Nineteen DNA samples that carry integrated hepatitis B virus (HBV) DNA were isolated from seven independent human hepatomas by molecular cloning, and their structures were determined. The results, combined with reported data, were analyzed so that one can obtain insights into the mechanisms of integration of this virus DNA and possible rearrangements that occur subsequently. Recent studies have shown that the HBV genome carries four coding frames, all of which are located on the same DNA strand. The genome also carries a unique ~Present address: Institute for Bioscience, Nippon Zeon Co., Kawasaki, 210 Japan.

15-Deoxy-D 12,14 -prostaglandin J 2 (15d-PGJ 2 ) has been identified as a natural ligand for peroxisome proliferatoractivated receptor (PPAR) c to promote adipogenesis. However, it remains elusive about the ability of PPARc-expressing adipocytes to produce PGJ 2 series and the role in the life cycle of adipocytes. Here, we developed an enzyme-linked immunosorbent assay specific for 15d-PGJ 2 . The analysis using this method revealed the increase in the endogenous synthesis of immunoreactive 15d-PGJ 2 in cultured adipocytes during the maturation phase. Further studies using cyclooxygenase inhibitors clarified the contribution of endogeous 15d-PGJ 2 produced by mature adipocytes to upregulation of fat storage in an autocrine manner.

Integrated hepatitis B virus (HBV) DNA is found in hepatocellular carcinomas which develop in HBV carriers. Presented here are the results of analyses of four integrants that show chromosomal rearrangements associated with the integrated HBV DNA. Two clones (p4 and C15) were found to have large inverted repeating structures, each consisting of HBV genome along with flanking cellular sequences. The structure must have arisen by duplication of the primary integrant, including the flanking cellular DNA, followed by recombination within the viral DNA. One of the two viral arms in each clone joins to the other viral arm at the "cohesive end region." Two clones (DA2-2 and DA2-6) were found to have integrated HBV sequences, each flanked by cellular DNAs from different chromosomes (chromosome X joined to 17 and chromosome 5 joined to 9). They must be the products of cellular DNA translocations using the integrated HBV DNA as the switch point. The viral DNA in each clone is a continuous stretch of a single virus genome with one end in the cohesive end region. These complex structures seem to have been produced by activation of the cohesive end of an integrant viral genome, followed by its recombination with another chromosomal DNA.

Although some eicosanoids serve as potent natural ligands to activate peroxisome proliferator-activated receptor (PPARgamma), the ability of adipocytes to produce eicosanoids and regulate PPARgamma remains unclear. Here, adipogenic 3T3-L1 cells were employed to determine the gene expression of isoforms of biosynthetic enzymes in the arachidonate cyclooxygenase (COX) pathway and the synthesis of prostaglandins (PGs). The expression of COX-2 was induced transiently in a biphasic manner upon the triggering of the differentiation and maturation phases while COX-1 was constitutive. The exclusive expression of lipocalin-type PGD synthase occurred and gradually increased during the maturation process along with the stable expression of PPARgamma. Moreover, we confirmed the formation of PGD2 from arachidonic acid by the mature adipocytes, suggesting conversion into PGJ2 derivatives. Even though cytosolic and membrane-associated subtypes of PGE synthase were expressed at relatively constant levels, the ability of preadipocytes to produce PGE2 was greater than that of mature adipocytes in the cell response. The treatment of the mature adipocytes with exogenous PGD2, 15-deoxy-delta12,14-PGJ2 and PGE2, in the presence of aspirin, enhanced the adipogenesis. These findings imply the specific roles of prostanoids produced by the mature adipocytes in the maintenance of terminal differentiation through an autocrine control mechanism.

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To assess oxidative stress (OS) induced by endurance exercise, concentrations of serum reactive oxygen species (ROS) were determined in 70 Japanese male amateur runners completing a two-day ultra-marathon race. Serum ROS levels were analyzed at three time points: before the race (baseline), after the 1st day race (mid-race), and after the 2nd day race (goal) (post-race). The means (SE) of ROS were 151.4(3.7) (U. CARR.), 168.7(4.4), and 156.8(4.4), respectively. Significant positive trends were noted between age and serum ROS concentrations at the three race points (p<0.05 for all). After adjusting for age, BMI and average monthly running distance, the baseline serum ROS concentrations were positively associated with completion times of the first-day race, in particular (p<0.05), suggesting that the concentrations may predict physical performance. The ROS production increased at mid-race (p<0.05), but the levels returned to baseline levels at post-race, indicating that an antioxidant defense system may develop post-race to reduce OS.

Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF1α, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF1α. According to the typical calibration curve of our ELISA, 6-keto-PGF1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF2α. The resulting ELISA was applied to the quantification of 6-keto-PGF1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF1α during the maturation phase of 4-6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF1α is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.