Chronopharmacological study of furosemide; (V). Influence of pretreatment with 6-hydroxydopamine

Our previous indirect evidences suggested that the adrenergic nervous system is involved in the mechanisms responsible for the time-dependent changes in the effects of furosemide in Wistar rats. In the present study, the role of this system was examined more directly by means of 6-hydroxydopamine-induced sympathectomy. Thirty mg/kg of 6-hydroxydopamine hydrobromide (6-OH-DA) (n = 9) or its vehicle alone (n = 9) was injected intra-arterially (i.a.) twice in Wistar rats. Furosemide (5 mg/kg) was administered i.a. at 1000 hrs (03HALO*) or at 2200 hrs (15HALO). Urine was collected for 60 min after the drug and urinary excretion of sodium and furosemide were determined respectively. Urine volume and urinary excretion of sodium and furosemide were significantly greater at 1000 hrs (03HALO) than at 2200 hrs (15HALO) in the vehicle-injected rats as observed in the previous study. However these administration-time-dependent changes in the effects of furosemide disappeared in the rats with 6-OH-DA. Thus, the present study provides more direct evidence and supports our original hypothesis concerning the mechanisms of this chronopharmacological phenomenon of the agent. Since 6-OH-DA does not penetrate the central nervous system from the blood stream, the present data also indicate that the peripheral adrenergic system is involved in this event.     

Chronopharmacological study of furosemide; (VI). Influence of prolonged exposure to continuous light

We have previously reported that a time-dependent variability is observed in the diuretic effects of furosemide in rats. The present study was undertaken to examine the influence of prolonged exposure to continuous light on chronopharmacological profiles of furosemide in Wistar rats. In study I, rats were maintained for more than 2 weeks under conditions of light (0700-1900 hrs) and dark (1900-0700 hrs) (L-D). Furosemide (30 mg/kg) was orally given at 1200 hrs or at 2400 hrs. Urine was collected for 8 hours after the drug and urinary excretion of sodium and furosemide were determined respectively. Thereafter, these rats were exposed to continuous light (L-L) for the next 4 weeks, and were again maintained under the L-D cycle. The identical trial of study I was repeated at the end of the L-L (study II) and the second L-D (study III) conditions. Urine volume and urinary excretion of sodium and furosemide following the drug were significantly greater at 1200 hrs than at 2400 hrs under conditions of L-D (study I and III). However these administration time-dependent changes in the effects of furosemide and its urinary amount disappeared with L-L condition (study II). These findings indicate that the mode of the time-dependent changes in the effects of furosemide is altered by prolonged exposure to continuous light.     

Preventive effect of platelet-activating factor antagonist, Y-24180, against cyclosporine-induced acute nephrotoxicity

To evaluate the effect of Y-24180, a potent and long-acting antagonist to platelet-activating factor (PAF) receptors, on cyclosporine A (CsA)-induced acute renal failure, the influence of its pretreatment on the CsA-induced alterations in renal hemodynamics was examined in male Wistar rats under anesthesia. CsA decreased the clearances of inulin and p-aminohippuric acid (PAH) in a dose-dependent manner. Y-24180 (3 mg/kg, i.v.) tended to attenuate the CsA-induced reduction in inulin clearance. Y-24180 (0.3 and 3 mg/kg) significantly prevented the reductions in PAH clearance and the increase in calculated renal vascular resistance (RVR) in a dose-dependent manner. Serum endothelin-1 (Et) concentration in the CsA-treated group was higher than that in the vehicle-treated group. Y-24180 did not influence such the elevated Et concentration. Serum thromboxane B2 (TxB2) concentration did not increase by treatment with CsA. A significant correlation was observed between RVR and Et, but not TxB2 concentration. The present study showed that a PAF receptor antagonist, Y-24180, has the preventive effect against CsA-induced acute renal failure, which indicates that PAF may partly be involved in the mechanism of renal vasoconstriction induced by CsA. The present findings also support the idea that Et contributes to the CsA-induced acute nephrotoxicity.     

Purification and Characterization of Lactobacillus bulgaricus Diaphorase

γ-Isomer of 1,2,3,4,5,6-hexachlorocyclohexane (BHC) showed greater decomposition on γ or UV irradiation of five isomers of BHC in crystalline state or in 2-propanol solution. The α- and δ-isomer of BHC and known la, 2a, 3e, 4e, 5e-pentachlorocyclohexane were separated from the irradiation product of crystalline γ-BHC. Four compounds were isolated from the irradiation product of γ-BHC in 2-propanol. Two compounds were tetrachloro-cyclohexenes (C₆H₆C1₄): γ-isomer (mp 86 ~87°C) and ?-isomer (mp 99 ~ 100°C). The other two were isomers of pentachlorocyclohexane (C₆H₇C1₅). One of them (mp 78 ~ 8.5°C) was consistent with known meso-1e,2a,3a,4a,5e isomer. The molecular structure of the other (mp 75°C) established by X-ray crystal structure analysis was 1α, 2α, 3α, 4β, 5α configuration or le 2a 3e 4e 5e conformation of CI atoms. A reaction mechanism was proposed that included a radical chain reaction and chlorine atom migration.     

Change in distribution of the vascular plant Sasa palmata in Sarobetsu Mire between 1977 and 2003

In recent decades, the extent of Sasa palmata-dominant communities has increased in Sarobetsu Mire in northern Hokkaido, Japan, replacing the original Sphagnum bog vegetation. However, this marked increase in distribution of Sasa in the mire has not been formally documented or investigated in detail. Using aerial photo-interpretation, the present study updated the distribution maps of Sasa communities, showing the changes that have occurred to these communities between 1977 and 2003. The results revealed that the extent of Sasa communities has increased by 15.8 % from 6.60 km² in 1977 to 7.64 km² in 2003. The most marked increase occurred on the ground associated with drainage channels, although the oldest channels were constructed more than half a century ago, suggesting that some responses to the drainage of peat bog ecosystems may take a considerable period of time before becoming particularly evident.     

A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.     

Mapping QTLs influencing rice floral morphology using recombinant inbred lines derived from a cross between <Emphasis Type="Italic">Oryza sativa</Emphasis> L. and <Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff

To understand the genetic basis of floral traits associated with the mating system in rice, we analyzed pistil, stamen and glume traits using a recombinant inbred line population, derived from a cross between an Asian cultivated rice ( Oryza sativa L.), Pei-kuh, and a wild rice ( Oryza rufipogon Griff.), W1944. Quantitative trait loci (QTLs) affecting floral morphology were detected by composite interval mapping using a linkage map constructed using 147 markers, mostly RFLPs. A total of 7, 4, 14 and 6 QTLs were detected for traits related to pistil, stamen, and size and shape of the glume, respectively. Comparison of 31 QTLs affecting these organs revealed ten QTLs affecting the different organs in four adjacent regions on chromosomes 2, 4, 5 and 10, but most QTLs (68%) were located separately on the whole chromosomes. Although four QTLs for stigma breadth, anther length and thickness of lemma and palea explained more than 25% of the total phenotypic variance, most QTLs (87%) had smaller effects. These results suggest that quantitative variation observed for pistil, stamen and glume traits is controlled by several distinct genes with small effects.     

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

Background

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.

Methodology/Principal Findings

Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.

Conclusions/Significance

We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.     

Comprehensive proteomics analysis of autophagy-deficient mouse liver

Autophagy is a bulk protein degradation system for the entire organelles and cytoplasmic proteins. Previously, we have shown the liver dysfunction by autophagy deficiency. To examine the pathological effect of autophagy deficiency, we examined protein composition and their levels in autophagy-deficient liver by the proteomic analysis. While impaired autophagy led to an increase in total protein mass, the protein composition was largely unchanged, consistent with non-selective proteins/organelles degradation of autophagy. However, a series of oxidative stress-inducible proteins, including glutathione S-transferase families, protein disulfide isomerase and glucose-regulated proteins were specifically increased in autophagy-deficient liver, probably due to enhanced gene expression, which is induced by accumulation of Nrf2 in the nuclei of mutant hepatocytes. Our results suggest that autophagy deficiency causes oxidative stress, and such stress might be the main cause of liver injury in autophagy-deficient liver.     

Diverse effects of cyclosporine on hepatitis C virus strain replication

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.