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Biomedical subjects

E Ferrari

Publications and source records attributed to E Ferrari.

At least 91 records · Page 5Linked to original sources
PubMed1999

Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli.

Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

Amino Acid Sequence↗
PubMed1999

A phase I study of paclitaxel and epirubicin, without and with filgrastim, for the treatment of platinum-resistant advanced ovarian cancer.

The aim of the study was to determine the maximum tolerated dose (MTD) of epirubicin combined with a fixed dose of paclitaxel, without and with support of filgrastim, in patients with platinum resistant or refractory ovarian cancer. Paclitaxel (150 mg/m2) and epirubicin (starting dose 90 mg/m2, 15 mg/m2 escalation per level) were given on day 1, every 28 days for 4-6 cycles. Filgrastim (F) (5 microg/kg/die) was given in case of grade 4 leukopenia (levels without support) or from day 4 up to leukocyte count >10,000/mm3 after nadir (levels with support). Cohorts of 3 patients were enrolled at each level and further 3 patients were planned if 1 or 2 unacceptable toxic events (UTE) were registered. MTD was determined first without and then with filgrastim. Four levels were studied (90, 90+F, 105+F, 120+F) with 4, 6, 5 and 4 patients enrolled, respectively. UTE (grade 4 neutropenia) were observed in 3 patients at level 1. Thus, 90 mg/m2 was the MTD for epirubicin without filgrastim. MTD of epirubicin with filgrastim was not reached at 120 mg/m2. Hematological toxicity was mild. Grade 3 mucositis was reported in 1 patient. Among the 14 patients with measurable or evaluable disease, 3 objective responses were observed (1 complete and 2 partial) for an overall response rate of 21.4%. The combination of paclitaxel 150 mg/m2 and epirubicin at 120 mg/m2 with filgrastim is a feasible therapy. Grade 4 leukopenia is the dose limiting toxicity using epirubicin at 90 mg/m2 without filgrastim.

Adult↗
PubMed1999

Phase II study of cisplatin and vinorelbine as first-line chemotherapy in patients with carcinoma of the uterine cervix.

PURPOSE: To evaluate the activity and toxicity of the combination of cisplatin (80 mg/m2 day 1) and vinorelbine (25 mg/m2 days 1 and 8) in patients with carcinoma of the uterine cervix that has not been previously treated with chemotherapy. PATIENTS AND METHODS: Fifty patients with cervical cancer were enrolled onto this study (27 stage IB-III, 23 stage IVB-recurrent). A two-stage optimal Simon design was applied. Thirteen responders of 29 treated patients were required to proceed beyond the first stage, and 28 responders were needed overall. RESULTS: Hematologic toxicity was mild, with neutropenia being the most frequent side effect. Nonhematologic toxicity was frequent but never severe; one patient had grade 3 peripheral neurotoxicity. Objective responses were recorded for 32 patients (64%): 11 patients (22%) achieved a complete response (CR) and 21 patients (42%) achieved a partial response (PR). The response rate was 81.5% in patients with IB-III stage (25.9% CR rate) and 43.5% in patients with IVB-recurrent disease (17.4% CR rate). Responses were seen both in stage IVB patients (one CR and two PRs, for an overall rate of 37.5%) and in patients with recurrent disease (three CRs + four PRs, for an overall rate of 46.7%). CONCLUSION: The combination of cisplatin and vinorelbine is an active regimen in the treatment of patients with early-stage and advanced carcinoma of the uterine cervix. The hematologic and nonhematologic toxicity of this combination is mild.

Adult↗
PubMed1999

Changes of serum allopregnanolone levels in the first 2 years of life and during pubertal development.

Allopregnanolone is the best characterized among neurosteroids, and its role in the control of neuroendocrine axes has attracted increasing interest recently. However, there is no available information about circulating levels of allopregnanolone during infancy, childhood and puberty. We studied two groups of children: 1) those aged between 0 and 2 y (n = 72), and 2) those aged between 6 and 18 y, at different Tanner's stages (n = 82). In each of these patients, serum allopregnanolone, progesterone, cortisol, and dehydroepiandrosterone levels were evaluated after informed consent; allopregnanolone was measured by RIA after acid extraction on cartridge. There was no significant variation of serum allopregnanolone levels either in male and female children during the first 2 y of life. Furthermore, although serum dehydroepiandrosterone levels showed a significant decrease, inversely correlated with age of the children (p < 0.01), serum cortisol and progesterone levels showed a significant age-related increase during the first 2 y of life. Cortisol and allopregnanolone levels were positively correlated (p < 0.01). During puberty, we observed a progressive increase in serum allopregnanolone levels in both boys and in girls, which were higher at Tanner' s stage IV-V (0.7+/-0.01 nM; mean +/- SEM) than at stages I-II (0.32+/-0.02 nM; p < 0.01); mean levels were significantly higher at puberty than in the first 2 y of life (p < 0.01). Furthermore, during puberty, serum progesterone and dehydroepiandrosterone levels also increased progressively with age in both boys and girls. Allopregnanolone and dehydroepiandrosterone levels were positively correlated throughout puberty. The present results indicate that serum allopregnanolone levels do not change during the first 2 y of life but increase during pubertal development, suggesting that this steroid may be involved in the adaptive neuroendocrine mechanisms related to puberty.

Adolescent↗
PubMed1999

Dehydroepiandrosterone sulfate enhances natural killer cell cytotoxicity in humans via locally generated immunoreactive insulin-like growth factor I.

Experimental and clinical investigations suggest the hypothesis that dehydroepiandrosterone sulfate (DHEAS) can positively influence natural killer (NK) immunity via locally produced insulin-like growth factor I (IGF-I) from NK cells. In the present study, the NK cell cytotoxicity (NKCC) and IGF-I levels in the supernatant of NK cells were studied at baseline and after exposure to various molar concentrations of DHEAS (from 10(-5)-10(-8) mol/L x mL/7.75 x 10(6) NK cells) in healthy subjects of young and old age. DHEAS-induced NKCC was also determined after DHEAS coincubation with somatostatin-14 (10-6) mol/L x mL/7.75 x 10(6) NK cells) and with interleukin-2 (IL-2; 100 IU/mL x 7.75 x 10(6) NK cells). NK cells were previously isolated by Ficoll-Hypaque density gradient and then by immunomagnetic procedure; the purity obtained was 97 +/- 1%. NKCC was determined against K562 tumoral targets. We observed that the increase in NKCC after DHEAS exposure was dose dependent and was correlated with the amount of IGF-I released in the supernatant of cultured NK cells. NKCC and IGF-I generation from NK cells were more elevated in healthy elder subjects than in healthy young subjects. The coincubation of DHEAS with somatostatin-14 significantly suppressed NKCC and IGF-I release from NK in both groups, whereas higher NKCC was found after DHEAS plus IL-2 exposure than after incubation with DHEAS alone. Taken together, this study suggests a role for NK-generated IGF-I in the modulation of NKCC by DHEAS in humans. Although DHEAS may contribute to the IL-2-mediated NKCC, its activity on NK cytolytic function can be dependent on a autocrine mechanism (IGF-I-mediated), probably independent of cytokine activation. The higher NKCC response to DHEAS found in old subjects than in younger might counterbalance the age-dependent decline in circulating DHEAS, thus contributing to maintain the pattern of NK immunity during aging.

Adult↗
PubMed1999

Travel as a risk factor for venous thromboembolic disease: a case-control study.

BACKGROUND: The link between travel and the risk of venous thromboembolic disease (VTED) has been widely suspected. However, only cases or series of cases have been reported in the literature. STUDY OBJECTIVES: By means of a case-control study, we sought to confirm this relationship and to determine the main features, if any, of these posttravel VTEDs. DESIGN: The history, in particular the history of recent travel, of 160 patients presenting in our department with VTED was scrupulously investigated. All journeys undertaken during the preceding 4 weeks and lasting > 4 h by whatever means of transport were considered. The same questionnaire was submitted to a control group. RESULTS: When the two groups of patients are compared, a history of recent travel is found almost four times more frequently in the VTED group (p < 0.0001). The odds ratio for having a VTED in patients who traveled was 3.98 (95% confidence interval, 1.9 to 8.4). Means of travel used included the train in 2 cases, airplane in 9, and car in 28. Mean duration of travel was 5.4+/-2.1 h. These posttravel VTEDs are not confined to a specific location, seem to involve no particular predisposition, and are more often "idiopathic." This fact supports the hypothesis that travel alone can produce vein clot formation. CONCLUSIONS: A history of recent travel is a risk factor for VTED. Posttravel venous thrombotic events can occur after short journeys in patients with no other risk factors or concomitant disease

Aged↗
PubMed1999

Toxicokinetics of 1,2-diethylbenzene in male Sprague-Dawley rats-part 1: excretion and metabolism of [(14)C]1,2-diethylbenzene.

The excretion and metabolism of neurotoxic 1,2-diethylbenzene (1, 2-DEB) was studied in male Sprague-Dawley rats after i.v. (1 mg/kg) or oral (1 or 100 mg/kg) administration of 1,2-diethyl[U-(14)C]benzene ([(14)C]1,2-DEB). Whatever the treatment, radioactivity was mainly excreted in urine (65-76% of the dose) and to a lower extent in feces (15-23% of the dose), or via exhaled air (3-5% of the dose). However, experiments with rats fitted with a biliary cannula demonstrated that about 52 to 64% of the administered doses (1 or 100 mg/kg) were initially excreted in bile. Biliary metabolites were extensively reabsorbed from the gut and ultimately excreted in urine after several enterohepatic circulations. Insignificant amounts of unchanged 1,2-DEB were recovered in the different excreta (urine, bile, and feces). As reported previously, presence of 1-(2'-ethylphenyl)ethanol (EPE) was confirmed in urine and demonstrated in bile and feces. The two main [(14)C]1,2-DEB metabolites accounted for 57 to 79% of urinary and biliary radioactivity, respectively. Beta-Glucuronidase hydrolysis and electron impact mass spectra results strongly supported their glucuronide structure. Additionally, these two main metabolites were thought to be the glucuronide conjugates of the two potential enantiomers of EPE. The results indicate that the main initial conversion step of the primary metabolic pathway of 1,2-DEB appears to be the hydroxylation of the alpha-carbon atom of the side chain. The presence of two glucuronide conjugates of EPE in the urine in a ratio different from one suggests that the metabolic conversion of 1, 2-DEB is under stereochemical control.

Administration, Oral↗
PubMed1998

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1.

The p21Cdn1 protein (cip1/waf1/sdi1) plays an important role as an inhibitor of mammalian cell proliferation in response to DNA damage. By interacting with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues, even though this pathway is PCNA-dependent. We investigated features of p21-resistant NER using human cell extracts. A direct end-labelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, filling of the approximately 30 nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap filling was uncoupled from incision. Binding p21 to PCNA could also inhibit filling of model 30 nt gaps by both purified DNA polymerases delta and epsilon. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved with time. This incubation gives p21 the opportunity to associate with other targets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap filling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an efficient inhibitor of NER synthesis in cell extracts.

Cell Extracts↗
PubMed1998

Usefulness of endocoronary electrocardiogram in the detection of myocardial viability and comparison with single-photon emission computed tomography Tl-201.

In 20 patients who had recently had an acute myocardial infarction, we compared endocoronary electrocardiographic modifications recorded during angioplasty with thallium-201 for the detection of myocardial viability. Our data demonstrate that endocoronary electrocardiography can be an easy and reliable tool to assess viability, with sensitivity, specificity, and positive and negative predictive values of 100%, 80%, 94%, 100%, respectively.

Aged↗
PubMed1998

Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.

We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

3T3 Cells↗
PubMed1998

Involvement of hormonal circadian secretion in the growth of HIV-infected children.

OBJECTIVE: To evaluate the circadian secretion of hormones involved in the regulation of growth in childhood, namely growth hormone, insulin-like growth factor (IGF)-I, cortisol, adrenocorticotropin hormone (ACTH), and thyroid-stimulating hormone (TSH) in HIV-infected children. DESIGN: The circadian secretory pattern of growth hormone, IGF-I, cortisol, ACTH and TSH was evaluated in 14 HIV-infected children; 13 healthy age- and sex-matched children were chosen as controls. METHODS: Sampling was performed every 4 h from 0400 h to 2000 h and every 2 h from 2000 h to 0400 h. Rhythmometric data were analysed by single and population mean cosinor methods and by analysis of variance. RESULTS: A statistically significant circadian rhythm for growth hormone, IGF-I and cortisol was detectable in HIV-seropositive children, but the mean basal IGF-I levels were below the normal range for age in 12 patients. A statistically significant circadian rhythm was not detectable for ACTH or TSH. CONCLUSION: These results show that there is a loss of the physiological regulation of growth hormone-IGF-I axis and a modification of 24 h TSH profile in our HIV-infected children. These abnormalities might be involved in the altered growth mechanism leading to the failure to thrive that is a peculiar feature of HIV-infected children.

Adolescent↗
PubMed1998

DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by these two essential DNA polymerases. DNA ligase I inhibited replication factor C-independent DNA synthesis by polymerase delta. Our results suggest that the inhibition may be due to DNA ligase I interaction with proliferating cell nuclear antigen (PCNA) and not to a direct interaction with the DNA polymerase delta itself. Strand displacement activity by DNA polymerase delta was also affected by DNA ligase I. The DNA polymerase delta holoenzyme (composed of DNA polymerase delta, PCNA, and replication factor C) was inhibited in the same way as the DNA polymerase delta core, strengthening the hypothesis of a PCNA interaction. Contrary to DNA polymerase delta, DNA synthesis by DNA polymerase epsilon was stimulated by DNA ligase I in a PCNA-dependent manner. We conclude that DNA ligase I displays different influences on the two multipotent DNA polymerases delta and epsilon through PCNA. This might be of importance in the selective involvement in DNA transactions such as DNA replication and various mechanisms of DNA repair.

DNA↗
PubMed1998

Central motor conduction after magnetic stimulation in diabetes.

Central motor conduction times (CMCTs), obtained by means of magnetic stimulation of the motor cortex and spinal roots, were studied in 138 patients affected by diabetes mellitus but with no signs or symptoms of central nervous system (CNS) involvement. CMCTs were significantly increased in diabetic patients (p < 0.001, t-test) with respect to normal controls, with values exceeding upper confidence limits (mean +/- 2.5 SD of controls) in about 30% of patients. There was no correlation between CMCT delay and type of diabetes (insulin-dependent or non-insulin-dependent), patient age, disease duration, degree of metabolic control compensation, presence or absence of retinopathy or nephropathy, and presence or absence of peripheral or autonomic neuropathy. CNS involvement in diabetes mellitus is discussed. Particular emphasis is given to the sensitivity and reliability of CMCTs obtained by means of magnetic stimulation as a tool in the early diagnosis of CNS functional alterations in diabetes mellitus.

Adult↗
PubMed1998

Evidence for receptor bound endothelin in renal but not in cardiac tissues from normal rats.

OBJECTIVES: The stoichiometric binding model (Frelin C, Guedin D. Cardiovasc Res 1994;28:1613-1622) implies that most endothelin in tissues is bound onto receptors rather than in a free state. The objective of this study was to assay receptor bound endothelins in normal rat tissues. METHODS: We first defined acidic conditions that promoted a mild and reversible denaturation of ETA and ETB receptors and that allowed dissociation of bound [125I]endothelin-1. The action of an acid wash on [125I]endothelin-1 binding to cell or tissue homogenates was then investigated. RESULTS: Acid washing of homogenates prepared from rat brain capillary endothelial cells that express prepro endothelin-1 mRNAs unmasked receptor sites. Acid washing of cardiac, lung, brain or liver homogenates did not increase [125I]endothelin-1 binding. An acid wash of kidney homogenates increased 2.2 fold [125I]endothelin-1 binding. Experiments using BQ-123 further indicated that the acid treatment of renal homogenates mainly unmasked ETA receptors. Masked renal ET receptors were mainly localized in the medulla. Treatment of rats with phosphoramidon decreased the density of masked ET receptors in kidney homogenates. CONCLUSION: As much as 50% of endothelin receptors in renal tissues are masked by endogenous endothelins. Most cardiac receptors are free of bound endothelins. These suggest that endothelins act as local rather than systemic mediators.

Animals↗