Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

Isoproterenol produces a rapid increase in sialidase activity in rat heart tissue and cardiomyocyte-derived H9c2 cells in culture

The effects of isoproterenol on sialidase activity in rat cardiomyocytes were examined. Administration of isoproterenol to rats (0.2 or 2 mg/kg body weight) produced an increase in sialidase activity in total membrane fraction of heart tissue within 120 min (121+/-13% of the control at 120 min after administration of 0.2 mg isoproterenol/kg, n=5, P<0.05). Sialidase activity in cardiomyocyte-derived H9c2 cells was also increased by treatment with isoproterenol (10 microM) for 60 min. The effect of isoproterenol on sialidase activity was amplified by the addition of 3-isobutyl-1-methylxanthine (IBMX). Sialidase activity in H9c2 cells was elevated by treatment with dibutyryl cAMP plus IBMX without isoproterenol. The content of N-acetylneuraminic acid in cells decreased by 22% after treatment with isoproterenol plus IBMX. These results suggest that sialidase activity in rat cardiomyocytes is regulated by beta-adrenergic stimulators via a cAMP-dependent process. The increased activity of sialidase may account for the reduction of sialic acid content of cells.     

The effects of hedonic properties of odors and attentional modulation on the olfactory event-related potentials

The purpose of this study was to investigate the influence of the hedonic properties of odors and the attention of subjects on components of the olfactory event-related potentials (OERP). The subjects were seven healthy male students. Two odors (orange and eugenol) of different hedonic properties were presented to the subjects via a constant-flow olfactometer during an oddball paradigm under ignore and attend conditions, and the OERP were then established. The latencies of the OERP were not affected by the qualitatively different odors, whereas the amplitude of late positive component (P3) during the presentation of orange was significantly larger than that during the presentation of eugenol. On the other hand, the allocation of a subject's attention led to a decrease in the latency and to an increase in the amplitude of P3. Moreover, the amplitude of P3 increased significantly when the pleasant odor (orange) in the rare stimulus was presented under the attend condition. These results suggested that hedonic property, distribution of attention, and the interaction between these factors may influence the OERP components.     

Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.     

G-protein signaling abnormalities mediated by CD95 in salivary epithelial cells

Salivary epithelial cells from patients with primary Sj?gren's syndrome (SS) undergo Fas-mediated apoptosis. Bcl-2 and Bcl-xL are apoptosis suppressing oncogenes. Very little is known about the role of these oncogene molecules in salivary epithelial cells. To investigate the possible prevention of salivary glandular destruction in SS by Bcl-2 and Bcl-xL, stable transfectants expressing these molecules were made from HSY cells, a human salivary epithelial cell line. HSY cells were transfected with an expression vector for human Bcl-2 or Bcl-xL. Stable transfectants were selected and apoptosis was induced by anti-Fas antibody. Apoptosis was quantified by propidium iodide staining followed by flow cytometry. Caspase activity was detected by immunohistochemical analysis and enzyme cleavage of DEVD-AMC, a fluorescent substrate. Response to carbachol, a muscarinic receptor agonist, and EGF was measured by Ca2+ mobilization and influx. Fas-mediated apoptosis was significantly inhibited in Bcl-2 and Bcl-xL transfectants compared to wild-type and control transfectants (empty vector). Surprisingly, caspase activity was not inhibited in Bcl-2 and Bcl-xL transfectants. Activation of the Fas pathway in the Bcl-2 and Bcl-xL transfectants by antibody also inhibited carbachol and EGF responsiveness (i.e., Ca2+ mobilization and/or influx) by 50-60%. This Fas-mediated inhibition of cell activation was partially or completely restored by specific peptide interference of caspase enzyme activity. The prevention of Fas-mediated apoptosis by the overexpression of Bcl-2 and Bcl-xL in salivary gland epithelial cells results in injured cells expressing caspase activity and unable to respond normally to receptor agonists. Such damaged cells may exist in SS patients and could explain the severe dryness out of proportion to the actual number of apoptotic cells seen on salivary gland biopsy.