Biomedical subjects

M Kuro-o

Publications and source records attributed to M Kuro-o.

At least 19 recordsLinked to original sources

PubMed2006

Molecular cytogenetic characterization and chromosomal distribution of the satellite DNA in the genome of Oxya hyla intricata (Orthoptera: Catantopidae).

The genomic DNA of the grasshopper (Oxya hyla intricata) was subjected to electrophoresis after digestion with HaeIII, and the result showed two bands of highly repetitive DNA, approximately 200 and 400 bp in length. The 200-bp HaeIII-digested fragment was cloned and characterized by sequencing and fluorescence in situ hybridization (FISH). The results showed the presence of two distinct satellite DNA (stDNA) families: one consisting of a 169-bp repeated element having an A+T content of 60.9% and the other consisting of a 204-bp repeated element having an A+T content of 53.9%. No significant homology between the two stDNA families was observed. FISH showed that the chromosomal locations of these families are different from each other. The 169-bp element was located in the C-band-positive regions of the short arms of most of the chromosomes, whereas the 204-bp element was located in the centromeric regions of three chromosome pairs. These results imply that the origins of these two DNA families are different. The results of zoo-blot hybridization to the genomic DNA from four Oxya species, O. hyla intricata, O. japonica japonica, O. chinensis formosana, and O. yezoensis, suggest that the two stDNA families found in the present study are species-specific for O. hyla intricata.

Animals↗

PubMed2005

Karyotypic evolution and organization of the highly repetitive DNA sequences in the Japanese shrew-moles, Dymecodon pilirostris and Urotrichus talpoides.

The karyological relationship and organization of highly repetitive DNA sequences in Japanese shrew-moles were studied by zoo-blot hybridization and fluorescence in situ hybridization (FISH). When the genomic DNA of the eastern race of Urotrichus talpoides was digested with PstI, three fragments of highly repetitive DNA sequences, approximately 0.7, 0.9, and 1.4 kb in length, were observed as distinct bands. The results of FISH in the eastern race of U. talpoides using these three fragments separately as probes showed that the 0.7-kb PstI fragment was distributed in the centromeric regions of most chromosomes, and that the 0.9- and 1.4-kb fragments were predominantly located in the C-heterochromatin region of chromosome 13p. Although the western race of U. talpoides also had three PstI fragments, 0.9- and 1.4-kb PstI fragments were more ambiguous than those of the eastern race. The PstI- digested genomic DNA in Dymecodonpilirostris produced only a faint 0.9-kb band, and its signal patterns obtained by zoo-blot hybridization were clearly different from those of U. talpoides. The 0.7-kb fragment of U. talpoides hybridized strongly with the 0.9-kb fragment of D. pilirostris. In a FISH analysis, the 0.9-kb fragment of D. pilirostris hybridized with highly repetitive DNA in the centromeric regions of most chromosomes from both D. pilirostris and U. talpoides. Zoo-blot hybridization and FISH analyses suggest that the 0.9- and 1.4-kb PstI fragments were generated specifically in the genome of U. talpoides after the common ancestor differentiated into two extant shrew-mole species. A difference in the length of the centromeric elements between U. talpoides and D. pilirostris might be observed due to certain modifications of the repeating unit.

Animals↗

PubMed2002

Cytogenetic studies of Hynobiidae (Urodela)XVIII. A ZZ/ZW sex-determining mechanism in a hynobiid salamander species, Hynobius tokyoensis Tago.

The karyotype of Hynobius tokyoensis (2n = 56) was analyzed using three kinds of banding methods to determine the morphological differentiation of the sex chromosomes of this species. Salamanders and egg sacs were collected from seven localities around Tokyo, Japan. Of 28 chromosome pairs, microchromosome No. 21 was identified as a ZZ/ZW-type sex chromosome. The Z chromosome was acrocentric, whereas the W chromosome was submetacentric, with a heterochromatic, elongated short arm. Interestingly, the W chromosome is of three distinct types, W(A), W(B), and W(C), based on R-banding and Ag-NOR patterns. W(A) was detected in five populations from southern habitats, whereas W(B) and W(C) were detected in one population each from northern habitats. W(A), W(B), and W(C) were all found to carry Ag-NORs on their heterochromatic short arms. Considering the karyotypes of other species belonging to the same genus, we discuss the evolution of the sex chromosomes of H. tokyoensis.

Animals↗

PubMed2001

Connection between B lymphocyte and osteoclast differentiation pathways.

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-kappaB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL(-/-)), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.

Aging↗

PubMed2001

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.

Alternative Splicing↗

PubMed2001

Disease model: human aging.

Very little is known about the molecular mechanisms of human aging. This, at least in part, derives from a paucity of appropriate animal models of aging. Until recently, the senescence-accelerated mouse was the only mammalian model of aging. However, novel mouse models that exhibit multiple aging phenotypes have been developed in the past few years by disruption of the klotho gene, the telomerase gene and the genes involved in premature aging syndromes. These mouse models are expected to be important tools for aging research.

Aging↗

PubMed2001

Troglitazone improves endothelial function and augments renal klotho mRNA expression in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with multiple atherogenic risk factors.

Targeted disruption of the klotho gene induces multiple phenotypes characteristic of human aging, including arteriosclerosis, pulmonary emphysema and osteoporosis. Moreover, we previously observed that insufficient klotho expression in mice leads to endothelial dysfunction. In the present study, we used Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which exhibit hypertension, obesity, severe hyperglycemia and hypertriglyceridemia, and are thus considered an animal model of atherogenic disease, to test the effects of oral administration of troglitazone (200 mg/kg) on renal klotho mRNA expression and endothelial function. Systolic blood pressure, body weight, plasma glucose and triglyceride levels were all significantly higher in 30-week-old OLETF rats than in controls (LETO; Long-Evans Tokushima Otsuka) (p<0.05, n=7). In addition, endothelium-dependent relaxation of the aorta in response to 10(-5) M acetylcholine was significantly attenuated in OLETF rats (p<0.05, n=7), as was renal expression of klotho mRNA. Administration of troglitazone for 10 weeks significantly reduced systolic blood pressure, plasma glucose and triglyceride levels in OLETF rats, while augmenting endothelium-dependent aortic relaxation and renal klotho mRNA expression. These findings suggest that troglitazone protects the vascular endothelium against damage caused by the presence of multiple atherogenic factors.

Animals↗

PubMed2000

In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome.

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes (e.g., arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, and short life span). We have previously shown that mice heterozygous for a defect in the klotho gene upon parabiosis with wild-type mice show improved endothelial function, suggesting that the klotho gene product protects against endothelial dysfunction. In the present study, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat which demonstrates multiple atherogenic risk factors (e.g., hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia) and is thus considered an experimental animal model of atherosclerotic disease, we show that adenovirus-mediated klotho gene delivery can (1) ameliorate vascular endothelial dysfunction, (2) increase nitric oxide production, (3) reduce elevated blood pressure, and (4) prevent medial hypertrophy and perivascular fibrosis. Based on these findings, klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function (e.g., hypertension and vascular remodeling). Our findings establish the basis for the therapeutic potential of klotho gene delivery in atherosclerotic disease.

Adenoviridae↗

PubMed2000

Establishment of the anti-Klotho monoclonal antibodies and detection of Klotho protein in kidneys.

A novel gene, klotho (kl), which is involved in the development of a syndrome resembling human aging in mice, was recently identified. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. There also exists a splice variant of kl mRNA which encodes a putative secreted protein in both human and mouse. In this study, to characterize the physiological roles of Klotho protein, we established three monoclonal antibodies (mAbs) against the recombinant human Klotho protein. The mAbs are named KM2076 (rat IgG(2)a), KM2119 (rat IgG(2)b), and KM2365 (mouse IgG(1)). In Western blots, KM2076 and KM2119 specifically recognized a 130 kDa Klotho protein in the mouse and human kidney membrane fractions. To detect the human Klotho protein, the sandwich-type ELISA system with KM2076 and KM2365 was established. Using the ELISA system, we detected the human Klotho protein as low as 20 ng/ml in the supernatant of Chinese hamster ovary cells (CHO cells), introduced the human klotho gene. KM2076 and KM2119 specifically gave a positive staining by immunohistochemical staining in paraffin or frozen sections of the kidneys from wild-type mice but not in those from kl mice. Strong staining was observed especially in cortical renal tubules of the mouse kidney, where expression of klotho transcripts overlaps. KM2076 also showed a similar reaction pattern in the paraffin sections of rat and human kidneys. The mAbs established in this paper will serve as useful analytical, pathological, and diagnostic tools to disclose the role of Klotho protein in the suppression of a syndrome resembling human aging.

Aging↗

PubMed2000

Decreased insulin production and increased insulin sensitivity in the klotho mutant mouse, a novel animal model for human aging.

We have recently identified a novel gene, klotho (kl), which may suppress several aging phenotypes. A defect of kl gene 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 kl mutation (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/kl mice versus wild-type mice. The insulin content of the pancreas was significantly lower in kl/kl mice compared with wild-type mice. Decreased insulin production was also supported by Northern blot analysis showing lower levels of insulin mRNA in kl/kl mice. To examine how lower blood glucose levels may exist in kl/kl mice despite decreased insulin production, insulin tolerance tests (ITTs) were performed. The glucose decline following insulin injection was more severe in kl/kl mice versus wild-type mice, suggesting that insulin sensitivity was higher in kl/kl mice versus wild-type mice. In kl/kl mice, 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 klotho mouse, a novel animal model for human aging.

Adrenocorticotropic Hormone↗

PubMed2000

Increases in intracellular calcium of arterial smooth muscle cells in transgenic mice overexpressing Na+/H+ exchanger.

We produced transgenic mice overexpressing Na+/ H+ exchanger as a model of salt-sensitive hypertension and reported that dietary salt loading elevates blood pressure in these transgenic mice. We speculate that this blood pressure elevation may be attributed to the elevation of intraarterial smooth muscle Ca2+ concentration through Na+/Ca2+ exchange. To test this hypothesis, we measured the isometric tension of aortic rings and intracellular free calcium ([Ca2+]i) of cultured smooth muscle cells. In the transgenic mice, the aortic ring contraction induced by 5 mM caffeine (percentage of 60 mM K-induced contraction) was significantly greater than control mice (60.1 +/- 5.5% vs. 44.8 +/- 3.1%). The mean [Ca2+]i in vascular smooth muscle cells (VSMCs) of transgenic mice (123.1 +/- 19.7 nM) was higher than those in VSMCs of control mice (66.6 +/- 7.2 nM). These observations suggest that dietary salt loading increases the concentration of calcium in arterial smooth muscle cells in this transgenic mice. These findings are helpful in tracing the causes of salt-sensitive hypertension.

Animals↗

PubMed1999

BTEB2, a Krüppel-like transcription factor, regulates expression of the SMemb/Nonmuscle myosin heavy chain B (SMemb/NMHC-B) gene.

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a lambdagt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione S-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.

Age Factors↗

PubMed1999

Independent impairment of osteoblast and osteoclast differentiation in klotho mouse exhibiting low-turnover osteopenia.

We recently identified a new gene, klotho, which is involved in the suppression of multiple aging phenotypes. The mouse homozygous for a disruption of the klotho locus (kl/kl) exhibited multiple pathological conditions resembling human aging. Histomorphometric analysis revealed low-turnover osteopenia in kl/kl mice. The decrease in bone formation exceeded that of bone resorption, resulting in a net bone loss. The number of osteoblast progenitors determined by ex vivo bone marrow cultures was reduced in kl/kl mice. In addition, cultured osteoblastic cells derived from kl/kl mice showed lower alkaline phosphatase activity and matrix nodule formation than those from wild-type mice. Osteoclastogenesis in the coculture of marrow cells and osteoblastic cells was decreased only when marrow cells originated from kl/kl mice independently of the origin of osteoblastic cells. We also found that the expression of osteoprotegerin, an osteoclastogenesis inhibitor, was significantly upregulated in kl/kl mice. We conclude that a defect in the klotho gene expression causes the independent impairment of both osteoblast and osteoclast differentiation, leading to low-turnover osteopenia. Because this state represents a characteristic feature of senile osteoporosis in humans, kl/kl mice can be regarded as a useful model for investigating cellular and molecular mechanisms of age-related bone loss.

Aging↗

PubMed1999

Dietary salt loading increases nitric oxide synthesis in transgenic mice overexpressing sodium-proton exchanger.

We studied the role of nitric oxide (NO) synthesis in amelioration of blood pressure elevation during dietary salt loading in transgenic mice overexpressing sodium proton exchanger. Systolic blood pressure rose after starting salt loading only in the high-salt group of transgenic mice. However, this elevation of blood pressure was not continued. Urinary excretion of inorganic nitrite and nitrate in the high-salt group of transgenic mice was significantly higher than in the high-salt group of control mice. These results suggest that increased NO synthesis in response to salt loading is one of the anti-hypertensive mechanisms in transgenic mice overexpressing sodium proton exchanger.

Animals↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

Downregulation of the Klotho gene in the kidney under sustained circulatory stress in rats.

We recently reported the isolation of the klotho gene, which in predominantly expressed in the kidney and involved in human aging phenotypes. In our previous studies, we demonstrated that the Klotho protein or its metabolites may possibly function as humoral factor(s) and protect against endothelial dysfunction because acetylcholine-mediated NO production in arteries was impaired in heterozygous klotho deficient mice (kl/+). However, the pathophysiological significance of the Klotho protein has not been clarified yet. In the present study, we examined expression of the klotho gene in the kidney of the following rat models for human diseases: (1) spontaneously hypertensive rat, (2) deoxycorticosterone acetate-salt hypertensive rat, (3) 5/6 nephrectomized rat, (4) non-insulin-dependent diabetes mellitus rat (the Otsuka Long-Evans Tokushima Fatty rat), and (5) rat with acute myocardial infarction. The expression levels of klotho mRNA in the kidney in these models were significantly lower than controls except for MI rats. This is the first report showing the expression of the klotho gene in the kidney is regulated under sustained circulatory stress such as long-term hypertension, diabetes mellitus, and chronic renal failure.

Aging↗

PubMed1998

Klotho protein protects against endothelial dysfunction.

Arteriosclerosis caused by aging is recognized to be a crucial risk factor of cardiovascular disease. We recently established klotho mouse which causes age-related disorders including arteriosclerosis. However, no information on endothelial function of klotho mouse or the physiological role of klotho protein as a circulating factor is available. In this report, we demonstrate that 50% effective dose of aortic relaxation in response to acetylcholine in heterozygous klotho mice is significantly greater (4 x 10(-5) M) than in wild-type mice (8 x 10(-6) M, n = 7, p < 0.05) and that the vasodilator response of arterioles to acetylcholine is significantly attenuated in heterozygous (20% effective dose; 2 x 10(-6) M) and homozygous klotho mice (>1 x 10(-5) M) as compared with wild-type mice (1 x 10(-7) M, n = 7, p < 0.05). Nitric oxide metabolites (NO-2 and NO-3) in urine are significantly lower in heterozygous klotho mice (142 +/- 16 nmol/day) than wild-type mice (241 +/- 28 nmol/day, n = 13, p < 0.05). Parabiosis between wild-type and heterozygous klotho mice results in restoration of endothelial function in heterozygous klotho mice. We conclude that the klotho protein protects the cardiovascular system through endothelium-derived NO production by humoral pathways.

Acetylcholine↗

PubMed1998

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.

Activating Transcription Factor 2↗