Chronopharmacological study of interferon-a in mice

ABSTRACT The influence of dosing time on the pharmacological effects (fever and antiviral activity) and the pharmacokinetics of interferon-␣ (IFN-␣) was investigated in ICR male mice under lightdark (12:12) cycle. There was a significant circadian rhythm in rectal temperature, as an index of fever, at 0.5 hr after IFN-␣ (10.0 MIU/kg i.v.) injection. The rhythmic pattern resembled overall the rhythm that occurs in the nondrugged state. However, the percent change from basal level of rectal temperature varied according to the dosing time. The rhythmicity corresponded to the dosing time-dependent difference of PGE 2 levels in thalamus after IFN-␣ injection, but it did not correspond to that of plasma IFN-␣ concentrations. A significant dosing time-dependent difference was also demonstrated for 2Ј-5Јoligoadenylate synthetase activities, as an index of antiviral activity, in plasma and liver at 24 hr after IFN-␣ injection. It was related to the rhythmicity in plasma IFN-␣ concentrations that was caused by the rhythmicity in clearance of IFN-␣. The choice of the most appropriate time of day for drug administration may help to achieve rational chronotherapeutics of IFN-␣ in certain experimental and clinical situations. A large number of physiological rhythmic variables are demonstrated in the CNS, in hormone secretion and so on Interferons, which belong to a group of cytokines, have been widely used as antiviral and antitumor agents in the human. However, interferons cause unavoidable adverse effects such as fever, fatigue, headache, rigors and myalgias. In particular, fever is an indispensable side effect in nearly all patients during the early phase of interferon treatment. Administration of IFN-␣ in cancer patients is better tolerated in evening than in morning Rectal temperature and immune functions show significant circadian rhythms in mammals under both nondrugged and drugged conditions The purpose of this study was to examine the diurnal change of IFN-␣ induced fever and antiviral activity in mice. The mechanisms underlying these phenomena were also investigated from the perspective of IFN-␣ pharmacokinetics. Methods Animals and treatments. Male ICR mice (5 weeks old) were purchased from Charles River Japan Inc. (Kanagawa, Japan). Mice were housed 6 or 10 per cage in a light-controlled room (light on from 07:00 to 19:00) at a room temperature of 24°C Ϯ 1°C and a humidity of 60% Ϯ 10% with food and water ad libitum. All mice were adapted to their light-dark cycle for 2 weeks before the experiments. In order to study the fever induced by IFN-␣ (Sumiferon, Sumitomo Seiyaku Co., Osaka, Japan), groups of six mice injected i.v. with 1.0, 5.0 or 10.0 MIU/kg IFN-␣ or sterilized saline at the same circadian phase (09:00). IFN-␣ was diluted by sterilized saline to adjust the concentration to 0.2, 1.0 and 2.0 MIU/ml. The volume of injection was 0.05 ml/10.0 g b.wt. The drug solutions were used within 30 min after preparation in order not to decrease their biologic activity. Rectal temperature was continuously determined before, and at 0.5, 1.0, 2.0 and 4.0 hr after, IFN-␣ or saline injection. In the study of the circadian rhythms of IFN-␣-induced fever and plasma IFN-␣ concentrations, groups of 8 to 10 mice were injected i.v. with 10.0 MIU/kg IFN-␣ or saline at one of six times: 09:00, 13:00, 17:00, 21:00, 01:00 or 05:00. Rectal temperature was determined before, and at 0.5, 1.0, 1.5 and 2.0 hr after, IFN-␣ or saline injection. Percent change of rectal temperature (%) from basal level was calculated as follows: % ϭ ([rectal temperature after IFN-␣ injection Ϫ rectal IFN-␣ before Received for publication December 27, 1996. ABBREVIATIONS: IFN-␣, interferon-␣; 2Ј-5ЈOAS, 2Ј-5Јoligoadenylate synthetase; CL, clearance; V c , central volume of distribution; K 12 , distribution rate constant from central to peripheral compartment; K 21 , distribution rate constant from peripheral to central compartment

Molecular basis for the dosing time-dependency of anti-allodynic effects of gabapentin in a mouse model of neuropathic pain

<p>Abstract</p> <p>Background</p> <p>Neuropathic pain is characterized by hypersensitivity to innocuous stimuli (tactile allodynia) that is nearly always resistant to NSAIDs or even opioids. Gabapentin, a GABA analogue, was originally developed to treat epilepsy. Accumulating clinical evidence supports the effectiveness of this drug for diverse neuropathic pain. In this study, we showed that the anti-allodynic effect of gabapentin was changed by the circadian oscillation in the expression of its target molecule, the calcium channel α2δ-1 subunit.</p> <p>Results</p> <p>Mice were underwent partial sciatic nerve ligation (PSL) to create a model of neuropathic pain. The paw withdrawal threshold (PWT) in PSL mice significantly decreased and fluctuated with a period length about 24 h. The PWT in PSL mice was dose-dependently increased by intraperitoneal injection of gabapentin, but the anti-allodynic effects varied according to its dosing time. The protein levels of α2δ-1 subunit were up-regulated in the DRG of PSL mice, but the protein levels oscillated in a circadian time-dependent manner. The time-dependent oscillation of α2δ-1 subunit protein correlated with fluctuations in the maximal binding capacity of gabapentin. The anti-allodynic effect of gabapentin was attenuated at the times of the day when α2δ-1 subunit protein was abundant.</p> <p>Conclusions</p> <p>These findings suggest that the dosing time-dependent difference in the anti-allodynic effects of gabapentin is attributable to the circadian oscillation of α2δ-1 subunit expression in the DRG and indicate that the optimizing its dosing schedule helps to achieve rational pharmacotherapy for neuropathic pain.</p

MDM2 is a novel E3 ligase for HIV-1 Vif

The human immunodeficiency virus type 1 (HIV-1) Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G). Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3) complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug

Time-Dependent Interaction between Differentiated Embryo Chondrocyte-2 and CCAAT/Enhancer-Binding Protein ␣ Underlies the Circadian Expression of CYP2D6 in Serum- Shocked HepG2 Cells

ABSTRACT Differentiated embryo chondrocyte-2 (DEC2), also known as bHLHE41 or Sharp1, is a pleiotropic transcription repressor that controls the expression of genes involved in cellular differentiation, hypoxia responses, apoptosis, and circadian rhythm regulation. Although a previous study demonstrated that DEC2 participates in the circadian control of hepatic metabolism by regulating the expression of cytochrome P450, the molecular mechanism is not fully understood. We reported previously that brief exposure of HepG2 cells to 50% serum resulted in 24-h oscillation in the expression of CYP3A4 as well as circadian clock genes. In this study, we found that the expression of CYP2D6, a major drug-metabolizing enzyme in humans, also exhibited a significant oscillation in serum-shocked HepG2 cells. DEC2 interacted with CCAAT/enhancer-binding protein (C/EBP␣), accompanied by formation of a complex with histone deacetylase-1, which suppressed the transcriptional activity of C/EBP␣ to induce the expression of CYP2D6. The oscillation in the protein levels of DEC2 in serum-shocked HepG2 cells was nearly antiphase to that in the mRNA levels of CYP2D6. Transfection of cells with small interfering RNA against DEC2 decreased the amplitude of CYP2D6 mRNA oscillation in serumshocked cells. These results suggest that DEC2 periodically represses the promoter activity of CYP2D6, resulting in its circadian expression in serum-shocked cells. DEC2 seems to constitute a molecular link through which output components from the circadian clock are associated with the time-dependent expression of hepatic drug-metabolizing enzyme

The habu genome reveals accelerated evolution of venom protein genes

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition

Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

publication en ligne. Article dans revue scientifique avec comité de lecture. nationale.National audienceThe human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology