Sildenafil inhibits duodenal contractility via activation of the NO-K+ channel pathway

Phosphodiesterase type-5 (PDE5) specifically cleaves cyclic guanosine monophosphate (cGMP), a key intracellular secondary messenger. The PDE5 inhibitor sildenafil is a well-known vasodilator that also has gastrointestinal myorelaxant properties. In the present study, we further investigated sildenafil-induced myorelaxation in rat isolated duodenum, assessing its interaction with nitric oxide (NO) synthase and K+ channel opening. The spontaneous contractions of duodenal strips were reversibly inhibited by sildenafil (0.1-300 mu M) in a concentration-dependent manner [mean (95% confidence interval); EC50 = 6.8 (2.7-17.3) mu M]. The sildenafil-induced myorelaxation was significantly decreased by the NO synthase inhibitor N-nitro-L-arginine methyl ester [increasing the EC50 value to 41.9 (26.1-67.3) mu M]. Sodium nitroprusside or forskolin pretreatments enhanced the sildenafil-induced myorelaxation. In isolated strips pretreated with BaCl2 (0.2 mM), 4-aminopyridine (4-AP, 3 mM), or glybenclamide (1 mu M), the sildenafil-induced EC50 value was significantly increased to 32.8 (19.1-56.4), 27.1 (15.2-48.3) and 20.1 (16.4-24.7) mu M, respectively. Minoxidil (50 mu M) or diazoxide (100 mu M) also significantly attenuated the sildenafil-induced potency. In conclusion, the NO synthase/cyclic nucleotide pathway activation is involved in sildenafil-induced inhibition of spontaneous duodenal contractions. Its pharmacological action seems to be influenced by K+ channel opening, especially the voltage-sensitive ones, being inhibited by 4-AP and K-ATP channels, sensitive to glybenclamide.

Correlation between beta-2-glycoprotein I gene polymorphism and anti-beta-2 glycoprotein I antibodies in patients with multibacillary leprosy

Antiphospholipid antibodies, such as anti-beta 2-glycoprotein I (beta 2GPI), are present in multibacillary leprosy (MB) patients; however, MB patients do not usually present with antiphospholipid antibody syndrome (APS), which is characterized by thromboembolic phenomena (TEP). Rare cases of TEP occur in leprosy patients, but the physiopathology of this condition remains unclear. In this case-control study, we examined whether single-nucleotide polymorphisms (SNPs) of the beta 2GPI gene contributed to the risk of leprosy and APS co-morbidity. SNPs Ser88Asn, Leu247Val, Cys306Gly and Trp316Ser were identified in 113 Brazilian leprosy patients. Additionally, anti-beta 2GPI antibodies and plasma concentrations of beta 2GPI were quantified. The Ser88Asn, Cys306Gly and Trp316Ser SNPs were not risk factors for APS in leprosy. A higher frequency of Val/Val homozygosity was observed in leprosy patients compared to controls (36 vs. 5%; P < 0.001). Forty-two percent of MB and 17% of paucibacillary leprosy patients were positive for anti-beta 2GPI IgM (P = 0.014). There was no correlation between SNP Ser88Asn or Cys306Gly and anti-beta 2GPI antibody levels. In MB patients with positive anti-beta 2GPI IgM, the frequency of Val/Val homozygosity was higher than in controls (32 vs. 15%; P = 0.042). The frequency of the mutant allele Ser316 was higher in MB patients with positive rather than negative anti-beta 2GPI IgM levels (6 vs. 0%; P = 0.040) and was greater than in the control group (6 vs. 1%; P = 0.034). The studied polymorphisms did not influence the plasma concentrations of beta 2GPI. These results suggest that Leu247Val and Trp316Ser SNPs may represent genetic risk factors for anti-beta 2GPI antibody production in MB patients.

Interleukin-18 and interferon-gamma polymorphisms in Brazilian human immunodeficiency virus-1-infected patients presenting with lipodystrophy syndrome

Cytokines play important roles in the pathogenesis of lipodystrophy syndrome (LS). Single nucleotide polymorphisms (SNPs) at positions -607(C/A) and -137(C/G) in the promoter region of the interleukin-18 (IL-18) gene and at position +874(T/A) of the interferon-gamma (IFN-gamma) gene are related to the expression of these cytokines. To examine whether IL-18 and IFN-gamma polymorphisms are associated with LS, these SNPs were genotyped in 88 human immunodeficiency virus (HIV)-infected patients presenting LS, 79 HIV-infected without LS, and 133 healthy controls. The -607A allele, -607AA genotype, and -137G/-607A and -137C/-607A haplotypes in the IL-18 gene were over-represented in HIV patients presenting LS. The -137G/-607C haplotype was associated with protection against LS. These results indicate that the -607(C/A) SNP is associated with LS development in HIV-infected patients.

TAC3/TACR3 Mutations Reveal Preferential Activation of Gonadotropin-Releasing Hormone Release by Neurokinin B in Neonatal Life Followed by Reversal in Adulthood

Context: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. Objective: A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. Design and Setting: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. Patients or Other Participants: 345 probands, 18 family members, and 292 controls were studied. Intervention: Reproductive phenotypes throughout reproductive life and before and after therapy were examined. Main Outcome Measure: Rare sequence variants in TAC3/TACR3 were detected. Results: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism. Conclusions: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time. (J Clin Endocrinol Metab 95: 2857-2867, 2010)

Assessing Individual Interethnic Admixture and Population Substructure Using a 48-Insertion-Deletion (INSEL) Ancestry-Informative Marker (AIM) Panel

Estimating the proportions of different ancestries in admixed populations is very important in population genetics studies, and it is particularly important for detecting population substructure effects in case-control association studies. In this work, a set of 48 ancestry, informative insertion, deletion polymorphisms (INDELs) were selected with the goal of efficiently measuring the proportions of three different ancestries (sub-Saharan African, European, and Native American) in mixed populations. All selected markers can be easily analyzed via multiplex PCR and detected with standard capillary electrophoresis. A total of 593 unrelated individuals representative of European, African, and Native American parental populations were typed, as were 380 individuals from three Brazilian populations with known admixture patterns. As expected, the interethnic admixture estimates show that individuals from southern Brazil present an almost exclusively European ancestry; Afro-descendant communities in the Amazon region, apart from the major African contribution, present some degree of admixture with Europeans and Native Americans; and a sample from Belem, in the northeastern Amazon, shows a significant contribution of the three ethnic groups, although with a greater European proportion. In summary, a panel of ancestry-informative INDELs was optimized and proven to be a variable tool for estimating individual and global ancestry proportions in admixed populations. The ability to accurately infer interethnic admixtures highlights the usefulness of this marker set for assessing population substructure in association studies, particularly those conducted in Brazilian and other Latin American populations sharing trihybrid ancestry patterns. Hum Mutat 31:184-190, 2010. (C) 2009 Wiley-Liss, Inc.

Frequency distribution of XbaIG > T and HaeIIIT > C GLUT1 polymorphisms among different Brazilian ethnic groups

GLUT is the major glucose transporter in mammalian cells. Single nucleotide polymorphisms (SNP) at GLUT1 promoter and regulatory regions have been associated to the risk of developing nephropathy in different type 1 and type 2 diabetic populations. It has been demonstrated that differences in allelic and genotypic frequencies of GLUT1 gene (SLC2A1) polymorphisms occur among different populations. Therefore, ethnic differences in distribution of GLUT1 gene polymorphisms may be an important factor in determining gene-disease association. In this study, we investigated the XbaIG > T and HaeIIIT > C polymorphisms in six different Brazilian populations: 102 individuals from Salvador population (Northern Brazil), 56 European descendants from Joinville (South Brazil), 85 Indians from Tiryi tribe (North Brazil) and 127 samples from Southern Brazil: 44 from European descendants, 42 from African descendants and 41 from Japanese descendants. Genotype frequencies from both sites did not differ significantly from those expected under the Hardy-Weinberg equilibrium. We verified that the allele frequencies of both polymorphisms were heterogeneous in these six Brazilian ethnic groups.

Characterization and pharmacological evaluation of febrile response on zymosan-induced arthritis in rats

Kanashiro A, Pessini AC, Machado RR, Malvar DC, Aguiar FA, Soares DM, Vale ML, Souza GEP. Characterization and pharmacological evaluation of febrile response on zymosan-induced arthritis in rats. Am J Physiol Regul Integr Comp Physiol 296: R1631-R1640, 2009. First published February 25, 2009; doi:10.1152/ajpregu.90527.2008.-The present study investigated the febrile response in zymosan-induced arthritis, as well as the increase in PGE(2) concentration in the cerebrospinal fluid (CSF), along with the effects of antipyretic drugs on these responses in rats. Zymosan intra-articularly injected at the dose of 0.5 mg did not affect the body core temperature (Tc) compared with saline (control), whereas at doses of 1 and 2 mg, zymosan promoted a flattened increase in Tc and declined thereafter. The dose of 4 mg of zymosan was selected for further experiments because it elicited a marked and long-lasting Tc elevation starting at 3 1/2 h, peaking at 5 1/2 h, and remaining until 10 h. This temperature increase was preceded by a decrease in the tail skin temperature, as well as hyperalgesia and edema in the knee joint. No febrile response was observed in the following days. In addition, zymosan-induced fever was not modified by the sciatic nerve excision. Zymosan increased PGE2 concentration in the CSF but not in the plasma. Oral pretreatment with ibuprofen (5-20 mg/kg), celecoxib (1-10 mg/kg), dipyrone (60-240 mg/kg), and paracetamol (100-200 mg/kg) or subcutaneous injection of dexamethasone (0.25-1.0 mg/kg) dose-dependently reduced or prevented the fever during the zymosan-induced arthritis. Celecoxib (5 mg/kg), paracetamol (150 mg/kg), and dipyrone (120 mg/kg) decreased CSF PGE2 concentration and fever during zymosan-induced arthritis, suggesting the involvement of PGE2 in this response.

Uridine adenosine tetraphosphate-induced contraction is increased in renal but not pulmonary arteries from DOCA-salt hypertensive rats

Matsumoto T, Tostes RC, Webb RC. Uridine adenosine tetraphosphate-induced contraction is increased in renal but not pulmonary arteries from DOCA-salt hypertensive rats. Am J Physiol Heart Circ Physiol 301: H409-H417, 2011. First published May 6, 2011; doi:10.1152/ajpheart.00084.2011.-Uridine adenosine tetraphosphate (Up(4)A) was reported as a novel endothelium-derived contracting factor. Up(4)A contains both purine and pyrimidine moieties, which activate purinergic (P2)X and P2Y receptors. However, alterations in the vasoconstrictor responses to Up(4)A in hypertensive states remain unclear. The present study examined the effects of Up(4)A on contraction of isolated renal arteries (RA) and pulmonary arteries (PA) from DOCA-salt rats using isometric tension recording. RA from DOCA-salt rats exhibited increased contraction to Up(4)A versus arteries from control uninephrectomized rats in the absence and presence of N(G)-nitro-L-arginine (nitric oxide synthase inhibitor). On the other hand, the Up(4)A-induced contraction in PA was similar between the two groups. Up(4)A-induced contraction was inhibited by suramin (nonselective P2 antagonist) but not by diinosine pentaphosphate pentasodium salt hydrate (Ip5I; P2X(1) antagonist) in RA from both groups. Furthermore, 2-thiouridine 5`-triphosphate tetrasodium salt (2-Thio-UTP; P2Y(2) agonist)-, uridine-5`-(gamma-thio)-triphosphate trisodium salt (UTP gamma S; P2Y(2)/P2Y(4) agonist)-, and 5-iodouridine-5`-O-diphosphate trisodium salt (MRS 2693; P2Y(6) agonist)-induced contractions were all increased in RA from DOCA-salt rats. Protein expression of P2Y(2)-, P2Y(4)-, and P2Y(6) receptors in RA was similar between the two groups. In DOCA-salt RA, the enhanced Up(4)A-induced contraction was reduced by PD98059, an ERK pathway inhibitor, and Up(4)Astimulated ERK activation was increased. These data are the first to indicate that Up(4)A-induced contraction is enhanced in RA from DOCA-salt rats. Enhanced P2Y receptor signaling and activation of the ERK pathway together represent a likely mechanism mediating the enhanced Up(4)A-induced contraction. Up(4)A might be of relevance in the pathophysiology of vascular tone regulation and renal dysfunction in arterial hypertension.

PYK2/PDZ-RhoGEF Links Ca(2+) Signaling to RhoA

Objective-Ras homolog gene family member A (RhoA)/Rho-kinase-mediated Ca(2+) sensitization is a critical component of constrictor responses. The present study investigates how angiotensin II activates RhoA. Methods and Results-Adenoviral vectors were used to manipulate the expression of regulator of G protein signaling (RGS) domain containing Rho-specific guanine exchange factors (RhoGEFs) and proline-rich tyrosine kinase 2 (PYK2), a nonreceptor tyrosine kinase, in primary rat vascular smooth muscle cells. As an evidence of RhoA activation, RhoA translocation and MYPT1 (the regulatory subunit of myosin light chain phosphatase) phosphorylation were analyzed by Western blot. Results showed that overexpression of PDZ-RhoGEF, but not p115-RhoGEF or leukemia-associated RhoGEF (LARG), enhanced RhoA activation by angiotensin II. Knockdown of PDZ-RhoGEF decreased RhoA activation by angiotensin II. PDZ-RhoGEF was phosphorylated and activated by PYK2 in vitro, and knockdown of PDZ-RhoGEF reduced RhoA activation by constitutively active PYK2, indicating that PDZ-RhoGEF links PYK2 to RhoA. Knockdown of PYK2 or PDZ-RhoGEF markedly decreased RhoA activation by A23187, a Ca(2+) ionophore, demonstrating that PYK2/PDZ-RhoGEF couples RhoA activation to Ca(2+). Conclusions-PYK2 and PDZ-RhoGEF are necessary for angiotensin II-induced RhoA activation and for Ca(2+) signaling to RhoA. (Arterioscler Thromb Vasc Biol. 2009;29:1657-1663.)

The A61 G EGF polymorphism is associated with development of extraaxial nervous system tumors but not with overall survival

Epidermal growth factor can activate several signaling pathways, leading to proliferation, differentiation, and tumorigenesis of epithelial tissues by binding with its receptor. The EGF protein is involved in nervous system development, and polymorphisms in the EGF gene on chromosome band 4q25 are associated with brain cancers. The purpose of this study was to investigate the association between the single-nucleotide polymorphism of EGF + 61 G/A and extraaxial brain tumors in a population of the southeast of Brazil. We analyzed the genotype distribution of this polymorphism in 90 patients and 100 healthy subjects, using the polymerase chain reaction restriction fragment length polymorphism technique. Comparison of genotype distribution revealed a significant difference between patients and control subjects (P < 0.001). The variant genotypes of A/G and G/G were associated with a significant increase of the risk of tumor development, compared with the homozygote A/A (P < 0.0001). When the analyses were stratified, we observed that the genotype GIG was more frequent in female patients (P = 0.021). The same genotype was observed more frequently in patients with low-grade tumors (P = 0.001). Overall survival rates did not show statistically significant differences. Our data suggest that the EGF A61 G polymorphism can be associated with susceptibility to development of these tumors. (C) 2010 Elsevier Inc. All rights reserved.

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

Association study of an epidermal growth factor gene functional polymorphism with the risk and prognosis of gliomas in Brazil

Epidermal growth factor (EGF) plays an important role in cancer. A functional single nucleotide polymorphism (SNP) in the 5`-untranslated region of the EGF gene (+61 A>G) may influence its expression and contribute to cancer predisposition and aggressiveness. Aiming to investigate the role of EGF +61 A>G in the susceptibility to glioma and its prognosis, we performed a case-control study with 165 patients and 200 healthy controls from Brazil. Comparisons of genotype distributions and allele frequencies did not reveal any significant differences between the groups. The mean overall survival was 9.2 months for A/A, 8.2 months for A/G, and 7.7 months for GIG. When survival curves were plotted we found that the +61G allele is associated with poor overall survival (p=0.023) but not with disease-free survival (p=0.527). Our data suggest that, although there is no association between the EGF +61 A>G genotype and glioma susceptibility, this SNP is associated with shorter overall survival of glioma patients in the Brazilian population. Nevertheless, future studies utilizing a larger series are essential for a definitive conclusion. (Int J Biol Markers 2009; 24: 277-81)

WRN Cys1367Arg SNP is not associated with risk and prognosis of gliomas in Southeast Brazil

Werner syndrome (WS) is a premature aging disorder characterized by early onset of symptoms related to normal aging and by a high predisposition to various types of cancer, including gliomas. WS is caused by inherited recessive mutations in the WRN gene, which encodes a helicase considered a caretaker of the genome. Aiming to study the role of WRN Cys1367Arg in glioma susceptibility and oncologic prognosis of patients, we investigated the genotype distribution of this single nucleotide polymorphism in 94 glioma patients and 100 healthy subjects. Comparisons of genotype distributions and allele frequencies did not reveal any significant difference between the groups. Overall and disease-free survival rates were calculated, but no statistically significant difference was observed. Our data suggest that WRN Cys1367Arg SNP is not involved either in susceptibility to developing gliomas or in patient survival, at least in the Brazilian population.

A single amino acid substitution in one of the lipases of Aspergillus nidulans confers resistance to the antimycotic drug undecanoic acid

A plausible approach to evaluate the inhibitory action of antifungals is through the investigation of the fungal resistance to these drugs. We describe here the molecular cloning and initial characterization of the A. nidulans lipA gene, where mutation (lipA1) conferred resistance to undecanoic acid, the most fungitoxic fatty acid in the C(7:0)-C(18:0) series. The lipA gene codes for a putative lipase with the sequence consensus GVSIS and WIFGGG as the catalytic signature. Comparison of the wild-type and LIP1 mutant strain nucleotide sequences showed a G -> A change in lipA1 allele, which results in a Glu(214) -> Lys substitution in LipA protein. This ionic charge change in a conserved LipA region, next to its catalytic site, may have altered the catalytic properties of this enzyme resulting in resistance to undecanoic acid.

Polymorphisms of Lewis and Secretor genes are related to breast cancer and metastasis in axillary lymph nodes

ABH and Lewis antigen expression has been associated with cancer development and prognosis, tumor differentiation, and metastasis. Considering that invasive ductal breast carcinoma (IDC) presents multiple molecular alterations, the aim of the present study was to determine whether the polymorphism of ABO, Lewis, and Secretor genes, as well as ABO phenotyping, could be associated with tumor differentiation and lymph nodes metastasis. Seventy-six women with IDC and 78 healthy female blood donors were submitted to ABO phenotyping/genotyping and Lewis and Secretor genotyping. Phenotyping was performed by hemagglutination and genotyping by the polymerase chain reaction with sequence-specific primers. ABO, Lewis, and Secretor genes were classified by individual single nucleotide polymorphism at sites 59, 1067, 202, and 314 of the Lewis gene, 428 of the Secretor gene, and 261 (O1 allele), 526 (O2 and B allele), and 703 (B allele). No association was found between breast cancer and ABO antigen expression (P = 0.9323) or genotype (P = 0.9356). Lewis-negative genotype was associated with IDC (P = 0.0126) but not with anatomoclinical parameters. Nonsecretor genotype was associated with axillary lymph node metastasis (P = 0.0149). In conclusion, Lewis and Secretor genotyping could be useful to predict respectively breast cancer susceptibility and axillary lymph nodes metastasis.