Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice

Genes of the DAZ family play critical roles in germ cell development in mammals and other animals. In mice, Dazl mRNA is first observed at embryonic day 11.5 (E11.5), but previous studies using Dazl-deficient mice of mixed genetic background have largely emphasized postnatal spermatogenic defects. Using an inbred C57BL/6 background, we show that Dazl is required for embryonic development and survival of XY germ cells. By E14.5, expression of germ cell markers (Mvh, Oct4, Dppa3/Stella, GCNA and MVH protein) was reduced in XY Dazl-/- gonads. By E15.5, most remaining germ cells in XY Dazl-/- embryos exhibited apoptotic morphology, and XY Dazl-/- gonads contained increased numbers of TUNEL-positive cells. The rare XY Dazl-/- germ cells that persisted until birth maintained a nuclear morphology that resembled that of wildtype germ cells at E12.5-E13.5, a critical developmental period when XY germ cells lose pluripotency and commit to a spermatogonial fate. We propose that Dazl is required as early as E12.5-E13.5, shortly after its expression is first detected, and that inbred Dazl-/- mice of C57BL/6 background provide a reproducible standard for exploring Dazl's roles in embryonic germ cell development.     

Genetic basis of HDL variation in 129/SvImJ and C57BL/6J mice: importance of testing candidate genes in targeted mutant mice

To evaluate the effect of genetic background on high-density lipoprotein cholesterol (HDL) levels in Soat1(-/-) mice, we backcrossed sterol O-acyltransferase 1 (Soat1)(-/-) mice, originally reported to have elevated HDL levels, to C57BL/6 mice and constructed a congenic strain with only a small region (3.3Mb) of 129 alleles, specifically excluding the nearby apolipoprotein A-II (Apoa2) gene from 129. HDL levels in these Soat1(-/-) mice were no different from C57BL/6, indicating that the passenger gene Apoa2 caused the previously reported elevation of HDL in these Soat1(-/-) mice. Because many knockouts are made in strain 129 and then subsequently backcrossed into C57BL/6, it is important to identify quantitative trait loci (QTL) that differ between 129 and C57BL/6 so that one can guard against effects ascribed to a knockout but really caused by a passenger gene from 129. To provide such data, we generated 528 F(2) progeny from an intercross of 129S1/SvImJ and C57BL/6 and measured HDL concentrations in F(2) animals first fed chow and then atherogenic diet. A genome wide scan using 508 single-nucleotide polymorphisms (SNPs) identified 19 QTL, 2 of which were male specific and 2 were female specific. Using comparative genomics and haplotype analysis, we narrowed QTL on chromosomes 3, 5, 8, 17, and 18 to 0.5, 6.3, 2.6, 1.1, and 0.6 Mb, respectively. These data will serve as a reference for any effort to test the impact of candidate genes on HDL using a knockout strategy.     

Riccardin C: a natural product that functions as a liver X receptor (LXR)alpha agonist and an LXRbeta antagonist

Liver X receptors (LXRs) alpha and beta share considerable sequence homology and several functions, respond to the same endogenous and synthetic ligands, and play critical roles in maintaining lipid homeostasis. In this study, liverwort-derived riccardin C (RC) and F (RF) were identified as an LXRalpha agonist/LXRbeta antagonist and an LXRalpha antagonist, respectively. RC and RF bound to LXRs, but had different abilities to recruit a coactivator and thereby induce transactivation. Despite its unique subtype-selective activity, RC enhanced ABCA1 and ABCG1 expression and cellular cholesterol efflux in THP-1 cells. RC may provide a novel tool for identifying subtype-function and drug development.     

Neospora caninum: high susceptibility to the parasite in C57BL/10ScCr mice

C57BL/10ScCr mice, lack Toll-like receptor 4 and a functional Interleukin-12 receptor. Taking this into account, susceptibility of these mice to Neospora caninum infection was assessed comparatively to that of immunocompetent C57BL/10ScSn mice. C57BL/10ScCr mice inoculated intraperitoneally with 5x10(5)N. caninum tachyzoites showed a high susceptibility to this parasite. All infected C57BL/10ScCr mice were dead by day 8 post-infection whereas all control C57BL/10ScSn mice survived this parasitic challenge. Immunohistochemical analysis of infected C57BL/10ScCr mice showed N. caninum tachyzoites spread in the pancreas, liver, lung, intestine, heart and brain whereas no parasites were detected in similarly infected C57BL/10ScSn controls. The higher susceptibility of C57BL/10ScCr mice to neosporosis correlates with reduced interferon-gamma mRNA expression and increased IL-4 mRNA expression, comparatively to C57BL/10ScSn controls, detected in the spleen after the parasitic challenge. C57BL/10ScCr mice could thus be used as a new experimental model where to study immunobiological mechanisms associated with host susceptibility to neosporosis.     

Molecular determinants of the interactions between SRC-1 and LXR/RXR heterodimers

Liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers have been shown to perform critical functions in cholesterol and lipid metabolism. Here, we have conducted a comparative analysis of the contributions of LXR and RXR binding to steroid receptor coactivator-1 (SRC-1), which contains three copies of the NR box. We demonstrated that the coactivator-binding surface of LXR, but not that of RXR, is critically important for physical and functional interactions with SRC-1, thereby confirming that RXR functions as an allosteric activator of SRC-1-LXR interaction. Notably, we identified NR box-2 and -3 as the essential binding targets for the SRC-1-induced stimulation of LXR transactivity, and observed the competitive in vitro binding of NR box-2 and -3 to LXR.

Structured summary

MINT-7986678, MINT-7986639, MINT-7986700, MINT-7986720, MINT-7986736, MINT-7986760, MINT-7986787: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) and RXR (uniprotkb:P19793) by pull down (MI:0096)MINT-7986596, MINT-7986621: SRC1 (uniprotkb:Q15788) physically interacts (MI:0915) with LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986555, MINT-7986575: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) by two hybrid (MI:0018)MINT-7986808, MINT-7986907, MINT-7986890: SRC1 (uniprotkb:Q15788) binds (MI:0407) to LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986822, MINT-7986848, MINT-7986865: SRC1 (uniprotkb:Q15788) binds (MI:0407) to RXR (uniprotkb:P19793) by pull down (MI:0096)     

The role of hydrophobic and negatively charged surface patches of lipid-free apolipoprotein A-I in lipid binding and ABCA1-mediated cholesterol efflux

Recent models of lipid-free apolipoprotein A-I, including a cross-link/homology model and an X-ray crystal structure have identified two potential functionally relevant “patches” on the protein surface. The first is a hydrophobic surface patch composed of leucine residues 42, 44, 46, and 47 and the second a negatively charged patch composed of glutamic acid residues 179, 191, and 198. To determine if these domains play a functional role, these surface patches were disrupted by site-directed mutagenesis and the bacterially expressed mutants were compared with respect to their ability to bind lipid and stimulate ABCA1-mediated cholesterol efflux. It was found that neither patch plays a significant functional role in the ability of apoA-I to accept cholesterol in an ABCA1-dependent manner, but that the hydrophobic patch did affect the ability of apoA-I to clear DMPC liposomes. Interestingly, contrary to previous predictions, disruption of the hydrophobic surface patch enhanced the lipid binding ability of apoA-I. The hydrophobic surface patch may be important to the structural stability of lipid-free apoA-I or may be a necessary permissive structural element for lipid binding.     

Leishsmania (Leishmania) amazonensis infection: Muscular involvement in BALB/c and C3H.HeN mice

Recent studies have provided some insights into Leishsmania (Leishmania) amazonensis muscular infection in dogs, although, muscular disease due to leishmaniasis has been poorly documented. The aim of our study was to evaluate involvement of Leishmania in muscular infection of two distinct mouse strains (BALB/c and C3H.He), with different genetic backgrounds. BALB/c mice, susceptible to Leishmania infection, showed, at the beginning of infection, a great number of infected macrophages among muscle fibers; however, in C3H.He resistant mice, muscle fibers were less damaged than in BALB/c mice, but some parasitized macrophages could be seen among them. A follow up of the infection showed an intense inflammatory infiltrate mainly composed of infected macrophages in BALB/c muscles and the presence of amastigotes within muscle fibers; while C3H.He mice exhibited a moderate inflammatory infiltrate among skeletal muscle fibers and an absence of amastigotes. Total destruction of muscles was observed in BALB/c mice in the late phase of infection (day 90) while C3H.He mice showed a process of muscle repair. We concluded that: (1) the muscles of BALB/c mice were more affected by leishmaniasis than those of C3/H.He mice; (2) Leishmania amastigotes are capable of infecting muscular fibers, as observed in BALB/c mice; (3) as inflammatory infiltrate is less intense in C3H.He mice these animals are capable of restoring muscular fibers.     

Deleterious impact of elaidic fatty acid on ABCA1-mediated cholesterol efflux from mouse and human macrophages

Consumption of trans fatty acids (TFA) increase cardiovascular risk more than do saturated FA, but the mechanisms explaining their atherogenicity are still unclear. We investigated the impact of membrane incorporation of TFA on cholesterol efflux by exposing J774 mouse macrophages or human monocyte-derived macrophages (HMDM) to media enriched or not (standard medium) with industrially produced elaidic (trans-9 18:1) acid, naturally produced vaccenic (trans-11 18:1) acid (34 h, 70 μM) or palmitic acid. In J774 macrophages, elaidic and palmitic acid, but not vaccenic acid, reduced ABCA1-mediated efflux by ~ 23% without affecting aqueous diffusion, SR-BI or ABCG1-mediated pathways, and this effect was maintained in cholesterol-loaded cells. The impact of elaidic acid on the ABCA1 pathway was weaker in cholesterol-normal HMDM, but elaidic acid induced a strong reduction of ABCA1-mediated efflux in cholesterol-loaded cells (− 36%). In J774 cells, the FA supplies had no impact on cellular free cholesterol or cholesteryl ester masses, the abundance of ABCA1 mRNA or the total and plasma membrane ABCA1 protein content. Conversely, TFA or palmitic acid incorporation induced strong modifications of the membrane FA composition with a decrease in the ratio of (cis-monounsaturated FA + polyunsaturated FA):(saturated FA + TFA), with elaidic and vaccenic acids representing each 20% and 13% of the total FA composition, respectively. Moreover, we demonstrated that cellular ATP was required for the effect of elaidic acid, suggesting that it contributes to atherogenesis by impairing ABCA1-mediated cholesterol efflux in macrophages, likely by decreasing the membrane fluidity, which could thereby reduce ATPase activity and the function of the transporter.     

Migration defects by DISC1 knockdown in C57BL/6, 129X1/SvJ, and ICR strains via in utero gene transfer and virus-mediated RNAi

Disrupted-in-Schizophrenia 1 (DISC1) is a promising genetic risk factor for major mental disorders. Many groups repeatedly reported a role for DISC1 in brain development in various strains of mice and rats by using RNA interference (RNAi) approach. Nonetheless, due to the complexity of its molecular disposition, such as many splice variants and a spontaneous deletion in a coding exon of the DISC1 gene in some mouse strains, there have been debates on the interpretation on these published data. Thus, in this study, we address this question by DISC1 knockdown via short-hairpin RNAs (shRNAs) against several distinct target sequences with more than one delivery methodologies into several mouse strains, including C57BL/6, ICR, and 129X1/SvJ. Here, we show that DISC1 knockdown by in utero electroporation of shRNA against exons 2, 6, and 10 consistently results in neuronal migration defects in the developing cerebral cortex, which are successfully rescued by co-expression of full-length DISC1. Furthermore, lentivirus-mediated shRNA also led to migration defects, which is consistent with two other methodologies already published, such as plasmid-mediated and retrovirus-mediated ones. The previous study by Song’s group also reported that, in the adult hippocampus, the phenotype elicited by DISC1 knockdown with shRNA targeting exon 2 was consistently seen in both C57BL/6 and 129S6 mice. Taken together, we propose that some of DISC1 isoforms that are feasible to be knocked down by shRNAs to exon 2, 6, and 10 of the DISC1 gene play a key role for neuronal migration commonly in various mouse strains and rats.     

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-γ and iNOS expression in the CNS compared to BALB/c mice. The CNS of C57BL/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite.     

Maternal n-3 PUFA supplementation promotes fetal brown adipose tissue development through epigenetic modifications in C57BL/6 mice

Brown adipose tissue (BAT) is a crucial regulator of energy expenditure. Emerging evidence suggests that n-3 PUFA potentiate brown adipogenesis in vitro. Since the pregnancy and lactation is a critical time for brown fat formation, we hypothesized that maternal supplementation of n-3 PUFA promotes BAT development in offspring. Female C57BL/6 mice were fed a diet containing n-3 PUFA (3%) derived from fish oil (FO), or an isocaloric diet devoid of n-3 PUFA (Cont) during pregnancy and lactation. Maternal n-3 PUFA intake was delivered to the BAT of neonates significantly reducing the n-6/n-3 ratio. The maternal n-3 PUFA exposure was linked with upregulated brown-specific gene and protein profiles and the functional cluster of brown-specific miRNAs. In addition, maternal n-3 PUFA induced histone modifications in the BAT evidenced by 1) increased epigenetic signature of brown adipogenesis, i.e., H3K27Ac and H3K9me2, 2) modified chromatin-remodeling enzymes, and 3) enriched the H3K27Ac in the promoter region of Ucp1. The offspring received maternal n-3 PUFA nutrition exhibited a significant increase in whole-body energy expenditure and better maintenance of core body temperature against acute cold treatment. Collectively, our results suggest that maternal n-3 PUFA supplementation potentiates fetal BAT development via the synergistic action of miRNA production and histone modifications, which may confer long-lasting metabolic benefits to offspring.     

Impaired ATP-binding cassette transporter A1-mediated sterol efflux from oxidized LDL-loaded macrophages

We investigated the interaction of oxidized low density lipoprotein (OxLDL) with the ATP-binding cassette A1 (ABCA1) pathway in J774 macrophages. Cellular efflux to apolipoprotein AI (apo-AI) of OxLDL-derived cholesterol was lower than efflux of cholesterol derived from acetylated low density lipoprotein (AcLDL). ABCA1 upregulation by 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP) or 22 (R)-hydroxycholesterol (22-OH) and 9-cis retinoic acid (9cRA) increased the efflux to apo-AI of cellular sterols derived from AcLDL, but not of those from OxLDL. AcLDL, but not OxLDL, induced ABCA1 protein content and activity in J774. However, OxLDL did not influence J774 ABCA1 upregulation by cpt-cAMP or 22-OH/9cRA. We conclude that sterols released to cells by OxLDL are available neither as substrate nor as modulator of ABCA1.     

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids

Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC₅₀ = 33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC₅₀ = 8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K_iapp = 10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K_iapp = 1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.     

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.     

Estrogenic restoration of functional pancreatic islet cytoarchitecture in diabetes (db/db) mutant C57BL/KsJ mice: relationship to estradiol localization,systemic glycemia,and persistent hyperinsulinemia

Cell and Tissue Research - The diabetes (db/db) genotype mutation induces a hyperglycemic–hyperinsulinemic endometabolic state in C57BL/KsJ mice, manifesting a type 2 NIDDM diabetes-obesity...     

The stimulating effect of 3′,5′-(cyclic)adenosine monophosphate and lipolytic hormones on 3-O-methylglucose transport and Ca release in adipocytes and skeletal muscle of the rat

(1) In order to assess the possible role of 3′,5′-(cyclic)adenosine monophosphate (cAMP) in the control of glucose transport, the effect of the nucleotide or agents known to increase its intracellular concentration on sugar transport or ⁴⁵Ca²⁺ washout were characterized in epididymal fat pads, free fat cells and soleus muscles of the rat. (2) When added to the incubation medium, cAMP (0.1–2.0 mM) stimulated 3-O-[¹⁴C]methylglucose washout from fat pads. This effect was abolished by cytochalasin B, and additive to that induced by submaximal (10–25 μU/ml), but not by supramaximal (10 mU/ml) concentrations of insulin. (3) cAMP (2 mM) stimulated the conversion of [U-¹⁴C]glucose into CO₂ and triacylglycerols. This effect was additive to that of insulin (100 μU/ml). (4) ACTH, glucagon, adrenaline, noradrenaline and salbutamol, which are all known to increase the cAMP content of adipose tissue, stimulated the washout of 3-O-[¹⁴C]methylglucose and ⁴⁵Ca²⁺ from preloaded fat pads. The fractional losses of the two isotopes were significantly correlated (P < 0.001, r = 0.73). (5) In free fat cells, adrenaline (10⁻⁶ M) and salbutamol (10⁻⁵ M) stimulated the uptake of 3-O-[¹⁴C]methylglucose, and salbutamol (10⁻⁵ M) did not interfere with the stimulating effect of insulin (25 μU/ml) on sugar uptake. (6) In rat soleus muscles, adrenaline and salbutamol produced a dose-dependent stimulation of the washout of 3-O-[¹⁴C]methylglucose and ⁴⁵Ca²⁺. The effect of adrenaline on sugar efflux was abolished by propranolol. (7) It is concluded that the activation of the glucose transport system by insulin is unlikely to be mediated by a drop in the cellular concentration of cAMP. An increase in cAMP brought about by β-adrenoceptor agonists or lipolytic hormones may induce a mobilization of calcium ions from cellular pools into the cytoplasm, which in turn leads to the activation of the glucose transport system demonstrated in the present as well as in several earlier studies.     

Remodeling of phosphatidylglycerol in Synechocystis PCC6803

The phosphatidylglycerol deficient ΔpgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization–Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/ΔpgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A₁ and A₂ or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.     

Macrophage mitochondrial damage from StAR transport of 7-hydroperoxycholesterol: Implications for oxidative stress-impaired reverse cholesterol transport

StAR family proteins in vascular macrophages participate in reverse cholesterol transport (RCT). We hypothesize that under pathophysiological oxidative stress, StARs will transport not only cholesterol to macrophage mitochondria, but also pro-oxidant cholesterol hydroperoxides (7-OOHs), thereby impairing early-stage RCT. Upon stimulation with dibutyryl-cAMP, RAW264.7 macrophages exhibited a strong time-dependent induction of mitochondrial StarD1 and plasma membrane ABCA1, which exports cholesterol. 7α-OOH uptake by stimulated RAW cell mitochondria (like cholesterol uptake) was strongly reduced by StarD1 knockdown, consistent with StarD1 involvement. Upon uptake by mitochondria, 7α-OOH (but not redox-inactive 7α-OH) triggered lipid peroxidation and membrane depolarization while reducing ABCA1 upregulation. These findings provide strong initial support for our hypothesis.     

Allele frequency distribution of CYP2C9*2 and CYP2C9*3 polymorphisms in six Mexican populations

Allele frequency differences of functional CYP2C9 polymorphisms are responsible for some of the variation in drug response observed in human populations. The most relevant CYP2C9 functional variants are CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910). These polymorphisms show variation in allele frequencies among different population groups. The present study aimed to analyze these polymorphisms in 947 Mexican-Mestizo from Mexico City and 483 individuals from five indigenous Mexican populations: Nahua, Teenek, Tarahumara, Purepecha and Huichol. The CYP2C9*2 allele frequencies in the Mestizo, Nahua and Teenek populations were 0.051, 0.007 and 0.005, respectively. As for CYP2C9*3, the allelic frequencies in the Mestizo, Nahua and Teenek populations were 0.04, 0.005 and 0.005, respectively. The CYP2C9*2 and CYP2C9*3 alleles were not observed in the Tarahumara, Purepecha and Huichol populations. These findings are in agreement with previous studies reporting very low allele frequencies for these polymorphisms in American Indigenous populations.     

Molecular modeling based approach, synthesis and in vitro assay to new indole inhibitors of hepatitis C NS3/4A serine protease

In an attempt to identify potential HCV NS3 protease inhibitors lead compounds, a series of novel indoles (10a-g) was designed. Molecular modeling study, including fitting to a 3D-pharmacophore model of the designed molecules (10a-g), with HCV NS3 protease hypothesis using catalyst program was fulfilled. Also, the molecular docking into the NS3 active site was examined using Discovery Studio 2.5 software. Several compounds showed significant high simulation docking score and fit values. The designed compounds with high docking score and fit values were synthesized and biologically evaluated in vitro using an NS3 protease binding assay. It appears that most of the tested compounds reveal promising inhibitory activity against NS3 protease. Of these, compounds 10a and 10b demonstrated potent HCV NS3 protease inhibitors with IC₅₀ values of 9 and 12 ??g/mL, respectively. The experimental serine protease inhibitor activities of compounds 10a-g were consistent with their molecular modeling results. Inhibitors from this class have promising characteristics for further development as anti-HCV agents.