Kiichi Miyashita

Hepatic AdipoR2 signaling plays a protective role against progression of nonalcoholic steatohepatitis in mice

It is unclear how hepatic adiponectin resistance and sensitivity mediated by the adiponectin receptor, AdipoR2, contributes to the progression of nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the roles of hepatic AdipoR2 in NASH, using an animal model. We fed C57BL/6 mice a methionine‐deficient and choline‐deficient (MCD) diet for up to 8 weeks and analyzed changes in liver pathology caused by either an AdipoR2 short hairpin RNA–expressing adenovirus or an AdipoR2‐overexpressing adenovirus. Inhibition of hepatic AdipoR2 expression aggravated the pathological state of NASH at all stages: fatty changes, inflammation, and fibrosis. In contrast, enhancement of AdipoR2 expression in the liver improved NASH at every stage, from the early stage to the progression of fibrosis. Inhibition of AdipoR2 signaling in the liver diminished hepatic peroxisome proliferator activated receptor (PPAR)‐α signaling, with decreased expression of acyl‐CoA oxidase (ACO) and catalase, leading to an increase in lipid peroxidation. Hepatic AdipoR2 overexpression had the opposite effect. Reactive oxygen species (ROS) accumulation in liver increases hepatic production of transforming growth factor (TGF)‐β1 at all stages of NASH; adiponectin/AdipoR2 signaling ameliorated TGF‐β–induced ROS accumulation in primary cultured hepatocytes, by enhancing PPAR‐α activity and catalase expression. Conclusion: The adiponectin resistance and sensitivity mediated by AdipoR2 in hepatocytes regulated steatohepatitis progression by changing PPAR‐α activity and ROS accumulation, a process in which TGF‐β signaling is implicated. Thus, the liver AdipoR2 signaling pathway could be a promising target in treating NASH. (HEPATOLOGY 2008;48:458–473.)

Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary

Recent studies point to a key role for the estrogen synthesizing enzyme P450 aromatase (P450 arom) in ovary determination in fish, birds and reptiles. It is unclear whether estrogen synthesis is important in sex determination of Xenopus gonad. To determine whether the aromatase gene is transcribed in the gonads of Xenopus tadpoles during the sex determination, we cloned a P450 arom cDNA and examined the level of P450 arom and estrogen receptor (ER) gene expression in association with estrogen activity. cDNA clones for P450 arom were isolated from a Xenopus ovarian cDNA library. There was an open reading frame (ORF) of 1500 bp from the ATG start to TAA stop codons encoding 500 predicted amino acids. cDNAs for P450 arom have previously been cloned from various vertebrates. The homology between the Xenopus P450 aromatase and the human P450 arom was higher. The expression of the P450 arom gene was mainly limited to reproductive organs. To determine the beginning of estrogen activity in gonads of embryos, expression of the aromatase and ER gene was also examined by RQ-RT-PCR. Both Xenopus aromatase and ER mRNA was detected at stage 51 in gonads. These observations are consistent with estrogens having a key role in ovarian development in various other vertebrates.

SRY-related genes in Xenopus oocytes☆

SRY-related genes are known as Sox (Sry-box) genes. Two Sox cDNAs from Xenopus oocytes were analyzed. The deduced product of the Xenopus Sox gene (xSox-11) consisted of the standard domains of an HMG box, glycine/alanine-rich region and glutamic acid/aspartic acid-rich region and may be involved in the control of transcription. The other Sox gene (xSox-11-D) had a deletion of 262 nucleotides at one end of the HMG box in the xSox-11 cDNA. The deletion resulted in a frame-shift and in variations in base pair composition and the length of the trinucleotide repeat in the C-terminal coding region. The amino acid sequence of the C-terminal domain encoded by xSox-II-D was highly basic and might be involved, together with the HMG box, in the binding to DNA.

Mechanism of the protective effect of heteropolyoxotungstate against herpes simplex virus type 2

The effects of heteropolyoxotungstate (K7[PTi2W10O40]· 6H2O; PM-19) on the replication of herpes simplex virus type 2 (HSV-2) were examined using a semiquantitative polymerase chain reaction of intracellular viral DNA established by us and also other methods. Vero cells were infected with HSV-2 strains: either the standard strain 169, or the acyclovir-resistant strain YS-4C-1. PM-19 was added at various stages during the replication of HSV-2. PM-19 strongly inhibited the synthesis of viral genomic DNA when it was added at the time of infection. The addition of PM-19 60–90 min after viral inoculation time-dependently decreased the antiviral activity and increased the relative yield of viral DNA, and the addition of PM-19 was completely ineffective at times later than 90 min. These results suggested that PM-19 inhibited viral penetration but did not affect the synthesis of viral DNA. Furthermore, PM-19 strongly inhibited a second round of infection.

Analysis of inhibition of topoisomerase IIα and cancer cell proliferation by ingenolEZ

We previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase inhibitory activity and/or inhibitory activity of cell proliferation. The inhibitory effects of 20‐O‐(2′E,4′Z‐decadienoyl) ingenol and 3‐O‐(2′E,4′Z‐decadienoyl)‐ingenol among these compounds on topoisomerase II activity and on the cell proliferative activity and arrest phase of the cell cycle were studied using a mouse breast cancer (MMT) cell line. Although 20‐O‐ingenolEZ exerted inhibitory effects on both topoisomerase II activity and cell proliferative activity, 3‐O‐ingenolEZ exerted inhibitory activity on neither. The 20‐O‐ingenolEZ‐induced cell arrest of MMT‐cell proliferation led to a cell cycle arrest in the G2/M phase. Topoisomerase II inhibition can be divided into the poison and catalytic inhibitor types. A checkpoint mechanism is activated when cells are treated with these topoisomerase II inhibitors. Poison‐type inhibition occurs via induction of the DNA damage checkpoint and the catalytic‐type inhibition occurs via induction of the DNA‐decatenation checkpoint, suggestive of distinct checkpoint reactions. 20‐O‐ingenolEZ inhibited topoisomerase IIα activity through inhibition of ATPase, and induced DNA‐decatenation checkpoint without signaling for phosphorylation of H2AX. (Cancer Sci 2010; 101: 374–378)

Quantitation of herpes simplex viral DNA in Vero cells for evaluation of an antiviral agent using the polymerase chain reaction.

A method for quantitation of the DNA of Herpes simplex virus type 2 (HSV-2)-infected Vero cells by the polymerase chain reaction (PCR) was developed. This method allowed recognition of several molecules of viral DNA among the total DNA extracted from cells. The method could be applied to a very large range (10(-0)-10(-7)) of initial amounts of template. Products of PCR were collected after each cycle for kinetic analysis. Products were subjected to electrophoresis and amplified bands were stained with ethidium bromide. The intensity of fluorescence of each band was measured with a charge-coupled device (CCD) image analyzer. The time course of increases in the relative yield of viral DNA was determined. Two-fold amplification of viral DNA occurred each 6-h cycle from 7 h after infection. Using this method, the yields of viral DNA after treatment with the drug acyclovir (ACV) at 0.1 and 2 microg/ml were about 1/10 and 1/80 of those from nontreated infected cells, respectively. These results indicate that this method makes clear the inhibitory effect of ACV on the synthesis of viral DNA.