Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.     

Examination of the method for measuring soil respiration in cultivated land: Effect of carbon dioxide concentration on soil respiration

An acceleration of soil respiration with decreasing CO₂ concentration was suggested in the field measurements. The result supporrs that obtained in laboratory experiments in our previous study. The CO₂ concentrations in a chamber of the alkali absorption method (the AA-method) were about 150–250 parts/10⁶ lower than that in the atmosphere (about 350 parts/10⁶), while those observed in the open-flow IRGA method (the OF-method) were nearly equal to the soil surface CO₂ levels. The AA-method at such low CO₂ levels in the chamber appears to overestimate the soil respiration. Our results showed that the rates obtained by the AA-method were about twice as large as those by the OF-method in field and laboratory measurements. This finding has important consequences with respect to the validity of the existing data obtained by the AA-method and the estimation of changes in the terrestrial carbon flow with elevated CO₂     

Mass propagation of ornamental gentian in liquid medium

An efficient system for clonal mass propagation in liquid culture was established for the propagation of ornamental gentian. In a test of the requirements for three cytokinins [6-benzylaminopurine, N-(2-chloro-4-pyridyl)-N′-phenylurea and N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ)] in combination with 1-naphthaleneacetic acid (NAA), we found that effective propagation of shoots occurred with 0.01 mg l^–1 TDZ in a 300 ml conical flask that contained 100 ml of medium. The propagation of shoots was also affected by the concentrations of macronutrients (KNO₃, NH₄NO₃ and CaCl₂) and sucrose in Murashige and Skoog's (MS) medium, and it was influenced to some extent by the speed of agitation on an orbital shaker. The most efficient propagation of shoots was achieved in full-strength MS medium supplemented with 0.01 mg l^–1 TDZ and 20 g l^–1 sucrose with agitation at 150 rpm. The propagation of shoots was maximal after 6 weeks of culture (140 shoots from five nodal segments in one flask). Large-scale propagation in a 5-l fermenter was attempted using 3 l of MS medium that contained 0.01 mg l^–1 TDZ and 20 g l^–1 sucrose. More than 2,000 shoots were obtained in the fermenter in 5 weeks following the initial cultivation of five nodal segments for 6 weeks in one 300-ml flask. The shoots that had propagated in the fermenter were transferred directly to soil without prior rooting in vitro and were easily acclimatized within 1 month. Received: 7 October 1997 / Revision received: 16 January 1998 / Accepted: 30 January 1998     

cDNA cloning and biochemical characterization of S-adenosyl-L-methionine: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase,a critical enzyme of the legume isoflavonoid phytoalexin pathway

Formononetin (7-hydroxy-4'-methoxyisoflavone, also known as 4'-O-methyldaidzein) is an essential intermediate of ecophysiologically active leguminous isoflavonoids. The biosynthetic pathway to produce 4'-methoxyl of formononetin has been unknown because the methyl transfer from S-adenosyl-L-methionine (SAM) to 4'-hydroxyl of daidzein has never been detected in any plants. A hypothesis that SAM: daidzein 7-O-methyltransferase (D7OMT), an enzyme with a different regiospecificity, is involved in formononetin biosynthesis through its intracellular compartmentation with other enzymes recently prevails, but no direct evidence has been presented. We proposed a new scheme of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor and subsequent dehydration. We now cloned a cDNA encoding SAM: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) through the screening of functionally expressed Glycyrrhiza echinata (Fabaceae) cDNAs. The reaction product, 2,7-dihydroxy-4'-methoxyisoflavanone, was unambiguously identified. Recombinant G. echinata D7OMT did not show HI4'OMT activity, and G. echinata HI4'OMT protein free from D7OMT was partially purified. HI4'OMT is thus concluded to be distinct from D7OMT, and their distant phylogenetic relationship was further presented. HI4'OMT may be functionally identical to (+)-6a-hydroxymaackiain 3-OMT of pea. Homologous cDNAs were found in several legumes, and the catalytic function of the Lotus japonicus HI4'OMT was verified, indicating that HI4'OMT is the enzyme of formononetin biosynthesis in general legumes.     

Radical production and DNA damage induced by carcinogenic 4-hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus

4-Hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus, is carcinogenic to rodents. To clarify the mechanism of carcinogenesis, we investigated DNA damage by 4-hydrazinobenzoic acid using ³²P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes. 4-Hydrazinobenzoic acid induced Cu(II)-dependent DNA damage especially piperidine-labile formation at thymine and cytosine residues. Typical hydroxyl radical scavengers showed no inhibitory effects on Cu(II)-mediated DNA damage by 4-hydrazinobenzoic acid. Bathocuproine and catalase inhibited the DNA damage, indicating the participation of Cu(I) and H₂O₂ in the DNA damage. These findings suggest that H₂O₂ generated by the autoxidation of 4-hydrazinobenzoic acid reacts with Cu(I) to form reactive oxygen species, capable of causing DNA damage. Interestingly, catalase did not completely inhibit DNA damage caused by a high concentration of 4-hydrazinobenzoic acid (over 50 μM) in the presence of Cu(II). 4-Hydrazinobenzoic acid induced piperidine-labile sites frequently at adenine and guanine residues in the presence of catalase. 4-Hydrazinobenzoic acid increased formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in calf thymus DNA, whereas 4-hydrazinobenzoic acid did not increase the formation of 8-oxodG in the presence of catalase. ESR spin-trapping experiments showed that the phenyl radical was formed during the reaction of 4-hydrazinobenzoic acid in the presence of Cu(II) and catalase. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF/mass) spectrometry analysis showed that phenyl radical formed adduct with adenosine and guanosine. These results suggested that 4-hydrazinobenzoic acid induced DNA damage via not only H₂O₂ production but also phenyl radical production. This study suggests that both oxidative DNA damage and DNA adduct formation play important roles in the expression of carcinogenesis of 4-hydrazinobenzoic acid.     

Epithelial-specific histone modification of the <Emphasis Type="Italic">miR-96/182</Emphasis> locus targeting <Emphasis Type="Italic">AMAP1</Emphasis> mRNA predisposes p53 to suppress cell invasion in epithelial cells

Background

TP53 mutations in cancer cells often evoke cell invasiveness, whereas fibroblasts show invasiveness in the presence of intact TP53. AMAP1 (also called DDEF1 or ASAP1) is a downstream effector of ARF6 and is essential for the ARF6-driven cell-invasive phenotype. We found that AMAP1 levels are under the control of p53 (TP53 gene product) in epithelial cells but not in fibroblasts, and here addressed that molecular basis of the epithelial-specific function of p53 in suppressing invasiveness via targeting AMAP1.

Methods

Using MDA-MB-231 cells expressing wild-type and p53 mutants, we identified miRNAs in which their expression is controlled by normal-p53. Among them, we identified miRNAs that target AMAP1 mRNA, and analyzed their expression levels and epigenetic statuses in epithelial cells and nonepithelial cells.

Results

We found that normal-p53 suppresses AMAP1 mRNA in cancer cells and normal epithelial cells, and that more than 30 miRNAs are induced by normal-p53. Among them, miR-96 and miR-182 were found to target the 3′-untranslated region of AMAP1 mRNA. Fibroblasts did not express these miRNAs at detectable levels. The ENCODE dataset demonstrated that the promoter region of the miR-183-96-182 cistron is enriched with H3K27 acetylation in epithelial cells, whereas this locus is enriched with H3K27 trimethylation in fibroblasts and other non-epithelial cells. miRNAs, such as miR-423, which are under the control of p53 but not associated with AMAP1 mRNA, demonstrated similar histone modifications at their gene loci in epithelial cells and fibroblasts, and were expressed in these cells.

Conclusion

Histone modifications of certain miRNA loci, such as the miR-183-96-182 cistron, are different between epithelial cells and non-epithelial cells. Such epithelial-specific miRNA regulation appears to provide the molecular basis for the epithelial-specific function of p53 in suppressing ARF6-driven invasiveness.

     

Dissection of genotype-phenotype associations in rice grains using metabolome quantitative trait loci analysis

A comprehensive and large‐scale metabolome quantitative trait loci (mQTL) analysis was performed to investigate the genetic backgrounds associated with metabolic phenotypes in rice grains. The metabolome dataset consisted of 759 metabolite signals obtained from the grains of 85 lines of rice (Oryza sativa, Sasanishiki × Habataki back‐crossed inbred lines). Metabolome analysis was performed using four mass spectrometry pipelines to enhance detection of different classes of metabolites. This mQTL analysis of a wide range of metabolites highlighted an uneven distribution of 802 mQTLs on the rice genome, as well as different modes of metabolic trait (m‐trait) control among various types of metabolites. The levels of most metabolites within rice grains were highly sensitive to environmental factors, but only weakly associated with mQTLs. Coordinated control was observed for several groups of metabolites, such as amino acids linked to the mQTL hotspot on chromosome 3. For flavonoids, m‐trait variation among the experimental lines was tightly governed by genetic factors that alter the glycosylation of flavones. Many loci affecting levels of metabolites were detected by QTL analysis, and plausible gene candidates were evaluated by in silico analysis. Several mQTLs profoundly influenced metabolite levels, providing insight into the control of rice metabolism. The genomic region and genes potentially responsible for the biosynthesis of apigenin‐6,8‐di‐C‐α‐l‐ arabinoside are presented as an example of a critical mQTL identified by the analysis.     

Inflammation-induced protein carbonylation contributes to poor prognosis for cholangiocarcinoma

Carbonylation is an irreversible and irreparable protein modification induced by oxidative stress. Cholangiocarcinoma (CCA) is associated with chronic inflammation caused by liver fluke infection. To investigate the relationship between protein carbonylation and CCA progression, carbonylated proteins were detected by 2D OxyBlot and identified by MALDI-TOF/TOF analyses in pooled CCA tissues in comparison to adjacent nontumor tissues and normal liver tissues. We identified 14 highly carbonylated proteins in CCA tissues. Immunoprecipitation and Western blot analyses of individual samples confirmed significantly greater carbonylation of serotransferrin, heat shock protein 70-kDa protein 1 (HSP70.1), and α1-antitrypsin (A1AT) in tumor tissues compared to normal tissues. The oxidative modification of these proteins was significantly associated with poor prognoses as determined by the Kaplan-Meier method. LC-MALDI-TOF/TOF mass spectrometry identified R50, K327, and P357 as carbonylated sites in serotransferrin, HSP70.1, and A1AT, respectively. Moreover, iron accumulation was significantly higher in CCA tissues with, compared to those without, carbonylated serotransferrin. We conclude that carbonylated serotransferrin-associated iron accumulation may induce oxidative stress via the Fenton reaction, and the carbonylation of HSP70.1 with antioxidative property and A1AT with protease inhibitory capacity may cause them to become dysfunctional, leading to CCA progression.     

Nitrogen resorption in senescing leaf blades of rice exposed to free-air CO<Subscript>2</Subscript> enrichment (FACE) under different N fertilization levels

Background and Aims

The effects of Sb(V), alone or combined with Se, on the growth and root development of plants are unknown. The aim of this study is to investigate the interaction between selenite and different forms of Sb and the effects on their uptake in rice and on rice root morphology.

Methods

A hydroponic experiment was conducted that contained fourteen treatments. The treatment levels for Se were 0.5 and 1 mg L^?1, and the treatment levels for Sb(III) and Sb(V) were 5 and 15 mg L^?1.

Results

Sb(V) alone significantly reduced the surface area, mean diameter and volume of the roots, whereas Sb(III) alone reduced the values of most parameters of root morphology. The addition of 1 mg L^?1 Se significantly enhanced the surface area, number of medium roots, and Sb concentration in the roots subjected to 15 mg L^?1 Sb(V), but it decreased the number of root forks, the number and proportion of fine roots, and the shoot Sb concentration under exposure to 15 mg L^?1 Sb(III). When the plants were subjected to 1 mg L^?1 Se, the addition of 15 mg L^?1 Sb(III) markedly reduced the shoot and root Se concentrations and the number of root tips, root forks, and fine roots and increased the mean root diameter. However, the addition of Sb(V) did not significantly affect the root and shoot Se concentrations but significantly decreased the number of root forks and fine roots and increased the proportion of medium roots.

Conclusions

Se and Sb(III) showed antagonistic effects on uptake in the shoots, but not in the roots, of paddy rice. A range of Se concentrations could stimulate the uptake of Sb in both the shoots and roots of paddy rice exposed to Sb(V).

     

Morphological differences between larvae and in vitro-cultured adults of Anisakis simplex (sensu stricto) and Anisakis pegreffii (Nematoda: Anisakidae)

Proper identification of Anisakis species infecting host fishes is very important to both human health and fish disease diagnosis. The foremost problem in the identification of Anisakis larvae in fishes is that L3 larvae cannot be easily differentiated morphologically, especially between A. simplex (sensu stricto) (s.s.) (Rudolphi, 1809) and A. pegreffii Campana-Rouget et Biocca, 1955. Instead, molecular means such as allozyme, mitochondrial DNA (mtDNA) cox2 region and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses had been successfully used. In this study, morphological differences of L3 larvae collected from fishes and in vitro-cultured L4 larvae and adult A. simplex (s.s.) and A. pegreffii were evaluated. Anisakis larvae were collected from 7 different host fishes within Japan. Undamaged A. simplex (s.s.) and A. pegreffii collected from Oncorhynchus keta (Walbaum) and Scomber japonicus Houttuyn, respectively, were used for in vitro-culture in order to obtain L4 and adult stages. Species identification was confirmed by PCR-RFLP analysis of the ITS region (ITS1-5.8S-ITS2) of ribosomal DNA and by mtDNA cox2 gene sequencing. Results revealed that L3, L4 and adult stages of A. simplex (s.s.) and A. pegreffii are morphologically distinguishable based on ventriculus length, wherein the former has longer ventriculus (0.90–1.50 mm) than the latter (0.50–0.78 mm). For oesophagus/ventriculus ratio, these two species are distinguishable only during L4 and adult stages. Also, adult male A. simplex (s.s.) and A. pegreffii were found to be distinguishable by differences in the distribution pattern of the caudal papillae, particularly the 3rd pair of distal papillae.