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M Kusakabe

Publications and source records attributed to M Kusakabe.

At least 37 records · Page 2Linked to original sources
PubMed2000

The TGF-beta family member derrière is involved in regulation of the establishment of left-right asymmetry.

Although a number of genes that are involved in the establishment of left-right asymmetry have been identified, earlier events in the molecular pathway developing left-right asymmetry remain to be elucidated. Here we present evidence suggesting that the transforming growth factor-beta family member derrière is involved in the development of left-right asymmetry in Xenopus embryos. Ectopic expression of derrière on the right side can fully invert cardiac and visceral left-right orientation and nodal expression, and expression of a dominant-negative form of derrière on the left side can partially randomize the left-right orientation and nodal expression. Moreover, while expression of the dominant-negative derrière does not inhibit the activity of Vg1 directly, it can rescue the altered left-right orientation induced by Vg1. Vg1 can induce derrière in animal cap explants. These results suggest that derrière is involved in earlier molecular pathways developing the left-right asymmetry.

Animals↗
PubMed2000

Mouse strain susceptibility to diethylnitrosamine induced hepatocarcinogenesis is cell autonomous whereas sex-susceptibility Is due to the micro-environment: analysis with C3H <--> BALB / c sexually chimeric mice.

In man, liver cancer is on the increase, especially in males. Sex differences also exist in rodent models. To elucidate the mechanisms, chimeric mice were produced by amalgamation of early embryos from high and low hepatocarcinogen-susceptible strains, C3H and BALB / c. Tumor formation was initiated with 10 mg / kg of diethylnitrosamine at the ages of 7 and 14 days and mice were sacrificed at 30 and 45 weeks. The chimeras were classified into XY <--> XY, XY <--> XX, XX <--> XY, and XX <--> XX in terms of sex chromosomes by means of polymerase chain reaction-simple sequence length polymorphism analysis (SSLP) using Y chromosome-specific Sry primers in combination with the D3Mit21 marker. Liver lesions were analyzed histopathologically, by immunostaining using a C3H strain-specific antibody and by DNA in situ hybridization with the Y chromosome-specific digoxigenin-labeled Y353 / B probe. Sex and strain genotyping by SSLP analysis matched histological observations, confirming the reliability of our system. The strain differences in liver tumor numbers of each strain type in XY <--> XY and XX <--> XX subtypes of C3H <--> BALB / c chimeras were retained well (P < 0. 0001 and P < 0.001, respectively), indicating a minimum influence of the C3H or BALB / c surrounding milieu on development of individual lesions. On the other hand, significant promotion of XX cell tumors was evident in phenotypically male sexually chimeric XY <--> XX and XX <--> XY chimeras for both C3H (P < 0.02) and BALB / c (P < 0.01) lesions compared to the XX <--> XX case. The results suggest the presence of hormonal or micro-environmental factors specific for males, which are not caused cell-autonomously. Basic strain differences, however, are determined by intrinsic genetic factors rather than the strain-dependent micro-environment.

Animals↗
PubMed2000

Mutation induction by heavy ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a lambda phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.

Animals↗
PubMed1999

Identification of two Smad4 proteins in Xenopus. Their common and distinct properties.

Smad family proteins have been identified as mediators of intracellular signal transduction by the transforming growth factor-beta (TGF-beta) superfamily. Each member of the pathway-restricted, receptor-activated Smad family cooperates and synergizes with Smad4, called co-Smad, to transduce the signals. Only Smad4 has been shown able to function as a common partner of the various pathway-restricted Smads in mammals. Here we have identified a novel Smad4-like molecule in Xenopus (XSmad4beta) as well as a Xenopus homolog of a well established Smad4 (XSmad4alpha). XSmad4beta is 70% identical to XSmad4alpha in amino acid sequence. Both of the Xenopus Smad4s can cooperate with Smad1 and Smad2, the pathway-restricted Smads specific for bone morphogenetic protein and TGF-beta, respectively. However, they show distinct properties in terms of their developmental expression patterns, subcellular localizations, and phosphorylation states. Moreover, XSmad4beta, but not XSmad4alpha, has the potent ability to induce ventralization when microinjected into the dorsal marginal region of the 4-cell stage of the embryos. These results suggest that the two Xenopus Smad4s have overlapping but distinct functions.

Amino Acid Sequence↗
PubMed1999

Tenascin-C in the cochlea of the developing mouse.

Tenascin-C is a glycoprotein of the extracellular matrix that acts in vitro as both a permissive and a nonpermissive substrate for neurite growth. We analyzed, by immunocytochemistry, the distribution of tenascin-C along neural growth pathways in the developing mouse cochlea. In the spiral lamina, tenascin-C coexists in a region where nerve bundles arborize. In the organ of Corti, tenascin-C lines the neural pathways along pillar and Deiters' cells before and during the time of nerve fiber ingrowth. By embryonic day 16, tenascin-C is abundant on the pillar side of the inner hair cell but does not accumulate on the modiolar side until about birth, a time after the arrival of afferent fibers. The synaptic zones beneath outer hair cells are strongly labeled during the time when early events in afferent synaptogenesis are progressing but not during the time of efferent synaptogenesis. At the age when most neural growth ceases, tenascin-C immunoreactivity disappears. Faint tenascin-C immunolabeling of normal hair cells, strong tenascin immunolabeling in pathological hair cells of Bronx waltzer (bv/bv) mice, and staining for beta-galactosidase, whose gene replaces tenascin in a "knockout" mouse, indicate that hair cells supply at least part of the tenascin-C. The changing composition of the extracellular matrix in the synaptic region during afferent and efferent synaptogenesis is consistent with a role for tenascin in synaptogenesis. The presence of tenascin-C along the growth routes of nerve fibers, particularly toward the outer hair cells, raises the possibility that growth cone interactions with tenascin-C helps to guide nerve fibers in the cochlea.

Aging↗
PubMed1999

Spatial and temporal changes in chondroitin sulfate distribution in the sclerotome play an essential role in the formation of migration patterns of mouse neural crest cells.

We have examined the roles of pertinent extracellular matrix molecules in the formation of the neural crest cell migration patterns in the sclerotome of the mouse embryo. The present data indicate that permissiveness for migration is inversely correlated with chondroitin sulfate content. Experimental removal of chondroitin sulfate proteoglycans in the embryo causes neural crest cells to migrate even within the posterior half of the somite, which they do not invade ordinarily. Moreover, three different sclerotomal regions defined by the presence or absence of the ventromedial and/or ventrolateral pathways are present along the anteroposterior axis and undergo systematic temporal changes that affect migration patterns. The most anterior portion of the sclerotome is conducive to both ventromedial and ventrolateral migration (Anterior Region). The intermediate portion is conducive to ventromedial migration only (Intermediate Region). No neural crest cells are seen within the posterior portion of the sclerotome (Posterior Region). At this level, they are observed exclusively in the dorsolateral space adjacent to the roof of the neural tube. With advancing embryonic development, the rostrocaudal length of the Anterior Region decreases and is accompanied by a corresponding enlargement of the Intermediate Region. These results suggest that temporal and regional differences in the sclerotome contribute to the neural crest cell migration patterns in the mouse. To refine our understanding of the underlying mechanisms, regional differences and temporal changes in the distribution of extracellular matrix molecules have been examined during migration. In the sclerotome, chondroitin sulfate displays distinct distribution patterns that are closely correlated with the migration patterns of mouse neural crest cells. Furthermore, their migration patterns are altered in embryos treated with the inhibitors of chondroitin sulfate proteoglycan biosynthesis, sodium chlorate, and beta-D-xyloside. In inhibitor-treated embryos, neural crest cell migration occurs even in the posterior portion of the sclerotome. The metameric organization of dorsal root ganglia is disturbed in these embryos. Our observations provide novel evidence for the importance of sclerotomal chondroitin sulfate distribution patterns in mouse crest cell migration patterns. We conclude that systematic spatiotemporal changes in the distribution of chondroitin sulfate proteoglycans are a key requisite for the formation of migration patterns of mouse neural crest cells in the sclerotome.

Animals↗
PubMed1999

Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain.

Recent in vitro studies have shown that the periventricular subependymal zone (SEZ) of the rodent brain is capable of de novo generation of neurons and glia. There is less information available on neurogenesis in the adult human brain, and no study has shown the clonal generation of neurons and glia from in vitro-generated "neurospheres." Here we describe the isolation of proliferative stem/progenitor cells within neurospheres from two different regions, the SEZ and the hippocampus, from surgical biopsy specimens of adult (24-57 years) human brain. Using light and electron microscopy; immunocytochemistry for a variety of neuronal, glial, and developmental (including extracellular matrix; ECM) markers; and the reverse transcriptase polymerase chain reaction to demonstrate different gene transcripts found in neurospheres, it is shown that the adult human brain harbors a complex population of stem/progenitor cells that can generate neuronal and glial progeny under particular in vitro growth conditions. These methods also show that these neurospheres contain both neurons and glia and demonstrate regional similarities at the mRNA level, indicating common stem/progenitor cell types within two different neurogenic regions of the adult human brain. In addition to the synthesis of developmentally regulated molecules such as the ECM protein tenascin-C, a variety of other genes (e.g., Pax 6) and proteins (e.g. , Bcl-2) involved in cell survival and differentiation are expressed by adult human brain neurospheres.

Adolescent↗
PubMed1999

Chromosomal localization of a gene responsible for vestibulocochlear defects of BUS/Idr mice: identification as an allele of waltzer.

Mice of the bustling mutant strain BUS/Idr have vestibulocochlear defects. bus/bus homozygotes, but not heterozygotes, are hyperactive and display an abnormal behavior such as circling, head bobbing and head tilting. To characterize BUS mice further, the auditory brain-stem response of the mutant was examined. In +/bus heterozygotes as well as control animals, the auditory brain-stem response was developmentally first recorded as early as 11 days of age and heterozygous and normal adults showed typical auditory brain-stem responses with five peaks in a threshold of 40-45 dB SPL. In contrast, bus/bus homozygotes showed no auditory brain-stem response at any age in response to stimuli up to 130 dB SPL, indicating that they are deaf throughout life. Linkage analysis revealed that the responsible gene, originally designated as bus, maps on chromosome 10, 1.09+/-0.9 cM distal to D10Mit127 and D10Mit59, and 0.72+/-0.51 cM proximal to three markers, D10Mit48, D10Mit112 and D10Mit258, at a site indistinguishable from that of the Albany waltzer, v(A/b). The results of allelism tests between BUS and Albany waltzer indicated that bus is allelic with v(Alb). From these data, we propose here that the bus mutation could represent another allele of waltzer, now designated v(bus).

Alleles↗
PubMed1999

Spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin during the development of chronic contact dermatitis.

In order to elucidate how chronic inflammation affects the organization of the extracellular matrix in the skin, a prolonged allergic contact dermatitis was induced in a mouse by repeated application to the ear of 2,4-dinitrofluorobenzene every 3 d for 66 d. Subsequently, the spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin were examined. In the acute phase of inflammation (day 3-day 12), the amount of fibronectin and tenascin-C increased markedly and were degraded, whereas the amount of fibulin-2 changed slightly. Abundant deposition of tenascin-C was observed in the connective tissue. Fibulin-1 and fibulin-2 distributed as fine fibrils. In contrast, the amounts of fibronectin and tenascin-C decreased and their degradation was suppressed in the chronic phase (day 15-day 66), but the amount of fibulin-2 increased. Tenascin-C was observed mainly at and underneath the epidermal basement membrane. In the subepidermal region, many fibulin-2-positive microfibrils were distributed. The amount and distribution of fibulin-1 did not change markedly in either phase. MMP-like enzymes of 62 kDa, probably activated MMP-2, were upregulated in the chronic phase, whereas components of 92, 85, or 67 kDa were highly induced in the acute phase. These results suggest that chronic inflammation in allergic contact dermatitis is associated with temporal changes in the expression, deposition, and degradation of inducible extracellular matrix components.

Animals↗
PubMed1999

Identification and immunohistochemical localization of annexin II in rat cornea.

PURPOSE: We have identified annexin II mRNA expression in the rat cornea and demonstrated immunolocalization of annexin II in normal and injured corneas. Furthermore, to investigate possible interaction between annexin II and its extracellular ligand tenascin during corneal wound healing, we also examined tenascin expression simultaneously. METHODS: Total RNA was extracted from the corneal tissue of male Wistar rats as well as from the cell cultures of corneal epithelial cells and keratocytes. cDNA was obtained by reverse transcription (RT). Annexin II mRNA expression was examined by polymerase chain reaction (PCR). Western blot analysis of annexin II protein was performed with protein samples that were obtained from the corneal tissue and cell cultures. In addition, the localization of annexin II and that of tenascin were clarified by immunohistochemical analysis, using both uninjured and epithelial scraped corneas. RESULTS: RT-PCR analysis revealed that annexin II mRNA was expressed in corneal tissues, epithelial cells and keratocytes. Western blot analysis of corneal epithelium and keratocytes showed a 38kDa band that corresponded to the molecular weight of annexin II. Immunohistochemical study showed that annexin II was present in keratocytes as well as the basal cells of corneal epithelium in the central cornea, and basal/suprabasal cells of the limbal epithelium. Positive annexin II immunoreactivity is translocated from cytoplasm to the cell periphery/extracellular position during the epithelial wound healing process. Annexin II and tenascin are coexpressed on the basal surface of limbal epithelium and the leading edge of the healing epithelium. The reappearance of cytoplasmic annexin II staining at the periphery of the cornea correlated with the epithelial cell proliferation. CONCLUSIONS: Annexin II is abundantly expressed in corneal tissue. The translocalization of annexin II protein suggests its role in the corneal epithelial migration during wound healing. The colocalization of annexin II and tenascin in migrating and proliferating corneal epithelial cells suggests that annexin II-tenascin interaction may play a role in epithelial wound healing.

Animals↗
PubMed1999

Clonality of urogenital organs as determined by analysis of chimeric mice.

Though the first mammalian chimera was reported in 1961, suitable markers for different animal strains which are easily detectable in histological sections of all or most organs have not existed. Chimeric mice were produced having an excellent histological marker, the C3H antigen, which is strain-specific and fulfills all the criteria for an ideal strain-specific histological marker. Using male and female C3H-Balb/c chimeric mice we examined epithelial cells of urogenital organs and their morphological or functional units, such as the glomerulus, to determine whether individual organs and their morphological subunits were monoclonal or polyclonal in origin. We found that the epithelial parenchyma of most male and female urogenital organs (the prostate, seminal vesicle, epididymis, ovaries, vagina, kidney, ureter and bladder) and their morphological subdivisions were derived from cells of both input strains, indicating a polyclonal origin for each organ and/or organ component. A notable exception was the uterus in which all individual uterine glands examined (n = 403) were found to be either entirely Balb/c or entirely C3H, indicating a monoclonal origin. The clonality of urogenital structures is discussed in terms of the morphogenesis of the urogenital system.

Animals↗
PubMed1999

Cone-beam CT angiography of the thorax: an experimental study.

The authors recently developed a cone-beam computed tomography (CT) scanner and this report presents their evaluation of its potential for thoracic vascular imaging. An X-ray tube and a video-fluoroscopic system were rotated around the objects and 360 projected images were collected in a 12-s scan. Each image was digitized and a 3 dimensional (D) image (256x256x256 voxel volume with a voxel dimension of 0.9x0.9x0.9 mm) was reconstructed. Two different 3D-CT angiographies were investigated in 2 pigs: right atriography and thoracic aortography. Each pig was anesthetized, mechanically ventilated and positioned within the scanner. Contrast agent was infused through the right atrium or the aortic root at a rate of 3 ml/s during the scan. The right atriography scan clearly delineated the anatomy of the pulmonary artery, heart chambers and thoracic aorta. The thoracic aortography scan also clearly delineated the aortic anatomy including the internal thoracic and intercostal arteries. In conclusion, cone-beam CT angiography is potentially useful for thoracic vascular imaging.

Angiography↗
PubMed1999

Corneal wound healing in tenascin knockout mouse.

PURPOSE: Tenascin (TN) is a large hexameric extracellular matrix glycoprotein that is expressed in developing organs and tumors. It has also been reported that TN is expressed in the embryonic cornea and during corneal wound healing. However, the role of TN in the cornea is not fully known. In this study, the role of TN in corneal wound healing was examined using the TN knockout (KO) mouse. METHODS: Two different injuries (a linear perforation wound and two 10-0 nylon suture wounds) were made separately on the corneas of both TNKO and congenic wild-type mice. The corneal wound healing was compared histologically, and the expression of TN and fibronectin (FN) on the injured cornea was examined immunohistochemically and by immunoblot analysis. RESULTS: Based on histologic analysis, there was no significant difference in the wound healing process between wild-type and TNKO mice in the linear incision experiment. However, the corneal stromata of TNKO mice were compressed prominently and devoid of migrating keratocytes in suture injury, which induced a more significant amount of TN than perforation wounds. Although FN expression on the sutured corneas of TNKO mice was upregulated during suture injury, the amount of FN protein was smaller than that of wild-type mice at the same time points after injury. CONCLUSIONS: In suture wounds, TN appears to enhance the amount of FN expression, and a lack of TN may impair stromal cell migration. TN plays a significant role in corneal wound healing, especially for wounds with mechanical stress.

Animals↗
PubMed1999

Development of vascular networks during the morphogenesis of intestinal villi in the fetal mouse.

The formation of villi was investigated in the proximal small intestine of fetal mice between 12 and 19 days of gestation in association with the development of vascular networks. Three-dimensional vascular networks were observed with confocal laser scanning microscopy after perfusing the fetuses with RITC-labelled gelatin. Some specimens were simultaneously stained for laminin by immunofluorescence to examine the epithelio-mesenchymal boundary. At 12 and 13 days of gestation, the epithelium of small intestine formed cylindrical tubes, and a loose plexus of large vessels formed in the mesenchyme. At 14 days of gestation, a dense plexus of smaller vessels was formed inside of the loose plexus and some capillary branches from this dense plexus developed beneath the epithelium. The epithelium began to make ingrowths in places where no capillaries were situated beneath the epithelium. The capillaries formed a loop in a concavity made between the ingrowths. As development proceeded, this concavity deepened and formed a villus. The top of the capillary loop always places at the top of the mesenchyme in the villus and the capillary loop then developed into a capillary network within the villus. These results suggest that the initial formation of intestinal villi in the fetal mouse occurs by an ingrowth of the epithelium and the developing vascular nets appear to play some role in villi formation. Throughout the villus formation, the top of the capillary loop in a villus would be anchored to the epithelial basement membrane or to the adjoining matrixes.

Animals↗
PubMed1998

Characterization of gene expression in mouse blastocyst using single-pass sequencing of 3995 clones.

To study the gene expression profile in the mouse blastocyst and to identify embryonic stage-specific genes, we randomly selected cDNAs derived from mouse blastocysts and sequenced a total of 3995 clones from one or both ends. Excluding the uninformative clones, 3395 clones were grouped as 937 different kinds of genes. Among these, 465 and 406 species showed similarity to known genes and expressed sequence tags (ESTs), respectively, whereas 66 species showed no significant similarity to any genes in known databases. Analysis of these cDNAs revealed that this library contained a variety of functional genes as well as genes that have not been detected in the human EST database; it should provide us with a useful resource for molecular analysis of developmental mechanisms. Although the human EST project is considered to represent roughly half of all genes, our findings indicate that many early stage developmental genes remain to be identified.

Animals↗
PubMed1998

A methylation imprint mark in the mouse imprinted gene Grf1/Cdc25Mm locus shares a common feature with the U2afbp-rs gene: an association with a short tandem repeat and a hypermethylated region.

We identified a sperm-specific methylation imprint mark (Site II) associated with a short tandem repeat sequence and a site/region methylated in both gametes (Site I) in the Grf1 locus on mouse chromosome 9, which shared a common feature with the U2afbp-rs gene. Sites or regions of gamete-specific methylation in imprinted genes are strong candidates for carrying information regarding the parental origin of alleles. The gamete-specific methylation pattern of Sites I and II was conserved after fertilization, but attained the somatic cell pattern by the blastocyst stage. In primordial germ cells, Site I was methylated, but Site II was unmethylated in both male and female embryos, suggesting that the sperm-specific methylation imprint mark in Site II was established during spermatogenesis. These common features in methylation imprint regions may be a clue to identifying regions carrying primary information for the imprinting regulation.

Animals↗