Genetic markers distinguishing between the two subspecies of the rosy bitterling,Rhodeus ocellatus (Cyprinidae)

Eleven populations of the rosy bitterling,Rhodeus ocellatus, from different localities in Japan, were genetically compared at 16 protein-coding loci using starch-gel electrophoresis. Two loci,Ldh-2 andPgdh, were demonstrated as diagnostic markers for the identification of two subspecies;R. ocellatus kurumeus, which is native to Japan, andR. ocellatus ocellatus, which was introduced from China. The two subspecies were distinguished by the complete substitution of different alleles between them. Population ofR. ocellatus kurumeus occurring in Yao City, Osaka, and in Kanzaki, Saga Prefecture were genetically closely related to each other (genetic distance: D=0.056) but distantly so toR. ocellatus ocellatus from Saitama Prefecture (D=0.202 or 0.265). Electrophoretic analyses also elucidated the existence of hybrid populations of the two subspecies. The populations ofR. ocellatus kurumeus in Yao City had lower genetic variability and a lower incidence of white coloration on the ventral fins than populations of the same in Saga Prefecture. The present study strongly implies that the introduction of the foreign freshwater fishes with subspecific differentiation, into the original range of indigenous subspecies, should be averted not to bring the genetic pollution.     

Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA.

A low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-1α) has been extensively purified from pig liver to give an apparently homogeneous preparation, which seemed to be analogous to the bacterial elongation factor, EF-Tu (Iwasaki, K., Nagata, S., Mizumoto, K., and Kaziro, Y. (1974) J. Biol. Chem. 249, 5008). Thus, the interaction of the purified EF-1α with guanine nucleotides as well as aminoacyl-tRNA has been investigated and the following results have been obtained. (1) EF-1α when kept in the absence of glycerol lost its activity to promote the binding of aminoacylt-RNA to ribosomes though it retained the ability to bind guanine nucleotides. However, the former activity could be stabilized by the addition of 25% ($\text{v}\text{v}$) glycerol to the solution. (2) EF-1α formed a binary complex with guanine nucleotides such as GTP, GDP, 5′-guanylyl methylenediphosphonate or 5′-guanylyl imidodiphosphate. The molar ratio of EF-1α to GTP or GDP in the binary complex was shown to be 1. (3) The presence of a ternary complex containing EF-1α, GTP and aminoacyl-tRNA was demonstrated by several methods, i.e., (i) an increased heat stability of EF-1α in the presence of GTP and Phe-tRNA, (ii) a decrease in the amount of the EF-1α·GTP complex in the presence of aminoacyl-tRNA, (iii) a protection of the ester linkage of Phe-tRNA from hydrolysis at alkaline pH by the presence of both EF-1α and GTP, and (iv) the isolation of the complex by gel filtration.     

Improved composting of Undaria pinnatifida seaweed by inoculation with Halomonas and Gracilibacillus sp. isolated from marine environments

Composting of the Undaria pinnatifida (wakame) seaweed was conducted after inoculation with 6×10(8) CFU g(-1)Halomonas sp. AW4 and the alginate-degrading bacterium Gracilibacillus sp. A7. Inoculation with strains A7 and AW4 resulted in 27.8% and 24.7% degradation of U. pinnatifida dry mass after 168 h, whereas only 17.5% degradation occurred in the uninoculated control. The C/N ratio decreased in the A7 and AW4 inoculated compost by 7.0% and 9.2% after 72 h, but increased by 11.5% in the control. Inoculation with A7 resulted in 2.8 times faster degradation of alginate and 1.2 and 1.6 times higher levels of reducing sugars and unsaturated sugars than inoculation with AW4. The compost produced from the inoculation with A7 had low plant toxicity as measured by germination experiment. The results suggest that inoculation of wakame with alginate-degrading bacteria not only shortened the length of composting but also created seaweed compost with good fertilizer qualities.     

Synthesis of Very-Long-Chain Fatty Acids in the Epidermis Controls Plant Organ Growth by Restricting Cell Proliferation

Plant organ growth is controlled by inter-cell-layer communication, which thus determines the overall size of the organism. The epidermal layer interfaces with the environment and participates in both driving and restricting growth via inter-cell-layer communication. However, it remains unknown whether the epidermis can send signals to internal tissue to limit cell proliferation in determinate growth. Very-long-chain fatty acids (VLCFAs) are synthesized in the epidermis and used in the formation of cuticular wax. Here we found that VLCFA synthesis in the epidermis is essential for proper development of Arabidopsis thaliana. Wild-type plants treated with a VLCFA synthesis inhibitor and pasticcino mutants with defects in VLCFA synthesis exhibited overproliferation of cells in the vasculature or in the rib zone of shoot apices. The decrease of VLCFA content increased the expression of IPT3, a key determinant of cytokinin biosynthesis in the vasculature, and, indeed, elevated cytokinin levels. These phenotypes were suppressed in ipt3;5;7 triple mutants, and also by vasculature-specific expression of cytokinin oxidase, which degrades active forms of cytokinin. Our results imply that VLCFA synthesis in the epidermis is required to suppress cytokinin biosynthesis in the vasculature, thus fine-tuning cell division activity in internal tissue, and therefore that shoot growth is controlled by the interaction between the surface (epidermis) and the axis (vasculature) of the plant body.     

Protective Effect Against 17β-Estradiol on Neuronal Apoptosis in Hippocampus Tissue Following Transient Ischemia/Recirculation in Mongolian Gerbils via Down-Regulation of Tissue Transglutaminase Activity

We analyzed the protective effect of 17β-estradiol (17β-ED) injection against delayed neuronal death in the hippocampus tissue of the brain in Mongolian gerbils after transient ischemia/recirculation treatment, especially in relation with bcl-2 gene expression and enzymatic activity changes of caspase-3 and tissue transglutaminase (tTGase). Daily intraperitoneal injection of 17β-ED to the animal after the ischemia stimulated the expression of an apoptosis suppressor gene, bcl-2, in the hippocampal tissue for a week. The gradually increasing apoptotic enzyme activity of caspase-3 and increased number of TUNEL positive fragmented neuronal nuclei caused by ischemic attack in the gerbil brain were clearly suppressed by 17β-ED administration. The reduced activity and enzyme protein of tTGase, a neurodegenerative marker of apoptosis in the hippocampus after ischemia, were also restored to nearly normal levels by 17β-ED injection. These results suggest that daily 17β-ED administration to the gerbil after transient ischemic insult with progressing neuronal deteriorative changes in hippocampus tissue can effectively prevent apoptotic changes through a molecular cascade involving gene expression regulation.     

Incorporation of tritiated thymidine into mitochondrial DNA of the liver and kidney cells of chickens and mice in tissue culture

Summary ³H-thymidine incorporation into mitochondrial DNA of the liver and the kidney cells of chick embryos and newborn mice in tissue culture was shown by means of electron microscope radioautography with accurate localization. In these cells, about 20% of all the mitochondria were labeled at their matrices between the cristae within 4 hours in contact with the radioisotope, which were removed by DN'ase.From the results, it is clear that the mitochondria of avian and mammalian cells in tissue culture synthesize DNA.     

X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T-->T:A transversion and G:C-->T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T-->T:A transversion and G:C-->T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis.     

Enhancement of secretion of human procollagen I in mouse HSP47-expressing insect cells

We previously demonstrated that insect cells were able to synthesize recombinant human procollagen I as triple-helical heterotrimers when transfected with cDNAs of both proalpha1(I) and proalpha2(I) chains. However, most of the heterotrimers were retained within the cells, unlike in the case of mammalian cells [Tomita, M., Kitajima, T., and Yoshizato, K. (1997) J. Biochem. 1061-1069]. In an attempt to improve the secretion of the heterotrimers, we introduced the putative collagen-specific chaperone HSP47 into this insect expression model. Mouse HSP47 produced by the insect cells bound intracellularly to both human proalpha1(I) and proalpha2(I) chains and enhanced the secretion of procollagen I heterotrimers. HSP47 was also coexpressed with either proalpha1(I) chains or proalpha2(I) chains, which showed that it enhanced the secretion of the former but not the latter. This selective effect of HSP47 was similarly observed in the cells treated with inhibitors of procollagen triple helix formation, indicating that HSP47 can also accelerate the secretion of non-helical procollagens. HSP47 did not change the intracellular solubility of proalpha1(I) and proalpha2(I) chains in 1% NP-40, eliminating the possibility that it prevents proalpha chains from aggregating into insoluble forms within the insect cells. We concluded that HSP47 can play a role in the secretion of alpha1(I)-procollagen chains in the insect cell model. The present study also demonstrated the dissimilarity in the mechanism of folding and secretion of the expressed procollagen I between the insect and mammalian cells.