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M Kusakabe

Publications and source records attributed to M Kusakabe.

At least 73 records · Page 4Linked to original sources
PubMed1997

TSC-36 (follistatin-related polypeptide) gene expression in estrogen receptor positive osteoblastic cell line, CDO7F.

Using an osteoblastic cell line (CDO7F) that was selected as an estrogen receptor positive cell by immunocytochemical detection and reverse transcription-polymerase chain reaction (RT-PCR) subtractive cDNA cloning using oligo(dT)30-Latex and PCR was performed to isolate a gene that is induced by estrogen. A cDNA clone was isolated and its nucleotide sequence identified it as TSC 36, previously called follistatin-related polypeptide. Northern blot analysis showed that the TSC-36 gene expression was certainly upregulated by estrogen. Tamoxifen also upregulated this gene expression. These findings indicate that TSC-36 may be one of the estrogen-regulated genes in osteoblastic cells.

Animals↗
PubMed1997

A note on the preparation of whole mount samples suitable for observation with the confocal laser scanning microscope.

The effects of fixatives and pretreatment on the immunofluorescence of whole mount specimens prepared for confocal laser scanning microscopy (CLSM) were examined. Intact villi were obtained from the proximal small intestine of mice fixed with 4% paraformaldehyde (4P) or 0.5% paraformaldehyde and 15% of saturated picric acid (PPa). Before immunostaining for laminin and tenascin, each specimen was pretreated with deoxycholate, while some 4P-fixed specimens received further pepsin pretreatment. Regardless of the fixatives employed, laminin and tenascin showed adequate immunofluorescence. Without pepsin pretreatment, the 4P-fixed specimens emitted conspicuous background fluorescence, and immunofluorescence was weak in the lamina propria. Pepsin pretreatment reduced the background fluorescence, but also diminished the immunofluorescence, especially that of tenascin. The PPa-fixed specimens displayed intense immunofluorescence of laminin and tenascin with very little background, even deeply within the lamina propria. When the PPa-fixed specimens were immunostained for vasoactive intestinal peptide, immunopositive nerve fibres were observed within the lamina propria. Ultrastructural investigation of the PPa-fixed specimens revealed that membranous structures in all cells were almost lost while tissue architecture was well preserved. These results indicate that PPa fixation and pretreatment with deoxycholate are suitable for preparing whole mount specimens for CLSM studies.

Animals↗
PubMed1997

Preprotachykinin A and cholecystokinin mRNAs in tenascin-gene knockout mouse brain.

Tenascin (TN), an extracellular matrix glycoprotein, exhibits various functions in the developmental stage of the mammalian brain. TN-gene deficient mice show abnormal behavior such as hyperlocomotion and poor swimming ability, and this abnormal behavior may derive from a low level of dopamine transmission in the striatum or hippocampus of the TN-gene disrupted mouse brain. We assayed preprotachykinin A (PPT) and cholecystokinin (CCK) mRNAs in the terminal fields of the dopamine neuron. The levels of PPT mRNA were significantly higher in the striatum, and the expression of CCK mRNA was markedly augmented in the hippocampus of the TN-knockout mice, compared to the wild or heterozygous mice. One possible explanation of the changes of PPT and CCK mRNA expressions is functional compensation against the low level of dopamine turnover rate in the TN-knockout mouse brain.

Animals↗
PubMed1997

Ion implantation and protein coating of detachable coils for endovascular treatment of cerebral aneurysms: concepts and preliminary results in swine models.

OBJECTIVE: Complete anatomic obliteration remains difficult to achieve with endovascular treatment of wide-necked aneurysms using Guglielmi detachable platinum coils (GDCs). Ion implantation is a physicochemical surface modification process resulting from the impingement of a high-energy ion beam. Ion implantation and protein coating were used to alter the surface properties (thrombogenicity, endothelial cellular migration, and adhesion) of GDCs. These modified coils were compared with standard GDCs in the treatment of experimental swine aneurysms. METHODS: In an initial study, straight platinum coils were used to compare the acute thrombogenicity of standard and modified coils. Modified coils were coated with albumin, fibronectin, or collagen and underwent Ne+ ion implantation at a dose of 1 x 10(15) ions/cm2 and an energy of 150 keV. Coils were placed in common iliac arteries of 17 swine for 1 hour, to evaluate their acute interactions with circulating blood. In a second study, GDCs were used to treat 34 aneurysms in an additional 17 swine. GDCs were coated with fibronectin, albumin, collagen, laminin, fibrinogen, or vitronectin and then implanted with ions as described above. Bilateral experimental swine aneurysms were embolized with standard GDCs on one side and with ion-implanted, protein-coated GDCs on the other side. The necks of aneurysms were evaluated macroscopically at autopsy, by using post-treatment Day 14 specimens. The dimensions of the orifice and the white fibrous membrane that covered the orifice were measured as the fibrous membrane to orifice proportion. Histopathological evaluation of the neck region was performed by light microscopy and scanning electron microscopy. RESULTS: Fibronectin-coated, ion-implanted coils showed the greatest acute thrombogenicity (average thrombus weight for standard coils, 1.9 +/- 1.5 mg; weight for fibronectin-coated coils, 8.6 +/- 6.2 mg; P < 0.0001). By using scanning electron microscopy, an intensive blood cellular response was observed on ion-implanted coil surfaces, whereas this was rare with standard coils. At Day 14, greater fibrous coverage of the necks of aneurysms was observed in the ion-implanted coil group (mean fibrous membrane to orifice proportion of 69.8 +/- 6.2% for the ion-implanted coil group, compared with 46.8 +/- 15.9% for the standard coil group; P = 0.0143). CONCLUSION: The results of this preliminary experimental study indicate that ion implantation combined with protein coating of GDCs improved cellular adhesion and proliferation. Future application of this technology may provide early wound healing at the necks of embolized, wide-necked, cerebral aneurysms.

Animals↗
PubMed1997

Three-dimensional distribution of extracellular matrix in the mouse small intestinal villi. Laminin and tenascin.

To investigate the three-dimensional distribution of laminin and tenascin in the villi of the mouse small intestine, we designed a new tissue preparation procedure for confocal laser scanning microscopy. Fixed tissue was pretreated with sodium deoxycholate and immunostained. The specimens displayed intense immunofluorescence even in the deeper sites of the lamina propria. Laminin was distributed like a sheet in the basement membrane of the epithelium as well as that of the capillaries. Additionally, it was associated with some tubular structures in the lamina propria. Many pores were found in the laminin sheet. By comparison, tenascin was distributed at the epithelial basement membrane and in the underlying connective tissue exhibiting a striped pattern. Tenascin was observed wrapping around the capillary wall in a spiral but was absent at the tip of the villi except in the capillary wall. Pores in the staining pattern of tenascin were also found as was the case for laminin. This three-dimensional reconstruction made it possible to observe more clearly the localization of these two extracellular matrix molecules.

Animals↗
PubMed1996

Primary monoclonal and secondary polyclonal growth of colon neoplastic lesions in C3H/HeN<-->BALB/c chimeric mice treated with 1,2-dimethylhydrazine immunohistochemical detection of C3H strain-specific antigen and simple sequence length polymorphism analysis of DNA.

To determine the clonality and cellular origin of colon pre-neoplastic and neoplastic lesions, C3H/HeN<-->BALB/c chimeric mice treated with 1,2-dimethylhydrazine (DMH) were investigated immunohistochemically using a specific antibody to C3H strain-specific antigen (CSA) enabling immunohistochemical discrimination of C3H cells in histological sections of chimeric mouse tissues. To confirm the results of immunostaining, simple sequence length polymorphism (SSLP) analysis was performed on DNA samples extracted from histological sections of adenocarcinomas. C3H/HeN<-->BALB/c chimeric mice were produced by an aggregation procedure and together with BALB/c and C3H/HeN animals were given weekly s.c. injections of 20 mg/kg body weight DMH for up to 20 weeks. At weeks 20 and 35 animals were killed and autopsied. In normal colonic mucosa of the chimeras, each gland was composed entirely of either CSA-positive or -negative cells and no mixed glands were found. Cells of all focal atypias in chimeric mice were, in each case, homogeneous for one or another of the parental types. Of 91 adenomas in chimeric mice, only one comprised both types of cell. Among 119 adenocarcinomas, 12 contained cells of both parental types. In these tumors, however, the 2 phenotypes were not mixed together at random but arranged in discrete areas, with intermingling limited to the junctions. SSLP analysis demonstrated DNAs extracted from CSA-positive and -negative tumors to exhibit the polymorphic patterns of C3H and BALB/c, respectively, while mixed CSA-positive and -negative tumors showed mixtures of both polymorphic DNA types.

1,2-Dimethylhydrazine↗
PubMed1996

Abnormal behavior and neurotransmissions of tenascin gene knockout mouse.

To examine the role of tenascin (TN) in vivo, we have produced mice in which the TN gene is inactivated. In behavioral studies, TN-knockout mice showed abnormal behavior such as hyperlocomotion and poor swimming ability. Biochemical analysis revealed that serotonin (5-HT) and dopamine (DA) transmission was decreased in the cerebral cortex, the hippocampus, or the striatum of TN-knockout mouse brain. The intraperitoneal administration of the DA receptor agonist, LY171555 (0.5 mg/kg, BW), inhibited the hyperlocomotion, and swimming behavior was transiently improved by the treatment with the 5-HT receptor agonist, 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride. These findings suggest that TN may play an important role in neurotransmissions related to behavior.

Animals↗
PubMed1996

Differential expression of tenascin in the skin during hapten-induced dermatitis.

Tenascin is a large extracellular matrix glycoprotein which is found in limited regions of normal adult tissues including the skin. We investigated the induction of tenascin expression in mouse skin during hapten-induced dermatitis. In the dorsal skin, hapten application first induced a transient expression of tenascin in deeper regions of the skin. Its distribution then spread over the whole dermis corresponding to the infiltration of Mac-2-positive macrophages. In the ear, tenascin was consistently found in the subcutaneous tissue on the inner side, but very little was seen on the outer side. Tenascin did appear transiently, however, on both sides under hapten treatment. In the early phase of allergic contact dermatitis, no apparent induction of tenascin expression was observed in the swollen ear. However, there was an abundant tenascin expression on both sides during healing. Tenascin expressed under normal conditions was mostly the 180-kDa isoform, while the 230-kDa isoform was markedly induced during healing of the dermatitis. These results suggest that tenascin, particularly the larger 230-kDa isoform, may play important roles in the pathogenesis and healing of hapten-induced dermatitis.

Animals↗
PubMed1996

Tenascin: growth and adhesion modulation--extracellular matrix degrading function: an in vitro study.

Tenascin (TN), a recently characterised extracellular matrix protein, largely confined to the process with the development of embryo in areas of epithelial-mesenchymal interactions and in areas where there are morphogenetic movements and tissue patterning, has a highly restricted expression in adult tissues. The expression of TN is enhanced in a variety of human neoplastic lesions. However, function(s) and molecular mechanisms of enhanced expression in neoplastic lesions remain unclear. We employed human tongue carcinoma cells (SCCKN), human salivary gland adenocarcinoma cells (SGT-1), normal mouse embryonic fibroblasts (NIH3T3-3) and K-ras-2 transformed fibroblasts (Cle-H3) in an in vitro study to elucidate the biological roles of TN. In in vitro studies, all the cell lines examined had enhanced secretion of TN in the presence of transforming growth factor-beta in a dose-dependent manner and TN itself was found to possess a growth-enhancing activity. Moreover, studies on adhesion of the cell lines on coated substrates of fibronectin (FN), laminin (LN), tenascin (TN), TN/FN and TN/LN showed that all the cells adhere and spread well on FN and LN. However, on TN they attach poorly and remain rounded. The relative concentrations of TN and FN affected the cellular adhesion and morphology. In SCCKN and SGT-1, but not in NIH3T3 and Cle-He3 fibroblasts, a higher concentration of TN inhibited cellular adhesion on fibronectin, suggesting that cells attach poorly on TN, it may interfere with the action of fibronectin, and the relative concentrations of TN, FN or LN may affect cellular adhesion and morphology which may differ in different cell types. When TN was added in the growth medium of exponentially growing cells, the cells lost their cell to cell contact and were seen to be separating. The presence of these extracellular matrix proteins were further tested to determine whether they could modulate the secretion of proteolytic enzymes responsible for extracellular matrix degradation by tumour cells, when the neoplastic cells but not the non-neoplastic cells grown on FN/TN substrate showed positive immunofluorescence for collagenase. FN, LN or TN alone did not induce collagenase in the tumour cells. If the same is true in vivo, although a number of factors and interactions may implicate the ultimate outcome, the enhanced expression of TN in neoplastic lesions may have potential implications for tumour growth, differentiation, cellular adhesion, invasion and metastasis.

3T3 Cells↗
PubMed1996

Monoclonal development of squamous cell carcinomas from polyclonal papillary or nodular hyperplasias in the forestomach of C3H/HeN<-->BALB/c chimeric mice treated with N-methyl-N-nitrosourea or diethylnitrosamine.

The clonality of epithelial proliferative lesions of forestomach carcinogenesis was immunohistochemically investigated in C3H/HeN<-->BALB/c chimeric mice using a specific antibody to C3H strain specific antigen (CSA) and as well as in terms of microsatellite DNA polymorphism patterns. The C3H/HeN<-->BALB/c chimeric mice were produced by an aggregation procedure. Male chimeric, C3H/HeN, and BALB/c animals were given N-methyl-N-nitrosourea (MNU) 0.5 mg/mouse once a week for a total of 10 times by intragastric intubation or 30 p.p.m. diethylnitrosamine (DEN) in their drinking water for 20 weeks. Those treated with MNU were killed at weeks 11, 25 and 45 and with DEN at week 35. Normal chimeric forestomach epithelium was found to demonstrate mixtures of epithelial cell groups composed of either CSA positive or negative cells. The same was the case for all simple hyperplasias. Papillary and nodular (PN) hyperplasias increased with time even after cessation of MNU treatment and many of them consisted of both CSA positive and negative cell groups. In one case, a CSA positive and a negative cancer were observed to have developed independently in the same PN-hyperplasia consisting of both parental cell types. In 28 tumor bearing chimeric mice, all squamous cell carcinomas (SCCs) were composed entirely of either CSA positive or negative tumor cells. However, in two animals with advanced CSA positive cancers and negative cancers, tiny cancer nests composed of both parental type cells were found in association. Microsatellite DNA polymorphism patterns of DNAs sampled from histological sections completely conformed with the outcomes of immunohistochemical staining. The results suggest that PN-hyperplasias are aggregates (polyclonal) of preneoplastic changes from which monoclonal SCCs are derived. Polyclonal cancers may also arise secondarily at low incidence during progression, due to two or more lesions coalescing.

Animals↗
PubMed1996

Dysfunction of the Orleans reeler gene arising from exon skipping due to transposition of a full-length copy of an active L1 sequence into the skipped exon.

We examined the genomic structure of the reeler gene in Orleans reeler mouse mutant. Exon skipping of the reeler gene caused a 220 bp deletion in the transcript, resulting in a frame shift of the reeler gene which disrupts the 8th EGF-like motif of the reeler product. Surprisingly, the skipped exon was inserted by the 7104 bp L1 element which carried the full-length stretch of the mouse L1 sequence, consisting of a 212 bp F-type tandem repeat, open reading frame 1 (ORF1), ORF2, the polyadenylation signal and a poly A stretch. The transposed L1 sequence was flanked by 13 bp of the target sequence at both ends. ORF1 and ORF2 of this L1 repeat element are thought to encode a component of the RNP particle and the reverse transcriptase, respectively. Orleans reeler was originally established by spontaneous mutation caused by L1 insertion, and this L1 sequence is considered to be potentially active for transposition in mouse genome.

Amino Acid Sequence↗
PubMed1996

Distinct stages of melanocyte differentiation revealed by anlaysis of nonuniform pigmentation patterns.

The injection of an antagonistic anti-murine c-kit monoclonal antibody ACK2 during mouse embryonic development produced three distinctive pigmentation patterns on the coat of the offspring. Pattern 1 consisted of pigmentation in craniofacial and caudal regions and was induced by an ACK2 injection between 9.5 and 11.5 days post coitum (dpc). In pattern 2, the entire coat was unpigmented and was induced by the injection at around 13.0 dpc. Pattern 3 consisted of pigmented patches spreading ventrolaterally from the dorsoanterior trunk regions towards the anterior and posterior directions and it was induced by ACK2 administered at 14.5-15.0 dpc. We investigated the embryological basis of these nonuniform pigmentation patterns to elucidate the process of melanoblast differentiation between lineage commitment and colonization into developing hair follicles. The results showed the following. (1) Melanocyte differentiation at the embryonic stage from 10.5 to 12.5 dpc progresses in a spatially nonuniform fashion, being faster in the craniofacial and caudal regions than in the trunk; pattern 1 reflects this. (2) Melanoblasts are activated to proliferate synchronously upon entering into the epidermis; pattern 2 correlates with this process. (3) c-kit functions as a survival signal for proliferating melanoblasts in the epidermis. (4) The melanoblasts that enter developing hair follicles can survive without a c-kit signal; pattern 3 essentially represents the hair follicles colonized by these cells. Analysis of the melanoblast distribution of ls/ls embryos that bear a loss-of-function mutation in the endothelin 3 gene suggested that endothelin 3 is required for early melanoblast differentiation before entering into the epidermis, whereas proliferation in the epidermis takes place without this molecule. Based on these data, we propose 4 distinct steps of embryonic melanocyte differentiation: (1) migration in the dermis, which requires both c-kit and endothelin 3; (2) a state before epidermal entry that is resistant to anti-c-kit mAb; (3) cell proliferation after entering the epidermal layer, which requires c-kit and endothelin receptor B but not endothelin 3 and (4) integration into developing hair follicles, which renders melanoblasts resistant to anti-c-kit mAb. Thus, melanoblast differentiation proceeds by alternately repeating c-kit -dependent and c-kit-independent stages and c-kit functions as a survival factor for the proliferating melanoblasts.

Animals↗
PubMed1996

Isolation of a germline-transmissible embryonic stem (ES) cell line from C3H/He mice.

We have isolated three embryonic stem (ES) cell lines from C3H/He mice using mouse STO cells as a feeder layer. One ES cell line (H-1) was male, and two (H-2 and H-3) were female, as determined by polymerase chain reaction, in situ hybridization, and karyotype analyses. All were immunocytochemically reactive with a C3H strain-specific antibody. Injection of cells from the female ES H-3 line into C57BL/6 blastocysts yielded four chimeras with slight coat color chimerism. All chimeras were male, and as expected, no germline-transmission was observed. By contrast, when male ES H-1 cells were injected into the perivitelline space of 8-cell C57BL/6 embryos, one male mouse with overt coat color chimerism was recovered, and it produced ES H-1-derived offspring exclusively. This germline-transmissible C3H/He cell line represents a novel addition to those ES lines currently employed for gene manipulation studies of development.

Animals↗
PubMed1995

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.

Animals↗
PubMed1995

Tenascin in breast cancer development--is epithelial tenascin a marker for poor prognosis?

(1) In mouse mammary gland development, immunoreactive tenascin (TN) is expressed in the dense mesenchyme surrounding the epithelial component of 14-day embryos, endbuds at puberty, and tumors. (2) Cells that produce TN are myofibroblastic and are characterized by nuclear invaginations, rough endoplasmic reticulum, and pinocytotic vesicles. These cells are not normally present in the stroma of mammary glands but present in cancer stroma, originating probably from fibroblasts differentiated under the influence of TGF-beta 1 stimulation. (3) Breast cancer cells are capable of synthesing TN under certain conditions. TN-non-producing MCF7 cells can produce TN when co-cultured with embryonic fibroblasts or with their conditioned medium. (4) Nine primary human breast cancers were examined for TN expression by in situ hybridization. TN mRNA was expressed in all nine cases in the stroma and in four cases in carcinoma cells as well. (5) Immunohistochemistry for TN was performed in human breast cancers, and it was found that the five-year survival after surgery was markedly lower in the group whose cancer cells were positive [corrected] for TN. TN expression in cancer cells appears to indicate poor prognosis.

Biomarkers, Tumor↗
PubMed1995

Germ line chimera produced by transfer of cultured chick primordial germ cells.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.

Animals↗