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Jose M Silva

Publications and source records attributed to Jose M Silva.

15 recordsLinked to original sources
PubMed2005

Second-generation shRNA libraries covering the mouse and human genomes.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Animals↗
PubMed2005

Discovery of a substituted 8-arylquinoline series of PDE4 inhibitors: structure-activity relationship, optimization, and identification of a highly potent, well tolerated, PDE4 inhibitor.

The discovery and SAR of a new series of substituted 8-arylquinoline PDE4 inhibitors are herein described. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of emesis to efficacy in several animal models. Typical optimized compounds from this series are potent inhibitors of PDE4 (IC(50)<1nM) and also of LPS-induced TNF-alpha release in human whole blood (IC(50)<0.5microM). The same compounds are potent inhibitors of ovalbumin-induced bronchoconstriction in conscious guinea pigs (EC(50)<0.1mg/kg ip) but require a dose of about 10mg/kg po in the squirrel monkey to produce an emetic response. From this series of compounds, 23a (L-454,560) was identified as an optimized compound.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

The DISCOVERY PENTA study: a DIrect Statin COmparison of LDL-C Value--an Evaluation of Rosuvastatin therapY compared with atorvastatin.

BACKGROUND: International guidelines emphasize the need to achieve recommended low-density lipoprotein cholesterol (LDL-C) levels in order to reduce morbidity and mortality associated with coronary heart disease (CHD). However, many patients with hypercholesterolemia fail to achieve LDL-C goals on treatment. OBJECTIVE: The primary objective was to compare the efficacy of rosuvastatin and atorvastatin for enabling patients to achieve National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) LDL-C goals. Secondary objectives were European LDL-C goal achievement, changes in the lipid profile, and safety. RESEARCH DESIGN AND METHODS: This 12-week, multicenter, multinational, randomized, open-label trial compared the efficacy and safety of rosuvastatin 10 mg with atorvastatin 10 mg in statin-naïve and switched patients with primary hypercholesterolemia from Brazil, Colombia, Mexico, Portugal, and Venezuela. RESULTS: A total of 1124 patients with similar baseline characteristics were randomized to the two treatment groups. After 12 weeks of treatment, a significantly greater percentage of patients receiving rosuvastatin 10 mg compared with atorvastatin 10 mg achieved NCEP ATP III LDL-C goals (71.2% vs 61.4%, p < 0.001), 1998 European LDL-C goals (73.5% vs 59.2%, p < 0.001) and 2003 European LDL-C goals (58.9% vs 44.6%, p < 0.001). Rosuvastatin treatment was associated with significant reductions in LDL-C and total cholesterol (TC) and, in statin-naïve patients, a significant increase in high-density lipoprotein cholesterol (HDL-C) compared with atorvastatin treatment. Both treatments were well tolerated with a similar incidence of adverse events. Clinically significant elevations in creatinine, creatine kinase or hepatic transaminases were low and similar between treatment groups. CONCLUSIONS: Rosuvastatin 10 mg is significantly more effective at achieving NCEP ATP III and European LDL-C goals, lowering LDL-C and TC in both naïve and switched patients and increasing HDL-C in naïve patients than atorvastatin 10mg, with a similar safety and tolerability profile. This study also provides evidence regarding the comparative effects of rosuvastatin versus atorvastatin in Latin American and Portuguese populations.

Adolescent↗
PubMed2004

RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells.

RNA interference (RNAi) is a biological process in which a double-stranded RNA directs the silencing of target genes in a sequence-specific manner. Exogenously delivered or endogenously encoded double-stranded RNAs can enter the RNAi pathway and guide the suppression of transgenes and cellular genes. This technique has emerged as a powerful tool for reverse genetic studies aimed toward the elucidation of gene function in numerous biological models. Two approaches, the use of small interfering RNAs and short hairpin RNAs (shRNAs), have been developed to permit the application of RNAi technology in mammalian cells. Here we describe the use of a shRNA-based live-cell microarray that allows simple, low-cost, high-throughput screening of phenotypes caused by the silencing of specific endogenous genes. This approach is a variation of "reverse transfection" in which mammalian cells are cultured on a microarray slide spotted with different shRNAs in a transfection carrier. Individual cell clusters become transfected with a defined shRNA that directs the inhibition of a particular gene of interest, potentially producing a specific phenotype. We have validated this approach by targeting genes involved in cytokinesis and proteasome-mediated proteolysis.

Animals↗
PubMed2004

A resource for large-scale RNA-interference-based screens in mammals.

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.

Animals↗
PubMed2003

Prevalence of aberrant methylation of p14ARF over p16INK4a in some human primary tumors.

The INK4a/ARF locus encodes two unrelated tumor suppressor proteins, p16INK4a and p14ARF, which participate in the two main cell-cycle control pathways, p16-Rb and p14-p53. Methylation of CpG promoter islands has been described as a mechanism of gene silencing. Exon 1 of the p16INK4a gene and the p14ARF promoter gene reside within CpG islands. Therefore, both can become methylated de novo and silenced. It has recently been proposed that the methylation changes in certain genes could be used as molecular markers for the detection of almost all forms of human cancer. Here, we analyzed concomitantly in each tumor sample and normal tissue the methylation status of p16INK4a and p14ARF by methylation-specific PCR (MSP) in 100 breast, 95 colon and 27 bladder carcinomas. A series of clinicopathological parameter were obtained from the medical records of the patients, p14ARF showed a higher rate of hypermethylation than p16INK4a in all three tumor types. p16INK4a and p14ARF aberrant methylation was significantly correlated with poor prognosis clinicopathological parameters of the three tumor types. We conclude that both p16INKa and p14ARF hypermethylation may be involved in breast, colon and bladder carcinogenesis, with special emphasis on the role of the lesser studied p14ARF gene, and that tumors with aberrant methylation in the two genes were associated with worse prognosis.

Breast Neoplasms↗
PubMed2003

A micrococcal nuclease homologue in RNAi effector complexes.

RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)--a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain--is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.

Animals↗
PubMed2003

Retention of transporter activities in cryopreserved, isolated rat hepatocytes.

The success of cryopreservation of isolated hepatocytes with existing methodologies is assessed with respect to the retentivity of cell integrity/viability (defined by trypan blue) and metabolic activities upon thawing in comparison to those of freshly prepared cells. But the ability of the cryopreserved cells to transport xenobiotics relative to that of freshly prepared cells has not been investigated. In this study, we optimized our previous methodology for cryopreservation and evaluated the metabolism and transport of thawed hepatocytes. Half of the freshly, isolated rat hepatocytes prepared by collagenase perfusion were immediately used for studies of transport of [(14)C]taurocholate, [(3)H]estrone sulfate and [(3)H]estradiol 17beta-D-glucuronide (1 microM) and metabolism of 7-hydroxy-4-(trifluoromethyl)-coumarin (100 microM), (3,4-difluorobenzyloxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one (250 microM), bufuralol (100 microM), and tolbutamide (100 microM), probes for UDP-glucuronyl transferase (UGT) and CYP3A, CYP2D, and CYP2C, respectively. The remaining half was cryopreserved using an optimized, programmed-freezing protocol, which was developed to minimize the prolonged release of latent heat during freezing. With the exception of the UGT probe, no significant difference (P > 0.05) was found in both metabolism and transport with freshly isolated versus cryopreserved hepatocytes upon thawing. In conclusion, we have demonstrated for the first time that thawed rat hepatocytes cryopreserved by a programmed-freezing protocol retain drug transport activities.

Animals↗
PubMed2003

Intratumoral heterogeneity in microsatellite alterations in BRCA1 and PTEN regions in sporadic colorectal cancer.

BACKGROUND: Chromosome regions 17q21 (BRCA1) and 10q23 (PTEN) have been found deleted in colorectal cancer. METHODS: We studied the frequency of loss of heterozygosity (LOH) in these 2 regions in 214 patients with only 1 sample per tumor and in 100 patients with several samples per tumor. Three microsatellite markers of each region were used for the LOH test. The polymerase chain reaction product was electrophoresed in 8% polyacrylamide gels, and band intensity was shown by silver staining. RESULTS: The proportions of LOH in the two regions were 38.4% for 17q21 and 30.8% for 10q23 in the group of 214 and were 47.7% for 17q21 and 34.7% for 10q23 in the group of 100. We found a high correlation between the LOH in both regions (P <.001), where 81% of LOH in 10q23 region was matched by concomitant LOH in 17q21. In the group of tumors with several samples (group of 100), 39% and 68% did not present LOH in the 17q21 and 10q23 regions, respectively, in all of their tumor samples. However, in the 20 patients with LOH in both regions in the group of 100 (several samples per tumor), all samples with LOH in 10q23 also had LOH in 17q21, whereas not all samples with LOH in 17q21 had LOH in 10q23. CONCLUSIONS: These results show that colorectal cancer is highly heterogeneous, at least for these tumors markers, and suggest a sequential acquisition pattern of these anomalies during tumor growth, in which changes in 17q21 could occur before those in 10q23.

Aged↗
PubMed2002

p14ARF promoter hypermethylation in plasma DNA as an indicator of disease recurrence in bladder cancer patients.

PURPOSE: Several genes are reported to be implicated in bladder carcinogenesis, including p53, p16INK4a, pRb, erbB-2, Cyclin D1, H-ras, EGFR, and c-myc. Gene alterations in plasma DNA identical to those observed within the tumor have been detected in various types of neoplasia. EXPERIMENTAL DESIGN: We analyzed loss of heterozygosity in six microsatellite markers (D17S695, D17S654, D13S310, TH2, D9S747, and D9S161), p53 and K-ras mutations, and the promoter status of p14ARF and p16INK4a in the mononuclear normal blood cells, tumor, and plasma DNA of 27 bladder cancer patients. We also studied the distribution of several clinicopathological parameters in these patients in regard to molecular alterations. RESULTS: Seventeen (63%) cases displayed the same alteration in plasma and tumor DNA (some patients showed more than one alteration simultaneously). Plasma p14ARF promoter hypermethylation was associated with the presence of multicentric foci (P = 0.03), larger tumors (P = 0.01), and relapse of the disease (P = 0.03). Plasma loss of heterozygosity was also linked to disease recurrence (P = 0.02). CONCLUSIONS: The results indicate that p14ARF aberrant promoter methylation could be involved in bladder carcinogenesis and that plasma DNA is a potential prognostic marker in urinary bladder cancer.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2002

Tumor DNA in plasma at diagnosis of breast cancer patients is a valuable predictor of disease-free survival.

PURPOSE: We examine prospectively whether the presence of plasma DNA with tumor characteristics before mastectomy is a predictive factor related to recurrence and disease-free survival (DFS). EXPERIMENTAL DESIGN: A series of 147 patients with breast carcinomas, selected sequentially, was analyzed. The characterization of plasma DNA, based on similar alterations in tumor and plasma DNA, was achieved with six polymorphic markers (D17S855, D17S654, D16S421, TH(2), D10S197, and D9S161) and mutations in the TP53 gene. Recurrence, DFS, overall survival, and 12 other clinicopathological parameters were obtained. Univariate and Cox's multivariate studies were performed. RESULTS: A total of 142 patients were eligible for study. A total of 104 tumors (73.2%) showed at least one molecular alteration. In 61 patients (42.9%), a similar molecular alteration was detected in plasma DNA and tumor DNA. No alterations were found in the plasma DNA of the remaining 81 patients (57%). During the follow-up period (median, 22 months; range, 1-46 months), we observed 23 recurrences (16%), the distribution of which was significantly different (P = 0.005) with regard to plasma DNA [17 patients (74%) with circulating tumor DNA and 6 patients (26%) without tumor plasma DNA]. Univariate statistical analysis confirmed the prognostic significance of the already known parameters (tumor size, lymph node metastases, and stage) and demonstrated that tumor plasma DNA was a predictor of DFS. In multivariate analysis, an independent borderline significance was observed for tumor plasma DNA. CONCLUSIONS: Tumor DNA in plasma at diagnosis in breast cancer patients can predict DFS, and its determination could be used as a prognostic factor in these patients.

Adult↗
PubMedDate not supplied

Persistence of tumor DNA in plasma of breast cancer patients after mastectomy.

BACKGROUND: We investigated tumor DNA changes before and after mastectomy in the plasma of breast cancer patients with no disseminated disease and eventually investigated these changes' relationship to specific pathological parameters of the tumors. METHODS: We studied 41 patients. DNA extracted from tumor and normal breast tissues, mononuclear blood cells, and plasma was used for molecular studies. Alterations in the microsatellite markers D17S855, D17S654, D16S421, TH2, D10S197, and D9S161, as well as point mutations in the p53 gene and aberrant methylation of p16(INK4a), were used to identify and characterize tumor and plasma DNA. A number of tumor clinicopathological parameters were analyzed in each patient. RESULTS: We found that 18 (44%) of the 27 patients with alterations in tumor DNA presented the same plasma DNA alteration before mastectomy, and persistence of the same molecular features was detected in plasma DNA 4 to 6 weeks postmastectomy in 8 (19.5%) patients. Patients with vascular invasion, more than three lymph node metastases, and higher histological grade at diagnosis displayed plasma DNA after mastectomy with a significant difference. CONCLUSIONS: Persistence of plasma DNA with features of tumor DNA may be present after mastectomy in breast cancer patients, and its relation to bad-prognosis histological parameters may suggest undetectable micrometastatic disease.

Biomarkers, Tumor↗