Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates

Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK _Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK _Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK _Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK _Ec or glpK _Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK _Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.     

Determination of prenatal sex ratio in man

Summary The sex of a conceptus at the early embryonic stage was diagnosed in 1000 induced abortions. Specimens were obtained from women who terminated their pregnancies within 12 menstrual weeks on socio-economic indications. By making use of the triple checking procedures, such as the karyotypic analysis of Giemsa-stained slides, the fluorescent Y chromosome analysis and the Y-body test in interphase nuclei, the sex ratio was determined as 106.6 (516 males/484 females). The sex distribution in the chromosomally normal embryos was 481 in males to 448 in females; that gave the ratio of 107.4. A slight excess of males was already present at this stage of pregnancy. When the ratios were calculated in relation to the maternal age, the lower sex ratio was noted for embryos born to mothers over 30 years. Taking a Y-bearing embryo as male, the ratio in 71 chromosomally aberrant embryos was 97.2 (35 males/36 females). The sex ratio in the cases of chromosome abnormalities was not statistically different from that of the normal embryos.     

Cyclophosphamide-Induced Bacterial Translocation in Escherichia coli C25-Monoassociated Specific Pathogen-Free Mice

The kinetics of bacterial translocation (BTL) from the intestine to the mesenteric lymph nodes (MLN) and the number of peripheral white blood cells (WBC), macrophages in Peyer's patches (PP) and M-cells on the surface of cecal PP after cyclophosphamide (CY) injection were examined in penicillin-G and streptomycin sulfate decontaminated and Escherichia coli C25-monoassociated specific pathogen-free mice. WBC were counted to confirm the immunological state of the mice. Until 8 days after CY injection, the number of WBC, bacteria in MLN and macrophages in PP decreased, but then significantly increased on day 14. The levels again decreased to the control levels on day 16. Although the number of M-cells decreased up to day 8, it did not return to the control level on day 16. These results indicate that BTL is stimulated in an immunopotentiated state after CY injection, and this phenomenon may be closely related to the number of macrophages in the blood and PP.     

Comparative Analyses of Thymocyte and Thymic Low-Density Adherent Cell Functions

Thymocytes which have developed in the C3H thymus showed depressed proliferative responses to stimulation with anti-CD3 antibody as compared with those which have developed in the thymus of other strains of mice (i.e. AKR). The present study was conducted to analyze immunological functions of the thymic stromal cell population (low-density adherent cells, LDAC) in the C3H mice using allogeneic bone marrow (BM) chimeras established by BM transplantation in the reciprocal combination of AKR and C3H mice as donor or recipient. The thymic LDAC from C3H mice or the [AKR(donor)→C3H(recipient)] chimeras contained a high proportion of Mac-1⁺ cells as compared to AKR mice or the [C3H→AKR] chimeras. The proportion of Mac-1⁺ cells paralleled the IL-1- and PGE₂-secreting ability of the LDAC cultured either in the presence or absence of LPS and also paralleled the antigen-presenting cell functions of the LDAC. Furthermore, after anti-CD3 stimulation the PGE₂ inhibited more profoundly proliferative responses of [AKR→C3H] or normal C3H thymocytes than those of the [C3H→AKR] chimera or normal AKR thymocytes. A PGE₂ inhibitor, indomethacin, reversed the depressed responses of the thymocytes which had developed in the C3H thymus. These findings suggest that the lower responsiveness of thymocytes from [AKR→C3H] chimeras to anti-CD3 stimulation may be attributable to large amounts of PGE₂ secreted by LDAC and/or to increased sensitivity of thymocytes themselves to PGE₂.     

Correlation of the level of β-citryl-l-glutamic acid with spermatogenesis in rat testes

β-Citryl-l-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.     

Regulation of chondrocytic phenotype by micro RNA 18a: Involvement of Ccn2/Ctgf as a major target gene

We searched for miRNAs that were down-regulated in chondrocytic cells and predicted to target CCN2/connective tissue growth factor (CCN2/CTGF) that promotes endochondral ossification. Among them, expression of miR-18a was most strongly repressed in chondrocytic cells. Reporter gene analysis confirmed the functionality of an miR-18a target in the 3′-untranslated region of Ccn2 mRNA, which was predicted in silico. Indeed, introduction of miR-18a efficiently repressed the CCN2 production from chondrocytic cells. Finally, transfected miR-18a significantly repressed the mature chondrocytic phenotype. Our present study revealed a regulatory role for miR-18a in chondrocytic differentiation through CCN2.