Sesquiterpenoids of Torilis japonica fruit

From the methanolic extract of Torilis japonica D. C. fruit (Umbelliferae), two eudesmane-type sesquiterpenoids were isolated together with five previously described sesquiterpenoids. From the results of spectral analyses, they were characterized as 4(15)-eudesmene-1beta,5alpha-diol and 4alpha,15-epoxyeudesmane-1beta,6alpha-diol, respectively. The absolute stereostructures of these sesquiterpenoids were elucidated by the modified Mosher's method.     

Dual roles of photosynthetic electron transport in photosystem I biogenesis: light induction of mRNAs and chromatic regulation at post-mRNA level

Light regulation of photosystem I (PSI) biogenesis was studied in a unicellular green alga, Chlamydomonas reinhardtii. When Chlamydomonas cells were transferred from darkness to the light, mRNAs for both nuclear- and chloroplast-encoded PSI subunits were induced in concert. This light induction was inhibited by photosynthetic electron transport (PET) inhibitors, 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6 isopropyl-p-benzoquinone, but not by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone. This indicated that PET plays a pivotal role in the light induction of PSI subunit mRNAs, but that photophosphorylation is not necessary. When we irradiated the Chlamydomonas cells with PSI-light (695 nm) or PSII-light (644 nm), which makes the plastoquinone pool oxidative and reductive, respectively, PSII-light caused the accumulation of PSI proteins more abundantly than did PSI-light. However, there was no difference for the PSI subunit mRNA levels between these light sources. From these results, we conclude that PET plays dual roles in the regulation of PSI biogenesis in Chlamydomonas: when cells are illuminated, PET first induces the PSI subunit mRNAs irrespective of the redox state of the intersystem electron carriers, and then their redox state fine-tunes PSI biogenesis at translational and/or post-translational steps to fulfil the chromatic adaptation.     

Transgenic mouse model of tauopathies with glial pathology and nervous system degeneration

Frontotemporal dementias (FTDs), including corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), are neurodegenerative tauopathies characterized by widespread CNS neuronal and glial tau pathologies, but there are no tau transgenic (Tg) mice that model neurodegeneration with glia tau lesions. Thus, we generated Tg mice overexpressing human tau in neurons and glia. No neuronal tau aggregates were detected, but old mice developed Thioflavin S- and Gallyas-positive glial tau pathology resembling CBD astrocytic plaques. Tau-immunoreactive and Gallyas-positive oligodendroglial coiled bodies (similar to CBD and PSP), glial degeneration, and motor deficits were associated with age-dependent accumulations of insoluble hyperphosphorylated human tau and tau immunopositive filaments in degenerating glial cells. Thus, tau-positive glial lesions similar to human FTDs occur in these Tg mice, and these pathologies are linked to glial and axonal degeneration.     

Differential expression of tissue factor and tissue factor pathway inhibitor in metastatic melanoma lesions

Tissue factor (TF) is involved in tumor progression and metastatic potency in some malignant tumors and its function is regulated by tissue factor pathway inhibitor (TFPI) therefore the interaction of both molecules is crucial for their functional role. We evaluated the clinical relevance of TF and TFPI expression in benign and malignant melanocytic lesions. Expression of both was examined by immunoperoxidase staining using serial tissue sections in 16 nevi, 34 primary and 15 metastatic melanoma lesions. TF and TFPI were ubiquitously expressed in benign and malignant melanocytic lesions. This finding was confirmed by Western blot analysis using cultured human melanocytes, nevi cells (NCN) and melanoma cell lines. Although TF expression was not associated with malignant transformation and disease progression, TFPI expression in primary and metastatic melanoma lesions was significantly lower and weaker than that in nevi lesions in terms of intensity and percentage of stained cells. In addition, TFPI expression in metastatic lesions was significantly lower and weaker than that of TF. These results suggest that the relative expression of TF to TFPI may play a crucial role in the malignant transformation and metastatic potency in melanocytic cells.     

Effects of Cl2MDP-encapsulating liposomes in a murine model of Pseudomonas aeruginosa-induced sepsis

Pseudomonas aeruginosa is a pathogen that frequently causes acute lung injury, bacteremia and sepsis in critically ill patients. As tissue macrophages are a major producer of inflammatory mediators that contribute to septic physiology, and are essential for eliminating bacteria from the circulation, we investigated the role of tissue macrophages in the generation of both inflammatory and anti-inflammatory cytokines in septic shock by using our mouse model of P. aeruginosa pneumonia. To see the effects of tissue macrophage depletion, we intravenously injected dichloromethylene-diphosphonate (Cl2MDP)-encapsulating liposomes in mice. Two days after the liposome injection, we instilled cytotoxic P. aeruginosa (PA103) into the lung that disseminates and causes septic shock. After the infection, we collected blood and bronchoalveolar lavage fluids. The samples were then analyzed for TNF-alpha, MIP-2, and IL-10 concentration. We compared these results to control mice that received either liposomes without Cl2MDP or phosphate buffered saline alone. Plasma TNF-alpha, MIP-2, and IL-10 levels were significantly decreased in the tissue macrophage-depleted mice compared to the control groups of mice. Although depletion of tissue macrophages by Cl2MDP-liposome administration did not affect the severity of bacteremia or the survival of infected mice, these results imply that tissue macrophages have a major role in the production of both proinflammatory and anti-inflammatory cytokines in the circulation and in the causing septic physiology associated with P. aeruginosa pneumonia.     

Use of 13C-labeled glucose for measuring exogenous glucose oxidation in mice

The author modified a respiratory gas analyzer to analyze the respiratory 13CO2 of 12 small laboratory animals all at once. To investigate the practical use of this system, mice were orally (OR) or intravenously (i.v.) given glucose solutions containing three different amounts of 13C-labeled glucose. Expired 13CO2 derived from exogenous glucose was detected within 10 minutes after administration in OR mice, but about 30 minutes in i.v. mice. The height of the peak of 13CO2 expiration was correlated with the administered 13C-glucose mass.     

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.     

Complete deoxygenation from a hemoglobin solution by an electrochemical method and heat treatment for virus inactivation

Hemoglobin (Hb) has been widely studied as a raw material for various types of oxygen carriers. In the purification of Hb from red blood cells including virus inactivation and denaturation of other proteins and the long-term storage of Hb vesicles (HbV), a deoxygenation process is one of the important processes because of the high stability of deoxygenated Hb to heating and metHb formation. Though an oxygenated Hb solution can be deoxygenated with an artificial lung, it is difficult to reduce the oxygen partial pressure of the Hb solution to less than 10 Torr. We developed an electrochemical system for complete deoxygenation of the Hb solution at the cathode compartment using hydrogen containing nitrogen gas at the anode compartment. Oxygen in the Hb solution was reduced to OH(-) at the cathode compartment within several minutes at a potential value of -1.67 V and was finally converted to water by neutralization with H(+) from the anode in the whole system. The resulting completely deoxygenated Hb could tolerate heat treatment at 62 degrees C for 10 h with no denaturation of deoxygenated Hb. The metHb formation rate of reoxygenated Hb at 37 degrees C was not changed after heat treatment. Furthermore, vesicular stomatitis virus (VSV) could be inactivated at an inactivation degree of more than 5.96 log by heat treatment.     

Pseudodactylogyrus kamegaii sp. n. (Monogenea: Pseudodactylogyridae) from wild Japanese eel,Anguilla japonica

The monogenean Pseudodactylogyrus kamegaii sp. n. is described, based on specimens collected from the gills of wild Japanese eel Anguilla japonica caught in Chiba Prefecture, Japan. This species is the most similar to P. anguillae (Yin and Sproston, 1948), but different in the shape and measurements of the male copulatory organ, vagina and marginal hook. This new species was collected from the eel in brackish waters, while P. anguillae and P. bini, the other known pseudodactylogyrids of Japanese eel, have been recorded only in fresh waters.     

Dietary-fiber-degrading enzymes from a human intestinal Clostridium and their application to oligosaccharide production from nonstarchy polysaccharides using immobilized cells

The secretion of nonstarchy polysaccharide-degrading enzymes from an anaerobic human intestinal bacterium, Clostridium butyricum- beijerinckii (isolated from human feces), was investigated. Growth of the bacterium was found when laminarin, konjac glucomannan, and pectic acid were added separately to the culture media as sole carbon source. The corresponding degrading enzymes for these dietary fibers, laminarinase (endo-1,3- beta-glucanase), endo-1,4-beta-mannanase, endo- and exo-pectate lyases, and pectin methylesterase, were then purified and characterized. These extracelluar enzymes, which were secreted by the bacterium in the human large intestine, were considered to contribute to digestion of the ingested dietary fibers to their oligosaccharides, following by short-chain fatty acid fermentation by the bacterium. We have developed cell immobilization techniques of the bacterium on cellulose-foam carriers that are effective for continuous production of the oligosaccharides from the dietary fibers in a fed-batch reactor system. From 9 g of pectic acid, a total of 3.96 g of 4,5-unsaturated digalacturonic acid was produced over 40 h in four 500-ml batchcultures. In the same manner, the corresponding oligosaccharides were obtained from konjac glucomannan and laminarin with average conversion rates of around 30-40%.