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Biomedical subjects

D Pinto

Publications and source records attributed to D Pinto.

At least 19 recordsLinked to original sources
PubMed2003

Association between CYP2E1 polymorphisms and susceptibility to prostate cancer.

Several genetic alterations have been associated with sporadic prostate cancer (PCa). In this study, the association between RsaI and DraI polymorphisms of CYP2E1 and PCa risk was analysed in a case-control study of 227 individuals using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Regarding DraI polymorphisms, the DD genotype is over-represented in PCa cases when compared with the control group (odds ratio (OR) 2.12; 95% confidence interval (CI) 1.11-4.05; P=0.022). Regarding the RsaI polymorphism, no significant differences were found. The results of this study indicate that DraI polymorphisms of the CYP2E1 gene may be associated with a twofold increased risk for the development of PCa.

Adult↗
PubMed2002

Endothelial nitric oxide synthase gene polymorphisms and genetic susceptibility to prostate cancer.

The endothelial cell-specific form of nitric oxide synthases (ecNOS) is localized at 7q35-q36 and is involved in vascular development and tumour growth in human prostate cancer. We have conducted a case-control study to investigate the prevalence of two polymorphisms at intron 4 (ecNOS4a/b) and exon 7 (Glu-Asp298) of ecNOS gene in 125 prostate cancer (PCa) patients and in 153 controls. We observed that the a-allele (aa or ab genotypes from ecNOS4a/b) was over-presented in the group of PCa with Gleason histological grade >or=7 (P=0.041). With regard to the Glu-Asp298 polymorphism, patients with the T-allele were younger than patients with no T-allele (P=0.037), and a statistically significant difference was noted in the Glu-Asp298 genotype distribution between cases with advanced disease and cases with localized disease (P=0.0013). When comparing cases and controls with logistic regression analysis we observed that the presence of the a-allele is associated with prostate cancer risk (odds ratio (OR) 1.83; 95% confidence interval (CI) 1.06-3.17; P=0.029), to high histological grade (Gleason >or=7) of PCa (OR 2.18; 95% CI 0.95-4.98; P=0.062) and with the risk of progression of the cancer disease (OR 2.85; 95% CI 1.19-6.82; P=0.018). Furthermore, we found that carriers with the combination of the a-allele (aa and ab ecNOS4a/b genotypes) and T-allele (GT and TT from Glu-Asp298) have a threefold increase in prostate cancer risk (OR 3.13; 95% CI 1.41-6.91, P=0.004). In summary, we have identified an NO-related genetic risk factor for prostate cancer that may help in understanding the molecular mechanism involved in the individual susceptibility to prostate cancer.

Adult↗
PubMed2001

Nanoflow gradient generator coupled with mu-LC-ESI-MS/MS for protein identification.

The large-scale identification of proteins from proteomes of complex organisms, and the availability of various types of protein and DNA databases, increasingly require the additional information provided by tandem mass spectrometry. HPLC and microLC coupled to ESI-MS/MS presently dominate the field of protein identification by tandem mass spectrometry and database searching. The analysis of protein digests is typically performed using HPLC or LC columns with 50-100-microm diameters, requiring the delivery of solvent gradients at low to mid nanoliter per minute flow rates. This has been typically achieved using expensive generic HPLC pumping systems for the delivery of microliter per minute gradients that were either flow-split or sampled. Here we present an alternative system for the delivery of nanoliter per minute gradients. The inexpensive nanoflow gradient generator (etagrad) described here can be modulated to reproducibly deliver selected gradients. The performance of the etagrad on-line with a microLC-ESI-MS/MS system has been demonstrated for the identification of standard protein digests. Moreover, the performance of the etagrad-microLC-ESI-MS/MS system, with protein prefractionation by IPG isoelectric focusing, was also evaluated for rapid study of yeast and human proteomes.

Chromatography, Liquid↗
PubMed2001

Proteomics on a chip: promising developments.

The field of proteomics is expanding rapidly due to the completion of the human genome and the realization that genomic information is often insufficient to comprehend cellular mechanisms. This considerable expansion of proteomics towards high-throughput platforms is stressing its current technical capabilities. In recent years, technologies in microfluidic and array technologies have appeared for proteomics. These novel approaches might help solve current technical challenges in proteomics. This review presents a general survey of the recent development in microfluidic and array technologies from a proteomics perspective.

Amino Acid Sequence↗
PubMed2000

Increased cytogenetic damage in outdoor painters.

Painters are exposed to an extensive variety of hazardous substances such as organic solvents, lead-containing pigments and residual plastic monomers. In this particular case, workers used commercially available exterior paints and occasionally gasoline or thinner as solvents. The application or removal of paints was performed without protection (masks or gloves). To determine occupational exposure risk, a monitoring study was designed. Group selection was made after a questionnaire administration, which included questions about lifestyle and medical history to exclude exposure to other potential sources of genotoxics. Smoking and drinking habits were also considered. Blood and buccal cell samples were obtained from 25 public building male painters and from a similar number of age- and gender-matched controls. Lead levels were measured in paint samples and in individuals' blood. Organic solvents and/or its metabolites were also determined in blood. Chromosomal aberrations (CA) and sister chromatid exchanges (SCE) were determined in peripheral blood lymphocyte cultures. Also, the frequency of micronuclei (MN) in buccal cells was investigated. Painters had higher lead levels in blood (p<0.05); CA and SCE in lymphocytes and MN in epithelial cells were also elevated (p<0.05). Cytogenetic damage was significantly associated with occupational exposure time but not with the levels of lead found in blood.

Adolescent↗
PubMed2000

[Magnetic resonance angiography of the peripheral vessels with automatic moving bed infusion tracking].

PURPOSE: To investigate the actual diagnostic reliability of the Mobitrack technique with a slow intravenous infusion of paramagnetic contrast agent (CA) in MR Angiography of the peripheral arterial district. MATERIAL AND METHODS: Twelve healthy volunteers (mean age: 34) with no personal or family history of peripheral arterial pathologic conditions, underwent MR Angiography of the peripheral vascular district. A 1.5 T superconductive magnet equipped with automatic table feed was used. In the preliminary phase, the circulation time at the abdominal aorta was optimized and customized for each patient. This was done by performing a pre-targeting Fast Field Echo 2D (FFE 2D) sequence with intravenous administration of 1-2 mL CA by an injector, to evaluate the delay time. A 2D Time of Flight (2D TOF) sequence was then performed for topographic purposes. The partially overlapping volumes were acquired using Fast T1-weighted sequences, intravenous CA administration and an automatic table feed of 10 mm/s. A Fast Field Echo 3D (FFE 3D) T1-weighted sequence with TR/TE/FA: of 6.3/1.6/40 degrees and a slice thickness of 1.5 mm were also performed. Thirty to forty mL CA were slowly administered intravenously at a rate of 0.3-0.6 mL/s. Two blinded readers independently evaluated the images giving one of three diagnostic judgements: 1) arteries were well visualized, 2) heterogeneous arteries with(out) the presence of veins, and 3) arteries seen poorly or not at all. The first two judgements were considered diagnostic. The readers considered 19 different anatomical districts for each patient, giving a total of 190 evaluations. RESULTS: A blinded evaluation of the readers judgements did not indicate a statistically significant difference (agreement: 100%). Overall, 82% of the images were rated as 1, 8.5% as 2 and 8.4% as 3. DISCUSSION: In 174/190 judgements the vessels were rated as 1 or 2, that is of diagnostic value. Judgement 3 was always due to the inability to visualize the medial and/or distal third of the arterial circulation of the leg. CONCLUSIONS: The results of this technique optimization study confirm the overall validity of the Mobitrack technique. However they also indicate that further technical advances are required to ensure maximum diagnostic accuracy in this vascular district.

Adult↗
PubMed1999

Regulatory sequences of the mouse villin gene that efficiently drive transgenic expression in immature and differentiated epithelial cells of small and large intestines.

Villin is an early marker of epithelial cells from the digestive and urogenital tracts. Indeed villin is expressed in the stem cells and the proliferative cells of the intestinal crypts. To investigate the underlying molecular mechanisms and particularly those responsible for the restricted tissue specificity, a large genomic region of the mouse villin gene has been analyzed. A 9-kilobase (kb) regulatory region of the mouse villin gene (harboring 3.5 kb upstream the transcription start site and 5.5 kb of the first intron) was able to promote transcription of the LacZ reporter gene in the small and large intestines of transgenic mice, in a transmissible manner, and thus efficiently directed subsequent beta-galactosidase expression in epithelial cells along the entire crypt-villus axis. In the kidney, the transgene was also expressed in the epithelial cells of the proximal tubules but is likely sensitive to the site of integration. A construct lacking the first intron restricted beta-galactosidase expression to the small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and differentiated epithelial cells of the small and/or large intestinal mucosa.

Animals↗
PubMed1998

Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection.

We report a method for the analysis of picomolar concentration proteins using electrophoretically mediated microanalysis (EMMA) to label proteins on-column with a fluorogenic reagent. Labeling is followed by capillary zone electrophoresis separation and postcolumn detection based on laser-induced fluorescence. The method provides a concentration detection limit (3 sigma) of 3 x 10(-13) M for conalbumin. The method provides separation efficiency of 300,000 theoretical plates. Protein extract from a human colon adenocarcinoma cell line generated a dozen major components and many minor components in a 12-min separation; the protein extract from 2.5 cells was used for this analysis. When compared to UV absorbance detection, the EMMA method provides 7,000,000-fold improvement in detection limit.

Conalbumin↗
PubMed1998

Flavopiridol, a novel cyclin-dependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis.

Flavopiridol (HMR 1275) has been identified recently as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Flavopiridol inhibits most cyclin-dependent kinases (cdks) and displays unique anticancer properties. Here, we investigated whether this compound was effective against head and neck squamous cell carcinomas (HNSCC). Exposure of HNSCC cells to flavopiridol diminished cdc2 and cdk2 activity and potently inhibited cell proliferation (IC50 43-83 nM), which was concomitant with the appearance of cells with a sub-G1 DNA content. Moreover, DNA fragmentation and TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) reaction confirmed that flavopiridol induces apoptosis in all cell lines, even on certain HNSCC cells that are insensitive to apoptosis to DNA-damaging agents (gamma-irradiation and bleomycin). A tumorigenic HNSCC cell line was used to assess the effect of flavopiridol in vivo. Treatment (5 mg/kg per day, intraperitoneally) for 5 d led to the appearance of apoptotic cells in the tumor xenografts and caused a 60-70% reduction in tumor size, which was sustained over a period of 10 wk. Flavopiridol treatment also resulted in a remarkable reduction of cyclin D1 expression in HNSCC cells and tumor xenografts. Our data indicate that flavopiridol exerts antitumor activity in HNSCC, and thus it can be considered a suitable candidate drug for testing in the treatment of refractory carcinomas of the head and neck.

Animals↗
PubMed1998

On-line nonaqueous capillary electrophoresis and electrospray mass spectrometry of tricyclic antidepressants and metabolic profiling of amitriptyline by Cunninghamella elegans.

An on-line nonaqueous capillary electrophoresis-electrospray mass spectrometry (ESI-MS) technique was developed using a commercial ion spray interface. The nonaqueous capillary electrophoresis ESI-MS system was used to profile tricyclic antidepressants of similar structures and mass-to-charge ratios. We found that pure methanol can be used as a sheath liquid to obtain stable ion spray from nonaqueous capillary electrophoresis. The flow rate of the coaxial nebulizing gas affected baseline signals, separation efficiency, and migration times. Other nonaqueous capillary electrophoresis operating conditions and electrospray parameters were optimized for enhanced baseline separation and high sensitivity detection. The effect of sample stacking on separation and detection was evaluated. The calculated detection limits were approximately 3 pg injected onto the capillary. ESI mass spectra of tricyclic antidepressants from a single quadrupole MS were obtained and elucidated. The information was used to propose fragmentation pathways of the tricyclic antidepressants. The method was also used to analyze the metabolites of amitriptyline produced by the fungus Cunninghamella elegans. Sixteen metabolites were detected and most of them were tentatively identified as demethylated and/or hydroxylated, and/or N-oxidized products.

Amitriptyline↗
PubMed1998

I-SceI-induced gene replacement at a natural locus in embryonic stem cells.

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 x 10[-6]) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.

Animals↗
PubMed1997

Analysis of the allelic diversity of a (CA)n repeat polymorphism among alpha 1-antitrypsin gene products from northern Portugal.

The level of molecular heterogeneity associated with alpha 1-antitrypsin gene products was assessed in the population of northern Portugal using three restriction fragment length polymorphisms (RFLPs) corresponding to specific amino acid substitutions and a highly variable (CA)n repeat polymorphism located at the 5' end of the PI gene. The allelic affinities inferred from the analysis of the DNA polymorphisms essentially agree with the evolutionary pattern proposed for the PI gene products on the basis of their amino acid sequences. PI*Z can be considered the most recent common PI allele and was found to be associated with the same predominant haplotype previously reported in northern European populations, thus confirming the hypothesis that most European Z alleles are derived from a single mutation. However, a rare deficient variant that is the likely result of a recurrent Z mutation on an M2 or M4 background was additionally observed. PIS was also found to be associated with a strongly predominant haplotype and seems to be the second most recent PI common allele, while M2 and M3 show weaker associations, suggesting more ancient origins of their corresponding mutations. M1Ala213 and M1Vat213 display more homogeneous (CA)n allele frequency distributions, M1Ala213 representing the most ancient PI allele as inferred from its highest variance in (CA)n allele length.

Alleles↗
PubMed1997

Long-term follow-up and post-relapse survival in patients with non-metastatic osteosarcoma of the extremity treated with neoadjuvant chemotherapy.

BACKGROUND: Most of the studies of the treatment of non-metastatic osteosarcoma of the extremity have reported results in terms of probability of survival up to five years with a minimum follow-up of less than two to three years. Definition of reliable indicators of prognosis and predictive factors for survival require mature data derived from a long-term survival analysis. PATIENTS AND METHODS: A review of 127 patients with non-metastatic osteosarcoma of the extremity, treated between March 1983 and June 1986, was performed. The treatment protocol consisted of primary chemotherapy with MTX (randomization to high vs. moderate dosages) and CDP followed by surgery. Postoperatively, patients with < 60% tumor necrosis received ADM and BCD; those with tumor necrosis > or = 60% < 90% (Fair Responders FR) were given MTX, CDP and ADM. Up to January 1984, patients with tumor necrosis > 90% received MTX and CDP only, and after then they were given the same treatment as for FR. A multivariate analysis to test predictive factors for survival was performed. RESULTS: With a median follow-up of 134 months (range 114-153), the 12-year DFS was 46%. A good histologic response, an LDH baseline value within the normal range, and the use of high-dose MTX were positive predictive factors for DFS. With a median time of observation for survivors of 130 months, the 12-year overall survival was 53%. None of the patients who relapsed with local or distant recurrences other than lung metastasis are now alive. Patients with a relapse-free interval longer than 24 months had a significantly better post-relapse survival than those with a shorter relapse-free interval (40% vs. 7%; P = 0.0159). All of the patients who were not surgically treated had disease progression and died within 40 months after the first recurrence. The surgically-treated patients had a 30% post-relapse survival probability. CONCLUSIONS: In non-metastatic osteosarcoma of the extremity, chemotherapy-induced tumor necrosis, the baseline LDH serum value and the use of HDMTX are significant predictive factors for DFS. The relapse-free interval and the possibility of metastasectomy are significant factors conditioning the post-relapse survival.

Adolescent↗
PubMed1997

Reactions to managed care among psychologists and social workers.

Although the delivery system for mental health care is undergoing unprecedented changes with managed care, few empirical assessments of practitioners' responses exist. Directories of managed care providers were used to contact 139 providers of managed mental health care for an anonymous survey of experiences with case management and short-term therapy. The differential effects of managed care on the practices of doctoral-level psychologists, master's-level psychologists, and social workers were examined.

Adult↗
PubMed1997

[Magnetic resonance angiography in the study of pulmonary veins: TOF 3D versus 2D].

The aim of this work was to optimize the magnetic resonance angiography (MRA) technique for the selective study of the pulmonary veins. Twenty consecutive patients (13 men and 7 women; mean age: 30.5 years) were examined. MRA was performed with a 1 T superconductive magnet and the 3D time of flight (TOF) technique. Fast sequences (3D FISP: TR 58 ms, TE 6 ms, FA 20 degrees, matrix 192 x 256; and 2D FLASH: TR 44 ms, TE 10 ms, FA 30 degrees, matrix 192 x 256) were used. Coronal and sagittal images were submitted to MIP processing; presaturation pulses for the pulmonary arteries were located in the mediastinal region. In the right lung, 3D TOF on the coronal plane well showed 124 veins, while sagittal images showed 106 veins. In the left lung, 3D TOF on the coronal plane well showed 96 vessels, while sagittal images showed 44 vessels. In the right lung, 2D TOF on the coronal plane well showed 54 veins, while sagittal images showed 36 vessels. In the left lung, 2D TOF on the coronal plane well showed 22 vessels, while sagittal images showed 21 vessels. Therefore, 3D TOF yielded better results than 2D TOF (p < 0.05). To conclude, 3D TOF with contrast agent administration is a useful tool to study the pulmonary veins; those with a larger caliber are better depicted and the integration of coronal and sagittal images depicts more veins.

Adult↗
PubMed1996

Comparative effects of selective cyclooxygenase 1 and cyclooxygenase 2 inhibitors on myeloperoxidase and 3 alpha-hydroxysteroid dehydrogenase.

The clinical efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) is believed to result from the ability of these compounds to inhibit the inducible isoform of the enzyme cyclooxygenase, COX2. The gastrointestinal and renal side effects of these drugs, in contrast, are thought to relate to their ability to inhibit the constitutive isozyme, COX1. There is structural and pharmacological evidence that suggests that NSAIDs may also inhibit two unrelated enzymes, myeloperoxidase (MP) and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), potentially with untoward consequences for the patient. Our laboratories have been investigating a new structural class of potential COX inhibitors, the tri-cyclic aromatics. In this study we have examined the inhibitory potency of selected compounds for the enzymes human COX1, human COX2, human MP, and rat liver 3 alpha-HSD. The compounds selected span a range of COX isoform selectivities, from specific for COX2 to selective for COX1 only, and include three representative tri-cyclic aromatics. We found that compounds within the tri-cyclic aromatic class do not act as potent inhibitors of either myeloperoxidase or 3 alpha-HSD. These results demonstrate the unique inhibitor selectivity that can be achieved with the tri-cyclic aromatics. Examples of COX1 selective, and COX2 selective inhibitors within this structural class are presented.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

[Magnetic resonance angiography of the origin of the supraaortic trunks: the tridimensional time-of-flight with single volume technique].

This work was aimed at studying the origin of the epiaortic vessels with Magnetic Resonance Angiography (MRA). Twenty normal volunteers (mean age: 21.5 years) were examined with MRA with a superconductive magnet at 1 T (Impact). All volunteers were examined with a body coil and the time of flight (TOF) technique. The FISP 3D TONE sequence was used, with the following parameters: TR 46 ms, TE 10 ms, FA 20 degrees, ST 2.2 mm, matrix 192 x 256. The images, acquired on the axial plane with three presaturation pulses, were rotated according to the maximum intensity projection on the Z axis. To classify the images, a multiple choice card was used by a reader, with three diagnostic judgements for the different segments of the vessels (i.e., good, poor and not visualized). 3D TOF MRA permitted good visualization of the right and left common carotid in 80% and 77% of cases, of the right and left vertebral arteries in 55% and 50% of cases, of the left and right subclavian arteries in 47% and 62% of cases, and of the anonymous artery in 100% of cases. To conclude, our preliminary results indicate FISP 3D TOF MRA as the best noninvasive technique to study the origin of the epiaortic vessels.

Adult↗