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M Katsuki

Publications and source records attributed to M Katsuki.

At least 55 records · Page 3Linked to original sources
PubMed1999

Functional analysis of microtubule-binding domain of bovine MAP4.

Bovine microtubule-associated protein 4 (MAP4) consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain of MAP4 is further divided into three subdomains: a region rich in proline and basic residues (Pro-rich region), a region containing four repeats of an assembly-promoting (AP) sequence, which consists of 22 amino acid residues (AP sequence region), and a hydrophobic tail region (Tail region). The subdomain structure of MAP4 microtubule binding domain is similar to those of other MAPs (MAP2 and tau). In order to study the function of each subdomain per se of bovine MAP4 microtubule-binding domain, we purified a series of truncated fragments of MAP4, expressed in Escherichia coil. Binding affinity of the PA4T fragment (containing the Pro-rich region, the AP sequence region and the Tail region) is only four times higher than that of the A4T fragment (containing the AP sequence region and the Tail region), while the microtubule nucleating activity of the PA4T fragment is far greater. We propose that the Pro-rich region promotes the nucleation of microtubule assembly. The A4 fragment (corresponding to the AP sequence region) stimulated the assembly of tubulin into coldstable amorphous aggregates. The AP sequence region of MAP4 failed to promote microtubule assembly. On the other hand, the fragment has an activity to stimulate microtubule elongation. The function of the MAP4 Tail region is not clear at present. The A4T fragment (containing the AP sequence region and the Tail region) promote both microtubule nucleation and elongation step, but the A4 fragment only promotes microtubule elongation, suggesting that the Tail region is indispensable for the nucleation step. However, the fragment containing only the Tail region could not bind to microtubule. Although MAP4 was considered to be long, thin and flexible molecule, never the Tail region may contribute to be the proper folding of MAP4, and/or may interact with other molecules. We concluded that both the Pro-rich region and the AP sequence region take part in the promotion of tubulin polymerization, and that the former is important for the lateral protofilament-protofilament interaction, and the latter is important for the longitudinal affinity between each tubulin dimer in a protofilament.

Animals↗
PubMed1998

Rac1 is required for the formation of three germ layers during gastrulation.

The Rac1, a member of the Rho family proteins, regulates actin organization of cytoskeleton and cell adhesion. We used genetic analysis to elucidate the role of Rac1 in mouse embryonic development. The rac1 deficient embryos showed numerous cell deaths in the space between the embryonic ectoderm and endoderm at the primitive streak stage. Investigation of the primary epiblast culture isolated from rac1 deficient embryos indicated that Rac1 is involved in lamellipodia formation, cell adhesion and cell migration in vivo. These results suggest that Rac1-mediated cell adhesion is essential for the formation of three germ layers during gastrulation.

Animals↗
PubMed1998

Identification of novel pancreas-specific regulatory sequences in the promoter region of human pancreatic secretory trypsin inhibitor gene.

The human pancreatic secretory trypsin inhibitor (PSTI) genes introduced into mice are specifically expressed in pancreas. The 1.0 kilobase pairs of PSTI 5'-flanking sequence directed preferential expression of a linked reporter chloramphenicol acetyltransferase, which was active in a PSTI-expressing pancreatic cell line (AR42j) but not in a PSTI-nonexpressing fibroblast cell line (XC). Two positively acting elements were found, Region I (-161/-116) and Region II (-103/-74), as defined by transfection and binding assays with AR42j cells. Region II is sufficient for the pancreas-specific expression, but the presence of both Regions I and II is needed for the maximum activity. Sequence studies also revealed that these two elements differ from the previously identified recognition sequence for pancreas transcription factor 1 (PTF1). When the same set of experiments was done with XC cells, one negatively acting element was identified, Region IV (-154/-137). Interestingly, Regions I and IV share a core sequence (-149/-139), CAATCAATAAC. These results suggest that this novel element regulates the human PSTI gene expression positively in pancreatic cells but negatively in nonpancreatic cells.

Animals↗
PubMed1998

In vivo detection of mutations induced by aflatoxin B1 using human CYP3A7/HITEC hybrid mice.

CYP3A7-M10 mouse is a transgenic mouse carrying human CYP3A7 cDNA, in which CYP3A7 is expressed in the small intestine but not in the kidney. HITEC mouse is a transgenic mouse developed to detect mutagenic potency of various chemicals in vivo. The M10/HITEC mouse was established by crossmating of these two strains of mice. When a 9,000 x g supernatant fraction prepared from the small intestine was added to an incubation mixture for Ames test with Salmonella typhimurium TA98 strain to examine the mutagen-producing activity from aflatoxin B1 (AFB1), the mutagen-producing activities of the 9, 000 x g supernatant fraction from the small intestine was found to be 1.7-fold higher in the M10/HITEC mice than in HITEC mice. Such a difference in the capacity to activate AFB1 was not seen with the 9, 000 x g supernatant fraction from the kidney from both strains of mice. Male M10/HITEC mice of 8 weeks old were treated with a single i.p. injection of AFB1 ( 8 mg/kg body weight). The mutation of the introduced rpsL gene in the genomic DNA from the small intestine and the kidney was analyzed. The mutation frequency in the small intestine of M10/HITEC mice was significantly higher (p<0.05) than that of HITEC mice, while the mutation frequency in both strains was similar in the kidney. These results provide the first evidence for the toxicological function of CYP3A7 in vivo.

Aflatoxin B1↗
PubMed1998

Overexpression of a minor component of myelin basic protein isoform (17.2 kDa) can restore myelinogenesis in transgenic shiverer mice.

Shiverer (shi) mice, which are neurologically mutant, lack a large portion of the gene for the myelin basic proteins (MBPs), have virtually no myelin in their central nervous system (CNS), and shiver, undergo seizures, and die early. At least five types of MBPs (21.5, 18.5, 17.3, 17.2 and 14.0 kDa) are known to be generated through alternative splicing from a single MBP gene. We have produced transgenic shi mice carrying a cDNA encoding mouse 14-kDa MBP isoform, the most abundant form of MBPs, under control of a mouse MBP gene promoter, and showed that expression of the 14-kDa MBP can restore CNS myelination. To test whether the 17.2-kDa MBP isoform, one of the minor components of MBPs, can also elicit myelination in homozygous shi mutants, we produced seven independent transgenic shi mice carrying cDNA encoding the mouse 17.2-kDa MBP isoform, and the transcription of which was driven by a mouse MBP gene promoter. The axons in the cerebellum of one transgenic line, which exhibited the highest expression of transgene-derived mRNA ( approximately 50% of the level of total MBP mRNA in the normal mouse brain), were myelinated. This mouse exhibited nearly normal behavior. These findings indicate that the 17.2-kDa MBP isoform, even when the only 17.2-kDa MBP isoform is present, has the ability to elicit CNS myelination in transgenic shi mice. This transgenic strategy will be useful for elucidating the role of each type of MBP isoform in CNS myelinogenesis.

Alternative Splicing↗
PubMed1998

Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas.

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Actin Cytoskeleton↗
PubMed1998

Mice lacking smooth muscle calponin display increased bone formation that is associated with enhancement of bone morphogenetic protein responses.

BACKGROUND: Calponin is a calmodulin-and actin-binding protein expressed in smooth muscle. It promotes actin polymerization and inhibits actin-activated myosin ATPase activity. Despite the molecular and functional characterization of calponin in vitro, the physiological role of calponin in vivo has not been clarified. RESULTS: We investigated the in vivo function of smooth muscle calponin (also called basic calponin or calponin h1) by generating mice carrying a targeted mutation in both alleles of the calponin gene. Mice lacking basic calponin expression displayed enhanced ectopic bone formation in vivo, induced by recombinant human bone morphogenetic protein-2 (rhBMP-2), and an augmentation of the degree of osteoblastic differentiation of embryonic mesenchymal cells when they were stimulated by rhBMP-2. Basic calponin messenger RNA was shown to be expressed in developing and healing bone tissues, and in undifferentiated MC3T3-E1 osteoblasts. An examination of the skeletons of mutated mice showed an early onset of cartilage formation and ossification, and increased postnatal bone formation characterized by an increase in the number of activated periosteal osteoblasts. Bone fracture healing was accelerated in mutated mice. CONCLUSION: This is the first demonstration of animals with enhanced BMP responsiveness in host cells, suggesting that endogenous basic calponin may play a negative role in an osteogenic programme.

Animals↗
PubMed1998

The high susceptibility of heterozygous p53(+/-) mice to malformation after foetal irradiation is related to sub-competent apoptosis.

PURPOSE: To investigate the relationship between the incidence of radiation-induced malformations and the extent of p53-dependent apoptosis. MATERIALS AND METHODS: Wild-type p53(+ / +) and heterozygous p53(+ / -) mice were exposed to X-rays at the mid-gestational period. The incidence of anomalies and prenatal deaths, the extent of apoptosis, and the levels of p53 protein were assessed. RESULTS: After X-irradiation with 2 Gy, the incidence of malformation (corrected for control levels) was 0 and 30%, respectively, for p53(+ / +) and p53(+ / -). After irradiation of p53(+ / +) foetuses with 3 Gy, the frequency (F) of apoptotic cells rapidly peaked at 80% at 4 h and fell close to the control level at 48 h. The relationship between F 4h after irradiation and dose (D) (1-3Gy) is accurately expressed by a single-hit equation, F= 1 -exp ( -(a + bD)¿, where the radiation-induced apoptosis rate, b, is 0.47 for the wild type and 0.22 for the heterozygous mice. The X-irradiated foetuses showed no increase in the levels of p53 protein. CONCLUSION: The higher susceptibility of irradiated p53(+ / -) foetuses to malformation is related to a twofold lower rate of apoptosis; competent removal by apoptosis of damaged cells from irradiated tissues is impaired dramatically if one of two wild-type p53 alleles is lost. The frequency of apoptotic cells in the wild type reached a maximum 4h after foetal irradiation with no measurable increase in the level of p53 protein, indicating that radiation-induced p53-mediated foetal apoptosis depends on non-transcriptional events.

Alleles↗
PubMed1998

Production of chimeric mice from cryopreserved blastocysts.

Mouse blastocysts derived from in vivo fertilization were cryopreserved by a simple vitrification method. Most of the cryopreserved blastocysts survived while demonstrating a normal morphology after warming (88.1%). The rate of cryopreserved blastocysts which developed to term as normal young was 56.8%, and chimeric mice were also produced from cryopreserved blastocysts injected with ES cells. As a result, 3.4% of the injected embryos developed to term as germ line chimeric mice.

Animals↗
PubMed1997

The 'assembly-promoting sequence region' of microtubule-associated protein 4 failed to promote microtubule assembly.

In order to study the function of the bovine MAP4 microtubule-binding domain (the assembly-promoting (AP) sequence region), a fragment corresponding to the AP sequence region was prepared using an Escherichia coli expression system. When the fragment was mixed with purified tubulin at 37 degrees C, the fragment caused a time- and dose-dependent turbidity increase, and the fragment bound to tubulin. However, the products were cold-stable, and amorphous aggregates were observed by electron microscopy. Using axonemes as the seeds for microtubule assembly, the microtubule-elongating activity of the fragment was examined. A dose-dependent turbidity increase of the sample was observed, and electron microscopic observation revealed that microtubules were dose-dependently elongated from the axonemes. Consequently, the AP sequence region does not nucleate microtubules, but elongates them.

Adrenal Cortex↗
PubMed1997

Reinnervation of denervated muscle by transplantation of fetal spinal cord to transected sciatic nerve in the rat.

When motor neurons in the spinal cord are destroyed, regeneration of motor axons and muscle reinnervation cannot be expected. We attempted reinnervation of the denervated muscle, i.e. motor unit reconstruction, using transplantation of the fetal spinal cord to the peripheral nerve. The sciatic nerve of an adult rat was resected for 20 mm, and a cavity was prepared using an autologous femoral vein at the distal stump of the nerve. The fetal spinal cord was then transplanted into the venous cavity. After 3-6 months, no voluntary muscle contraction was observed due to the absence of communication with the central nervous system. However, reinnervation of the muscles via the sciatic nerve by the transplanted spinal neurons was demonstrated electrophysiologically and histochemically. This suggested that a motor unit can be reconstructed by fetal spinal cord transplantation even if the original motor neurons in the spinal cord are not available.

Animals↗
PubMed1997

Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a degenerative disorder of the skeletal muscle in human and is caused by mutations in the dystrophin gene. The mdx mouse is a spontaneous mutant and an animal model for DMD. It has a point mutation in exon 23 of the dystrophin gene that eliminates the expression of dystrophin. However, this mutation does not disrupt the expression of four other shorter isoforms that are also expressed from the dystrophin gene through differential promoter usage. We generated another mutant mouse by gene targeting. Exon 52 of the dystrophin gene was disrupted, because the deletion of this exon is known to result in the DMD phenotype in human. In this mouse (mdx52), Dp140 and Dp260, shorter dystrophin isoforms, were absent in addition to dystrophin. The skeletal muscles were hypertrophic and the histology exhibited variations in the fiber size with a necrotic and regenerating process. This mouse is thus considered to represent another model for DMD.

Animals↗
PubMed1997

A novel aortic smooth muscle cell line obtained from p53 knock out mice expresses several differentiation characteristics.

Here we report that we could obtain a highly differentiated smooth muscle cell line by screening the expression of a-smooth muscle actin from p53 knook out mice aorta. This cell revealed extended bipolar shape and expressed h-caldesmon and calponin as well as a-smooth muscle actin as protein markers of differentiated smooth muscle. Further intracellular calcium increase was induced by application of noradrenaline in a dose dependent manner and calcium oscillation was also observed in a higher dose (100 microM). Appropriate application of 5-azacytidine enhanced these tendencies and induced slow contraction by endothelin-1 and phenylephrine.

Animals↗
PubMed1997

K-ras is essential for the development of the mouse embryo.

ras genes encode members of the small GTP-binding proteins. Ras protein in highly conserved in various species from yeast to humans and plays a key role in signal transduction. Ras is related to cell proliferation and differentiation. While, in addition, mutations in the ras genes are implicated in a variety of tumors. However, the physiological functions and specific roles of each ras gene, H-ras, K-ras and N-ras, are still not fully understood. To clarify the role of the K-Ras in vivo, we generated K-ras mutant mice by gene targeting. In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term. At embryonic day 15.5, their ventricular walls are extremely thin. Besides, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord. Our results thus indicate K-Ras to be essential for normal development in mice and residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice.

Animals↗
PubMed1997

An oocyte-specific methylation imprint center in the mouse U2afbp-rs/U2af1-rs1 gene marks the establishment of allele-specific methylation during preimplantation development.

An oocyte-specific methylation imprint mark region, consisting of approximately 200 bp from the mouse imprinted gene U2afbp-rs, was identified within an area containing a CpG island and a short tandem repeat sequence. The oocyte-specific methylation was preserved in fertilized eggs and then expanded on the repressed maternal allele during preimplantation development until the adult methylation pattern was achieved by 12.5 days of embryonic development. These results indicate that the oocyte-specific imprinting mark region acts as a center in establishing the hypermethylated region on the repressed maternal allele. Furthermore, a region that is hypermethylated in both gametes was identified but its hypermethylation was conserved only on the maternal allele during preimplantation development, suggesting that some factor(s) inherited from oocytes may act to maintain hypermethylation on the maternal allele.

Alleles↗
PubMed1997

In vivo activation of aflatoxin B1 in C57BL/6N mice carrying a human fetus-specific CYP3A7 gene.

The in vivo activation of aflatoxin B1 (AFB1) was assessed by using two transgenic mouse lines, M2 and M10, in which the human fetus-specific CYP3A7 was expressed in the kidney (M2) and the liver (M10), respectively. Male mice of 8 weeks old from these two lines were treated with a single i.p. injection of AFB1 (4 mg/kg body weight). AFB1-N7-guanine adduct was quantified by high-performance liquid chromatography. DNA damage was measured using the alkaline elution technique 2 and 6 h after AFB1 treatment. Administration of AFB1 resulted in a significantly higher level of AFB1-N7-guanine in the livers of M10 transgenic mice compared with their nontransgenic littermates (16.5 +/- 4.2 versus 10.4 +/- 1.2 ng/mg DNA, P < 0.01). The level of this biomarker was also significantly higher in the kidney of the M2 mice compared with control mice (73.0 +/- 6.3 versus 50.2 +/- 9.5 ng/mg DNA, P < 0.01). Similar results were also observed with DNA damage expressed as normalized area above curve (NAAC) in the two transgenic lineages, e.g., NAAC values were significantly higher in the livers of M10 and the kidneys of M2 mice. A dose-response relationship of NAAC values was observed in the livers of M10 mice when treated with AFB1 at different doses ranging from 1 to 16 mg/kg body weight, whereas in nontransgenic mice, only slight but not statistically significant increases of NAAC values were observed. Both the mouse CYP3A11 and GST-Yc subunit were expressed at identical levels in these transgenic lines. The results of this study present further evidence that human fetuses are also under the carcinogenic attack of AFB1 as adults if exposed to this potent carcinogen.

Aflatoxin B1↗
PubMed1997

Positive and negative CD4+ thymocyte selection by a single MHC class II/peptide ligand affected by its expression level in the thymus.

The central event in thymic selection of T cells bearing alpha beta TCRs is their interaction with self-peptides bound to self-MHC molecules. With the use of transgenic mouse lines expressing a single peptide/MHC class II complex, we show that CD4+ T cells with the preferential usage of particular TCR V(alpha)s and V(beta)s were selected to mature on this complex in lines with the lower expression, whereas such CD4+ T cells were eliminated in the thymus in a line with the relatively high expression. When a low expressing line was crossed with a high expressing line, the frequency of CD4+ T cells selected by this complex markedly decreased. Thus, these results suggest that a single peptide/MHC class II complex, being affected by its cell surface density in the thymus, can serve as both positively and negatively selecting ligand in vivo.

Amino Acid Sequence↗