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Biomedical subjects

Y Nabeshima

Publications and source records attributed to Y Nabeshima.

At least 19 recordsLinked to original sources

A novel homeobox gene mediates the Dpp signal to establish functional specificity within target cells.

Morphogen gradients of secreted molecules play critical roles in the establishment of the spatial pattern of gene expression. During midgut development in Drosophila, secreted molecules of Decapentaplegic (Dpp) and Wingless (Wg) establish unique transcriptional regulation within target cells to specify the resultant cell types. Here we report the identification of a novel homeobox gene, defective proventriculus (dve), which is required for the midgut specification under the control of Dpp and Wg. In dve mutants, two distinct parts of the midgut, the proventriculus and middle midgut, are abnormally organized. The Wg signal regulates dve expression during proventriculus development. On the other hand, dve is a downstream target of Dpp in the middle midgut and defines the functional specificity of copper cells along with another Dpp target gene, labial. Thus, the dve gene acts under the two distinct extracellular signals at distant parts of the midgut primordia.

Amino Acid Sequence

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

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

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

Effective restoration of dystrophin-associated proteins in vivo by adenovirus-mediated transfer of truncated dystrophin cDNAs.

A series of truncated dystrophin cDNAs (3.1-4.2 kbp) containing only three, three, two or one rod repeats with hinge 1 and 4 (named deltaDysAX2, AX11, AH3, M3, respectively) or no rod repeat retaining either hinge 1 or 4 (named deltaDysH1, H4, respectively) were constructed. These cDNAs were introduced into skeletal muscle of adult mdx mice using the adenovirus vector with a strong CAG promoter. deltaDysAX2, AX11, AH3 and deltaDysM3 expressed themselves successfully and recovered dystrophin-associated proteins effectively. Especially 3.7 kbp cDNA for deltaDysM3 offers the possibility of an approach utilizing newly developed virus vectors, such as an adeno-associated virus vector, toward gene therapy of Duchenne muscular dystrophy.

Adenoviridae

Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein.

We previously established a novel mouse model for human aging and identified the genetic foundation responsible for it. A defect in expression of a novel gene, termed klotho (kl), leads to a syndrome resembling human aging in mice. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. In this report, we present the entire mouse kl gene organization. The mouse kl gene spans about 50 kilobases and consists of five exons. The promoter region lacks a TATA-box and contains four potential binding sites for SP1. We further show that two kl gene transcripts encoding membrane or secreted protein are generated through alternative transcriptional termination. These findings provide fundamental information for further study of the kl gene which may regulate aging in vivo.

Alternative Splicing

Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein.

Inactivation of the klotho (kl) gene in mice results in multiple disorders that resemble human aging. The mouse kl gene encodes a novel single-pass membrane protein with homology to beta-glucosidases. In this study, we have isolated a human homologue of the kl gene and determined its gene structure. The human kl gene is composed of 5 exons and ranges over 50 kb on chromosome 13q12. We have further identified two transcripts that encode a membrane or secreted protein. These transcripts arise from a single kl gene through alternative RNA splicing. Expression of the putative secreted form predominates over that of the membrane form. The present study provides fundamental information necessary for further analyses of the human kl gene and its functions.

Aging

Spatially- and temporally-restricted expression of meltrin alpha (ADAM12) and beta (ADAM19) in mouse embryo.

The cloning of the full-length cDNA encoding meltrin beta (ADAM19), one of the metalloprotease-disintegrins expressed in mouse myogenic cells, revealed that the meltrin beta gene encodes a membrane protein closely related to meltrin alpha (ADAM12) which participates in myotube formation in vitro. To delineate the functions of meltrin alpha and beta, we examined the expression patterns of their transcripts during embryogenesis. The meltrin alpha gene is activated in condensed mesenchymal cells that give rise to skeletal muscle, bones and visceral organs. Meltrin beta mRNA, in contrast, is markedly expressed in craniofacial and dorsal root ganglia and ventral horns of the spinal cord, where peripheral neuronal cell lineages differentiate. Heart, skeletal muscle, intestine and lung also express meltrin beta mRNA transiently. Although the meltrin alpha and beta transcripts exhibit distinct expression patterns during embryogenesis, both genes are mainly activated in mesenchymal cells that are derived from both mesoderm and ectoderm.

ADAM Proteins

Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates 'reserve cells'.

When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors. The differentiating part of the cell population followed a series of well-described events, including expression of myogenin, p21(WAF1), and contractile proteins, permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells did not initiate any of these events. Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression, which had been originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells were isolated and returned to the growth conditions, they progressed through the cell cycle and regained MyoD expression. These cells demonstrated identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture were designated 'reserve cells'. Upon serum deprivation, MyoD expression rapidly decreased as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells expressed myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells did not differentiate and became the reserve cells. We also demonstrated that ectopic expression of MyoD converted the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells.

Animals

Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions.

Asymmetric cell division is a general process used in many developmental contexts to create two differently fated cells from a single progenitor cell. Intrinsic mechanisms like the asymmetric transmission of cell-fate determinants during cell division, and extrinsic cell-interaction mechanisms, can mediate asymmetric divisions. During embryonic development of the Drosophila central nervous system, neural stem cells called neuroblasts divide asymmetrically to produce another multipotent neuroblast and a ganglion mother cell (GMC) of more restricted developmental potential. Intrinsic mechanisms promote asymmetric division of neuroblasts: for example, the transcription factor Prospero localizes to the basal cell cortex of mitotic neuroblasts and then segregates exclusively into the GMC, which buds off from the basal side of the neuroblast. In the GMC, Prospero translocates to the nucleus, where it establishes differential gene expression between sibling cells. Here we report the identification of a gene, miranda, which encodes a new protein that co-localizes with Prospero in mitotic neuroblasts, tethers Prospero to the basal cortex of mitotic neuroblasts, directing Prospero into the GMC, and releases Prospero from the cell cortex within GMCs. miranda thus creates intrinsic differences between sibling cells by mediating the asymmetric segregation of a transcription factor into only one daughter cell during neural stem-cell division.

Alleles

Laminin alpha2 chain-null mutant mice by targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)-deficient congenital muscular dystrophy.

Using the gene targeting technique, we have generated a new mouse model of congenital muscular dystrophy (CMD), a null mutant for the laminin alpha2 chain. These homozygous mice, designated dy3Kldy3K, are characterized by growth retardation and severe muscular dystrophic symptoms and die by 5 weeks of age. Light microscopy revealed that muscle fiber degeneration in these mice begins no later than postnatal day 9. In degenerating muscles, considerable amounts of TUNEL positive nuclei were detected as well as DNA laddering, suggesting increased apoptotic cell death was involved in the process of muscle fiber degeneration.

Animals

An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements.

The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this role in vivo. To directly test these questions, we disrupted the murine nrf2 gene in vivo. While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidant in vivo in both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygous nrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. The nrf2-deficient mice may prove to be a very useful model for the in vivo analysis of chemical carcinogenesis and resistance to anti-cancer drugs.

Animals

Extension injury of the thoracolumbar spine.

STUDY DESIGN: This is a report of a patient with a rare fracture in the thoracolumbar spine. OBJECTIVES: To illustrate the previously undescribed fracture in the thoracolumbar spine and to propose a new mechanism of this rare lesion with a literature review. SUMMARY OF BACKGROUND DATA: Extension injury of the thoracolumbar spine is uncommon. Although there have been several reports of hyperextension injuries in the thoracolumbar spine, the injury mechanism of the present case was different from those in the previously reported cases. METHODS: The clinical findings, roentgenographic appearance, treatment, and follow-up result were presented, and the mechanism of this lesion was analyzed. RESULTS: Physical examination revealed no neurologic deficit. Radiographs showed the oblique fracture line extended from the middle region of the L1-L2 disc to the posteroinferior edge of the L2 vertebral body, with the widening of bilateral L2-L3 facet joints. The patient was treated conservatively with a successful outcome. Two years after his injury, the patient had resumed his previous activity completely. CONCLUSIONS: The authors considered that the mechanism of this rare injury was a combination of hyperextension force and axial loading.

Adult

Identification of a chick homologue of Fringe and C-Fringe 1: involvement in the neurogenesis and the somitogenesis.

In Drosophila, Serrate plays an important role to the wing margin formation. A putative secretory protein, Fringe, is indispensable for the wing margin formation inducing Serrate and other genes. Recently, Xenopus homologues of Fringe were identified and one of them, lunatic Fringe (X-lFng), was demonstrated to be involved in mesoderm induction. We have identified two chick Fringe homologous genes by reverse transcription-polymerase chain reaction and cDNA library screening. One of them, C-Fringe 1, showed sequence similarity to X-lFng. In situ hybridization study of C-Fringe 1 has demonstrated its expression in the developing nervous system and in the presomitic mesoderm. The hindbrain and spinal cord showed the distinct stripe pattern expression which was complementary to that of C-Serrate, indicating the correlation between them in vertebrate.

Amino Acid Sequence

Still life, a protein in synaptic terminals of Drosophila homologous to GDP-GTP exchangers.

The morphology of axon terminals changes with differentiation into mature synapses. A molecule that might regulate this process was identified by a screen of Drosophila mutants for abnormal motor activities. The still life (sif) gene encodes a protein homologous to guanine nucleotide exchange factors, which convert Rho-like guanosine triphosphatases (GTPases) from a guanosine diphosphate-bound inactive state to a guanosine triphosphate-bound active state. The SIF proteins are found adjacent to the plasma membrane of synaptic terminals. Expression of a truncated SIF protein resulted in defects in neuronal morphology and induced membrane ruffling with altered actin localization in human KB cells. Thus, SIF proteins may regulate synaptic differentiation through the organization of the actin cytoskeleton by activating Rho-like GTPases.

Actins

Dp260 disrupted mice revealed prolonged implicit time of the b-wave in ERG and loss of accumulation of beta-dystroglycan in the outer plexiform layer of the retina.

Dp260 is a C-terminal isoform of dystrophin and is expressed specifically in the retina. Abnormal electroretinograms (ERG) in some Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients are likely linked to a disruption of Dp260. To clarify the importance of Dp260 in the retina, we examined dystrophin exon 52 knock-out mice, whose expression of Dp260 is impaired. We also confirmed the localization of Dp260 in the outer plexiform layer (OPL) of the retina. Disruption of Dp260 causes a change in the localization of beta-dystroglycan, which is normally found in the OPL of the retina. This suggests a requirement for Dp260 for normal formation of the dystrophin-dystroglycan complex in the retina. Dp71, also expressed in the retina, was, however, not detected in the OPL. The difference in localization of Dp260 and Dp71 implies that the two isoforms have different functions. The dystrophin exon 52 knock-out mice had a prolonged implicit time of the b-wave in ERG, although no significant change was observed in amplitude. These ERG findings differed from those of DMD and BMD patients, especially with regard to amplitude of the b-wave, but make it clear that Dp260 is required for normal electrophysiology.

Animals

Effect of maternal exercise on fetal umbilical artery waveforms: the comparison of IUGR and AFD fetuses.

OBJECTIVE: To determine the effect of maternal exercise on the fetal blood flow of normal and growth-retarded fetuses. METHODS: The effect of maternal exercise on fetal blood flow was investigated with 24 pregnant women between 36 and 42 weeks' gestation. Seventeen pregnancies were uncomplicated (normal group) and the other 7 were complicated with growth-retarded fetuses (IUGR group). They underwent graded walking on a treadmill with an upper limit of 150 bpm for the maternal heart rate. The systolic over diastolic velocity ratio (S/D ratio) of the umbilical artery was measured before exercise, and at 3, 5, 10, and 15 minutes after exercise. RESULTS: The mean S/D ratio of the IUGR group before exercise was significantly higher than that of the normal group (p < 0.05). The S/D ratio did not change significantly after exercise, in either the normal group or the IUGR group. CONCLUSION: This study suggests that maternal exercise at this level of intensity and duration does not have any harmful effect, even on growth-retarded fetuses that might have placental dysfunctions.

Diastole