Preferential biosynthesis of ribosomal structural proteins by free and loosely bound polysomes from regenerating rat liver.

Homologous cell-free systems were prepared using free, total bound, tightly bound or KCl-sensitive loosely bound (KCl-sensitive) polysomes from regenerating rat liver. [14C]Leucine was incubated with one kind of polysomes and [3H]leucine with another kind. The reaction mixtures were then combined, and ribosomal structural proteins were purified as described previously [4], using two-dimensional polyacrylamide gel electrophoresis as the final step [5]. The 3H to 14C ratios of the purified fractions were estimated to compare the activities of the two kinds of polysomes for biosynthesis of ribosomal structural proteins. The following results were obtained: (1) The activity of free polysomes for biosynthesis of ribosomal structural proteins was about 3.6 or 2.4 times higher than that of total bound polysomes in two experiments in which 14C and 3H labeling was reversed. The radioactivities incorporated by free polysomes into most of the proteins separated on two-dimensional gel were found to be definitely higher than those in the surrounding areas, suggesting that most of the ribosomal structural proteins were synthesized by free polysomes. The activity of free polysomes for biosynthesis of ribosomal structural proteins was about 7 times higher than that of tightly bound polysomes, which were prepared by washing the microsomal membrane fraction with 0.5 M KCl. The radioactivities incorporated by tightly bound polysomes into the proteins separated on two-dimensional gel were only slightly higher than those in the surrounding areas, indicating that these polysomes had very low synthetic activity. (2) Preferential synthesis of histones by free polysomes was also shown using the same procedures. (3) KCl-sensitive polysomes which were released by washing the microsomal membrane fraction with 0.5 M KCl, were shown to have definitely higher activity than tightly bound polysomes for biosynthesis of ribosomal structural proteins. (4) From these results, it is concluded that most of the ribosomal structural proteins are preferentially synthesized by free and KCl-sensitive polysomes in regenerating rat liver.

Molecular cloning and expression analyses of mouse betaklotho, which encodes a novel Klotho family protein.

We report here the identification of mouse betaklotho (betakl), which encodes a type I membrane protein with high resemblance to Klotho (KL). Both betaKL and KL consist of two internal repeats with homology to family 1 glycosidases, while these essential glutamates for the enzymatic activities were not conserved. The identical pattern of substitution and variation in the substituted amino acids between these two proteins indicate that they likely to form a unique family within the glycosidase family 1 superfamily. During mouse embryonic development, strong betakl expression was detected in the yolk sac, gut, brown and white adipose tissues, liver and pancreas, and in the adult, predominantly in the liver and pancreas. Despite the high structural similarity between betaKL and KL, their expression profiles were considerably different and betakl expression was not induced in kl-deficient mouse mutants.

Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice.

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.

Klotho-deficient mice are resistant to bone loss induced by unloading due to sciatic neurectomy.

Unloading induces bone loss as seen in experimental animals as well as in space flight or in bed-ridden conditions; however, the mechanisms involved in this phenomenon are not fully understood. Klotho mutant mice exhibit osteopetrosis in the metaphyseal regions indicating that the klotho gene product is involved in the regulation of bone metabolism. To examine whether the klotho gene product is involved in the unloading-induced bone loss, the response of the osteopetrotic cancellous bones in these mice was investigated. Sciatic nerve resection was conducted using klotho mutant (kl/kl) and control heterozygous mice (+/kl) and its effect on bone was examined by micro-computed tomography (microCT). As reported previously for wild-type mice (+/+), about 30% bone loss was induced in heterozygous mice (+/kl) by unloading due to neurectomy within 30 days of the surgery. By contrast, kl/kl mice were resistant against bone loss induced by unloading after neurectomy. Unloading due to neurectomy also induced a small but significant bone loss in the cortical bone of the mid-shaft of the femur in the heterozygous mice; no reduction in the cortical bone was observed in kl/kl mice. These results indicate that klotho mutant mice are resistant against bone loss induced by unloading due to neurectomy in both cortical and trabecular bone and indicate that klotho is one of the molecules involved in the loss of bone by unloading.

Expression of Na+/Ca(2+) exchanger (NCX1) gene in the developmental mouse embryo and adult mouse brain.

The Na(+)/Ca(2+) exchanger gene, NCX1, is widely expressed in many tissues, encoding several isoforms through alternative RNA splicing. NCX1 deficient mice are known to be lethal at embryonic day 9-10 (E9-10). However, its expression pattern during embryogenesis is largely unknown. Therefore, to identify and compare the localization and alternatively spliced isoforms of NCX1 mRNA expressed in the developmental stages, we analyzed the mouse embryo. Northern blot analysis demonstrated that NCX1 mRNA was expressed from the earliest stage examined, E7. In situ hybridization analysis revealed that NCX1 mRNA was expressed in the heart alone until E10.5. However, at E14.5 and 16.5, NCX1 mRNA was expressed not only in the heart, but also in neuronal cells. In addition, the expression of NCX1 mRNA in the adult brain was most abundant in the hippocampus. Using reverse transcription-polymerase chain reaction (RT-PCR), we also identified the alternatively spliced isoforms expressed during each developmental stage. The restricted expression of the NCX1 gene suggested that NCX1 may play an important role in the developing mouse embryo.

Identification of EPS8 as a Dvl1-associated molecule.

Dishevelled (Dsh) is involved in both Wingless (Wg) and Frizzled (Fz) signaling pathways. To further determine the function of Dsh, we have performed yeast two-hybrid screening and isolated several genes encoding the molecules associated with the PDZ domain of Dvl1, one of the murine Dsh homologs. During the screening, we found that EPS8, which is a substrate for activated EGF receptor (EGFR), specifically interacted with Dvl1. This interaction was also confirmed in vitro. Through transfection studies, we observed the mutual action between Dvl1 and EPS8. Dvl1 was hyperphosphorylated in the presence of EPS8, whereas the tyrosine phosphorylation of EPS8 by activated EGFR was inhibited in the presence of Dvl1. Immunohistochemistry showed that Dvl1 and EPS8 expression overlap in particular tissues during organogenesis. These results indicate that interaction between Dvl1 and receptor tyrosine kinase signal plays certain roles in developmental events.

Disruption of the klotho gene causes pulmonary emphysema in mice. Defect in maintenance of pulmonary integrity during postnatal life.

Homozygous mutant klotho (KL(-/-)) mice exhibit multiple phenotypes resembling human aging. In the present study, we focused on examining the pathology of the lungs of klotho mice and found that it closely resembled pulmonary emphysema in humans both histologically and functionally. Histology of the lung of KL(-/-) mice was indistinguishable from those of wild-type littermates up to 2 wk of age. The first histologic changes appeared at 4 wk of age, showing enlargement of the air spaces accompanied by destruction of the alveolar walls, and progressed gradually with age. In addition to these changes, we observed calcium deposits in type I collagen fibers in alveolar septa and degeneration of type II pneumocytes in 8- to 10-wk-old KL(-/-) mice. Pulmonary function tests revealed prolonged expiration time in KL(-/-) mice, which is comparable with the pathophysiology of pulmonary emphysema. The expression level of messenger RNA for type IV collagen, surfactant protein-A and mitochondrial beta-adenosine triphosphatase was significantly increased in KL(-/-) mice, which may represent a compensatory response to alveolar destruction. Additionally, the heterozygous mutant klotho mice also developed pulmonary emphysema late in life, around 120 wk of age. These findings indicate that klotho gene expression is essential to maintaining pulmonary integrity during postnatal life. The klotho mutant mouse is a useful laboratory animal model for examining the relationship between aging and pulmonary emphysema.

Mutation of the mouse klotho gene leads to a syndrome resembling ageing.

A new gene, termed klotho, has been identified that is involved in the suppression of several ageing phenotypes. A defect in klotho gene expression in the mouse results in a syndrome that resembles human ageing, including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema. The gene encodes a membrane protein that shares sequence similarity with the beta-glucosidase enzymes. The klotho gene product may function as part of a signalling pathway that regulates ageing in vivo and morbidity in age-related diseases.

Molecular cloning of rat klotho cDNA: markedly decreased expression of klotho by acute inflammatory stress.

We have recently identified a novel gene, termed klotho, that is involved in the suppression of several aging phenotypes. The gene encodes a membrane protein that shares sequence similarity with the beta-glucosidases of bacteria and plants. In this study, we isolated rat klotho cDNA and examined its tissue distribution in rats. The deduced amino acid sequence of rat Klotho protein was 1014 amino acids in length and 94 and 85% homologous to those of mouse and human Klotho proteins, respectively. Northern blot analysis using the rat klotho cDNA probe identified a single transcript of 5.2 kb in size expressed predominantly in the kidney, while RT-PCR detected low levels of expression also in the brain, lung, intestine, and ovaries. During development, klotho expression in the kidney was markedly augmented after birth. Chromosomal localization of rat klotho was mapped to 12q12. Northern blot analysis showed that expression of klotho was markedly decreased by lipopolysaccharide (LPS) in vivo, suggesting that expression of klotho is affected by acute inflammatory stress. The present study leads to a better understanding of the physiologic and pathophysiologic roles of Klotho.

UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells.

We have obtained a novel transcriptional cofactor, termed undifferentiated embryonic cell transcription factor 1 (UTF1), from F9 embryonic carcinoma (EC) cells. This protein is expressed in EC and embryonic stem cells, as well as in germ line tissues, but could not be detected in any of the other adult mouse tissues tested. Furthermore, when EC cells are induced to differentiate, UTF1 expression is rapidly extinguished. In normal mouse embryos, UTF1 mRNA is present in the inner cell mass, the primitive ectoderm and the extra-embryonic tissues. During the primitive streak stage, the induction of mesodermal cells is accompanied by the down-regulation of UTF1 in the primitive ectoderm. However, its expression is maintained for up to 13.5 days post-coitum in the extra-embryonic tissue. Functionally, UTF1 boosts the level of transcription of the adenovirus E2A promoter. However, unlike the pluripotent cell-specific E1A-like activity, which requires the E2F sites of the E2A promoter for increased transcriptional activation, UTF1-mediated activation is dependent on the upstream ATF site of this promoter. This result indicates that UTF1 is not a major component of the E1A-like activity present in pluripotent embryonic cells. Further analyses revealed that UTF1 interacts not only with the activation domain of ATF-2, but also with the TFIID complex in vivo. Thus, UTF1 displays many of the hallmark characteristics expected for a tissue-specific transcriptional coactivator that works in early embryogenesis.