilities have been analyzed from an equation written to emphasize that v = f([X]) is, in general and at a fixed [S], a hyperbolic function. Formulas for Su (the value of [S], different from zero, at which v is unaffected by the modifier) and v(su) (v at that particular [S]) were deduced. In Lineweaver-Burk plots, the straight lines related to different [X] generally cross in a point (P) with coordinates (Su, v(su)). In certain cases, point P is located in the first quadrant which implies that X acts as activator, as inhibitor, or has no effect, depending on [S]. Furthermore, we discuss: (1) the apparent Vmax and Km displayed by the enzyme in different situations; (2) the degree of effect (inhibition or activation) observed at different concentrations of substrate and modifier; (3) the concept of Ke, a parameter that depends on the concentration of substrate and helps to evaluate the effect of the modifier: it equals the value of [X] at which the increase or decrease in the reaction rate is half of that achieved at saturating [X]. Equations were deduced for the general case and for particular situations, and used to obtain computer-drawn graphs that are presented and discussed. Formulas for apparent Vmax, Km and Ke have been written in a way making it evident that these parameters can be expressed as pondered means.

Relationship between electron donor and microorganism on the dechlorination of carbon tetrachloride by an anaerobic enrichment culture

An investigation involving the supplement of different concentrations of substrates and microorganisms was carried out under anaerobic condition to assess the feasibility of bioremediation of carbon tetrachloride (CCl 4) with the amendment of low concentrations of auxiliary substrate and microorganisms. The concentrations of substrate and microorganisms ranged from 10 to 100 mg/l and from 3.7 × 10 4 to 3.7 × 10 6 cell/ml, respectively. The biotransformation rate of CCl 4 increased progressively with the increase in the concentrations of the substrate and microorganisms. In the low biomass-amended system (3.7 × 10 4cells/ml), 28–71% and 57–96% of CCl 4 removals were exhibited when 10–100 mg/l of acetate or glucose was supplemented, respectively, whereas nearly complete degradation of CCl 4 was observed in the heavily inoculated systems (3.7 × 10 6 cells/ml). An addition of electron donor in the low microbial activity batches enhanced greater efficiency in dechlorination than in the high microbial activity batches. The second-order rate constants ranged from 0.0059 to 0.0092 l/mg/day in high biomass input system, while a two- to four-fold increase in rate constant was obtained in the low microbial activity system. This study indicates that biomass was the more important environmental parameter than substrate affecting the fate of CCl 4. The addition of auxiliary substrates was effective only in low biomass-amended batches (0.56 mg-VSS/l) and diminished inversely with the increase of microbial concentration.

Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition.

The steady-state reaction of trimethylamine dehydrogenase (TMADH) with the artificial electron acceptor ferricenium hexafluorophosphate (Fc(+)) has been studied by stopped-flow spectroscopy, with particular reference to the mechanism of inhibition by trimethylamine (TMA). Previous studies have suggested that the presence of alternate redox cycles is responsible for the inhibition of activity seen in the high-substrate regime. Here, we demonstrate that partitioning between these redox cycles (termed the 0/2 and 1/3 cycles on the basis of the number of reducing equivalents present in the oxidized/reduced enzyme encountered in each cycle) is dependent on both TMA and electron acceptor concentration. The use of Fc(+) as electron acceptor has enabled a study of the major redox forms of TMADH present during steady-state turnover at different concentrations of substrate. Reduction of Fc(+) is found to occur via the 4Fe-4S center of TMADH and not the 6-S-cysteinyl flavin mononucleotide: the direction of electron flow is thus analogous to the route of electron transfer to the physiological electron acceptor, an electron-transferring flavoprotein (ETF). In steady-state reactions with Fc(+) as electron acceptor, partitioning between the 0/2 and 1/3 redox cycles is dependent on the concentration of the electron acceptor. In the high-concentration regime, inhibition is less pronounced, consistent with the predicted effects on the proposed branching kinetic scheme. Photodiode array analysis of the absorption spectrum of TMADH during steady-state turnover at high TMA concentrations reveals that one-electron reduced TMADH-possessing the anionic flavin semiquinone-is the predominant species. Conversely, at low concentrations of TMA, the enzyme is predominantly in the oxidized form during steady-state turnover. The data, together with evidence derived from enzyme-monitored turnover experiments performed at different concentrations of TMA, establish the operation of the branched kinetic scheme in steady-state reactions. With dimethylbutylamine (DMButA) as substrate, the partitioning between the 0/2 and 1/3 redox cycles is poised more toward the 0/2 cycle at all DMButA concentrations studied-an observation that is consistent with the inability of DMButA to act as an effective inhibitor of TMADH.

Inactivation of succinate–ubiquinone reductase in substrate mixture

Succinate–ubiquinone reductase plays an important role in the respiratory chain. Previous work showed that preparation of succinate–ubiquinone reductase was relatively stable. Though the enzyme catalysis has been extensively studied, the inactivation of succinate–ubiquinone reductase has never been reported. In the present study, the kinetic theory of the substrate reaction of irreversible inhibition described by Tsou (Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381–436) was applied to study the course of an unexpected slow inactivation of succinate–ubiquinone reductase in the substrate assay mixture containing different concentrations of substrates, succinate and 2,6-dichloroindophenol. The results showed that the inactivation of succinate–ubiquinone reductase in the substrate mixture is a first order reaction. The inactivation rate decreased with increasing concentration of succinate. The values of the micro rate constants for free and succinate bound enzyme were 0.22±0.01 and 0.052±0.002 min −1, respectively. Binding with 2-thenoyl-trifluroacetone, a inhibitor specially for the quinone binding site, slowed down the inactivation. However, the rate of inactivation did not change with increasing 2,6-dichloroindophenol concentration. The study showed that succinate–ubiquinone reductase was irreversibly inactivated in the substrate mixture. The results suggest that the inactivation was not due to dilution or dissociation of the enzyme, nor to complete usage of the substrate, inhibition of the yielded product or some possible trace component in the substrate mixture, nor to modification of the essential thiol group in the succinate binding site of succinate–ubiquinone reductase. The enzyme became more stable after binding with succinate.

Glucose Transporter Isoforms GLUT1 and GLUT3 Transport Dehydroascorbic Acid

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1-5 and SGLT1. The apparent Km of DHA transport via GLUT1 and GLUT3 was 1.1 +/- 0.2 and 1.7 +/- 0.3 mM, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2-4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, D-glucose, and 3-O-methylglucose among other hexoses while fructose and L-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-alpha-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2-8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.

Modeling of recombinant bacteria fermentation for enhanced productivity

Accurate kinetic models are necessary in order to scale biochemical processes which utilize recombinant organisms. In this work, the performance of a kinetic model proposed by Miao and Kompala (Biotechnol. Bioeng.,40, 787–796, 1992) has been tested against experimental data at different concentrations of substrates. The experimental results from the induction of the batch cultures with IPTG (isopropyl-β-D-thiogalactopyranoside) prove that the model equations are reliable in predicting the biomass and foreign protein concentrations as well as the optimum induction time.

Effects of the male antifertility agent ornidazole on sperm function in vitro and in the female genital tract.

Infertility is induced in male rats by oral administration of 400 mg ornidazole kg-1 body mass day-1 within 10 days, without drastic effect on the motility of epididymal spermatozoa obtained after 15 days of treatment. Spermatozoa were recovered from the female tract 9 h after mating with males treated with ornidazole for 10 days to identify the cause of infertility. The number and motility of spermatozoa in the uterus indicated normal seminal deposition. Spermatozoa could pass the utero-tubal junction but total numbers of spermatozoa and their velocities in the oviductal isthmus were significantly lower than those recovered from females mated to fertile, control males fed vehicle alone. However, the number of spermatozoa recovered from the ampulla was not different from control and the number of spermatozoa that had penetrated the cumulus mass was lower and none of the eggs was fertilized by spermatozoa from rats treated with ornidazole. The ability of cauda epididymal spermatozoa from ornidazole-fed rats to penetrate viscous media in vitro was lower than that of the controls. When incubated in media containing different concentrations of substrates and ions, a reduction in movement of spermatozoa was observed when glucose was the only exogenous substrate or after preincubation in substrate-free medium. These results indicate that the antifertility action of orally administered ornidazole occurs via damage to spermatozoa in the upper regions of the female tract, possibly reflecting a failure in capacitation as a result of reduced energy production.