Hyperthermophilic Thermotoga arginine repressor binding to full-length cognate and heterologous arginine operators and to half-site targets

The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar. However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgR(Tn)) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus. A high-resolution contact map of ArgR(Tn) binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgR(Tn) establishes all of its strong contacts with a single ARG box-like sequence of the operator only. Protein array and electrophoretic mobility-shift data demonstrate that ArgR(Tn) has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets. Moreover, the overall effect of L-arginine on the apparent K(d) of ArgR(Tn) binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors. We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgR(Tn), instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArR(Tn) was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth. We assume that ArgR(Tn), owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga     

Inhibition of InhA activity,but not KasA activity,induces formation of a KasA-containing complex in mycobacteria

Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase InhA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of InhA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The InhA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of InhA inhibition. In addition, in vitro assays using purified InhA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited InhA but not KasA activity. Moreover, several thermosensitive InhA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of InhA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with InhA being the primary target of INH.     

Food effects on the absorption and pharmacokinetics of cocoa flavanols

Macronutrients in food and gastric acid are known to have a pronounced effect on the metabolism of many xenobiotics, an effect that impacts their efficacy as bioactive agents. In this investigation we assessed the impact of select food treatments and the histamine H(2)-receptor antagonist Famotidine (Pepcid-AC) on flavanol absorption and metabolism. Four crossover intervention studies were conducted with 6 subjects each. Volunteers consumed sugar-free, flavanol-rich cocoa (0.125 g/kg body wt) alone, with macronutrient-rich foods (8.75 or 17.5 kJ/kg subject body wt) or Famotidine (Pepcid-AC). Blood samples were drawn at 5 time points including baseline. Plasma samples were analyzed for epicatechin and catechin flavanols by HPLC. Pharmacokinetic parameters were assessed using non-compartmental methodology. When provided at 17.5 kJ/kg subject body weight (approximately 4 kcal/kg), sugar and bread test meals increased flavanol area under the curve (AUC) values to 140% of control values (P < 0.05). A corresponding tendency for plasma antioxidant capacity to increase was observed for the cocoa treatment at 1.5 and 2.5 h (P < 0.17, P < 0.06, respectively). The ability of treatment meals to affect AUC values was positively correlated with treatment carbohydrate content (r = 0.83; P< 0.02). In contrast to carbohydrate rich meals, lipid and protein rich meals and Famotidine treatment had minimal effects on flavanol absorption. Based on C(max) and AUC values, this data suggests that the uptake of flavanols can be increased significantly by concurrent carbohydrate consumption.     

A haplotype implicated in schizophrenia susceptibility is associated with reduced COMT expression in human brain

The gene encoding catechol-O-methyltransferase (COMT) is a strong candidate for schizophrenia susceptibility, owing to the role of COMT in dopamine metabolism, and the location of the gene within the deleted region in velocardiofacial syndrome, a disorder associated with high rates of schizophrenia. Recently, a highly significant association was reported between schizophrenia and a COMT haplotype in a large case-control sample (Shifman et al. 2002). In addition to a functional valine-->methionine (Val/Met) polymorphism, this haplotype included two noncoding single-nucleotide polymorphisms (SNPs) at either end of the COMT gene. Given the role of COMT in dopamine catabolism and that deletion of 22q11 (containing COMT) is associated with schizophrenia, we postulated that the susceptibility COMT haplotype is associated with low COMT expression. To test this hypothesis, we have applied quantitative measures of allele-specific expression using mRNA from human brain. We demonstrate that COMT is subject to allelic differences in expression in human brain and that the COMT haplotype implicated in schizophrenia (Shifman et al. 2002) is associated with lower expression of COMT mRNA. We also show that the 3' flanking region SNP that gave greatest evidence for association with schizophrenia in that study is transcribed in human brain and exhibits significant differences in allelic expression, with lower relative expression of the associated allele. Our results indicate that COMT variants other than the Val/Met change are of functional importance in human brain and that the haplotype implicated in schizophrenia susceptibility is likely to exert its effect, directly or indirectly, by down-regulating COMT expression.     

Water plays a different role on activation thermodynamic parameters of alcoholysis reaction catalyzed by lipase in gaseous and organic media

The effect of water on the alcoholysis of methyl propionate and n-propanol catalyzed by immobilized Candida antarctica lipase B (CALB) has been compared in a continuous solid-gas reactor and in an organic liquid medium. The enthalpic and entropic contributions of water to the Gibbs free energy of activation in the gas phase were different from the ones in the organic phase, the inverse trends being observed for the variation of both DeltaH* and DeltaS* with water activity.Different phenomena were identified for their influence on the thermodynamic parameters. When increasing a(w), the enhanced flexibility of the enzyme was predominant in the gas phase whereas substrate-solvent interactions due to an increased polarity of the solvent affected mainly the thermodynamic parameters in the organic phase. The observed variations of DeltaG* with water activity were in accordance with kinetics results previously obtained in both reaction media.     

In vivo effect of epidermal growth factor,interleukin-1beta,and interleukin-1RA on equine preovulatory follicles

Paracrine factors have significant effects during folliculogenesis. Because of various morphological features, the mare is a convenient model to study in vivo the effects of factors involved in periovulatory events. In the present work, epidermal growth factor (EGF; experiment 1, n = 49 mares) and interleukin-1beta and interleukin-1RA (IL-1beta and IL-1RA, respectively; experiment 2, n = 80 mares) were injected intrafollicularly to evaluate the influence of these factors on in vivo maturation of equine preovulatory follicles. A transvaginal ultrasound-guided injection was performed when the diameter of the dominant follicle reached 30-34 mm. In experiment 1, the four experimental groups were 1) EGF group, intrafollicular (i.f.) injection of EGF (2 ml; 0.5 microg/ml) plus i.v. injection of physiological serum; 2) control group, no injection; 3) PBS group, i.f. injection of 2 ml of PBS plus i.v. injection of physiological serum; 4) crude equine gonadotropins (CEG) group, i.f. injection of PBS plus i.v. injection of CEG (20 mg). In experiment 2, groups 3 and 4 were the same as in experiment 1, but groups 1 and 2 were changed as follows: 1) IL-1beta group, i.f. injection of IL-1beta (2 ml; 0.5 microg/ml) plus i.v. injection of physiological serum; 2) IL-1RA group, i.f. injection of IL-1RA (2 ml; 0.5 microg/ml) plus i.v. injection of physiological serum. In each experiment, cumulus-oocyte complexes from dominant/injected follicles were collected by transvaginal ultrasound-guided aspiration 38 h after intrafollicular injection. Cumulus morphology and oocyte nuclear stage were assessed. Additionally, in experiment 2, 40 mares were used to determine the time of ovulation after treatments. Our results indicate that intrafollicular injection of EGF or PBS induced lower cumulus expansion and oocyte maturation rates compared with the CEG group (P < 0.05). In experiment 2, the IL-1beta and CEG groups showed the same expansion rate, the same oocyte maturation rate, and the same ovulation distribution. On the other hand, the intrafollicular injection of IL-1RA, as PBS, did not induce follicle and cumulus-oocyte complex (COC) maturation. In conclusion, we confirmed that the technique of intrafollicular injection can be used in the mare to study the role of specific molecules. We demonstrated for the first time in mares that the injection of EGF did not influence in vivo COC maturation. In contrast, IL-1beta injection into the dominant follicle induced in vivo oocyte maturation and the ovulation process whereas IL-1RA seemed to block these mechanisms.     

Neurovascular congruence results from a shared patterning mechanism that utilizes Semaphorin3A and Neuropilin-1

Peripheral nerves and blood vessels have similar patterns in quail forelimb development. Usually, nerves extend adjacent to existing blood vessels, but in a few cases, vessels follow nerves. Nerves have been proposed to follow vascular smooth muscle, endothelium, or their basal laminae. Focusing on the major axial blood vessels and nerves, we found that when nerves grow into forelimbs at E3.5-E5, vascular smooth muscle was not detectable by smooth muscle actin immunoreactivity. Additionally, transmission electron microscopy at E5.5 confirmed that early blood vessels lacked smooth muscle and showed that the endothelial cell layer lacks a basal lamina, and we did not observe physical contact between peripheral nerves and these endothelial cells. To test more generally whether lack of nerves affected blood vessel patterns, forelimb-level neural tube ablations were performed at E2 to produce aneural limbs; these had completely normal vascular patterns up to at least E10. To test more generally whether vascular perturbation affected nerve patterns, VEGF(165), VEGF(121), Ang-1, and soluble Flt-1/Fc proteins singly and in combination were focally introduced via beads implanted into E4.5 forelimbs. These produced significant alterations to the vascular patterns, which included the formation of neo-vessels and the creation of ectopic avascular spaces at E6, but in both under- and overvascularized forelimbs, the peripheral nerve pattern was normal. The spatial distribution of semaphorin3A protein immunoreactivity was consistent with a negative regulation of neural and/or vascular patterning. Semaphorin3A bead implantations into E4.5 forelimbs caused failure of nerves and blood vessels to form and to deviate away from the bead. Conversely, semaphorin3A antibody bead implantation was associated with a local increase in capillary formation. Furthermore, neural tube electroporation at E2 with a construct for the soluble form of neuropilin-1 caused vascular malformations and hemorrhage as well as altered nerve trajectories and peripheral nerve defasciculation at E5-E6. These results suggest that neurovascular congruency does not arise from interdependence between peripheral nerves and blood vessels, but supports the hypothesis that it arises by a shared patterning mechanism that utilizes semaphorin3A.     

Haplotype sharing refines the location of an imprinted quantitative trait locus with major effect on muscle mass to a 250-kb chromosome segment containing the porcine IGF2 gene

We herein describe the fine mapping of an imprinted QTL with major effect on muscle mass that was previously assigned to distal SSC2p in the pig. The proposed approach exploits linkage disequilibrium in combination with QTL genotyping by marker-assisted segregation analysis. By identifying a haplotype shared by all "Q" chromosomes, we map the QTL to an approximately 250-kb chromosome segment containing INS and IGF2 as the only known paternally expressed genes. This considerably reinforces the candidacy of these genes, justifying their detailed analysis.