Tohru Komiya

Expression of multiple troponin T variants in neonatal chicken breast muscle

The types of troponin-T (TNT) expressed in neonatal chicken breast muscle were examined by two-dimensional gel electrophoresis (2-D PAGE), immunoblotting, and peptide mapping. When troponin from neonatal chicken breast muscle or whole lysate of the muscle was displayed on 2-D PAGE, multiple spots were observed in the TNT region on the gel. They differed slightly from those in adult breast- and leg-type TNT, but were positively stained with the antibody specific for TN-T. These results indicate that multiple spots observed in the TNT region are all TNT isoforms. The TNT isoforms in the neonatal breast muscle were classified into two groups, based on size. Each group contained about five variants. The first group with a larger size was in the molecular weight range of adult breast TNT, while the smaller-sized second group was in the molecular weight range of adult leg TNT. Overall peptide map patterns of variants in the first group and also that of adult breast TNT resembled each other, whereas those of variants in the second group were similar to that of adult leg TNT. The TNT of adult breast-type appeared at about 2- to 3-weeks posthatch, and thereafter became a major TNT isoform.

Generation of multiple troponin T isoforms is a common feature of the muscles in various chordate animals

1. Troponin T (TNT) expressed in various vertebrate skeletal and ascidian smooth muscles was examined by two-dimensional electrophoresis in combination with immunoblotting. 2. A monoclonal anti-TNT antibody, NT-302, exhibited binding ability to various TNT variants in the vertebrate and protochordate animals. 3. TNT isoform pattern differed among the animals, but the existence of multiple TNT isoforms in a single muscle tissue was the general feature of all the animals examined.

Characterization of the 38 kDa protein lacking in gastrula-arrested mutant Xenopus embryos

We have reported elsewhere that offspring from the No. 65 female of Xenopus laevis cleaved normally, but their development was arrested at the onset of gastrulation, like the Ambystoma ova-deficient (o) mutant, irrespective of mating with different wild-type males, and that an acidic, 38 kDa protein present in wild-type eggs was lacking in eggs of the female. In the current study, we first determined the partial amino acid sequence (VANLE) of one of the well-separated tryptic peptides from the protein, which was found in elongation factor 1 delta (Ef1delta) in Xenopus, and finally identified the protein as one of the Ef1delta isoforms, Ef1delta2, by peptide mass spectrometry. RT-PCR analyses for Ef1delta2 and its close homolog Ef1delta1 in wild-type oocytes and embryos demonstrated that both transcripts are maternal and Ef1delta1 is present more abundantly than Ef1delta2 throughout the stages examined. Importantly, the amount of the Ef1delta2 transcript per embryo decreased gradually after gastrulation, in accordance with the gradual decrease of the 38 kDa protein per embryo reported in our earlier study. Because pharmacological inhibition of translation induces gastrulation arrest in wild-type embryos, it is reasonable to conclude that the mutant embryos arrest in development due to the lack of Ef1delta2 that is indispensable for translation. Thus, the present study provides the first molecular information on the cause of the gastrulation-defective mutation in Amphibia.

A novel method for microinjection into Xenopus eggs and embryos supported in methylcellulose solution

We have developed a novel method for microinjection into Xenopus eggs and embryos. Microinjection was performed into eggs or embryos that were placed in wells (ca. 2.5 mm x 2.5 mm x 0.8 mm for each well) at the bottom of a commercially available hybridoma dish, which was filled with 1.5% methylcellulose solution. Eggs or embryos, rotated to a desired orientation in the viscous methylcellulose solution with a hair loop, could remain in the orientation for more than twenty minutes. Accordingly, samples such as mRNAs, DNAs, proteins and antisense morpholino oligonucleotides could be easily and efficiently microinjected into any part (animal, vegetal, dorsal, lateral or ventral) of more than five hundred eggs and embryos in one day. In addition, methylcellulose did not interfere with the development of the injected eggs and embryos.

Actin isoforms of insect muscles: Two-dimensional electrophoresis studies

Abstract 1. Two-dimensional electrophoresis was carried out to determine the isoelectric points of actins from thoracic and leg muscles of various kinds of insects (Coleoptera, Orthoptera, Odonata, Hemiptera, Lepidoptera, Hymenoptera and Diptera). 2. The isoelectric points of insect muscle actin ranged from 5.6 to 5.9, appreciably more basic than rabbit skeletal muscle actin (α-actin). 3. There was usually one predominant isoform of insect muscle actin except that two isoforms were detected in fly and wasp thoracic muscles. 4. In a cicada, the isoelectric points of actins from thoracic, leg and sound organ were 5.65, 5.68 and 5.57, respectively. 5. Actins from spider thoracic muscle, and crab claw and crayfish claw muscles also showed the isoelectric points of 5.5–5.7. 6. Thus arthropod muscle actins appear to have more basic isoelectric points than vertebrate skeletal muscle ones.

Xenopus Vasa Homolog XVLG1 is Essential for Migration and Survival of Primordial Germ Cells

Xenopus vasa-like gene 1 (XVLG1), a DEAD-Box Helicase 4 (DDX4) gene identified as a vertebrate vasa homologue, is required for the formation of primordial germ cells (PGCs). However, it remains to be clarified when and how XVLG1 functions in the formation of the germ cells. To gain a better understanding of the molecular mechanisms underlying XVLG1 during PGC development, we injected XVLG1 morpholino oligos into germ-plasm containing blastomeres of 32-cell stage of Xenopus embryos, and traced cell fates of the injected blastomere-derived PGCs. As a result of this procedure, migration of the PGCs was impaired and the number of PGCs derived from the blastomeres was significantly decreased. In addition, TUNEL staining in combination with in situ hybridization revealed that the loss of PGCs peaked at stage 27 was caused by apoptosis. This data strongly suggests an essential role for XVLG1 in migration and survival of the germ cells.