Significant Competitive Inhibition Research Articles

A CAPRICE gene of Rosa rugosa (RrCPC) suppresses the trichome formation of Arabidopsis

Rosa rugosa is a spice plant with important economic value in the world. Its stem is densely covered with thorns. Its anatomical structure, as a special form of epidermal hair with developed secondary structure, has always gained attention. However, the molecular mechanism of its developmental regulation remains to be studied. In this research, rose prickle types were first investigated and classified, and R. rugosa ‘Zizhi’ with only non-glandular prickles excavated. The number of prickles on the primary, secondary, and tertiary branches of R. rugosa ‘Zizhi’ were gradually decreased and corresponding tissues were used for transcriptome sequencing. 4588 differentially expressed genes were identified and GO enriched terms of ‘highly intensified response to stimulus’, ‘cell periphery’, and ‘nucleic acid binding transcription factor activity’, as well as KEGG enriched pathway of ‘plant hormone signal transduction’, were closely related to the development of rose prickles. CAPRICE (RrCPC) gene which upregulated with the decreasing prickle number was decided as a candidate gene involved in the prickle development of R. rugosa. RrCPC ectopic expression significantly inhibited trichomes in different organs of Arabidopsis by down-regulation of GL2 and other endogenous genes. It indicated that overexpression of RrCPC posed a significant competitive inhibition on the MBW complex. Meanwhile, consistent enrichment GO terms shared by rose and Arabidopsis suggested that regulation genes involved in prickle development may be overlapped with that of trichome. These results revealed that RrCPC was a negative regulator of the trichomes but its correlation with number of R. rugosa prickle was ambiguous. This study proved the wide difference in physiological structure of prickles and trichomes, and provided a potential gene for further genetic analysis of R. rugosa prickle formation.

Enhanced activity of hyperthermostable Pyrococcus horikoshii endoglucanase in superbase ionic liquids

ObjectivesIonic liquids (ILs) that dissolve biomass are harmful to the enzymes that degrade lignocellulose. Enzyme hyperthermostability promotes a tolerance to ILs. Therefore, the limits of hyperthemophilic Pyrococcus horikoschii endoglucanase (PhEG) to tolerate 11 superbase ILs were explored.ResultsPhEG was found to be most tolerant to 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) in soluble 1% carboxymethylcellulose (CMC) and insoluble 1% Avicel substrates. At 35% concentration, this IL caused an increase in enzyme activity (up to 1.5-fold) with CMC. Several ILs were more enzyme inhibiting with insoluble Avicel than with soluble CMC. Km increased greatly in the presence ILs, indicating significant competitive inhibition. Increased hydrophobicity of the IL cation or anion was associated with the strongest enzyme inhibition and activation. Surprisingly, PhEG activity was increased 2.0–2.5-fold by several ILs in 4% substrate. Cations exerted the main role in competitive inhibition of the enzyme as revealed by their greater binding energy to the active site.ConclusionsThese results reveal new ways to design a beneficial combination of ILs and enzymes for the hydrolysis of lignocellulose, and the strong potential of PhEG in industrial, high substrate concentrations in aqueous IL solutions.

Effects of Additional Carbon Sources in the Biodegradation of 1,4-Dioxane by a Mixed Culture

A mixed culture utilizing 1,4-dioxane as a sole carbon and energy source was obtained from the activated sludge at a textile wastewater treatment plant. The biodegradation of 1,4-dioxane was characterized by a model based on the Monod equation. The effects of the presence of easily degradable carbon sources other than 1,4-dioxane were investigated using dextrose. Structural analogs commonly found in 1,4-dioxane-containing wastewater such as tetrahydrofuran (THF), 2-methyl-1,3-dioxolane, and 1,4-dioxene were also evaluated for their potential effects on 1,4-dioxane biodegradation. The presence of dextrose did not show any synergetic or antagonistic effects on 1,4-dioxane biodegradation, while the structural analogs showed significant competitive inhibition effects. The inhibitory effects were relatively strong with heptagonal cyclic ethers such as THF and 2-methyl-1,3-dioxolane, and mild with hexagonal cyclic ethers such as 1,4-dioxene. It was also shown that the treatment of 1,4-dioxane in the raw textile wastewater required 170% more time to remove 1,4-dioxane due to the co-presence of 2-methyl-1,3-dioxolane, and the extent of delay depended on the initial concentration of 1,3-doxolane.

Physiologically based pharmacokinetic modeling of CYP3A4 induction by rifampicin in human: Influence of time between substrate and inducer administration

The induction of cytochrome P450 enzymes (CYPs) is an important source of drug–drug interaction (DDI) and can result in pronounced changes in pharmacokinetics (PK). Rifampicin (RIF) is a potent inducer of CYP3A4 and also acts as a competitive inhibitor which can partially mask the induction. The objective of this study was to determine a clinical DDI study design for RIF resulting in maximum CYP3A4 induction.A physiologically based pharmacokinetic (PBPK) model was developed to project the dynamics and magnitude of CYP3A4 induction in vivo from in vitro data generated with primary human hepatocytes. The interaction model included both inductive and inhibitory effects of RIF on CYP3A4 in gut and liver and accounting for the observed RIF auto-induction. The model has been verified for 4 CYP3A4 substrates: midazolam, triazolam, alfentanil and nifedipine using plasma concentration data from 20 clinical study designs with intravenous (n=7) and oral (n=13) administrations. Finally, the influence of the time between RIF and substrate administration was explored for the interaction between midazolam and RIF.The model integrating in vitro induction parameters correctly predicted intravenous induction but underestimated oral induction with 30% of simulated concentrations more than 2-fold higher than of observed data. The use of a 1.6-fold higher value for the maximum induction effect (Emax) improved significantly the accuracy and precision of oral induction with 82% of simulated concentrations and all predicted PK parameters within 2-fold of observed data. Our simulations suggested that a concomitant administration of RIF and midazolam resulted in significant competitive inhibition limited to intestinal enzyme. Accordingly, a maximum induction effect could be achieved with a RIF pretreatment of 600mg/day during 5days and a substrate administration at least 2h after the last RIF dose. A period of 2weeks after RIF removal was found sufficient to allow return to baseline levels of enzyme.

β-oxidation modulates metabolic competition between eicosapentaenoic acid and arachidonic acid regulating prostaglandin E 2 synthesis in rat hepatocytes–Kupffer cells

The ability of n-3 PUFA to competitively inhibit the use of arachidonic acid (AA) for membrane phospholipid synthesis and prostaglandin E 2 (PGE 2) production has been well demonstrated in single cell models. In the present study, we investigated the metabolic competition between AA and eicosapentaenoic acid (EPA) for PGE 2 synthesis in a rat hepatocyte–Kupffer cell (HPC/KC) co-culture system when the cellular oxidation capacity was enhanced by exogenous l-carnitine. We demonstrate that in the absence of l-carnitine, 1) β-oxidation rates of EPA and AA were comparable in HPCs and in KCs; 2) AA and not EPA was preferentially incorporated into glycerolipids; and 3) addition of EPA significantly decreased AA-dependent PGE 2 synthesis in HPCs and cyclooxygenase-2 (COX-2) expression in co-cultured HPCs/KCs. However, enhancing the cellular oxidation capacity by the addition of l-carnitine 1) significantly increased β-oxidation of EPA in HPCs, but only marginally elevated the oxidation of AA in HPCs and the oxidation of both fatty acids in KCs; 2) decreased the esterification, but did not alter the preferential incorporation of AA into glycerolipids; and 3) alleviated the significant competitive inhibition of AA-dependent PGE 2 synthesis and COX-2 expression by EPA. Taken together, the results strongly suggest that l-carnitine affects competition between AA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA in HPCs. This implies that the beneficial effects of n-3 PUFA, especially EPA, are affected by the cellular oxidation capacity.

Multisubstrate biodegradation kinetics for binary and complex mixtures of polycyclic aromatic hydrocarbons

Biodegradation kinetics were studied for binary and complex mixtures of nine polycyclic aromatic hydrocarbons (PAHs): Naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, 2-ethylnaphthalene, phenanthrene, anthracene, pyrene, fluorene, and fluoranthene. Discrepancies between the observed biodegradation rates and those predicted by a sole-substrate model indicate that significant substrate interactions occurred in both the binary and complex-mixture experiments. For all compounds except naphthalene, biodegradation was enhanced. The observations were compared to predictions from two multisubstrate biodegradation kinetic models: One that accounts for competitive inhibition, and one that does not. Both models are fully predictive in that parameters had been determined from an independent set of sole-substrate experiments. In the binary experiments, the major multisubstrate effect was biomass enhancement as a result of growth on naphthalene. Substrate interactions were orders of magnitude larger for most compounds in the complex mixtures, but significant competitive inhibition effects counteracted some of the biomass enhancement effect. The present study has demonstrated that the sole-substrate model is inadequate to describe multisubstrate biodegradation kinetics for a broad range of PAH mixtures. Whereas the multisubstrate model without inhibition did an adequate job of predicting the observed effects in some cases, we advocate the use of the multisubstrate model with inhibition for similar modeling efforts in light of the evidence that the model was correct more often than not. Theory supports its use because of the common enzyme pathways for biodegradation of PAHs.

Effect of various doses of deoxynivalenol on liver xenobiotic metabolizing enzymes in mice

DON is one of the major mycotoxic contaminant of cereal grains throughout the world. The purpose of this investigation was to characterize the effects of a range of environmentally relevant doses of DON in mice exposed through a subchronic toxicological assay. Animals received 3 days per week for 4 weeks, 0.014, 0.071, 0.355 or 1.774 mg of toxin/kg b.w. All doses, except 0.014 mg/kg, provoked increases in plasma immunoglobulin A whereas there was no change in plasma biochemical parameters such as alkaline phosphatase, electrolytes or other immunoglobulins. Administration of 0.071 or 0.355 mg/kg doses led to increased liver microsomal pentoxyresorufin depentylase and cytosolic glutathione transferase activities. Examining protein modulation, western blot analyses liver fractions from mice receiving these doses revealed increased levels in both P450 2b, GST α and π isoenzymes without any change in P450 1a expression. A significant competitive inhibition of deoxynivalenol on CDNB conjugation in vitro suggests that the mycotoxin is a putative substrate for glutathione S-transferases. These changes in liver xenobiotic metabolizing enzymes are discussed by considering the structural nature of deoxynivalenol and previous reports on similar effects exerted by other trichothecenes. These results suggest that a subchronic exposure to low doses of deoxynivalenol causes changes in the normal liver metabolism of xenobiotics.

Inhibition of Friend Murine Leukemia Virus activity by guanosine/thymidine oligonucleotides

Oligonucleotides consisting of only deoxyguanosine and deoxythymidine were stable in culture and were able to significantly inhibit Friend Murine Leukemia Virus (FMLV) production in acute cell culture assay systems. The oligonucleotides did not share homology with, or possess any complementary (antisense) sequence motifs to the FMLV genome. The guanosine/thymidine-containing oligonucleotides (GTOs) which demonstrated anti-FMLV activity in acute infection assays were synthesized with natural phosphodiester (PD) linkages (backbones). The observed antiviral activities of these oligonucleotides increased significantly when the PD backbone was replaced with a phosphorothioate (PT) backbone. Experiments designed to investigate a potential antiviral mechanism of action demonstrated that oligonucleotides tested were capable of blocking virus adsorption. In addition, GTOs with PD backbones were competitive inhibitors of FMLV reverse transcriptase (RT). When the same experiments were performed using oligonculeotides with PT backbones, all compounds tested demonstrated significant competitive inhibition of FMLV RT. The measured inhibitory activity of all compounds tested in culture assays was enhanced by at least a factor of 10 when the PD linkages were replaced with PT. The enhanced antiviral activity exhibited by the sulfur group on the oligonucleotide backbone, and the lack of any designed, sequence-specific interactions, suggest that a large percentage of the reported antiviral activity of oligonucleotides containing a phosphorothioate backbone is due to factors other than rationally designed, sequence-specific interactions. The ability of GTOs to inhibit FMLV in culture, potentially via a number of different mechanisms, makes this a class of compounds which warrants investigation as therapeutic agents to be used against retroviral infections.

Export of mRNA from microinjected nuclei of Xenopus laevis oocytes.

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.

Hexose uptake in primary cultures of bovine brain microvessel endothetial cells. I. Basic characteristics and effects of d-glucose and insulin

The basic characteristics of hexose uptake and regulation of the glucose transporter (GLUT1) by d-glucose and insulin were studied in primary cultures of bovine brain microvessel endothelial cells (BMECs). A non-metabolizable glucose analog, 3- O-[ 3H]methyl- d-glucose ([ 3H]3MG), was used as a model substrate, and the uptake was studied using BMECs grown in tissue culture plates. Uptake of [ 3H]3MG was equilibrative, temperature-dependent, and independent of sodium. The uptake also decreased gradually with culture age from 7 to 13 days. Saturation kinetics were observed for [ 3H]3MG uptake and the apparent K m and V max values were determined to be 13.2 mM and 169 nmol/mg per min, respectively. Pre-incubation with high concentrations of d-glucose and 3MG accelerated [ 3H]3MG uptake by BMECs by a counter-transport mechanism. d-Glucose, 2-deoxy- d-glucose, d-mannose, d-xylose, d-galactose and d-ribose showed significant competitive inhibition with (3H]3MG, whereas l-glucose, d-fructose, and sucrose did not affect [ 3H]3MG uptake by BMECs. [ 3H]3MG uptake was inhibited significantly by cytochalasin B and phloretin but not by phlorizin, 2,4-dinitrophenol, or ouabain. d-Glucose starvation of BMECs by incubation with d-glucose-free media for 24 h resulted in a significant increase (40–70%) in uptake of [ 3H]3MG compared with control conditions (7.3 mM d-glucose). Low d-glucose treatments (2.43 and 1.83 mM) for 7 days induced a slight but significant increase (20%) in [ 3H]3MG uptake, while long-term high glucose treatments (25 mM) showed no significant effect on [ 3H]3MG uptake irrespective of exposure time. The increase in [ 3H]3MG accumulation following d-glucose starvation was dependent upon starvation time (12 to 48 hr) and protein synthesis. Refeeding of d-glucose (7.3 mM) to d-glucose-starved BMECs resulted in a return of [ 3H]3MG uptake to control levels in 48 h. The d-glucose-starvation-induced increase in [ 3H]3MG uptake was shown to result from an increase in V max; the K m remained constant. In addition, d-glucose-starved BMECs were shown to have an increased level of GLUT1 using an antibody against human GLUT1 and an enzyme-linked immunosorbent assay (ELISA). The increased uptake following d-glucose starvation was not significantly affected by the presence of l-glucose, was partially impaired by the presence of d-galactose, d-fructose, and d-xylose, and was completely inhibited by the presence of d-mannose and 3MG. Furthermore, preincubation of BMECs with insulin (10 μg/ml) for 20 min did not affect the uptake of [ 3H]3MG or 2-deoxy d-[ 3H]glucose ([ 3H]2DG). The present study demonstrated that hexoses can be taken up by cultured bovine BMECs by a carrier-mediated, facilitated diffusio mechanism, similar to the mechanism observed in the blood-brain barrier (BBB) and that the uptake by bovine BMECs was regulated by low levels of d-glucose but not by high levels of d-glucose or by insulin.

Characterization of the T cell-mediated cellular cytotoxicity during acute infectious mononucleosis.

Primary infection with EBV during acute infectious mononucleosis (IM) is associated with a cytotoxic response against allogeneic target cells. C depletion with anti-CD3 (OKT3) and anti-CD8 (OKT8) mAb decreased the allogeneic cytolysis of two EBV-infected lymphoblastoid cell lines (LCL) by 96% and 89%, respectively. Complement depletion with the NK cell-specific mAb Leu-11b and NKH-1a resulted in only a slight decrease (less than 35%) in the lysis of these LCL. mAb inhibition studies with OKT3 and OKT8 inhibited the allogeneic lysis of two LCL by 87% and 82%, respectively. The alloreactive cytotoxic response was strongly inhibited by mAb specific for MHC class I determinants (W6/32, 65% inhibition and BBM.1, 58% inhibition). Acute IM lymphocytes lysed the allogeneic EBV-negative cell lines HSB2 (45%) and HTLV-1 T cell lines (16%). NK cell-depleted lymphocytes from an acute IM patient demonstrated preferential lysis of K562 transfected with human HLA-A2 (73%) compared with the K562 transfected control (20%). Cold target competition studies with allogeneic and autologous target and competitor LCL demonstrated no significant competitive inhibition between allogeneic and autologous cells. We interpret these results as evidence that 1) the acute IM-alloreactive cytotoxic response is mediated primarily by CTL; 2) these alloreactive CTL lyse allogeneic target cells irrespective of EBV antigenic expression; 3) MHC class I expression is sufficient for allogeneic recognition and lysis of target cells; 4) distinct effector CTL populations mediate lysis of autologous and allogeneic target cells; and 5) during acute IM, EBV infection results in the induction of both virus-specific and alloreactive CTL populations.

GABA uptake is inhibited by thyroid hormones: Implications for depression

Studies of the effects of thyroid hormones on the uptake of neurotransmitters by homogenates of rat cerebral cortex have revealed a significant competitive inhibition of neuronal uptake of [ 3H]GABA by thyroid hormones (T 1 > T 4 > rT 4). The IC 50 for inhibition of GABA uptake by T 3 was estimated at 4 μM and that of T 4 at 11 μm. GABA uptake in homogenates of cerebral cortex from hypothyroid rats was significantly enhanced over that of controls; however, uptake in tissues from hyperthyroid rats was not significantly diminished.

Studies on mouse autoreactive cells: I. Role of H-2 antigens in mouse autologous rosette formation

A minority (1–2%) of normal mouse lymphoid cells bind autologous erythrocytes and form rosettes. In this study we examined the antigenic specificity involved in the formation of such rosettes. A significant difference in the incidence of rosettes formed, respectively, with autologous and allogeneic mouse erythrocytes is found. Moreover, preincubation of lymphoid cells with low concentrations of syngeneic erythrocytic ghosts causes significant competitive inhibition of subsequent rosette formation. Allogeneic ghosts obtained from nonrelated or from congenic resistant strains of mice do not display this inhibitory effect under the same conditions. It is thus suggested that mouse autologous rosette-forming cells bear receptors for syngeneic H-2 antigens that are involved in the binding of autologous erythrocytes. More precisely, compatibility between lymphocyte and erythrocyte restricted to K or D only is sufficient to ensure a level of rosettes similar to that obtained when complete identity occurs for K, I, and D regions.