5' and 3' UTR thymidylate synthase polymorphisms modulate the risk of colorectal cancer independently of the intake of methyl group donors

Thymidylate synthase, as a rate-limiting step in DNA synthesis, catalyses the conversion of dUMP into dTMP using 5,10-methylenotetrahydrofolate as the methyl donor. Two polymorphisms have been described in this gene: a repeat polymorphism in the 5' promoter enhancer region (3R versus 2R) and a 6 bp deletion in the 3' unstranslated region. Both of these may affect protein levels. The present case control study was aimed at investigating the influence of these two polymorphisms on the development of colorectal cancer (CRC), as well as their potential interaction with folate, vitamin B6 and vitamin B12 intake. A total of 196 cases and 200 controls, matched for age and sex distribution, were included in the study. No association was found between CRC and the 28 bp repeat polymorphism, but it was observed that individuals with the 6 bp/del and del/del genotypes had a significantly lower risk of developing the disease (OR=0.47; 95% CI 0.30-0.72). A combined genotype (2R/2R; 6 bp/del+del/del) was also found, which was associated with an even lower risk of developing of the disease (OR=0.42; 95% CI 0.26-0.69). No significant interaction between these polymorphisms and vitamin intake was observed. These results indicate for the first time that the 6 bp/del allele might be a protective factor in the development of CRC, independent of the intake of methyl group donors.

Lack of Association Between a 3 ` UTR VNTR Polymorphism of Dopamine Transporter Gene (SLC6A3) and ADHD in a Brazilian Sample of Adult Patients

Objective: To investigate a possible association between a 3`UTR VNTR polymorphism of the dopamine transporter gene (SLC6A3) and ADHD in a Brazilian sample of adult patients. Method: Study Case-control with 102 ADHD adult outpatients (DSM-IV criteria) and 479 healthy controls. The primers` sequence used were: 3`UTR-Forward: 5`TGT GGT GAT GGG AAC GGC CTG AG 3` and 3`UTR-Reverse: 5`CTT CCT GGA GGT CAC GGC TCA AGG 3`. Alleles of the 3`UTR were coded according to their number of repeats: 6- repeat 320 bp (allele 6), 8- repeat 400 bp (allele 8), 9-repeat 480 bp (allele 9), 10- repeat 480 bp (allele 10), and 11- repeat 520 bp (allele 11). Results: There were no allelic (chi(2) = 2.67, 5df, p = .75) and genotypic (chi(2) = 7.20, 1df, p = .61) association between adult ADHD and VNTR 3`UTR polymorphism of SLC6A3. Conclusion: Our findings do not support SLC6A3 as marker genetic susceptibility factor in adult ADHD. More comprehensive polymorphism coverage within the SLC6A3 region should be conducted in larger samples, including comparisons in clinical subgroups, and in samples with different ethnic backgrounds. (J. of Att. Dis. 2011; 15(4) 305-309)

Detection and Molecular Characterization of Gene 3 and 5 of Turkey Coronavirus from Turkeys with Severe Enteritis in Brazil

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3` UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3` UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

Post-transcriptional regulation of the GLI1 oncogene by the expression of alternative 5 ' untranslated regions

The oncogene GLI1 is involved in the formation of basal cell carcinoma and other tumor types as a result of the aberrant signaling of the Sonic hedgehog-Patched pathway. In this study, we have identified alternative GLI1 transcripts that differ in their 5' untranslated regions (UTRs) and are generated by exon skipping. These are denoted (alpha -UTR, beta -UTR, and gamma -UTR according to the number of noncoding exons possessed (three, two, and one, respectively). The alpha- and beta -UTR forms represent the major Gli1 transcripts expressed in mouse tissues, whereas the gamma -UTR is present at relatively low levels but is markedly induced in mouse skin treated with 12-O-tetradecanoylphorbol 13-acetate, Transcripts corresponding to the murine beta and gamma forms were identified in human tissues, but significantly, only the gamma -UTR form was present in basal cell carcinomas and in proliferating cultures of a keratinocyte cell line. Flow cytometry analysis determined that the gamma -UTR variant expresses a heterologous reporter gene 14-23-fold higher than the alpha -UTR and 5-13-fold higher than the beta -UTR in a variety of cell types. Because expression of the gamma -UTR variant correlates with proliferation, consistent with a role for GLI1 in growth promotion, up-regulation of GLI1 expression through skipping of 5' noncoding exons may be an important tumorigenic mechanism.

Papel de la región 3'UTR en la acumulación del RNA mensajero de VEGF-D en células PC12 que expresan constitutivamente el oncogen wnt-1

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Origin and evolution of flavivirus 5'UTRs and panhandles: trans-terminal duplications?

Flavivirus replication is mediated by interactions between complementary ssRNA sequences of the 5'- and 3'-termini that form dsRNA cyclisation stems or panhandles, varying in length, sequence and specific location in the mosquito-borne, tick-borne, non-vectored and non-classified flaviviruses. In this manuscript we manually aligned the flavivirus 5'UTRs and adjacent capsid genes and revealed significantly more homology than has hitherto been identified. Analysis of the alignments revealed that the panhandles represent evolutionary remnants of a long cyclisation domain that probably emerged through duplication of one of the UTR termini.

Hipersensibilidade e necrose sistêmica em Nicotiana benthamiana transformada com o gene de resistência Sw-5 de tomateiro

O gene Sw-5 do tomateiro confere resistência a várias espécies de tospovírus e codifica uma proteína contendo domínios de ligação a nucleotídeos e repetições ricas em leucina. Tomateiros com Sw-5 exibem reações necróticas nas folhas inoculadas com tospovírus. Estas reações e a estrutura da proteína Sw-5 indicam que a resistência ocorre por meio do reconhecimento do patógeno e desencadeamento da resposta de hipersensibilidade. A capacidade de Sw-5 de conferir resistência a tospovírus em tabaco selvagem (Nicotiana benthamiana Domin.) foi avaliada em plantas transgênicas. Uma construção com a seqüência aberta de leitura de Sw-5 e sua região 3 não-traduzida sob controle do promotor 35S do CaMV foi utilizada para transformação de N. benthamiana via Agrobacterium tumefaciens. Plantas de progênies R1 foram inoculadas com um isolado de tospovírus e avaliadas quanto à ocorrência de reação de hipersensibilidade e resistência à infecção sistêmica. em uma progênie com segregação 3:1 (resistente:suscetível), foi selecionada uma planta homozigota e sua progênie avaliada quanto ao espectro da resistência a tospovírus. Plantas com o transgene exibiram resposta de hipersensibilidade 48 h após a inoculação, sendo resistentes à infecção sistêmica. O fenótipo da resistência foi dependente do isolado viral e um isolado de Tomato chlorotic spot virus (TCSV) causou necrose sistêmica em todas as plantas inoculadas, enquanto que isolados de Groundnut ringspot virus (GRSV) e um isolado relacionado a Chrysanthemum stem necrosis virus (CSNV) ficaram restritos ao sítio de infecção. Comparações do espectro da resistência obtido neste trabalho com aquele observado em outros membros da família Solanaceae indicam que as vias de transdução de sinais e as respostas de defesa ativadas por Sw-5 são conservadas dentro desta família e polimorfismos genéticos nas vias de transdução de sinais ou em componentes das respostas de defesa podem resultar em diferentes níveis de resistência.

Pyrimidine-5'-nucleotidase Campinas, A New Mutation (p.r56g) In The Nt5c3 Gene Associated With Pyrimidine-5'-nucleotidase Type I Deficiency And Influence Of Gilbert's Syndrome On Clinical Expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Chemo-, Regio- And Stereoselective Heck Arylation Of Allylated Malonates: Mechanistic Insights By Esi-ms And Synthetic Application Toward 5-arylmethyl-γ-lactones.

We describe herein a general method for the controlled Heck arylation of allylated malonates. Both electron-rich and electron-poor aryldiazonium salts were readily employed as the aryl-transfer agents in good yields and in high chemo-, regio-, and stereoselectivity without formation of decarboxylated byproducts. Reaction monitoring via ESI-MS was used to support the formation of chelated Pd species through the catalytic cycle. Additionally, some Heck adducts were successfully used in the total synthesis of pharmacologically active γ-lactones.

Direct Adhesive Pin-retained Restorations For Severely Worn Dentition Treatment: A 1.5-year Follow-up Report.

Excessive occlusal surface wear can result in occlusal disharmony, functional and esthetic impairment. As a therapeutic approach, conventional single crowns have been proposed, but this kind of treatment is complex, highly invasive and expensive. This case report describes the clinical outcomes of an alternative minimally invasive treatment based on direct adhesive-pin retained restorations. A 64-year-old woman with severely worn dentition, eating problems related to missing teeth and generalized tooth hypersensitivity was referred for treatment. Proper treatment planning based on the diagnostic wax-up simulation was used to guide the reconstruction of maxillary anterior teeth with direct composite resin over self-threading dentin pins. As the mandibular remaining teeth were extremely worn, a tooth-supported overdenture was installed. A stabilization splint was also used to protect the restorations. This treatment was a less expensive alternative to full-mouth rehabilitation with positive esthetic and functional outcomes after 1.5 years of follow-up.

Synthesis And Antitumor Activity Of Novel 1-substituted Phenyl 3-(2-oxo-1,3,4-oxadiazol-5-yl) β-carbolines And Their Mannich Bases.

A series of novel 1-(substituted phenyl)-3-(2-oxo-1,3,4-oxadiazol-5-yl) β-carbolines (4a-e) and the corresponding Mannich bases 5-9(a-c) were synthesized and evaluated for their in vitro antitumor activity against seven human cancer cell lines. Compounds of 4a-e series showed a broad spectrum of antitumor activity, with GI50 values lower than 15μM for five cell lines. The derivative 4b, having the N,N-dimethylaminophenyl group at C-1, displayed the highest activity with GI50 in the range of 0.67-3.20μM. A high selectivity and potent activity were observed for some Mannich bases, particularly towards resistant ovarian (NCI-ADR/RES) cell lines (5a, 5b, 6a, 6c and 9b), and ovarian (OVCAR-03) cell lines (5b, 6a, 6c, 9a, 9b and 9c). In addition, the interaction of compound 4b with DNA was investigated by using UV and fluorescence spectroscopic analysis. These studies indicated that 4b interact with ctDNA by intercalation binding.

S-nitrosoglutathione Accelerates Recovery From 5-fluorouracil-induced Oral Mucositis.

Mucositis induced by anti-neoplastic drugs is an important, dose-limiting and costly side-effect of cancer therapy. To evaluate the effect of the topical application of S-nitrosoglutathione (GSNO), a nitric oxide donor, on 5-fluorouracil (5-FU)-induced oral mucositis in hamsters. Oral mucositis was induced in male hamsters by two intraperitoneal administrations of 5-FU on the first and second days of the experiment (60 and 40 mg/kg, respectively) followed by mechanical trauma on the fourth day. Animals received saline, HPMC or HPMC/GSNO (0.1, 0.5 or 2.0 mM) 1 h prior to the 5-FU injection and twice a day for 10 or 14 days. Samples of cheek pouches were harvested for: histopathological analysis, TNF-α and IL-1β levels, immunohistochemical staining for iNOS, TNF-α, IL-1β, Ki67 and TGF-β RII and a TUNEL assay. The presence and levels of 39 bacterial taxa were analyzed using the Checkerboard DNA-DNA hybridization method. The profiles of NO released from the HPMC/GSNO formulations were characterized using chemiluminescence. The HPMC/GSNO formulations were found to provide sustained release of NO for more than 4 h at concentration-dependent rates of 14 to 80 nmol/mL/h. Treatment with HPMC/GSNO (0.5 mM) significantly reduced mucosal damage, inflammatory alterations and cell death associated with 5-FU-induced oral mucositis on day 14 but not on day 10. HPMC/GSNO administration also reversed the inhibitory effect of 5-FU on cell proliferation on day 14. In addition, we observed that the chemotherapy significantly increased the levels and/or prevalence of several bacterial species. Topical HPMC/GSNO accelerates mucosal recovery, reduces inflammatory parameters, speeds up re-epithelization and decreases levels of periodontopathic species in mucosal ulcers.

Effects Of Pde Type 5 Inhibitors On Left Ventricular Diastolic Dysfunction In Resistant Hypertension.

Resistant hypertension (RHTN) is a multifactorial disease characterized by blood pressure (BP) levels above goal (140/90 mmHg) in spite of the concurrent use of three or more antihypertensive drugs of different classes. Moreover, it is well known that RHTN subjects have high prevalence of left ventricular diastolic dysfunction (LVDD), which leads to increased risk of heart failure progression. This review gathers data from studies evaluating the effects of phosphodiesterase-5 (PDE-5) inhibitors (administration of acute sildenafil and short-term tadalafil) on diastolic function, biochemical and hemodynamic parameters in patients with RHTN. Acute study with sildenafil treatment found that inhibition of PDE-5 improved hemodynamic parameters and diastolic relaxation. In addition, short-term study with the use of tadalafil demonstrated improvement of LVDD, cGMP and BNP-32 levels, regardless of BP reduction. No endothelial function changes were observed in the studies. The findings of acute and short-term studies revealed potential therapeutic effects of IPDE-5 drugs on LVDD in RHTN patients.A Hipertensão arterial resistente (HAR) é uma doença multifatorial caracterizada por níveis pressóricos acima das metas (140/90 mmHg), a despeito de tratamento farmacológico otimizado de 3 ou mais fármacos anti-hipertensivos de diferentes classes. Pacientes diagnosticados como hipertensos resistentes apresentam alta prevalência de disfunção diastólica do ventrículo esquerdo (DDVE) que proporciona risco aumentado para insuficiência cardíaca. Esta revisão reúne dados de estudos prévios avaliando os efeitos dos inibidores de fosfodiesterase-5 (PDE-5) (administração aguda de sildenafil e de curto prazo de tadalafil) na função diastólica e nos parâmetros bioquímicos e hemodinâmicos em pacientes com HAR. O estudo agudo com sildenafil demonstrou que a inibição da PDE-5 melhorou os parâmetros hemodinâmicos e de relaxamento diastólico. Além disso, o estudo curto prazo com o uso de tadalafil revelou melhora da DDVE e dos níveis de GMPc e BNP-32, independente de redução de pressão arterial. A função endotelial não apresentou alteração com ambos os tratamentos. Os resultados dos estudos agudo e de curto prazo sugerem efeitos terapêuticos potenciais dos fármacos inibidores da PDE-5 na disfunção diastólica em pacientes com HAR.

Indução assimétrica 1,5-Anti na adição de enolatos de boro de metilcetonas beta-oxigenadas a aldeídos

High levels of substrate-based 1,5-stereoinduction are obtained in the boron-mediated aldol reactions of beta-oxygenated methyl ketones with achiral and chiral aldehydes. Remote induction from the boron enolates gives the 1,5-anti adducts, with the enolate pi-facial selectivity critically dependent upon the nature of the beta-alkoxy protecting group. This 1,5-anti aldol methodology has been strategically employed in the total synthesis of several natural products. At present, the origin of the high level of 1,5-anti induction obtained with the boron enolates is unclear, although a model based on a hydrogen bonding between the alkoxy oxygen and the formyl hydrogen has been recently proposed.