Characterization of the carp myosin heavy chain multigene family

We isolated partial coding sequences for 29 carp myosin heavy chain genes (MyoHCs) and determined the nucleotide sequences around the region encoding the loop 2 of the myosin molecule. The predicted amino acid sequences from the isolated genes all showed very high similarity to those of skeletal and cardiac muscles from higher vertebrates, but not to those of smooth and non-muscle counterparts. Among all clones isolated, carp MyoHC10, MyoHCI-1-3 and MyoHC30 showed exon-nucleotide sequences identical to those of cDNAs encoding the loop 2 region of the 10 degrees C-, intermediate- and 30 degrees C-type fast skeletal isoforms [Hirayama and Watabe, Euro. J. Biochem. 246 (1997) 380-387]. The loop 2 of 28 types of carp MyoHCs was encoded by two exons separated by an intron corresponding to that of the 16th in higher vertebrate MyoHCs, whilst this intron was not found in carp MyoHC30. Although carp MyoHC30 had a gene organization different from those of higher vertebrates and other carp MyoHCs, its predicted amino acid sequence for loop 2 showed the highest homology to those of higher vertebrates among carp MyoHCs. In the 28 carp MyoHCs containing the intron, a combination of different nucleotide sequences for the two resulted in 14 distinct series for the combined coding sequence. These different nucleotide sequences encoded nine distinct amino acid sequences. Phylogenetic analysis for the present loop 2 and light meromyosin previously reported for carp MyoHCs [Imai et al., J. Exp. Biol. 200 (1997) 27-34] revealed that carp MyoHCs have recently diverged and are more closely related to each other than to MyoHCs from other species.     

Inhibitory effect of a SALMFamide neuropeptide on secretion of gonad-stimulating substance from radial nerves in the starfish Asterina pectinifera

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The SALMFamide family is also known to an echinoderm neuropeptide. The present study examined effect of SALMFamide 1 (S1) on oocyte maturation of starfish Asterina pectinifera. Unlike GSS, S1 did not induce spawning in starfish ovary. In contrast, S1 was found to inhibit GSS secretion from radial nerves by treatment with high K+ concentration. Fifty percent inhibition was obtained by 0.1 mM S1. S1 did not have any effect on GSS- and 1-MeAde-induced oocyte maturation. Following incubation with a S1 antibody and subsequently with rhodamine-conjugated second antibody, neural networks were observed in ovaries. The networks were restricted mainly to their surface with little evidence of immunoreactivity inside the basement membranes. This indicates that neural networks are distributed in the ovarian wall. The result further suggests that S1 plays a role in oocyte maturation to regulate GSS secretion from the nervous system.     

Artificial life simulation of self-assembly in bacteriophage by movable finite automata

This paper presents a model which is based on biological research using the movable finite automata (MFA) on a self-assembly of T4 phage, and exhibits the results of artificial life simulation. In the previous work, Thompson and Goel [Artificial Life, Addison Weley, 1989, pp. 317-340; Biosystems 18 (1985) 23; J. Theor. Biol. 131 (1988) 351] presented the movable finite automata (MFA) which has a capability of moving on finite automata, and simulated on a computer. They were represented individual rectangular boxes, however, the results of simulation was different from real T4 phage. We propose the sphere model as a protein structure, and simulate the self-assembly of the entire structure of the T4 phage on a computer.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Concerted and nonconcerted evolution of the Hsp70 gene superfamily in two sibling species of nematodes

We have identified the Hsp70 gene superfamily of the nematode Caenorhabditis briggsae and investigated the evolution of these genes in comparison with Hsp70 genes from C. elegans, Drosophila, and yeast. The Hsp70 genes are classified into three monophyletic groups according to their subcellular localization, namely, cytoplasm (CYT), endoplasmic reticulum (ER), and mitochondria (MT). The Hsp110 genes can be classified into the polyphyletic CYT group and the monophyletic ER group. The different Hsp70 and Hsp110 groups appeared to evolve following the model of divergent evolution. This model can also explain the evolution of the ER and MT genes. On the other hand, the CYT genes are divided into heat-inducible and constitutively expressed genes. The constitutively expressed genes have evolved more or less following the birth-and-death process, and the rates of gene birth and gene death are different between the two nematode species. By contrast, some heat-inducible genes show an intraspecies phylogenetic clustering. This suggests that they are subject to sequence homogenization resulting from gene conversion-like events. In addition, the heat-inducible genes show high levels of sequence conservation in both intra-species and inter-species comparisons, and in most cases, amino acid sequence similarity is higher than nucleotide sequence similarity. This indicates that purifying selection also plays an important role in maintaining high sequence similarity among paralogous Hsp70 genes. Therefore, we suggest that the CYT heat-inducible genes have been subjected to a combination of purifying selection, birth-and-death process, and gene conversion-like events.     

Carry-over effects of ozone and water stress on leaf phenological characteristics and bud frost hardiness of <Emphasis Type="Italic">Fagus crenata</Emphasis> seedlings

We examined the carry-over effects of ozone (O₃) and/or water stress on leaf phenological characteristics and bud frost hardiness of Fagus crenata seedlings. Three-year-old seedlings were exposed to charcoal-filtered air or 60 nl l^–1 O₃, 7 h a day, from May to October 1999 in naturally-lit growth chambers. Half of the seedlings in each gas treatment received 250 ml of water at 3-day intervals (well-watered treatment), while the rest received 175 ml of water at the same intervals (water-stressed treatment). All the seedlings were moved from the growth chambers to an experimental field on October 1999, and grown until April 2000 under field conditions. The exposure to O₃ during the growing season induced early leaf fall and reduction in leaf non-structural carbohydrates concentrations in the early autumn, as well as resulting in late bud break and reduction in the number of leaves per bud in the following spring. However, O₃ did not affect bud frost hardiness in the following winter. On the contrary, water stress did not affect leaf phenological characteristics, leaf and bud non-structural carbohydrates concentrations and bud frost hardiness. There were no significant synergistic or antagonistic effects of O₃ and water stress on leaf phenological characteristics, concentrations of leaf and bud non-structural carbohydrates and bud frost hardiness of the seedlings. These results show that the carry-over effects of O₃ can be found on the phenological characteristics and leaf non-structural carbohydrates concentrations, although there are almost no carry-over effects of water stress on phenological characteristics and winter hardiness of the seedlings.     

Stable isotope labeling of Arabidopsis thaliana for an NMR-based metabolomics approach

Nuclear magnetic resonance (NMR) will become a key technology in plant metabolomics with the use of stable isotope labeling and advanced hetero-nuclear NMR methodologies. To demonstrate the power of this approach, we performed multi-dimensional hetero-nuclear NMR analysis of metabolic movement of carbon and nitrogen nuclei in Arabidopsis thaliana. First, distinct ethanol-stress response was investigated using (13)C-labeled wild type and an ethanol-hypersensitive mutant plants. Furthermore, we followed nitrogen fluxes in (15)N-labeled seeds during the initiation of germination in vivo. The future role of stable isotope-labeling combined with advanced hetero-nuclear NMR in plant metabolomics is discussed.     

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.     

BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.