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P Valente

Publications and source records attributed to P Valente.

At least 19 recordsLinked to original sources
PubMed2004

Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl- conductance.

We show that dysregulation of the Cl- homeostasis mediates the staurosporine-induced apoptotic cell death in human ECV304 cells. A pronounced apoptotic volume decrease (AVD), and an increase in plasma membrane Cl- conductance were early (<1 h) events following staurosporine challenge. Both processes were involved in apoptotic death, as demonstrated by the observation that the Cl- channel blocker phloretin inhibited both the staurosporine-evoked Cl- current and AVD, and preserved cell viability. Prolonged incubation (>2 h) with staurosporine caused a decrease in intracellular pH, which, however, was not required for the progression of the apoptotic process, because inhibitors of proton extrusion pathways, which lowered cytoplasmic pH, failed to inhibit both caspase-3 activation and DNA laddering. Moreover, clamping the cytosolic pH to an alkaline value did not prevent the apoptotic cell death. Collectively, these data demonstrate that staurosporine-mediated apoptosis of ECV304 cells is caused by the upregulation of Cl- channel activity and subsequent AVD, but is independent of intracellular acidification.

Apoptosis↗
PubMed2003

TNF increases camptothecin-induced apoptosis by inhibition of NF-kappaB.

rHuTNF potentiates CPT-cytotoxicity in human ovarian A2780 cells. In this study, we examined the role of NF-kappaB in this potentiation. A pulse-labelled DNA study indicated that the combination CPT+TNF had little effect on the rate of DNA elongation at 6 h after drug removal, whereas CPT alone produced a complete inhibition for at least 6 h after drug removal. Flow cytometry analyses showed that CPT+TNF arrested cells in the G2-M phase, whereas CPT blocked cells in S phase. Looking at the persistence of the NF-kappaB complexes in cells, it appeared that they were still present at 24 h in TNF-treated cells. In contrast, in CPT-treated cells they persisted for 6 h. In CPT+TNF-treated cells, the NF-kappaB complexes disappeared quickly and became undetectable at 6 h. The induction of apoptosis was detected only in the CPT+TNF treated cells (using flow cytometry, a filter binding assay and ApopTag staining). These findings show that TNF, in combination with CPT, reduces the time that NF-kappaB complexes persist in cells likely resulting in the induction of apoptosis.

Antineoplastic Agents, Phytogenic↗
PubMed2003

Staurosporine induces apoptotic volume decrease (AVD) in ECV304 cells.

Incubation of ECV304 cells with 1 micro M staurosporine (STS) causes apoptotic cell death. In the present study, we investigate whether a significant apoptotic volume decrease (AVD) was apparent during the very early times (1 h) of the apoptotic process. Our data suggest that upregulation of Cl(-) (and possibly K(+)) channels by STS may be a very early primary event required for the subsequent onset of AVD, which results in apoptosis.

Apoptosis↗
PubMed2001

Heteroduplex mobility assay of the D1/D2 region of the 26S rDNA for differentiation of Saccharomyces species.

AIMS: We present the HMA method for Saccharomyces differentiation using the PCR amplified D1/D2 26S rDNA. METHODS AND RESULTS: This methodology is based on heteroduplex formation when two different DNAs are hybridized. We tested 11 type cultures of Saccharomyces, 27 different cultures of S. cerevisiae and four other ascomycetic genera. CONCLUSION: The method was capable of differentiating Saccharomyces species and was mainly very efficient for S. cerevisiae identification. HMA can probably be applied in other genera, where identification is sometimes difficult only by conventional traits, which are based on physiology and morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: HMA provides a rapid and relatively simple molecular tool, contributing for yeast taxonomy.

DNA, Fungal↗
PubMed2000

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems.

In 1998 we reported that an L-peptide derived from H1 of c-Myc (Int-H1-S6A,F8A), linked to an internalization sequence from the third a-helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro-inverso (RI-) peptides can assume a 3D conformation very close to their corresponding L-forms and can be recognized by the same monoclonal antibody. We synthesized a RI-peptide form of our original L-peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI-form of the original Antennapedia internalization sequence was perfectly capable of carrying a D-peptide into human cells. We have studied three different potentially active peptides. L-peptides: Int-H1wt, Int-H1-S6A,F8A. D-peptides: RI-Int -H1-S6A,F8A. We have also studied three presumed control peptides: Int and RI-Int (no H1 motif), H1-S6A,F8A (no internalization sequence). Both 'active' and 'control' peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 mM) of the control peptide RI-Int, non-Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c-Myc (and molecules of the family), we chose an iso-amphipathic modified peptide of the H1 motif, with a proximity coefficient >50% and where the major change was at position 7 (F-->A). From a family of 73 H1 motifs belonging to (H1-Loop-H2) hu man sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N-Myc, L-Myc, c-Myc, H1-S6A,F8A of c-Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int-H1isoamph and the corresponding RI-Int-H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF-7 cells) a colon cancer line (HCT-116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso-amphipathic-modified H1 sequence were always very clearly (3-10 times) less active than the corresponding peptides carrying a conserved "H1 of Myc" motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF-7 cells treated with L-peptides; MCF-7 cells treated with RI-peptides; HCT-116 cells treated with L-peptides; PBLs treated with L-peptides; PBLs treated with RI-peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF-7 cells. On a molar basis, RI-peptides were about 5-10 times more potent and 30-35 times more stable in complete culture medium, than their corresponding L-forms. RI-Int can probably internalize longer peptido-mimetic molecules (for instance molecules mimetic of (H1-Loop-H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors-two steps closer to a potential drug.

Amino Acid Motifs↗
PubMed1999

Sequencing as a tool in yeast molecular taxonomy.

The literature on sequencing as a tool for yeast molecular taxonomy is reviewed. Ribosomal DNA has been preferred for sequencing over other molecules such as mitochondrial DNA, and a large database is now available. rDNA consists of regions that evolve at different rates, allowing comparison of different levels of relationship among yeasts. Sequences of the 18S rDNA and the 25S rDNA have been largely used for yeast systematics and phylogeny, but the search for regions with increased resolving power has led to the study of the spacer regions of the rDNA. Few studies are concerned with signature sequences.

DNA, Fungal↗
PubMed1998

TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis.

The matrix metalloproteinase (MMP) inhibitor TIMP-2 has a high specificity for gelatinase A/MMP-2. An imbalance between gelatinase A and TIMP-2 in favor of enzymatic activity is linked to the degradation of the extracellular matrix (ECM) associated with several physiologic and pathologic events, including angiogenesis, invasion and metastasis. Since TIMPs are secreted molecules, they have the potential to be used for gene therapy of certain tumors. We transfected B16F10 murine melanoma cells, a highly invasive and metastatic cell line, with an expression vector harboring a cDNA encoding for human TIMP-2. The clones obtained were isolated and examined for TIMP-2 over-expression and changes in tumor cell phenotype. The amount of recombinant TIMP-2 produced correlated with a reduction in invasion. In an in vivo angiogenesis assay, TIMP-2-transfected clones showed reduced levels of blood vessel formation, and in vitro conditioned media from TIMP-2 transfectants showed diminished induction of endothelial cell migration and invasion. TIMP-2 over-expression limited tumor growth in vivo and neoangiogenesis when cells were injected subcutaneously in mice in the presence of Matrigel. However, TIMP-2 overexpressing clones were found to be more resistant to apoptosis than parental and control melanoma cells, while necrosis was increased. Our data confirm the role of TIMP-2 in the down-regulation of metastasis and angiogenesis but indicate a possible involvement in tumor cell survival.

Animals↗
PubMed1996

RB1 oncosuppressor gene over-expression inhibits tumor progression and induces melanogenesis in metastatic melanoma cells.

The retinoblastoma gene (RB1) is frequently deleted or mutated in many tumor types and in all cases of retinoblastoma. Apart from its role in regulation of the cell cycle, the RB1 gene product (p110RB1) appears to be involved in control of differentiation. Malignant metastatic cells show many properties of poorly differentiated cells, and are highly invasive in vitro and in vivo. We have transfected the human RB1 cDNA in an expression vector under the control of the beta-actin promoter into B16F10 murine melanoma cells. These cells highly overexpress RB1 mRNA and the p110RB1 product, show reduced growth rate and increased melanogenesis in vitro. Vector control transfectants showed no alteration of invasiveness. The p110RB1 over-expressing cells also had a reduced capacity to migrate and invade through an artificial basement membrane, key characteristics of metastatic cells. When injected into nude mice, the p110RB1 over-expressing cells showed reduced tumor growth and reduced metastatic potential. The few metastasis observed were predominantly melanotic. These data indicate that RB1 gene expression is involved in melanoma cell differentiation and plays a role in downregulation of migration, invasion and metastatic potential of these cells.

Animals↗
PubMed1996

PCR amplification of the rDNA internal transcribed spacer region for differentiation of Saccharomyces cultures.

The size of the internal transcribed spacer (ITS) region as measured by gel electrophoresis of PCR products, amplified by primers ITS1 and ITS4, was over 800 bp for all Saccharomyces sensu stricto species, but yeasts belonging to other Saccharomyces species had a shorter ITS region, making this characteristic potentially useful in the identification of Saccharomyces isolates. The ITS product length was homogeneous within the species Saccharomyces cerevisiae.

Base Sequence↗
PubMed1996

Eating pattern in the first year following adjustable silicone gastric banding (ASGB) for morbid obesity.

OBJECTIVE: To analyse the relationships between eating pattern, vomiting frequency, weight loss and the rate of band related complications in morbidly obese patients undergoing Adjustable Silicone Gastric Banding (ASGB). SUBJECTS: 80 morbidly obese patients (57 females and 23 males) consecutively operated by ASGB were evaluated both before and 3, 6 and 12 months after ASGB. Ten patients (12.5%) had binge eating disorder and were analysed separately. MEASUREMENTS: (1) weight loss expressed as percentage of overweight, (2) total daily energy intake, (3) percentage of energy as lipids, carbohydrates and proteins, (4) percent as liquid, soft or solid foods and (5) vomiting frequency. RESULTS: ASGB induced a highly significant reduction of total daily energy intake and percent as solid foods, without significant changes in macronutrient distribution. There was an inverse relationship between vomiting frequency and the intake of solid foods. Non-binge eaters with more vomiting ate less solid food and lost more weight than patients without vomiting. The frequency of neostoma stenosis was higher in patients with high vomiting frequency than in patients with no vomiting. Patients with binge eating disorder had a significantly higher vomiting frequency and a five-fold higher frequency of neostoma stenosis than patients without binge eating disorder. However, the percentage of overweight lost did not differ between patients with and without binge eating. CONCLUSIONS: Vomiting is a major determinant of global outcome after ASGB. The vomiting frequency in the first months after ASGB was associated with eating pattern, the frequency of neostoma stenosis and possibly the rate of weight loss during the first year of follow-up.

Adolescent↗
PubMed1996

Altered production of laminin and nidogen by high and low metastatic variants of murine melanoma cells.

An altered ability to interact with and degrade extracellular matrix molecules is a common feature of the malignant phenotype. Although changes in the expression of matrix proteins in metastases in vivo are relatively well documented, little is known about the changes in matrix production by malignant cells in culture. Here we have examined the synthesis of the basement membrane components laminin and nidogen (entactin) by low and high metastatic variants of the K-1735 murine melanoma cells. Protein deposition was examined by western blotting as well as immunofluorescence; protein synthesis was examined by immunoprecipitation with specific antibodies. Gene expression was also evaluated by measuring steady-state mRNA levels using cDNA probes on northern and dot-blots. Laminin gamma 1 levels appeared to be similar in both high and low metastatic lines; however, the high metastatic lines had reduced levels of the laminin beta 1 chain. On the contrary, nidogen expression was observed only in the high metastatic lines. Traces of a laminin alpha chain were present only in immunoprecipitates of the low metastatic cells and could not be detected in the high metastatic cells. Both high and low metastatic cells deposited an extracellular matrix of basement membrane components, with laminin deposition decreased in high metastatic cells. Modified expression, production, and deposition of basement membrane components in high metastatic melanoma cells could be involved in their altered interactions with the extracellular matrix.

Animals↗
PubMed1995

Trihalomethanes in drinking water and cancer: risk assessment and integrated evaluation of available data, in animals and humans.

In our study, we attempted to jointly consider THM concentration data collected from drinking waters and carcinogenic risk assessment derived from mathematical models commonly used in this field (multi-stage models for laboratory animal experimentation data, and 'unit risk' derived from the relative risk in the case of epidemiological data). In order to estimate the risks related to joint exposure to different THMs, in this study the risk additivity hypothesis is taken into account. Based on animal data for the various tumors, carcinogenic risk estimates for different THM combinations vary from 2.7 x 10(-7) to 4.6 x 10(-6) per micrograms/l in relation to different carcinogenic substances published in the literature or specifically calculated in this study. The carcinogenic risk parameters derived from experimental studies and from epidemiological data were substantially consistent. Our study uses also as an example some data on concentration levels of THMs for drinking water supplies in Sardinia. The area mean THM concentration values for each supply varied, for ground waters, from 8.1 to 13.6 micrograms/l and, for surface waters, from 52.8 to 168 micrograms/l. For the 1976-1989 period, bladder cancer standardized mortality rates in the water distribution system areas where the THMs were measured indicate values similar, but generally lower, than the national ones, except in the province of Cagliari where the values were not significantly different. The risk estimates derived from animal studies are of the same order of magnitude as the epidemiological data in literature.

Animals↗
PubMed1995

Relationship between energy expenditure and visceral fat accumulation in obese women submitted to adjustable silicone gastric banding (ASGB).

OBJECTIVE: To analyze the relationship between visceral fat accumulation and resting energy expenditure in obese women and to evaluate the effects of a severe weight loss both on energy expenditure and on fat distribution. DESIGN: Twelve premenopausal women, aged 19-50 years, undergoing adjustable silicone gastric banding (ASGB) for morbid obesity participated at the study. The patients were evaluated twice. The baseline evaluation was performed immediately before surgery. After surgery, a diet specifically developed for patients submitted to gastric restriction (2.5 MJ/day) was given to the patients. The second evaluation was performed 6 months after surgery. MEASUREMENTS: Resting metabolic rate (RMR) was determined by indirect calorimetry. Total fat area (TFA), visceral fat area (VFA) and subcutaneous fat area (SFA) were measured by abdominal computed tomography. Fat mass (FM) and fat free mass (FFM) were derived by bioelectrical impedance analysis. RESULTS: At baseline, RMR was positively related to VFA (r = 0.60, P < 0.05). ASGB induced a highly significant weight loss of 24.4 +/- 9.0 kg. This weight reduction was mainly due to a loss of FM (68.5 +/- 10.8 vs 48.5 +/- 9.2 kg, P < 0.001), whereas FFM was only slightly reduced (52.6 +/- 4.0 vs 47.9 +/- 4.6 kg, P < 0.05). The BMI reduction was positively related to the baseline BMI and FM values (r = 0.61, P < 0.05 and r = 0.55, P < 0.05, respectively). There was no significant correlation between the BMI reduction and the baseline variables of fat distribution, nor between the BMI reduction and the basal RMR. Weight loss was accompanied by modifications of fat distribution. In particular, the reduction of VFA after surgery was strictly related to the VFA values at baseline (r = 0.91, P < 0.001). Weight loss induced a significant reduction of RMR (7.96 +/- 1.77 vs 6.57 +/- 6.90 MJ/day; P < 0.01). The reduction of the RMR observed with weight loss was significantly related to the FFM loss (r = 0.63, P < 0.05), whereas no correlations were found between the changes of RMR and the FM loss. Regarding to fat distribution, the reduction of the RMR was significantly related to the visceral fat loss (r = 0.57, P < 0.05), but not to the modifications of total or subcutaneous fat area. The independent contribution of the modifications of FFM, FM, and visceral fat to the changes of RMR was analyzed by multiple regression analysis. In this model, both FFM and visceral fat changes resulted independently related to the RMR. CONCLUSIONS: (1) visceral fat accumulation was a significant predictor of RMR in the very obese woman; (2) visceral obese women lost more visceral fat than subcutaneous ones; (3) the reduction of the RMR observed during weight loss could partly be explained by a reduction of visceral fat mass.

Adipose Tissue↗
PubMed1993

Cancer mortality in a cohort of rural licensed pesticide users in the province of Rome.

The mortality of a cohort of 2310 male workers who obtained a licence to handle pesticides in the period 1973-1979 in the province of Rome was investigated. The cohort contributed 26,846 person-years of exposure. The vital status of the cohort was determined up to the end of 1988. The causes of death of the 207 who had died were ascertained from death certificates. Standardized mortality ratios (SMR) were computed using both the provincial and the national mortality rates. The two methods yielded very similar results. Using the national rates, the SMR for all causes was 56 (95% confidence interval (CI) : 45.3-59.8), for cardiovascular diseases 47 (95% CI : 37.1-59.1), and for all cancers 72 (95% CI : 57.8-89.3). A statistically significant excess was noted for brain cancer (SMR = 270, 95% CI : 108.6-556.9). In addition, the cohort experienced statistically significant lower lung cancer mortality (SMR = 57, 95% CI : 35.6-80.0).

Adolescent↗
PubMed1993

Mortality in a cohort of pesticide applicators in an urban setting.

The mortality of a cohort of 168 pesticide applicators employed in the disinfestation service of the city of Rome for an average of 20 years is examined. Exposure data were abstracted from work records, and causes of death for the 42 workers who had died, were obtained from death certificates. Standardized mortality ratios (SMR) for specific causes of death were computed on the basis of provincial mortality rates. An excess in mortality from cancer of the liver and bile ducts with four cases observed and 0.7 expected (SMR = 571, 95% confidence interval (CI): 154-1463) was found. Increased risk for other cancers was also observed, but the SMR were not statistically different from unity. An increased risk of liver cancer occurs in those exposed to organochlorine pesticides between 1960 and 1965.

Bile Duct Neoplasms↗
PubMed1991

The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule.

A heparan sulfate proteoglycan is a component of all basement membranes. This molecule consists of three heparan sulfate side chains linked to a large core protein of approximately 400 kDa. We have isolated seven overlapping murine cDNA clones that encode the entire mRNA sequence of 12.685 kilobases of this molecule. This sequence has a single open reading frame of 3,707 amino acids that encodes for a protein of 396 kDa. Identical or near identical matchups with nine peptide sequences derived from the core protein of the molecule isolated from the Engelbreth-Holm-Swarm tumor were found with the deduced sequence. Sequence analysis and data base comparison of the deduced sequence show the protein to consist of five different domains, most of which contain internal repeats. Domain I contains a start methionine followed by a typical signal transfer sequence and a unique segment of 172 amino acids that contains the three probable sites of heparan sulfate attachment, SGD. Domain II contains four cysteine- and acidic amino acid-rich repeats that are very similar to those found in the LDL receptor and proteins such as GP330. Domain III consists of cysteine-rich and globular regions, both of which show similarity to those in the short arm of the laminin A chain. Domain IV contains 14 repeats of the immunoglobulin superfamily that are most highly similar to the immunoglobulin-like repeats in the neural cell adhesion molecule. Domain V contains three repeats with similarity to the laminin A chain G domain that are separated by epidermal growth factor-like regions not found in the laminin A chain. As the primary structural data agree with the appearance of the molecule in the electron microscope as a series of globules separated by rods, or "beads on a string," we have adopted the name perlecan for this molecule. The variety of domains in perlecan suggest multiple interactions with other molecules.

Amino Acid Sequence↗
PubMed1990

Biotinylation of double stranded DNA after transamination.

The bisulfite catalyzed transamination of cytidine and cytosine has been reported to be single strand specific, but local thermal instabilities of the DNA double helix, coupled with the extreme sensitivity of the Biotin-Avidin revelation methods, allows the random labelling of cytosines in d.s. DNA to detectable levels for those purposes where the overall label can be very low. We have evaluated the use of this reaction to prepare double stranded DNA molecules containing N4-aminoethyl-cytosine (4-aeC). After this step 4-aeC residues can be conjugated to biotinyl-n-hydroxysuccinimide ester yielding biotinylated DNA. This reaction allows the massive production of biotinylated probes. Labelled DNA can serve as molecular weight marker and positive control in Southern-blots. Moreover it can be useful in the study of DNA-protein interaction and in the isolation of d.s. DNA-binding proteins through chromatographic procedures.

Amination↗