Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine-/choline-deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long-term feeding with a high-fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1–14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Masson's trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression—not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.

An estimated 170 million individuals worldwide are infected with hepatitis C virus (HCV), a serious cause of chronic liver disease. Current interferon-based therapy for treating HCV infection has an unsatisfactory cure rate, and the development of more efficient drugs is needed. During the early stages of HCV infections, various host genes are differentially regulated, and it is possible that inhibition of host proteins affords a therapeutic strategy for treatment of HCV infection. Using an HCV subgenomic replicon cell culture system, here we have identified, from a secondary fungal metabolite, a lipophilic long-chain base compound, NA255 (1), a previously unknown small-molecule HCV replication inhibitor. NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, thereby inhibiting serine palmitoyltransferase, and it disrupts the association among HCV nonstructural (NS) viral proteins on the lipid rafts. Furthermore, we found that NS5B protein has a sphingolipid-binding motif in its molecular structure and that the domain was able to directly interact with sphingomyelin. Thus, NA255 is a new anti-HCV replication inhibitor that targets host lipid rafts, suggesting that inhibition of sphingolipid metabolism may provide a new therapeutic strategy for treatment of HCV infection.

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2⌬ and homozygous chs3⌬ null mutants.Like Saccharomyces cerevisiae, Candida albicans harbors three chitin synthase genes, designated CHS1, CHS2, and CHS3 (2, 6, 13). In S. cerevisiae, it was demonstrated by gene disruption experiments that chitin synthase 1 (Chs1p) is involved in the repair of damaged chitin, Chs2p is required for primary septum formation, and Chs3p is responsible for all other chitin syntheses (5,12,14). More recently, Kollar et al. reported that CHS3 also contributes to the formation of linkage between chitin and ␤-1,3-glucan in S. cerevisiae (10). In order to gain more insights into the physiological roles of the chitin synthases of C. albicans, we have disrupted both CHS2 and CHS3 in C. albicans by means of the URA blaster protocol (1).The homozygous chs2⌬ null mutant and the homozygous chs3⌬ null mutant strains of C. albicans were obtained by transforming CAI-4 cells (ura3⌬::imm34/ura3⌬::imm34) with DNA fragments containing either CHS2 in which the hisG-URA3-hisG cassette was inserted at the unique XhoI site or CHS3 in which the 0.8-kb NcoI-ClaI region was replaced by the hisG-URA3-hisG cassette by the lithium acetate method (9). These DNA fragments were successfully integrated into one of the diploid CHS2 or CHS3 alleles, respectively, and the URA3 gene was efficiently eliminated by 5-fluoroorotic acid (5-FOA) selection (11) (Fig. 1). Then these DNA fragments were again transfected into cells in which one of the diploid CHS2 or CHS3 alleles was already flanked by the hisG sequence. Although the second allele of the CHS2 locus was efficiently targeted by the same DNA fragment used to disrupt the first allele, the remaining CHS3 allele was not easily disrupted by transfection of the same DNA fragment. Therefore, we constructed another plasmid in which the hisG-URA3-hisG cassette was inserted at the NcoI site of CHS3. We assumed that use of this DNA for the second round of transfection would increase the efficiency of homologous recombination between the transfected DNA and the remaining intact CHS3 allele because the 0.8-kb NcoI-ClaI region of CHS3 was missing in the already targeted CHS3 locus. As expected, in 3 of 24 uracil auxotrophs, both of the CHS3 alleles were found to be flanked by the hisG sequence after 5-FOA selection, resulting in the homozygous chs3⌬ null mutation (Fig. 1).Cells lacking functional CHS3 grew in a rich medium such as YPD (1% peptone, 2% yeast extract, and 2% dextrose), but their growth was somewhat slower than that of cells missing CHS2 or the parental strain CAI-4 (the doubling times for CAI-4, the homozygous chs2⌬ null mutant, and the homozygous chs3⌬ null mutant were about 70, 72, and 90 min, respectively)...

Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases. Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly. In addition to these amino acids, Asp441 was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme.