A transient rise in plasma beta-endorphin after a traditional 47 degrees C hot-spring bath in Kusatsu-spa, Japan.

To clarify the mechanism of the intoxicating feeling attained after a traditional 47 degrees C hot-spring bath called 'jikan-yu' in Kusatsu-spa, Japan, we examined the change in plasma levels of beta-endorphin and methionine enkephalin in 7 healthy subjects. The mean sublingual temperature rose from 36.8 degrees C to 38.6 degrees C and the plasma beta-endorphin level from 16.2 pg/ml to 49.5 pg/ml 2 minutes after completing a 3-minute bath in 47 degrees C hot-spring water. However, the plasma methionine enkephalin level was not changed. This feeling of intoxication may be explained by the transient rise in plasma beta-endorphin level.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).     

A clinical study of the efficacy of a single session of individual exercise for depressive patients,assessed by the change in saliva free cortisol level

Background

The efficacy of physical exercise as an augmentation to pharmacotherapy with antidepressants for depressive patients has been documented. However, to clarify the effectiveness of exercise in the treatment of depression, it is necessary to distinguish the effect of the exercise itself from the effect of group dynamics. Furthermore, an objective measurement for estimation of the effect is needed. Previous reports adopted a series of group exercises as the exercise intervention and mainly psychometric instruments for the measurement of effectiveness. Therefore, this clinical study was done to examine the effectiveness of a single session of individual exercise on depressive symptoms by assessing the change in saliva free cortisol level, which reflects hypothalamic-pituitary-adrenocortical axis function that is disturbed in depressive patients.

Method

Eighteen medicated patients, who met the DSM-IV-TR criteria for major depressive disorder, were examined for the change in saliva free cortisol levels and the change in subjective depressive symptoms before and after pedaling a bicycle ergometer for fifteen minutes. Within a month after the exercise session, participants conducted a non-exercise control session, which was sitting quietly at the same time of day as the exercise session.

Results

Depressed patients who participated in this study were in remission or in mild depressive state. However, they suffered chronic depression and had disturbed quality of life. The saliva free cortisol level and subjective depressive symptoms significantly decreased after the exercise session. Moreover, the changes in these variables were significantly, positively correlated. On the other hand, although the subjective depressive symptoms improved in the control session, the saliva free cortisol level did not change.

Conclusion

For the first time in depressive patients, we were able to show a decrease in the saliva free cortisol level due to physical exercise, accompanied by the improvement of subjective depressive symptoms. This identified a possible influence of exercise on the hypothalamic-pituitary-adrenal axis in depression.These results suggest the utility of assessing the effect of physical exercise by saliva free cortisol level in depressive patients who suffer from bio-psycho-social disability.

     

Regulation of natural killer cell-mediated swine endothelial cell lysis through genetic remodeling of a glycoantigen

The effect of remodeling of a glycoantigen such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes on natural killer (NK) cell-mediated direct cytotoxicity was investigated using human peripheral blood mononuclear cells (PBMC) or an NK-like cell line, YT cells, as an effector, and swine endothelial cells (SEC) as a target. Several SEC transfectants were established by transfection with the genes for beta1,4-N-acetylglucosaminyltransferase III, alpha2, 3-sialyltransferase and alpha1,2-fucosyltransferase. These transfections led to dramatic reductions in both direct and indirect NK cell-mediated cytotoxicity, by 72-94% in the case of PBMC and 27-72% in that of YT cells, in addition to an effective reduction in xenoantigenicity, which is substantially caused by the alpha-Gal epitope, to human natural antibodies. The NK cell-mediated direct cytotoxicity was remarkably blocked by an anti-alpha-Gal epitope monoclonal antibody or GSI lectin which preferentially binds to the epitope. Furthermore, treatment of the parental cells with alpha-galactosidase resulted in a significant reduction in cytotoxicity. These results suggest that the alpha-Gal epitope is involved not only in hyperacute rejection and acute vascular rejection, but also in NK cell-mediated direct cytotoxicity. Thus, the genetic remodeling of the alpha-Gal epitope and probably other glycoantigens as well can be expected to represent a new approach for overcoming not only indirect but also direct immunity to xenografts.     

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.     

Synthesis of a new Cre recombinase gene based on optimal codon usage for mammalian systems

The origin of the Cre recombinase gene is bacteriophage P1, and thus the codon usages are different from in mammals. In order to adapt this codon usage for mammals, we synthesized a "mammalian Cre recombinase gene" and examined its expression in Chinese hamster ovarian tumor (CHO) cells. Significant increases in protein production as well as mRNA levels were observed. When the recombination efficiency was compared using CHO cell transfectants having a cDNA containing loxP sites, the "mammalian Cre recombinase gene" recombined the loxP sites much more efficiently than the wild-type Cre recombinase gene.     

Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.     

Translocation and accumulation of nicotine via distinct spatio-temporal regulation of nicotine transporters in Nicotiana tabacum

In plants, secondary metabolites play important roles in adaptation to the environment. Nicotine, a pyridine alkaloid in Nicotiana tabacum, functions as chemical barrier against herbivores. Nicotine produced in the root undergoes long-distance transport and accumulates mainly in the leaves. Since production of such defensive compounds is costly, plants must regulate the allocation of the products to their tissues; however, the molecular mechanism of nicotine translocation remains unclear. Our recent studies identified a novel multidrug and toxic compound extrusion (MATE)-type nicotine transporter, JAT2 (jasmonate-inducible alkaloid transporter 2). This transporter is specifically expressed in leaves, localizes to the tonoplast, and transports nicotine as its substrate. The specific induction of JAT2 expression in leaves by methyl jasmonate (MeJA) treatment suggests that this transporter plays an important role in nicotine distribution to leaves, especially under herbivore attack, by transporting nicotine into the vacuole. Considering JAT2, together with the previously identified MATE transporters JAT1, MATE1, and MATE2, and the PUP (purine permease) transporter NUP1 (nicotine uptake permease1), we show a model of nicotine translocation and accumulation via distinct spatio-temporal regulation of nicotine transporter expression. Furthermore, we discuss the possible role of nicotine transporters in determining outcrossing rates and seed production.     

Selectivity improvement of an azole inhibitor of CYP707A by replacing the monosubstituted azole with a disubstituted azole

The plant growth-retardant uniconazole (UNI), a triazole inhibitor of gibberellin biosynthetic enzyme (CYP701A), inhibits multiple P450 enzymes including ABA 8′-hydroxylase (CYP707A), a key enzyme in ABA catabolism. Azole P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp² nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from adopting a distinct conformation and orientation for different active sites. Based on this hypothesis, we designed and synthesized novel UNI analogs with a disubstituted azole ring (DSI). These analogs were expected to have higher selectivity than UNI because the added functional group may interact with the active site to restrict orientation of the molecule in the active site. DSI-505ME and DSI-505MZ, which have an imidazolyl group with a methyl 5-acrylate, strongly inhibited recombinant CYP707A3, with no growth-retardant effect.