Biomedical subjects

A Costantino

Publications and source records attributed to A Costantino.

At least 19 recordsLinked to original sources

PubMed2005

Activation of endoplasmic reticulum stress response by hepatitis C virus proteins.

Flaviviruses utilize the endoplasmic reticulum (ER) as the main site for replication and protein synthesis and cause some level of ER stress. In the present study, we evaluated the ability of HCV proteins to induce ER stress response by using a tetracycline-regulated cell line expressing a region of HCV genome containing the structural genes. In this system different levels of HCV protein expression could be obtained by varying the concentration of tetracycline in the medium. Real Time PCR and Western blotting assay demonstrated that HCV mRNA and protein levels reach a maximum value at 24-48 h and decrease at 72 h postinduction. Cell proliferation analysis indicated that HCV synthesis causes cell growth inhibition. The effect was also observed in cells expressing lower levels of HCV proteins. The expression profile of specific genes, which are markers of ER stress response, revealed the upregulation of the chaperone GRP78 and the transcription factor GADD153. Induction of GADD153 correlates with the downregulation of the antiapoptotic Bcl-2 gene suggesting that synthesis of HCV proteins may influence cell fate through the activation of ER stress signaling pathway.

CCAAT-Enhancer-Binding Proteins↗

PubMed2005

Norethisterone enanthate plus testosterone undecanoate for male contraception: effects of various injection intervals on spermatogenesis, reproductive hormones, testis, and prostate.

The goal of this study was to find the most favorable injection interval of norethisterone enanthate (NETE) plus testosterone undecanoate (TU) in terms of gonadotropin, sperm suppression, and prostatic effects. Fifty normal men were randomly assigned to receive NETE 200 mg plus TU 1000 mg every 8 wk (n = 10), every 12 wk (n = 10), every 6 wk for 12 wk and then every 12 wk (n = 10), and every 6 wk for 12 wk and thereafter TU 1000 mg plus placebo every 12 wk (n = 10), and placebo plus placebo every 6 wk for 12 wk and then every 12 wk (n = 10) for 48 wk. Semen analyses, blood drawings, physical examinations, and prostate ultrasounds were performed throughout the study. Of the men in the 8-wk injection group, 90% (nine of 10) achieved azoospermia, compared with 37.5% (three of eight) in the 12-wk injection group (P = 0.019). TU plus placebo injected every 12 wk did not maintain sperm suppression. Prostate volumes did not change significantly in either group. In conclusion, these data suggest that the combined administration of NETE and TU at 8-wk intervals represents an effective hormonal contraceptive regimen.

Adolescent↗

PubMed2005

Short-term modification of sex hormones is associated with changes in ghrelin circulating levels in healthy normal-weight men.

The aim of this study was to evaluate the effect of selective and short-term sex hormone modifications on ghrelin levels in normal-weight eugonadal men undergoing hormonal contraceptive treatments. Seven men received an oral progestin [cyproterone acetate (CPA) or dienogest (DNG)] 10 mg/day for 3 weeks (CPA-DNG group), 7 CPA orally 5 mg/day in association with testosterone enanthate (TE) im 200 mg/week for 8 weeks (CPA-TE group), and 7 placebo (PLAC) for 8 weeks (PLAC group). Anthropometry and blood levels of LH, FSH, testosterone, estradiol, glucose, insulin and total ghrelin were evaluated. At baseline, no parameters differed among the three groups. After treatment, LH and FSH decreased in both CPA-DNG and CPA-TE groups, whereas they did not change in the PLAC group. Testosterone and estradiol decreased in the CPA-DNG group to the hypogonadal range, increased in the CPA-TE group to supraphysiological concentrations and, as expected, remained unchanged in the PLAC group. Total ghrelin levels increased in the CPA-DNG, decreased in the CPA-TE and did not change in the PLAC group. Ns modifications in the other parameters were observed in any group, demonstrating that the short-term changes of circulating sex hormones are able to modify ghrelin levels. These data, therefore, suggest that sex steroids are important regulators of ghrelin in normal-weight healthy men too.

Adult↗

PubMed2004

The transmembrane domain of hepatitis C virus E1 glycoprotein induces cell death.

The E1 protein of hepatitis C virus (HCV) shows the ability to induce cell lysis by the alteration of membrane permeability when expressed in Escherichia coli cells. This function seems to be an intrinsic property of a C-terminal hydrophobic region of E1 as permeability changes and cell lysis can be blocked by mutagenesis of specific amino acids in this domain. To establish whether the expression of E1 protein and its C-terminal domain was able to induce cell death also in eukaryotic cell, we cloned HCV sequences expressing the full-length E1 (E383), the C-terminal domain (SVP) and a mutant lacking the C-terminal region (E340) in the pRC/CMV expression vector. HepG2 cell line was co-transfected with empty vector or HCV expression plasmids and a reporter vector that expressed beta-galactosidase (beta-gal) to visualize co-transfected blue cells. At 60 h after transfection, the loss of blue cells, considered as a measure of cell death, was 31.5 and 64.3% for the E1 and SVP clones. On the contrary, the number of blue cells after transfection with E340 plasmid was similar to that observed with the control vector. The analysis by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay revealed an increased number of apoptotic cells at 48 h after transfection with E1 and SVP clones. Furthermore, cells transfected with SVP revealed a typical internucleosomal DNA fragmentation and the activation of caspase-3-like proteases as the specific inhibitor Ac-DEVD-CHO peptide partially blocked SVP apoptosis. These data indicate that the intracellular expression of HCV E1 protein and its C-terminal domain induces an apoptotic response in human hepatoma cell line.

Apoptosis↗

PubMed2003

Testosterone undecanoate maintains spermatogenic suppression induced by cyproterone acetate plus testosterone undecanoate in normal men.

In this study we evaluated whether testosterone undecanoate (TU), alone or combined with low dose cyproterone acetate (CPA), can maintain spermatogenic suppression induced by higher doses of CPA plus TU. Twenty-four men received for 12 wk 20 mg/d CPA plus 1000 mg/6 wk TU and then 1000 mg/8 wk TU plus 20 mg/d CPA (n = 8), 2 mg/d CPA (n = 8), or plus placebo (n = 8) for 32 wk. Blood samples, physical examinations, hormones, chemistry, hematology, semen analysis, and sexual/behavioral assessments were performed throughout the study. Sperm counts decreased to less than 1 million/ml in all subjects by wk 12, and 54% of them achieved azoospermia. Suppression of sperm counts was maintained until wk 44. Serum LH and FSH levels were suppressed by wk 12 of hormone administration and remained suppressed until wk 44. No significant changes in any biochemical parameters were detected at wk 44 in any group. There was a slight increase in total prostate volume to within the normal range at wk 44 that returned to baseline 1 yr after stopping hormone administration. In conclusion, TU alone or combined with lower doses of CPA maintains sperm suppression induced by higher dose CPA plus TU for 32 wk. This prototype regimen represents a promising male contraceptive regimen.

Adult↗

PubMed2002

Recent advances in hormonal male contraception.

Hormones inhibit fertility in men by suppressing sperm production. Testosterone-enanthate induced azoospermia has proven to provide optimal contraceptive protection. Preliminary studies have shown that testosterone alone or androgen-progestin combinations induce profound sperm suppression in the Eastern and white populations, respectively. Thus, these regimens may represent viable options for male contraception. New long-acting androgen formulations represent a major advancement in this field, allowing for the development of more acceptable and effective regimens.

Androgens↗

PubMed2002

Infection rate and spontaneous seroreversion of anti-hepatitis C virus during the natural course of hepatitis C virus infection in the general population.

BACKGROUND: Hepatitis C virus (HCV) infection is common worldwide but there are different prevalence rates in different countries. Data on the incidence of HCV in the general population are scarce. Spontaneous viral clearance occurs in 10-25% of infected individuals after acute infection yet controversy exists regarding the frequency of spontaneous clearance during the natural course of HCV infection in the general population. AIMS: Anti-HCV prevalence, HCV infection rate, and the kinetics of anti-HCV were studied in a cross section of the general population of central Italy. STUDY POPULATION AND METHODS: Anti-HCV prevalence (EIA-3 Ortho, RIBA-3 Ortho Chiron) was estimated in 3884 randomly selected individuals. Infection rate and antibody kinetics were estimated in 2032 participants for whom a second blood sample was taken after a median follow up of seven years. HCV-RNA determination by polymerase chain reaction was performed on follow up sera. RESULTS: The overall confirmed anti-HCV prevalence was 2.4%. Two participants seroconverted for anti-HCV, giving an overall infection rate of 1.4 cases per 10 000 person years (95% confidence interval 0.2-5.2 per 10 000 person years). Of the 36 individuals confirmed as anti-HCV positive at enrollment, seven (19.4%) showed complete seroreversion. Seven (87%) of the eight individuals with indeterminate results at enrollment were serologically non-reactive at the end of follow up. Of the 25 participants confirmed to be anti-HCV positive at both enrollment and follow up, 23 (92.0%) with stable serological profiles tested positive for HCV-RNA at the end of follow up. CONCLUSIONS: There is still a permanent risk, although low, of HCV spread in the general population in an area of low level endemicity. In this setting, a wide spectrum of modifications of viral and antibody patterns can be observed in HCV infected patients.

Adult↗

PubMed2001

Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity.

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed in Escherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331-383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced in E. coli by influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262-291) was examined by expressing different deletion mutants of E1 in an E. coli system that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg(339), Trp(368) and Lys(370) play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.

Cell Membrane Permeability↗

PubMed2000

Expression and membrane association of hepatitis C virus envelope 1 protein.

The expression of hepatitis C virus (HCV) E1 protein is toxic for Escherichia coli cells. For this reason, we have cloned the E1 gene in the pET3a vector and analyzed the inducible expression of the protein in two strains of E. coli characterised by a different level of reduction of basal synthesis. The results indicated that synthesis of E1 was supported only by the BL21(DE3)pLysS strain which provides a tightest control of protein expression before the induction. The BL21(DE3)pLysS cells were then used for the expression of E1 gene, varying at its carboxy terminus in order to retain (E1, aa 192-383) or delete (Elt, aa 192-340) a C-terminal hydrophobic region that may be involved in membrane association. Following cell fractionation, E1 protein was found associated with the membrane fraction. By contrast, the truncated mutant E1t, was identified in the soluble phase suggesting a direct role for the C-terminal domain in E1 membrane association.

Cell Membrane↗

PubMed1999

Quantification of phenylurea herbicides and their free and humic acid-associated metabolites in natural waters.

There is increasing interest in and demand for simultaneously monitoring pesticides as well as related degradation products (DPs) in natural waters, as the latter compounds can be even more toxic than the former ones. A method for determining parts per trillion levels of phenylurea herbicides and their DPs, that is their dealkylated forms and aromatic amines, is described. This method is based on solid-phase extraction with a Carbograph 4 cartridge followed by liquid chromatography (LC) with electrospray (ES) mass spectrometric detection. A study aimed at optimizing the response of the ES-MS detector for very weakly basic chloroanilines was conducted. Results showed that ion signal intensities of the above species were dependent on the composition of the LC mobile phase to an astonishing degree. At concentration levels of a few hundred ng/l, laboratory experiments showed that the aromatic amines considered here were mostly associated to dissolved humic acids (HAs) by both reversible and irreversible bindings. The addition of a reducing agent, i.e., NaBH4, succeeded in liberating that fraction of aromatic amines, which being reversibly bound to quinoidal structures of HAs are bioavailable. Analyte recoveries were better than 85% on extraction from 4 l of drinking water (spike level, 25 ng/l), 2 l of ground water (spike level, 50 ng/l) and 0.5 l of river water (spike level, 200 ng/l). Relative standard deviations ranged between 4.6 and 20% for drinking water, 4.3 and 15% for ground water, 5.9 and 13% for river water. Method detection limits calculated for drinking water, groundwater and surface water were between 3 and 11, 6 and 21, 36 and 75 ng/l, respectively.

Chromatography, Liquid↗

PubMed1999

Insulin receptor activation by IGF-II in breast cancers: evidence for a new autocrine/paracrine mechanism.

IGF-II, produced by breast cancer epithelial and stromal cells, enhances tumor growth by activating the IGF-I receptor (IGF-I-R) via autocrine and paracrine mechanisms. Previously we found that the insulin receptor (IR), which is related to the IGF-I-R, is overexpressed in breast cancer cells. Herein, we find that, in breast cancer the IR is activated by IGF-II. In eight human breast cancer cell lines studied there was high affinity IGF-II binding to the IR, with subsequent IR activation. In these lines, IGF-II had a potency up to 63% that of insulin. In contrast, in non malignant human breast cells, IGF-II was less than 1% potent as insulin. Via activation of the IR tyrosine kinase IGF-II stimulated breast cancer cell growth. Moreover, IGF-II also activated the IR in breast cancer tissue specimens; IGF-II was 10-100% as potent as insulin. The IR occurs in two isoforms generated by alternative splicing of exon 11; these isoforms are IR-A (Ex11-) and IR-B (Ex11+). IR-A was predominantly expressed in breast cancer cells and specimens and the potency of IGF-II was correlated to the expression of this isoform (P<0.0001). These data indicate, therefore, that the IR-A, which binds IGF-II with high affinity, is predominantly expressed in breast cancer cells and represents a new autocrine/paracrine loop involved in tumor biology.

Binding, Competitive↗

PubMed1999

Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells.

Insulin-like growth factor II (IGF-II) is a peptide growth factor that is homologous to both insulin-like growth factor I (IGF-I) and insulin and plays an important role in embryonic development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type 1 insulin-like growth factor receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to the alternative splicing of exon 11. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These differences in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal growth and cancer biology.

3T3 Cells↗

PubMed1999

Insulin and insulin-like growth factor-I (IGF-I) receptor overexpression in breast cancers leads to insulin/IGF-I hybrid receptor overexpression: evidence for a second mechanism of IGF-I signaling.

The insulin receptor (IR) form hybrids with the closely related insulin-like growth factor-I (IGF-I) receptor (IGF-I-R). Because most human breast carcinomas overexpress both the IR and the IGF-I-R, we evaluated whether the insulin/IGF-I hybrid receptor (Hybrid-R) is also overexpressed in these tumors and what role it plays in breast cancer biology. Using specific ELISAs and Western blots, we measured Hybrid-R content and function in 8 human cultured breast cancer cell lines and 39 human breast cancer specimens. Hybrid-R content and function were also compared to the content and function of the IR and the IGF-I-R. Hybrid-R content exceeded the IGF-I-R content in >75% of breast cancer specimens and was directly related to the molar ratio of both the IR and IGF-I-R content, suggesting that Hybrid-R formation occurred by random assembly of IR and IGF-I-R half-receptors. Hybrid-Rs became tyrosine autophosphorylated when breast cancer cells were exposed to IGF-I but not when they were exposed to insulin. In cells with an elevated Hybrid-R content, Hybrid-R autophosphorylation in response to IGF-I exceeded IGF-I-R autophosphorylation, suggesting that most of the IGF-I effect occurred via the Hybrid-R. Furthermore, Hybrid-Rs mediated growth in response to IGF-I, as indicated by experiments with blocking antibodies to the IGF-I-R. These data indicated therefore that: (a) Hybrid-Rs are present and play a major role in mediating the IGF-I signal in breast cancer; (b) their expression is directly related to IR overexpression; and (c) potential therapies designed to block IGF-I actions in breast cancer must take into account the role of these Hybrid-Rs.

Antibodies, Monoclonal↗

PubMed1998

Hepatitis C virus E1 protein induces modification of membrane permeability in E. coli cells.

The E1 gene of hepatitis C virus (HCV) has been cloned and expressed in BL21(DE3)pLys Escherichia coli strain by pET3a vector to analyze changes in membrane permeability produced by this protein. We showed that the expression of E1 (aa 192-383), as well as of two C-terminal fragments (aa 331-383 and aa 341-383) corresponding to the transmembrane (TM) region of this protein, induced a rapid lysis of cells. On the contrary, the expression of a mutant of E1 (aa 192-340), lacking the last 40 amino acids, did not cause cell lysis. The analysis of permeability changes revealed that modification of membrane permeability to several compounds were observed only in clones expressing E1 and C-terminal fragments, while the synthesis of the C-terminal-deleted mutant had little or no effect on permeability. These findings demonstrate that the TM domain of E1 protein has membrane-active properties that may be involved in some aspects of virus-cell interaction.

Bacteriolysis↗

PubMed1998

Overexpression of the RON gene in human breast carcinoma.

Constitutive activation of the RON gene, known to code for the tyrosine-kinase receptor for Macrophage Stimulating Protein (also known as Scatter Factor 2), has been shown to induce invasive-metastatic phenotype in vitro. As yet, nothing is known about the expression of this novel member of the MET-oncogene family in spontaneously occurring human cancers. Here we report that Ron is expressed at abnormally high levels in about 50% primary breast carcinomas (35/74 patients). Among these, the expression is increased more than 20-fold in 12 cases and the overexpressed protein is constitutively phosphorylated on tyrosine residues. Notably, Ron is only barely detectable in epithelial cells of the mammary gland, and its expression remains unchanged in benign breast lesions (including adenomas and papillomas). Overexpression was observed in different histotypic variants of carcinomas; it is associated with the disease at any stage and correlates with the post-menopausal status. In breast carcinoma cells grown in vitro, activation of the Ron receptor resulted in proliferation, migration and invasion through reconstituted basement membranes. Altogether, these data suggest a role for the RON gene in progression of human breast carcinomas to the invasive-metastatic phenotype.

Animals↗

PubMed1998

The relationships of chronic hepatitis and cirrhosis to alcohol intake, hepatitis B and C, and delta virus infection: a case-control study in Albania.

The present study examined the effect of hepatitis B virus (HBV) and alcohol intake, and the role of hepatitis delta virus (HDV) and hepatitis C virus (HCV) in the aetiology of chronic liver disease in Albania. A total of 106 cases of liver cirrhosis or chronic hepatitis were compared to 195 control patients without these or other liver diseases. Adjusted odds ratios were 52.7 (95% CI 22.7-122) for HBV surface antigen, 26.9 (95% CI 4.9-147) for anti-HCV, 26.2 (95% CI 3-1-221) for anti-HDV, 2.4 (95% CI 1.3-4.4) for lifetime alcohol intake and 2.3 (95% CI 1-5.5) for duration of alcohol intake. Although not significant, an interaction was suggested between HBsAg and anti-HCV and between HBsAg and alcohol intake. Our study underlines the role of hepatitis viruses in the development of chronic liver diseases. Additionally, it suggests that heavy alcohol intake may magnify the effect of HBV on these diseases. HBV vaccination and alcohol abstention appear to be important strategies to reduce the risk of liver cirrhosis and chronic hepatitis in Albania.

Adolescent↗

PubMed1998

Low dose of cyproterone acetate and testosterone enanthate for contraception in men.

After a control phase, 10 normal men received cyproterone acetate (CPA) at a dose of 25 mg/day (CPA-25; n=5) or 12.5 mg/day (CPA-12.5; n=5) plus testosterone enanthate (TE) 100 mg/week, for 16 weeks. Throughout the study sperm counts were performed every 2 weeks, and luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, biochemical and haematological tests were performed every 4 weeks. All five men in group CPA-25 and three men in group CPA-12.5 achieved azoospermia. One man in group CPA-25 was azoospermic by week 12 of hormone administration, but had a sperm count of 0.1 x 10(6)/ml at week 16. Time to azoospermia was 9.0+/-1.3 and 8.7+/-0.7 weeks in groups CPA-25 and CPA-12.5 respectively. Gonadotrophins were decreased by week 4 of hormone administration, remained around the minimum detectability of the assay for the duration of hormone administration and returned to baseline after stopping hormone administration. Testosterone values did not change. No change in any biochemical parameters was found. Haematological parameters were decreased at week 16 of hormone administration and returned to baseline after stopping hormone administration. In conclusion, these results suggest that an hormonal regimen consisting of testosterone plus a progestin with anti-androgenic properties holds promise as an effective, safe and reversible male contraceptive.

Adult↗

PubMed1998

Urinary excretion of d-amphetamine following oral doses in humans: implications for urine drug testing.

Seven healthy male volunteers received a single oral dose of 5 mg, 10 mg, or 20 mg of d-amphetamine. Urine was collected at 2, 4, 8, 12, 18, and 24 h post-dose. Total urine volume was measured, and pH and creatinine were determined. All specimens were analyzed by TDx Amphetamine/Methamphetamine II (TDx), Emit-d.a.u. Monoclonal Amphetamine/Methamphetamine (EM), and Emit II Amphetamine/Methamphetamine (EII) immunoassays at a cutoff value of 1000-ng/mL amphetamine. Quantitation of urinary amphetamine in all specimens was performed by gas chromatography-mass spectrometry. All urine testing results by the three immunoassays, EM, EII, and TDx, were in agreement; there were no discordant findings. Of the 42 total urine specimens collected following a 5-mg dose of amphetamine, only 8 (19%) screened positive by immunoassay. Twenty-four of 36 (67%) urine specimens yielded positive responses following a 10-mg dose, and 37 of 42 (88%) were positive by immunoassay following a 20-mg dose. These data demonstrate the present guideline for regulated forensic urine drug testing (FUDT) for amphetamine with a screening cutoff of 1000 ng/mL is too high to consistently detect the administration of a single 5-mg oral dose of d-amphetamine. There was considerable overlap of amphetamine concentrations in individual specimens following the various doses. Peak urinary amphetamine ranged from 620 to 3160 ng/mL following 5-mg doses. The time to peak concentration also varied widely at each dose, occurring in urines collected 2 to 18 h post-administration. The mean percent of dose excreted as unchanged amphetamine over 24 h at each dose ranged from 35 to 44%. The data demonstrated that amphetamine excretion increases with increasing urine flow and decreasing urine pH. Thus, a positive FUDT result for amphetamine means only that the individual was administered or self-administered amphetamine at some time prior to collection of the specimen.

Administration, Oral↗