Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants

We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".     

Production of functional spermatids from mouse germline stem cells in ectopically reconstituted seminiferous tubules

Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.     

Over-expression of 3-ketoacyl-ACP synthase III or malonyl-CoA-ACP transacylase gene induces monomer supply for polyhydroxybutyrate production in Escherichia coli HB101

A Pseudomonas sp. 61-3 malonyl-CoA-ACP transacylase gene (fabD _Ps) was cloned by Southern analysis using an equivalent gene of Escherichia coli (fabD _Ec) as a probe. Some recombinant E. coli HB101 strains harboring fabD _Ps, fabD _Ec, or E. coli 3-ketoacyl-ACP synthase III gene (fabH _Ec) with Aeromonas caviae polyhydroxyalkanoate synthase gene (phaC _Ac) were constructed and grown on one-stage cultivation in Luria-Bertani broth containing glucose as carbon source. These strains accumulated 5 to 11 wt% of poly (3-hydroxybutyrate) (PHB) within cells. Over-expression of fabH _Ec, fabD _Ec, or fabD _Ps has been suggested to lead the monomer-supply of (R)-3-hydroxybutyryl-CoA for PHB synthesis in E. coli cells.     

Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in the Biosynthesis of Phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.     

Geographical variation in the early regeneration process of Siebold's Beech (Fagus crenata BLUME) in Japan

The causes and timing of seed death in early regeneration process of Siebold's beech (Fagus crenata Blume) was studied at 15 sites along a snowfall gradient in Japan, in order to clarify why the seedling density of the species has geographic difference remarkably. Seed production did not significantly differ along the snowfall gradient. Pre-dispersal seed mortality by insect damage was higher at sites with light snowfall than at sites with heavy snowfall, but this only seemed to be a minor factor influencing the population. A large proportion of the viable nuts that fall in autumn ware killed in winter before germination. Winter mortality was much higher at sites with thin snow cover than that at sites with thick snow cover, and this factor was strongly correlated with the geographic variation of seedling regeneration probability. There was little seed mortality by winter desiccation. The main factor contributing to the geographic difference seemed to be a seed predation by rodents in winter. Deep snow cover may reduce the success of rodents finding seeds in winter. Thus the observed relationship between snowpack depth and early mortality may be due to an indirect effect through the process of seed predation.p>