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Biomedical subjects

E Ferrari

Publications and source records attributed to E Ferrari.

At least 37 records · Page 2Linked to original sources
PubMed2001

In vivo and in vitro percutaneous absorption of [(14)C]di-N-butylphthalate in rat.

This study evaluated the toxicokinetics of [(14)C]di-n-butylphthalate ([(14)C]DBP) after an intravenous administration (1 and 10 mg/kg, in Cremophor) or a topical application (10 microl/cm(2); 10 cm(2), neat) in haired male Sprague-Dawley rats. Additional in vivo and in vitro percutaneous penetration studies of [(14)C]DBP were conducted on male and female haired rats and male hairless rats. After intravenous administration, unchanged DBP disappeared rapidly from the plasma, following a two-exponential function (T1/2beta = 5-7 min). The peak levels of monobutylphthalate (MBP) and its glucuronide conjugate (MBP-Gluc) occurred 1 to 2 and 20 to 30 min after administration, respectively. These metabolites were intensively and rapidly excreted in urine (57% of the dose). However, about 35% of the dose recovered in urine was primarily excreted in bile (mainly as MBP-Gluc) and underwent hepatobiliary recycling. Unchanged DBP was barely detectable in excreta. DBP rapidly penetrated the skin, which constituted a reservoir. The absorption flux determined for 0.5 to 8 and 8 to 48 h of exposure were 43 and 156 microg/cm(2)/h, respectively. The higher flux may be due to radial diffusion of DBP in the stratum and/or epidermis. The in vivo and in vitro experiments revealed that DBP was intensively metabolized into the skin. In vivo percutaneous absorption flux was very similar in male and female haired rats. In contrast, the percutaneous absorption determined in vivo and in vitro was higher in hairless than in haired male rats. Absorption flux was accurately estimated from urinary excretion rate of MBP or MBP-Gluc.

Animals↗
PubMed2001

Toxicokinetics and metabolism of 1,2-diethylbenzene in male Sprague Dawley rats--part 2: evidence for in vitro and in vivo stereoselectivity of 1,2-diethylbenzene metabolism.

In a previous study, it was shown that the neurotoxic compound 1,2-diethylbenzene (1,2-DEB) is mainly hydroxylated in the alkyl chain to give 1-(2'-ethylphenyl)ethanol (1,2-EPE) and excreted in urine of rats as two glucuronide compounds (GA1 and GA2). Some findings have suggested that the two enantiomers of 1,2-EPE are formed in vivo. In the present study, a chiral high-performance liquid chromatography method was developed to separate the two enantiomers of 1,2-EPE from a synthesized racemic mixture. Absolute configuration of both enantiomers was determined after esterification with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid and analysis of their (1)H NMR spectra in CCl(4) added with Eu (fod)(3). The two main urinary metabolites, GA1 and GA2, from [(14)C]1,2-DEB-treated Sprague-Dawley rats (80 mg/kg, i.p.) were identified, after hydrolysis with beta-glucuronidase from Escherichia coli, as (R) and (S) glucuronide conjugates of 1,2-EPE, respectively. In vitro hydroxylation of 1,2-DEB and glucuroconjugation of 1,2-EPE were under stereoselective control in S9 fraction or microsomes from male Sprague-Dawley rat liver. The V(max) and K(m) constants for (R)1,2-EPE enantiomer formation determined in S9 fraction were greater than those for the (S) enantiomer. In the plasma of bile duct-cannulated rats, the ratio was 1.2 +/- 0.02 over the 1- to 4-h period after oral administration of [(14)C]1,2-DEB (100 mg/kg). In contrast, the glucuroconjugation rate of (S)1,2-DEB enantiomer was 4 times that of (R)1,2-EPE glucuroconjugation. A similar ratio of (R) to (S)1,2-EPE glucuronide conjugates was obtained in the plasma of bile duct-cannulated rats.

Animals↗
PubMed2001

[When is antivitamin K therapy indicated in the stable coronary patient?].

In the cardiac patient, there are clinical situations where antivitamin K is indicated more by the co-existing pathological associations or by a particular thrombogenic situation than by the cardiac disease itself. The presence of an embologenic abnormal rhythm, an apical thrombus or a large anterior akinesis are recognised as situations where antivitamin K must be discussed and, except for absolute contraindication, initiated. The studies undertaken for several decades are highly instructive and their contributions are considerable in the different questions which could be asked regarding the efficacy of antivitamin K. In particular they have the merit of signalling the correct directions to take and the errors to avoid. Concerning the evolution of cardiac disease, it must be admitted that the very good results of antivitamin K treatment alone at high dose are to be balanced against their haemorrhagic risk. The studies testing the association of low-dose aspirin with moderate-dose antivitamin K (INR 2 to 2.5) are to date very promising. The evaluation of the understanding of the treatment by patient education remains a major stage when initiating antivitamin K treatment in the cardiac patient.

4-Hydroxycoumarins↗
PubMed2001

[Duration of antivitamin K therapy in venous thromboembolic disease. Certainties and uncertainties].

The necessity of anticoagulant treatment after a pulmonary embolus or a deep venous thrombosis has been demonstrated. The modalities of this treatment have been well established, especially the usefulness of initial heparin therapy followed by a period of antivitamin K treatment with an "ideal target INR" between 2 and 3. One of the last questions in this therapeutic protocol is the duration of antivitamin K treatment. The choice of duration of treatment must be made on numerous criteria. It is necessary to distinguish the circumstances of the occurrence of the DVT or the PE and the context. It is thus possible for less than 3 months treatment in secondary venous thrombo-embolic disease, which occurs in precise, recognised circumstances for which the cause will have been controlled. Otherwise, in so-called idiopathic venous thrombo-embolic disease, which is distinguished by a higher prevalence of recurrences, it is known that long-term antivitamin K treatment is effective for the thrombo-embolic recurrences but at the price of a risk of haemorrhage. Finally thrombo-embolic recurrences also benefit from a long treatment. These circumstances of occurrence are thus important in order to decide the choice of treatment duration. But, in our opinion, the compliance to antivitamin K treatment remains the primary criterion to consider. The dilemma facing the prescriber is to evaluate the risk-benefit ratio of each patient, asking especially if the antivitamin K treatment surveillance of a particular patient will be done as well as in the randomised studies. For the future, long-term antivitamin K does not perhaps represent the only therapeutic option. The results of studies evaluating the durations of long-term treatment with less intense levels of anticoagulation (INR < 2) as well as therapeutic alternatives to antivitamin K (antiaggregants or other antithrombins) are awaited.

4-Hydroxycoumarins↗
PubMed2001

[Venous thromboembolism and cancer].

Malignant disease predisposes to deep venous thrombosis (DVT) or pulmonary embolism (PE) in several ways. One classical situation is that of DVT or PE with no apparent cause which may be the first sign of an occult cancer. In this domain, although the epidemiological data is well known, it is important to recognise the limitations of "blind" investigations. Another situation is more common. The patients have a diagnosed malignancy and thromboembolic disease is the main extra-cancer complication. The approach to this problem is changing, both in primary prevention where many trials have already reported encouraging results, and in the treatment after the event where classical therapeutic protocols are not always well adapted. Ancestral fears of the prescription of anticoagulants in cancer patients must cede to a more objective benefit/risk analysis which seems to be very favourable in some situations. Moreover, some publications have demonstrated a chance finding of a possible anticancer effect of antithrombotic agents.

Anticoagulants↗
PubMed2000

The PCNA from Thermococcus fumicolans functionally interacts with DNA polymerase delta.

We have cloned the gene encoding proliferating cell nuclear antigen (PCNA) from the hyperthermophilic euryarchaeote Thermococcus fumicolans (Tfu). Tfu PCNA contains 250 amino acids with a calculated M(r) of 28,000 and is 26% identical to human PCNA. Next, Tfu PCNA was overexpressed in Escherichia coli and it showed an apparent molecular mass of 33.5 kDa. The purified Tfu PCNA was tested first with recombinant Tfu DNA polymerase I (Tfu pol) and second with calf thymus DNA polymerase delta (pol delta). When tested with the homologous Tfu pol on bacteriophage lambda DNA, large amounts of Tfu PCNA were required to obtain two- to threefold stimulation. Surprisingly, however, Tfu PCNA was much more efficient than human PCNA in stimulating calf thymus pol delta. Our data suggest that PCNA has been functionally conserved not only within eukaryotes but also from hyperthermophilic euryarchaeotes to mammals.

Amino Acid Sequence↗
PubMed2000

Paternal concentrations of dioxin and sex ratio of offspring.

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin), is commonly considered the most toxic man-made substance. We have previously shown that high serum concentrations of TCDD in parents from Seveso, Italy, were linked to their having a relative increase in the number of female births after the parents exposure to a release of dioxin in 1976. We have continued the study to determine whether the parents' sex and/or age at exposure affected the sex ratio of their children. METHODS: We measured the TCDD concentrations in serum samples from potentially exposed parents collected in 1976 and 1977, and investigated the sex ratio of their offspring. FINDINGS: Serum samples were collected from 239 men and 296 women. 346 girls and 328 boys were born to potentially exposed parents between 1977 and 1996, showing an increased probability of female births (lower sex ratio) with increasing TCDD concentrations in the serum samples from the fathers (p=0.008). This effect starts at concentrations less than 20 ng per kg bodyweight. Fathers exposed when they were younger than 19 years of age sired significantly more girls than boys (sex ratio 0.38 [95% CI 0.30-0.47]). INTERPRETATION: Exposure of men to TCDD is linked to a lowered male/female sex ratio in their offspring, which may persist for years after exposure. The median concentration of dioxin in fathers in this study is similar to doses that induce epididymal impairments in rats and is about 20 times the estimated average concentration of TCDD currently found in human beings in industrialised countries. These observations could have important public-health implications.

Adolescent↗
PubMed2000

DNA polymerase switching: II. Replication factor C abrogates primer synthesis by DNA polymerase alpha at a critical length.

A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA primer by DNA polymerase alpha/primase to the pro?cessive elongation by DNA polymerase delta. In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA polymerase alpha were investigated. We show that RF-C stalls DNA polymerase alpha after synthesis of approximately 30 nucleotides, while not inhibiting the polymerase activity per se. This suggested that RF-C and the length of the primer may be two important factors contributing to the polymerase switch. Furthermore the DNA binding properties of RF-C were tested. Band shift experiments indicated that RF-C has a preference for 5' recessed ends and double-stranded DNA over 3' ends. Finally PCNA can be loaded onto a DNA template carrying a RNA primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp. Thus we propose a model where RF-C, upon binding to the RNA/DNA primer, influences primer synthesis and sets the conditions for a polymerase switch after recruiting PCNA to DNA.

Base Sequence↗
PubMed2000

Evaluation of the circadian profiles of serum dehydroepiandrosterone (DHEA), cortisol, and cortisol/DHEA molar ratio after a single oral administration of DHEA in elderly subjects.

Aging is associated with a selective decline in circulating levels of dehydroepiandrosterone (DHEA) and its sulfate, with no major changes in cortisol secretion. In young subjects, serum levels of both DHEA and cortisol are regulated according to a circadian rhythm, and an age-related attenuation of DHEA, but not cortisol, circadian rhythmicity has been reported. Several trials have evaluated the effects of DHEA supplementation in elderly subjects, although the results are still controversial. However, no data are available on the 24-hour profile of DHEA circulating levels in elderly subjects with DHEA administration. In the present study, we evaluated the circadian rhythms of DHEA, cortisol, and the cortisol/DHEA molar ratio in old subjects treated with either placebo (old-PL) or a single 50-mg dose of DHEA (old-D), both administered orally at 0700 hours. For each variable, the circadian profiles were compared with those obtained in young control subjects. The group of young subjects displayed a circadian rhythm for both DHEA and cortisol serum concentrations but no rhythm for the cortisol/DHEA molar ratio. In the old-PL group, the circadian rhythm of DHEA was completely abolished, whereas significant rhythms for both cortisol and the cortisol/DHEA molar ratio were observed. Particularly, at each time point, the cortisol/DHEA molar ratio was significantly higher in these subjects versus the young group. In the old-D group, the circadian rhythm of DHEA was completely restored and was comparable to that observed in the young group. Analogous to the observations in young subjects, the profile of the cortisol/DHEA molar ratio in old-D subjects did not display any circadian rhythmicity, the values being almost completely comparable to those observed in young controls. Our data demonstrate that the circadian rhythm of DHEA is totally abolished in elderly subjects. A single 50-mg dose of DHEA administered orally at 0700 hours restores the circadian rhythmicity of serum DHEA and almost completely normalizes the 24-hour profile of the cortisol/DHEA molar ratio in old subjects without affecting the cortisol circadian rhythm.

Administration, Oral↗
PubMed2000

Pineal and pituitary-adrenocortical function in physiological aging and in senile dementia.

The simultaneous evaluation of the circadian rhythm of plasma melatonin and ACTH and of serum cortisol and DHEAS represents a clinically reliable tool to appreciate the neuroendocrine changes occurring in physiological and pathological brain aging.A selective impairment of the nocturnal melatonin secretion has been observed in elderly subjects, being significantly related either to the age or to the severity of dementia. A significant increase of serum cortisol levels during evening- and night-times was found in elderly subjects, particularly if demented, when compared to young controls. Besides, both the circadian amplitude of cortisol rhythm and the nocturnal cortisol increase were significantly reduced in relation either to age or to cognitive impairment. By comparison to vascular dementia, patients with Alzheimer's disease exhibited the highest cortisol concentrations throughout the 24h. The sensitivity of the hypothalamic-pituitary-adrenal axis to the steroid feedback was significantly impaired in old subjects and particularly in the demented ones. The serum DHEAS levels were significantly lower in elderly subjects and even more in demented patients than in young controls. Consequently, a significant increase of the cortisol/DHEAS molar ratio was evident when going from young controls to healthy elderly subjects and to demented patients. In conclusion, the aging process affects many neuroendocrine functions resulting in subtle but clinically relevant consequences; the occurrence of senile dementia seems to play an additive role.

Adrenocorticotropic Hormone↗
PubMed2000

Phase I study with weekly cisplatin-paclitaxel and concurrent radiotherapy in patients with carcinoma of the cervix uteri.

BACKGROUND: Cisplatin and paclitaxel are active in cervical cancer and both are able to potentiate the effects of radiotherapy. In this study we evaluated the maximum-tolerated dose (MTD) of paclitaxel in combination with a fixed dose of cisplatin when given weekly concurrently with pelvic radiotherapy to patients with carcinoma of the cervix uteri. PATIENTS AND METHODS: Eighteen patients with cervical cancer were enrolled in this study. Cisplatin (30 mg/m2) and paclitaxel (starting dose 40 mg/m2; 5 mg/m2 escalation per level) were given on day 1 of radiotherapy and then weekly for six times. Radiotherapy was given to the pelvis with a four-field box technique for five days each week. Patients received 65 Gy in 1.8 Gy fractions. Cohorts of three patients were enrolled at each level and three further patients were included if one or two dose-limiting severe adverse events (SAE) were recorded. SAE was defined as grade 3 or 4 nonhematologic toxicity, excluding nausea or vomiting and alopecia, grade 4 neutropenia or thrombocytopenia, and prolonged (> 1 week) neutropenia or thrombocytopenia. RESULTS: Four levels were studied (paclitaxel 40, 45, 50, 55 mg/m2) with three, five, four and six patients enrolled, respectively. The MTD of paclitaxel was found at 50 mg/m2/wk and cisplatin 30 mg/m2/wk. Diarrhea was the dose-limiting toxicity. Thirteen patients were evaluable for response: seven complete and five partial responses were obtained with an overall response rate of 92.3%. CONCLUSIONS: The MTD of paclitaxel is 50 mg/m2/wk when associated to cisplatin 30 mg/m2/wk and concurrent pelvic radiotherapy. Diarrhea is the dose limiting side effect. Preliminary data suggest that concurrent chemoradiotherapy with paclitaxel and cisplatin could be a very active treatment for patients with locally advanced carcinoma of the cervix.

Adenocarcinoma↗
PubMed2000

RNA-dependent RNA polymerase activity encoded by GB virus-B non-structural protein 5B.

Phylogenetic analysis and polyprotein organization comparison have shown that GB virus-B (GBV-B) is closely related to hepatitis C virus (HCV). In this study, the coding region for GBV-B non-structural protein 5B (NS5B) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) from pooled serum of GBV-B-infected tamarins. Expression of soluble GBV-B NS5B protein in Escherichia coli was achieved by removal of a 19-amino acid hydrophobic domain at the C-terminus of the protein. The truncated GBV-B NS5B (NS5BDeltaCT19) was purified to homogeneity and shown to possess an RNA-dependent RNA polymerase (RdRp) activity in both gel-based and scintillation proximity assays. NS5BDeltaCT19 required the divalent cation Mn2+ for enzymatic activity, at an optimal concentration of 15 mM. Interestingly, Mg2+, at concentrations up to 20 mM, did not support the GBV-B NS5B activity. This differs from HCV NS5B where both Mn2+ and Mg2+ can support RdRp activity. Zn2+ was found to inhibit the activity of GBV-B NS5B, with a 50% inhibitory concentration (IC50) of 5-10 microM. Higher concentrations of monovalent salts (NaCl or KCl > 100 mM) and glycerol (> 3%) were also inhibitory. NS5BDeltaCT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis. Mutational analysis of GBV-B NS5B demonstrated the importance of several conserved sequence motifs for enzymatic activity. Based on sequence homology ( approximately 37% identity and 52% similarity) between GBV-B and HCV NS5B proteins, the active GBV-B RdRp provides a good surrogate assay system for HCV polymerase studies.

Amino Acid Sequence↗
PubMed2000

Overproduction of IFN-gamma and TNF-alpha from natural killer (NK) cells is associated with abnormal NK reactivity and cognitive derangement in Alzheimer's disease.

Alterations of natural killer (NK) function can be involved in the neuroimmune mechanism of neurodegeneration in dementia of the Alzheimer's type (DAT). NK cell cytotoxicity (NKCC) and the generation and release of IFN-gamma and TNF-alpha (spontaneous and modulated by IL-2) from pure NK cells (CD 16+, CD 56+, CD 3-) were studied together with circulating IFN-gamma and TNF-alpha levels and cognitive function in 22 old patients with DAT and 15 healthy old subjects. Higher (p < 0.001) IL-2 modulated NKCC (with IL-2 50 U/mL and 100 U/mL) was demonstrated in DAT patients (+35% and +99% from baseline) than in healthy subjects (+6% and +76% from baseline). Increased spontaneous and IL-2-induced release of IFN-gamma and TNF-alpha from NK cells were found in DAT patients compared to healthy subjects (p < 0.001), whereas no difference of serum IFN-gamma and TNF-alpha was demonstrated between DAT and control groups. Significant negative correlations among the spontaneous release of IFN-gamma and TNF-alpha from NK and the decrease of the score of cognitive function (MMSE) were found in patients with DAT. In conclusion, alterations of NKCC control and NK-derived cytokine release in DAT could be involved in the neuroinflammatory mechanism related to the progression of neurodegeneration and dementia.

Aged↗
PubMed2000

Variability of interactions between neuroendocrine and immunological functions in physiological aging and dementia of the Alzheimer's type.

A link between neuroendocrine and immunological changes has been suggested in the pathophysiology of dementia of the Alzheimer's type (DAT). Healthy young and old subjects and patients with DAT were recruited to evaluate the chrononeuroendocrine organization of cortisol, GH, and melatonin (MLT) secretions. The study was carried out together with the evaluation of natural killer (NK) cell function: cytotoxic activity (NKCC) and TNF-alpha and IFN-gamma release after exposure to IL-2 (100 U/mL). Moreover, a cerebral morphometric analysis of hippocampus and temporal lobe (MRI) was performed. The activation of hypothalamo-pituitary-adrenal (HPA) axis and the decrease of GH, and MLT nocturnal peaks were associated with normal NKCC and TNF-alpha/IFN-gamma in healthy elderly subjects, whereas in DAT patients the same neuroendocrine changes occurred together with abnormal NKCC (spontaneous and IL-2/IFN-beta-modulated) and with alterations of TNF-alpha/INF-gamma generation from NK. Moreover significant correlations among the increase of NKCC and TNF-alpha and the decrease of cognitive function were found in the DAT group. These correlations were associated with the impairment of nocturnal GH and MLT levels and with the relatively higher serum cortisol concentrations. Moreover, the impairment of cortisol suppression after dexamethasone (1 mg orally at 23:00) was significantly correlated with the increase of spontaneous release of TNF-alpha and with IL-2-modulated NKCC. Finally the imunoneuroendocrine alterations found in DAT were associated with the reduction of cerebral volume in hippocampus and temporal lobes. Taken together these data indicate that the immunoneuroendocrine balance is maintained in physiological aging, whereas NK immune dysregulation in DAT could contribute to altering the neuroendocrine functions and to extend the progression of neurodegeneration and dementia.

Adolescent↗
PubMed2000

Aspirin and sodium salicylate inhibit endothelin ETA receptors by an allosteric type of mechanism.

Aspirin is a commonly used drug with a wide pharmacological spectrum including antiplatelet, anti-inflammatory, and neuroprotective actions. This study shows that aspirin and sodium salicylate, its major blood metabolite, reverse contractile actions of endothelin-1 (ET-1) in isolated rat aorta and human mammary arteries. They also prevent the intracellular Ca(2+) mobilizing action of ET-1 in cultured endothelial cells but not those of neuromedin B or UTP. Inhibition of the actions of ET-1 by salicylates is apparently competitive. Salicylates inhibit (125)I-ET-1 binding to recombinant rat ETA receptors. Salicylic acid promotes dissociation of (125)I-ET-1 ETA receptor complexes both in the absence and the presence of unlabeled ET-1. It has no influence on the rate of association of (125)I-ET-1 to ETA receptors. Salicylates do not promote dissociation of (125)I-ET-1 ETB receptor complexes. Salicylates potentiate relaxing actions of receptor antagonists such as bosentan. It is concluded that salicylates are allosteric inhibitors of ETA receptors. The results also suggest that: 1) irreversible ET-1 binding probably limits actions of receptor antagonists in vivo, and 2) an association of salicylates and ETA receptor antagonists should be used to evaluate the physiopathological role of ET-1 and may be of therapeutic interest in the treatment of ischemic heart disease.

Allosteric Regulation↗
PubMed2000

Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase.

Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3' terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3' terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3' terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique beta-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.

Genome, Viral↗
PubMed2000

De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase.

RNA-dependent RNA polymerase (RdRp) encoded by positive-strand RNA viruses is critical to the replication of viral RNA genome. Like other positive-strand RNA viruses, replication of hepatitis C virus (HCV) RNA is mediated through a negative-strand intermediate, which is generated through copying the positive-strand genomic RNA. Although it has been demonstrated that HCV NS5B alone can direct RNA replication through a copy-back primer at the 3' end, de novo initiation of RNA synthesis is likely to be the mode of RNA replication in infected cells. In this study, we demonstrate that a recombinant HCV NS5B protein has the ability to initiate de novo RNA synthesis in vitro. The NS5B used HCV 3' X-tail RNA (98 nucleotides) as the template to synthesize an RNA product of monomer size, which can be labeled by ¿gamma-(32)Pnucleoside triphosphate. The de novo initiation activity was further confirmed by using small synthetic RNAs ending with dideoxynucleotides at the 3' termini. In addition, HCV NS5B preferred GTP as the initiation nucleotide. The optimal conditions for the de novo initiation activity have been determined. Identification and characterization of the de novo priming or initiation activity by HCV NS5B provides an opportunity to screen for inhibitors that specifically target the initiation step.

Hepacivirus↗