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Biomedical subjects

C Orlando

Publications and source records attributed to C Orlando.

At least 55 records · Page 3Linked to original sources
PubMed1998

Rapid, quantitative nonisotopic assay for telomerase activity in human tumors.

Telomerase is a ribonucleoprotein enzyme that adds TTAGGG repeats onto human telomeres, preventing their shortening. The activation of this enzyme is an important step in cell immortalization and carcinogenesis and seems to represent a new and promising marker in cancer diagnosis and management. Telomerase activity is usually detected in cellular protein extract by the telomeric repeat amplification protocol (TRAP) assay, which can provide only a qualitative (presence/absence) evaluation. Here we present a modification of this method that can provide quantitative information without requiring time-consuming post-PCR procedures such as gel electrophoresis with radioactive materials and autoradiography. The detection and measurement of telomerase activity is performed by evaluating the amount of double-stranded DNA generated in the telomerase reaction and PCR amplification, with the use of the sensitive DNA fluorescent dye PicoGreen. In a subset of tumors, the presence of telomerase activity was confirmed by the conventional TRAP assay. By this method we evaluated telomerase activity in unselected groups of breast (n = 15), ovarian (n = 12), endometrial (n = 12), gastric (n = 20), and renal (n = 12) carcinomas, in meningiomas (n = 8), and in pheochromocitomas (n = 10). The results indicate substantial differences of telomerase activity among cancer groups; however, a large variability among patients of the same group is observed. Kidney, ovarian, and breast carcinomas showed the highest mean values (31.8 +/- 28.9, 29.2 +/- 26.7, and 35.3 +/- 15.9 ng DNA/microg protein, respectively, mean +/- SD), whereas gastric and endometrial cancers had a lower activity (17.2 +/- 8.8 and 13.5 +/- 7.9 ng DNA/microg protein, respectively). Very low or no detectable telomerase activity was found in meningiomas (with the exception of one malignant atypical variant) and pheochromocitomas (9.7 +/- 12.9 and 2.8 +/- 2.1 ng DNA/microg protein, respectively). In conclusion, our method seems to be an accurate and reasonable procedure for measuring telomerase activity in human cancers.

Biomarkers, Tumor↗
PubMed1997

Retrospective evaluation of c-erbB-2 oncogene amplification using competitive PCR in collecting duct carcinoma of the kidney.

PURPOSE: To evaluate retrospectively c-erbB-2 oncogene amplification in paraffin embedded specimens of collecting duct carcinoma of the kidney (CDC) with competitive polymerase chain reaction (PCR). MATERIALS AND METHODS: Eleven CDC specimens were evaluated with a novel PCR procedure for oncogene amplification measurement, which provides sensitive and accurate results even in presence of low-quality DNA, unsuitable for Southern blot techniques. RESULTS: c-erbB-2 oncogene amplification was present in 5 out of 11 cases (45%) with a number of copies ranging from 4 to 12. All patients presenting oncogene amplification decreased within one year, while 50% (3/6) of those without amplification are alive with a mean follow-up of 42 months. CONCLUSIONS: The high incidence of c-erbB-2 oncogene amplification in CDC further characterizes this tumor as a separate entity from renal cell carcinoma, and shows some genetic characteristics in common with transitional cell carcinoma.

Adult↗
PubMed1997

Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification.

We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'-->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P < 0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.

Base Sequence↗
PubMed1997

Imaging of somatostatin receptors by indium-111-pentetreotide correlates with quantitative determination of somatostatin receptor type 2 gene expression in neuroblastoma tumors.

We reported previously that the relative level of gene expression for sst2, a subtype of somatostatin receptors, was positively related to patient outcome in the childhood tumor neuroblastoma (NB). Because sst2 binds with high-affinity octreotide and its scintigraphic derivative, 111In-pentetreotide, we tested the hypothesis of whether NB tumor imaging with 111In-pentetreotide gives similar information to ex vivo measurement of sst2 expression. We, therefore, studied simultaneously nine NB tumors with 111In-pentetreotide single photon emission computed tomography and competitive reverse transcription-PCR for sst2, along with other prognostic markers. To quantitate the relative abundance of 111In-pentetreotide binding to NB tumors, we developed a simple semiquantitative method, based on the mathematical analysis of 111In-pentetreotide association to cancer cell receptors at different time points (4 and 24 h). We indeed found that the ratio between the activity in a manually extracted region of interest from pathological (ROIT) and background (ROINT) area was increasing between early and late acquisition only in affected tissues. The rate of this pathological increase was quite different among patients and significantly (P < 0.01) related to the abundance of sst2 gene expression, as measured by competitive reverse transcription-PCR on ex vivo tumor samples. Because we demonstrated that in 26 NB patients the density of sst2 is strongly related to survival (P < 0.0005) and apparently independent from N-myc oncogene amplification (P < 0.05), we propose that NB tumor imaging with 111In-pentetreotide may have not only a diagnostic but also a prognostic value.

Adolescent↗
PubMed1996

Expression of endothelin-1 gene and protein in human granulosa cells.

Previous studies in animal models indicated an autocrine/paracrine action of endothelin-1 (ET-1) in the ovary. We now report evidence on the presence of ET-1 in human ovary during reproductive life. Immunohistochemical and in situ hybridization studies demonstrated a positive signal into cytoplasm of granulosa cells (GC) of follicles at different growth stages. The concentration of ET-1-like immunoreactivity (ET-1-LI) was also measured by a specific RIA in human follicular fluid (FF). FF samples were obtained from women in an in vitro fertilization program undergoing gonadotropin stimulation (group A; n = 24) or no treatment (group B; n = 7). The mean (+/-SD) ET-1-LI FF level in group A (4.85 +/- 2.06 pg/mL) was significantly higher than that in group B (1.29 +/- 0.43 pg/mL; P < 0.01), whereas the corresponding mean plasma levels were not significantly different and were not correlated to respective FF values. Our results indicate for the first time the presence of ET-1 and its messenger ribonucleic acid in the GC of the human ovary. The higher ET-1-LI levels found in the FF from women undergoing gonadotropin treatment suggest a modulation by gonadotropins and/or ovarian steroids of ET-1 production by GC.

Adult↗
PubMed1996

Detection of c-erbB-2 amplification in transitional cell bladder carcinoma using competitive PCR technique.

PURPOSE: We used competitive PCR to verify retrospectively the prognostic significance of c-erbB-2 oncogene amplification in transitional cell bladder carcinomas as a predictive index of patient survival with a maximum follow-up of nine years, and to investigate the variations of c-erbB-2 amplification during bladder carcinoma recurrence and/or progression from superficial to more invasive states. MATERIALS AND METHODS: Oncogene amplification was determined by an accurate and sensitive procedure based on competitive PCR. Measurements were performed in DNA extracted from fresh cancers or from formalin-fixed, paraffin-embedded tumor samples. RESULTS: The overall mean incidence of c-erbB-2 oncogene amplification was 26% (24/92), with a significant relationship with tumor grade (p < 0.0001). We did not find any statistical difference in survival probability between subjects with (20%) or without (30%) oncogene amplification. During tumor progression we observed a limited increase of tumors carrying oncogene amplification (2 of 20) whereas the mean degree of amplification was not affected. CONCLUSIONS: c-erbB-2 amplification seems to be a genetic event related to the degree of bladder tumor differentiation. However the presence and/or the degree of this genetic alteration do not seem predictive of tumor progression, recurrence and survival probability, at least in patients with advanced transitional cell bladder carcinoma. These data seem to indicate that the amplification of c-erbB-2 in bladder carcinoma could be considered as an epiphenomenon, present in a subset of tumors but apparently not related to the clinical outcome.

Aged↗
PubMed1996

Quantitation of somatostatin receptor type 2 gene expression in neuroblastoma cell lines and primary tumors using competitive reverse transcription-polymerase chain reaction.

We previously reported the presence of somatostatin (SS-14)-binding sites in a wide panel of human neuroblastoma (NB) tumor cell lines. Given that the adrenal gland and its relative embryonal and adult tumors express an abundance of mRNA for somatostatin receptor type 2 (sst2) mRNA, we studied the quantitative expression of sst2 in 6 NB cell lines and 15 primary tumors using competitive reverse transcription (RT)-PCR. This method uses an insertion mutant of the target gene as a competitor for the RT-PCR reaction, thus allowing exact quantitation of sst2 mRNA abundance. We found expression of specific transcripts for sst2 in all of the NB cell lines and tumors investigated (range, 9 x 10(5)-4 x 10(9) molecules/microg RNA). In NB cells, the expression of sst2 was highly correlated with SS-14-binding sites (R = 0.93). In primary tumors, sst2 was positively related to the expression of the neuroendocrine marker secretogranin II (P < 0.05) and negatively related to N-myc amplification (a poor prognostic factor, P < 0.005) and metastatic dissemination (P < 0.05). In addition, Kaplan-Meier curves indicate that sst2 expression is positively related to survival (P = 0.01). In a patient with stage IVs disease (a spontaneously regressing form), we found the highest sst2 expression (4 x 10(9) molecules/microgram RNA), a value relatively similar to that of normal adrenal. In conclusion, these data indicate that quantitation of sst2, as assessed with competitive RT-PCR, could represent a new prognostic tool in the neuroendocrine tumor NB. Since sst2 recognizes octreotide with high affinity, these findings could also have both diagnostic and therapeutic value.

Adult↗
PubMed1995

Proliferation and migration of endothelial cells is promoted by endothelins via activation of ETB receptors.

The growth and migration of endothelial cells are the prerequisites for vascular remodeling. The effects of endothelin (ET)-1 and -3 (ET-1, ET-3) on the proliferation and migration of endothelial cells isolated from bovine adrenal capillaries (BACE) and human umbilical veins (HUVEC) have been investigated. Cell proliferation (measured as DNA synthesis and as total cell number) and migration were significantly increased by ET-1 and ET-3. Dose-dependent proliferation was produced by ET-1 and ET-3 in both cell lines, with maximal effects at 0.1 nM concentration. ET-1 and ET-3 also stimulated BACE and HUVEC mobilization in a dose-dependent manner. The maximal responses were obtained at 10 nM concentration in both BACE and HUVEC. The full agonist for the ETB receptor, ET-(16--21), was able to reproduce the effects of ET on proliferation and migration of both cell lines. Modification of ET-(16--21) at Leu17 and Ile19 and amidation of the COOH-terminal were accompanied by loss of activity. The ETB-receptor antagonist IRL-1038 blocked the migration induced by ET-3 and ET-(16--21), whereas the ETA-receptor antagonist BQ-123 was not effective. We conclude that ETs, by favoring endothelial cell growth and mobilization, can contribute to neovascularization through an autocrine mechanism that requires ETB-receptor activation.

Adrenal Glands↗
PubMed1995

Gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2, and N-myc measured by quantitative PCR with a multiple competitor template.

We recently proposed a quantitative PCR procedure for the absolute measurement of c-erbB-2 oncogene amplification, based on the simultaneous polymerase chain reaction (PCR) amplification of the target gene and of a competitor DNA molecule acting as internal standard. To increase the number of assayable oncogenes and the accuracy of the quantitative comparison of gene amplification degree within the same tumor, we have now constructed a single synthetic competitor for int-2, c-myc, N-myc epidermal growth factor receptor, and c-erbB-2 genes, and for the reference gene beta-globin. This competitor was constructed by a two-step recombinant PCR procedure and inserted as a 297-bp sequence in a plasmid. The order of primer insertion was designed to obtain competitors of comparable sizes to those of the respective genomic targets, but still easily recognizable from the latter ones by gel electrophoresis. The clone competitor was tested to evaluate the linearity range for each assay. The present application of quantitative PCR based on a multiple competitor represents the first approach for the achievement of a single reagent for the evaluation of a panel of genes potentially amplified in human tumors.

Base Sequence↗
PubMed1994

Characterization, regulation, and function of specific cell membrane receptors for insulin-like growth factor I on bone endothelial cells.

It is now widely accepted that insulin-like growth factor-I (IGF-I) has a local regulatory role in bone remodeling. IGF-I has also been demonstrated to regulate proliferation of bone-derived endothelial cells. Such studies suggest a role of IGF-I in skeletal angiogenesis. Using BBE cells, a bovine bone endothelial cell line, we characterized the kinetics and chemical properties of IGF-I receptors and examined the effect of IGF-I on bone endothelium migration. Two classes of binding sites with high affinity for IGF-I were detected by binding experiments on bone endothelial cells. Both competition analyses and cross-linking studies revealed the presence of type I IGF receptor in bone endothelial cells. Moreover, these cells produced and released authentic IGF-I into the medium, as evidenced by radioimmunoassay analyses of gel-filtered conditioned media. Both IGF-I binding capacity and release decreased either with increases in cell number or after treatment with 17 beta-estradiol (17 beta E2) and parathyroid hormone (PTH). Both hormones also inhibited chemotactic responses of bone endothelial cells to IGF-I. Taken together, these results strongly suggest that IGF-I, a growth factor that promotes the proliferation of various bone cell types, also induces growth and chemotactic responses in bone endothelium acting through the type I IGF receptor. This may be part of a generalized response of bone cells to IGF-I that facilitates cell migration.

Analysis of Variance↗
PubMed1994

Adenosine triphosphate release by osmotic shock and hemoglobin A1C in diabetic subjects' erythrocytes.

We investigated the significance of adenosine triphosphate (ATP) release from diabetic subjects' red blood cells (RBCs) following osmotic shock (OS) and its possible relationship with hemoglobin A1C (HbA1C) and with the RBC membrane protein skeleton. RBCs from type I (insulin-dependent [IDDM]) and type II (non-insulin-dependent [NIDDM]) diabetic subjects and age- and sex-matched control subjects were submitted to OS using NaCl solutions (from 0.9% to 0.045% final concentration). ATP release values were determined by the bioluminescent method. For pattern study, they were expressed both as absolute values and as percentages (%) of ATP maximum release (at 0.045% NaCl solution). Twenty-seven IDDM and 25 NIDDM subjects and two control groups were investigated. ATP content in RBCs was 2.08 +/- 0.19 pmol/10(4) RBC in IDDM and 1.23 +/- 0.20 pmol/10(4) RBC in NIDDM subjects. The ATP content of IDDM subjects' RBCs was significantly higher than that of the corresponding control group. ATP release at 0.49% NaCI OS, both as absolute value and as percentage value, was significantly lower in both diabetic groups, and ATP% was inversely correlated with HbA1" (IDDM: r = -.489, P < .01; NIDDM: r = -.654, P < .01), suggesting a possible relationship between Hb glycation, RBC membrane protein skeleton glycation, and its influence on ATP release by OS. In conclusion, the proposed method seems useful for measuring RBC ATP content and, at the same time, for monitoring the leak effect of the RBC membrane before it bursts.

Adenosine Triphosphate↗
PubMed1994

Simultaneous measurement of sperm LDH, LDH-X, CPK activities and ATP content in normospermic and oligozoospermic men.

Total lactate dehydrogenase (LDH), LDH-X, creatine phosphokinase (CPK) activities and ATP content were measured in washed spermatozoa from 188 normospermic and 94 oligozoospermic men. These four biochemical parameters were significantly higher (p < 0.001) in spermatozoa from oligozoospermic men than in those from normospermic men and were related inversely to sperm concentration. In oligozoospermic men the activity of sperm LDH and LDH-X was related significantly to sperm CPK activity (r = 0.5486 and r = 0.4442, respectively), whereas this relationship was less evident in normospermic men (r = 0.4124 and r = 0.3844, respectively). Sperm ATP content was related weakly to sperm LDH, LDH-X and CPK activities both in oligozoospermic and normospermic men. After separation on a discontinuous (40% and 80%) Percoll gradient LDH, LDH-X, CPK activities and ATP content in 80% separated sperm fractions from 48 normospermic and 30 oligozoospermic men were found to be significantly lower than in the initial semen sample in both groups of subjects. The 80% separated sperm fractions from oligozoospermic and normospermic men did not differ in their LDH or LDH-X activities or ATP content, whereas sperm CPK activity was still significantly higher in oligozoospermic men. These data suggest that, of the four parameters studied, high levels of intracellular CPK activity are the most relevant biochemical feature of spermatozoa from oligozoospermic men.

Adenosine Triphosphate↗
PubMed1994

Measuring c-erbB-2 oncogene amplification in fresh and paraffin-embedded tumors by competitive polymerase chain reaction.

We present an original application of competitive polymerase chain reaction (PCR) for measuring oncogene amplification in DNA from human tumors by simultaneous PCR amplification of genomic DNA with fixed amounts of an internal standard (competitor DNA). Competitors share the same sequence as the target genes but contain an additional 15- to 20-base-pair insert, which allows resolution of the amplified products after polyacrylamide gel electrophoresis and ethidium bromide staining. The gene copy number is derived from the ratio between the intensities of the bands corresponding to the amplified products. Using this procedure, we measured c-erbB-2 amplification in breast and bladder carcinomas in both fresh tumor tissues and paraffin-embedded tissue samples and assessed the precision, sensitivity, and accuracy of the assay. Competitive PCR is a simple, reliable, and accurate method for the evaluation of c-erbB-2 amplification and is potentially suitable for use in the clinical laboratory.

Base Sequence↗
PubMed1993

Endothelin-1: a new autocrine/paracrine factor in rat testis.

Cultured Sertoli cells of 20-day-old rats were found to produce and release endothelin-1-like immunoreactivity (ET-1-LI) under follicle-stimulating hormone control. The elution profile of ET-1-LI from extracts of spent Sertoli cell culture medium corresponds to that of synthetic ET-1, suggesting a testicular production of authentic ET-1. In contrast, the conditioned medium from rat Leydig cells did not contain ET-1-LI. Immunohistochemical studies confirmed that, in 20-day-old rats, the positive staining was confined to some Sertoli cells, whereas interstitial cells were negative. In the adult rat testis the positivity was not limited to the tubular compartment (Sertoli cells) but was also present in the interstitium. A high concentration (13 pmol/mg protein) of high-affinity (dissociation constant = 0.6 nM) 125I-labeled ET-1 binding sites was present in Leydig cells. These sites bind ET-1 and ET-2 with 1,000-fold higher affinity than ET-3, suggesting that they correspond to the subtype ETA of the ET receptors. Specific 125I-ET-1 binding sites are present also in Sertoli cells but are 50-fold less concentrated than in Leydig cells. Our results suggest an autocrine/paracrine role for ET-1 in rat testis.

Animals↗
PubMed1993

Alterations of vibratory and thermal peripheral sensitivity in liver cirrhosis.

Peripheral neurological involvement, causing distal paresthesias and/or cramps has been described in liver cirrhosis, and its symptoms are quite frequent in decompensated patients under diuretic treatment. In this paper we reported thermal (TPT) and vibratory (VPT) sensations in 100 well-defined cirrhotics (43 in group A, and 63 in B and C of Child-Pugh classification) to define the prevalence of such alterations and the role of sex, age, severity and aetiology of the cirrhosis, and diuretic treatment. The TPT and VPT values were out of the normal range in 28% and 39% of the patients, respectively. Significantly, the alteration of VPT was associated both to alcoholic aetiology and decompensated cirrhosis, whereas the only parameter significantly associated to TPT alterations was alcoholic aetiology. Multiple factors such as inappropriate nutrition, hypercatabolic status, and portal systemic shunting may play a role in inducing trophic disturbances of nervous fibres in cirrhotics.

Adult↗
PubMed1992

A monoclonal antibody directed against the catalytic site of Bacillus anthracis adenylyl cyclase identifies a novel mammalian brain catalytic subunit.

A brain adenylyl cyclase was shown to contain an epitope closely related to that specified by a conserved sequence containing a nucleotide-binding consensus sequence GXXXXGKS and located in the catalytic sites of bacterial, calmodulin-dependent adenylyl cyclases [Goyard, S., Orlando, C., Sabatier, J.-M., Labruyere, E., d'Alayer, J., Fontan, G., van Rietschoten, J., Mock, M., Danchin, A., Ullmann, A., & Monneron, A. (1989) Biochemistry 28, 1964-1967]. A monoclonal antibody, mab 164, produced against a peptide corresponding to this conserved sequence specifically inhibited the Bordetella pertussis adenylyl cyclase. It also specifically inhibited rat and rabbit brain synaptosomal adenylyl cyclases. The extent of inhibition depended upon the type of enzyme purification, reaching 90% for the calmodulin-sensitive species of enzyme and 20-35% for the forskolin-agarose-retained species. The extent of inhibition in a given fraction also depended upon the effector present. mab 164 reacted on Western blots of forskolin-agarose-retained fractions with a 175-kDa component and did not recognize the Gs alpha stimulatory subunit. Consequently, the 175-kDa protein was considered as a good candidate for an adenylyl cyclase catalyst. The adenylyl cyclase activity contained in forskolin-agarose-retained fractions was further purified on calmodulin-Sepharose. On Western blots of such fractions, mab 164 reacted with a 140-kDa protein, a component that appeared to derive from the 175-kDa protein enriched in the previous step. The kcat of this 140-kDa presumptive adenylyl cyclase was estimated to be of the order of 600 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗