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We have recently identified a novel gene, klotho(kl), which may suppress several aging phenotypes. A defect of klgene expression in the mouse results in a syndrome resembling human aging, such as arteriosclerosis, skin atrophy, osteoporosis, and pulmonary emphysema. To determine whether mouse homozygotes for the klmutation (kl/ kl) show abnormal glucose metabolism, an oral glucose tolerance test (OGTT) was performed at 6 to 8 weeks of age. Blood glucose levels during the OGTT were significantly lower in kl/ klmice versus wild-type mice. The insulin content of the pancreas was significantly lower in kl/ klmice compared with wild-type mice. Decreased insulin production was also supported by Northern blot analysis showing lower levels of insulin mRNA in kl/ klmice. To examine how lower blood glucose levels may exist in kl/ klmice despite decreased insulin production, insulin tolerance tests (ITTs) were performed. The glucose decline following insulin injection was more severe in kl/ klmice versus wild-type mice, suggesting that insulin sensitivity was higher in kl/ klmice versus wild-type mice. In kl/ klmice, an augmented expression of GLUT4 in skeletal muscle was demonstrated by both Northern blot analysis and Western blot analysis. Thus, we conclude that insulin production is decreased and insulin sensitivity is increased in the klothomouse, a novel animal model for human aging.

Inactivation of the klotho gene in mice results in multiple disorders that resemble human aging after 3 weeks of age. Because hematopoiesis, especially B lymphopoiesis, is affected in humans and mice by aging, we analyzed the hematopoietic state in homozygous klotho (kl/kl) mice. The kl/kl mice showed thymic atrophy and a reduced number of splenocytes. These mice had almost the normal number of myeloid cells, erythroid cells, IL-3-responsive myeloid precursors and colony forming units in spleen (CFU-S) in bone marrow (BM), but had a substantially decreased number of B cells in BM and peripheral blood as compared with wild-type mice. IL-7-responsive B cell precursors and all of the maturation stages of B cells in BM were also reduced. However, the function of hematopoietic stem cells including their capacity of B lymphopoiesis in vivo and in vitro was normal. Early B cell development was also normal in neonates and young kl/kl mice until 2 weeks old without aging phenotypes. RT-PCR analysis revealed that the level of IL-7 gene expression was significantly reduced in freshly isolated kl/kl BM cells. However, injection of IL-7 in kl/kl mice could not rescue the B lymphopenia. These findings indicate that Klotho protein may regulate B lymphopoiesis via its influence on the hematopoietic microenvironment.

The chromosome number of a Chinese salamander, Batrachuperus pinchonii, was re-examined. Adults and embryonic specimens had a diploid number of 66, with 33 bivalents during meiosis, in contrast to previous reported results. Furthermore, when C-banding analysis was performed with embryos, chromosomes with banding patterns homoeologous to those of Salamandrella keyserlingiiand Hynobiusspecies were found. It appears, therefore, that Batrachuperus, Salamandrellaand Hynobiusmight be derived from a common ancestral species in eastern Asia.

The R-banding technique of Dutrillaux et al. (1973) was modified in order to analyze the chromosomes of salamanders in the genus Hynobius. Embryonic cells of Hynobius nigrescens Stejneger from Nakakubiki County, Niigata Prefecture, and Hakui County, Ishikawa Prefecture, were cultured in medium containing 5-bromodeoxyuridine (BrdU). Banded metaphases were obtained by the FPG (fluorescent-plus-Giemsa) technique (Perry and Wolff, 1974), with slight modifications. With this modified R-banding technique, multiple, clear DNA replication bands were obtained on the chromosomes, and 18 of 28 chromosome pairs could be identified easily by their replication patterns in embryos collected from Nakakubiki County. A distinct heteromorphism in banding pattern was detected on the long arm of chromosome 9 in these embryos, but the frequency of this variant was too low for chromosome 9 to be a sex chromosome. Chromosomes 1 14, except for 6, 12 (for which the data were not satisfactory), and 9 (the variant type from Nakakubiki County), had the same replication patterns in embryos obtained from Nakakubiki and Hakui Counties.

Chromosome analysis was done in salamanders belonging to the genus Hynobius using the R-banding (RBG) technique. The embryonic cells of H. leechii Boulenger from Korea and H. nebulosus (Schlegel), H. tokyoensis Tago, H. nigrescens Stejneger, and H. retardatus Dunn from Japan were cultured in medium containing 5-bromodeoxyuridine. R-banding patterns in these five species were analyzed, except for most of the small-sized chromosomes. Eighteen out of 28 chromosome pairs in H. leechii, H. nebulosus, H. tokyoensis, and H. nigrescens, and 16 out of 20 chromosome pairs in H. retardatus could be identified by their R-banding patterns. The R-banding patterns in H. leechii, H. nebulosus, H. tokyoensis, and H. nigrescens were very similar to one another as are their karyotypes. Among these four species, about 90% of the total lengths of the karyotypes in each species showed homoeologous R-banding patterns, while among all the five species, including H. retardatus, homoeologies decreased to about 65%. The results imply that H. leechii, H. nebulosus, H. tokyoensis, and H. nigrescens are extremely close to one another phylogenetically, and that H. retardatus is a near phylogenetic relation to the other species of this study.

Cytogenetic studies were performed on two types of Japanese hagfish (Eptatretus okinoseanus) that eliminate about 45% (type A) and 55% (type B) of their DNA from presumptive somatic cells during the differentiation of somatic cells. The observations revealed inter- and intraindividual variations in the number of chromosomes in germ cells of both types of hagfishes. Although the modal number of chromosomes in the germ cells was 54 in both types, the percentage of cells with the modal number was rather low (38.6% [51/132] in five specimens of type A and 22.7% [25/110] in eight specimens of type B). In addition, one of seven type B specimens clearly had a modal number of 62 chromosomes. Another specimen of type B had a bimodal distribution of chromosome numbers, with peaks of 54 and 59 chromosomes. The observation of interindividual variations was supported by data on the amount of DNA in germ cells of type B specimens. However, these variations were rarely observed in somatic cells. These results suggest that supernumerary (B) chromosomes are maintained in germ cells and are eliminated together with some other chromosomes and/or chromatin from somatic cells.

Adult rabbit smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2 which are generated through alternative RNA splicing from a single gene (Nagai, R., Kuro-o, M., Babij, P. & Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). We previously reported that the expression of SM1 and SM2 during vascular development is differentially regulated at the level of RNA splicing, whereby SM1 is constitutively expressed from early development but SM2 appear after birth (Kuro-o, M., Nagai, R., Tsuchimochi, H., Katoh, H., Yazaki, Y., Ohkubo, A. & Takaku, F. (1989) J. Biol. Chem. 264, 18272-18275). We also demonstrated that embryonic vascular smooth muscles contain a third type of MHC isoform, referred to as SMemb in this report, which comigrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SM2. In the present study we have isolated and characterized a cDNA clone (FSMHC34) for SMemb. FSMHC34 encodes the light meromyosin region including the carboxyl terminus and showed 70% amino acid sequence identity with SM1 or SM2. SMemb is a nonmuscle-type MHC and identical with brain MHC, but clearly distinct from 196-kDa nonmuscle MHC in cultured smooth muscle cells. The expression of SMemb was predominant in embryonic and perinatal aortas, but down-regulated with vascular development. Interestingly SMemb was reexpressed in proliferating smooth muscle cells of arteriosclerotic neointimas. These results suggest that smooth muscle proliferation is coupled to the expression of SMemb and that dedifferentiation of smooth muscles toward the embryonic phenotype is involved in the mechanisms underlying atherosclerosis.

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A.85, 1047–1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3′-carboxyl coding region and a portion of the 3′-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry27, 3807–3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem.264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.

C- and R-banding analyses were performed on chromosomes of Salamandrella keyserlingii, in which 20 of 31 chromosome pairs could be identified. Banding patterns were compared between S. keyserlingii and specimens of Hynobius species of the family Hynobiidae. Between S. keyserlingii and six Hynobius species (2n = 56), there were four homoeologous and two partially homoeologous chromosome pairs. Between S. keyserlingii and H. retardatus (2n = 40), there were two homoeologous and two partially homoeologous chromosome pairs.

Chromosomal characteristics of the salamander speciesHynobius abei, from Ohimya (Kyoto) were revealed by the techniques of R-and C-banding. The karyotype ofH. abeiwas characterized by the shortness of an R-negative (C-positive) band in the terminal region of the long arm of chromosome 2 and a band encompassing the whole short arm of chromosome 10. These two bands inH. abeiwere the shortest among those of the variousHynobiusspecies that have been examined. Otherwise no differences could be detected betweenH. abeiand seven other pond-type species ofHynobius(2n=56) in terms of the banding patterns of 18 specifically identifiable pairs of their chromosomes.