Biomedical subjects

K Watabe

Publications and source records attributed to K Watabe.

At least 37 records · Page 2Linked to original sources

PubMed2001

Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.

We isolated a novel gene, termed MLZE, from a B16-BL6 cDNA library after subtraction of B16-F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1 - 2, which contains the c-myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze-positive cases was significantly larger in Clark levels III, IV and V melanomas (6 / 11 = 55%) than in Clark levels I and II melanomas (2 / 15 = 13%). In two cases of hMlze-positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.

Adult↗

PubMed2001

Granulomatous tissue formation of shikon and shikonin by air pouch method.

"The extract of shikon" (SK) and shikonin play important roles in the development of granulomatous tissue formation. To reveal the augmenting effect of SK or shikonin on vascular endothelial growth factor (VEGF) production and neovascularization, we investigated murine granulomatous tissue induced by SK and shikonin, comparing them to pouches in which trehalose 6,6'-dimycolate (TDM) was injected. The development of granulomatous tissue formation was evaluated by the wet weight of pouch walls. At day 5 and 7 after SK and shikonin injection, prominent granulomatous tissue formation was detected. Histological observations on the development of granulomatous tissue showed that the pouch was formed in the submuscular connective tissue and necrotic tissue directly facing the cavity and granulomatous tissue developed in the connective tissue. At day 1, VEGF-positive neutrophils accumulated in the pouch wall. Granulomatous tissue formation and neovascularization by injection of SK or shikonin was not more prominent than TDM. However, the present results indicate that SK and shikonin induce neovascularization in granulomatous tissue.

Animals↗

PubMed2001

Adenoviral gene transfer of glial cell line-derived neurotrophic factor to injured adult motoneurons.

Glial cell line-derived neurotrophic factor (GDNF) strongly supports the survival of injured neonatal motoneurons, suggesting its potential uses in the treatment of motoneuron injury and motor neuron diseases. We examined neuroprotective effects of an adenoviral vector encoding GDNF (AxCAhGDNF) on the survival of lesioned adult rat facial and spinal motoneurons. The facial nerve or the seventh cervical segment (C7) ventral and dorsal roots of 3 month-old Fischer 344 male rats were avulsed and removed from the stylomastoid or vertebral foramen, respectively, and AxCALacZ (adenovirus containing beta-galactosidase gene), AxCAhGDNF, or PBS was inoculated in the lesioned foramen. One week after the avulsion and treatment with AxCALacZ, the animal showed expression of beta-galactosidase activity in lesioned facial and spinal motoneurons. Animals avulsed and treated with AxCAhGDNF showed intense immunolabeling for GDNF in lesioned facial and spinal motoneurons and expression of virus-induced human GDNF mRNA transcripts in the lesioned brain stem and spinal cord tissues. The treatment with AxCAhGDNF after avulsion significantly prevented the loss of lesioned facial and C7 spinal motoneurons, ameliorated choline acetyltransferase immunoreactivity, and suppressed the activity of nitric oxide synthase in these neurons. These results indicate that the adenovirus-mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Adenoviridae↗

PubMed2000

Rescue of ischemic brain injury by adenoviral gene transfer of glial cell line-derived neurotrophic factor after transient global ischemia in gerbils.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor (TGF)-beta superfamily, is one of the most potent neurotrophic factors and promotes survival of many populations of cells. We examined neuroprotective effect of an adenoviral vector encoding glial cell line-derived neurotrophic factor (AxCAhGDNF) on the transient global ischemia. Gerbils received administration of AxCAhGDNF or an adenoviral vector encoding bacterial beta-galactosidase gene (AxCALacZ) through the lateral ventricle. Two days later, occluding bilateral common carotid arteries for 5 min using aneurysm clips produced the transient global forebrain ischemia. Animals showed intense immunolabeling for GDNF in ependymal cells on 2, 4 and 7 days after the operation. The exogenous gene transducted by adenovirus in the same cells was detected by in situ hybridization. The treatment with AxCAhGDNF significantly prevented the loss of hippocampal CA1 pyramidal neurons 2 to 7 days after the operation, as compared to AxCALacZ treatment. Also terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) staining was markedly reduced in the case with AxCAhGDNF treatment at 7 days after the operation. These results indicated that the adenovirus-mediated gene transfer of GDNF might prevent the delayed neuronal death of stroke and other disorders of the cerebral vasculature.

Adenoviridae↗

PubMed2000

The yabG gene of Bacillus subtilis encodes a sporulation specific protease which is involved in the processing of several spore coat proteins.

The synthesis and proteolysis of the spore coat proteins, SpoIVA and YrbA, of Bacillus subtilis were analyzed using antisera. Almost no intact full-length proteins of either type were extracted from wild-type spores, while yabG mutant spores contained intact SpoIVA and YrbA proteins. We purified recombinant YrbA and YabG proteins from Escherichia coli transformants and found that YrbA was cleaved to the smaller moiety in the presence of YabG in vitro. These observations indicate that YabG is a protease involved in the proteolysis and maturation of SpoIVA and YrbA proteins, conserved with the cortex and/or coat assembly by B. subtilis.

Bacillus subtilis↗

PubMed2000

Activation of the tumor metastasis suppressor gene, KAI1, by etoposide is mediated by p53 and c-Jun genes.

KAI1 is a metastasis suppressor gene which is capable of inhibiting the processes of tumor metastasis without affecting tumorigenicity per se. We found that etoposide, a topoisomerase II inhibitor, is able to activate the expression of the KAI1 gene in a dose-dependent manner in human prostate cancer cell lines, ALVA, DU145, and PC-3 as well as in human lung carcinoma cell A549. The activation of the KAI1 gene was mainly mediated by the c-Jun gene in the PC-3 and DU145 cell lines, while it was mediated by both p53 and c-Jun genes in the A549 cell line. These results suggest that the augmentation of the KAI1 gene expression is independently controlled by p53 and c-Jun at the transcriptional level in the human cancer cell lines. Furthermore, treatment of these cell lines with etoposide resulted in significant reduction of cellular invasion measured by the Matrigel invasion chamber. Because etoposide has been shown to be effective on advanced prostate cancer when used in combination with other regimens, our results provide further rationale to use this drug as an antimetastatic agent.

5' Untranslated Regions↗

PubMed2000

Immunohistochemical localization of substance P receptors in the midline glia of the developing rat medulla oblongata with special reference to the formation of raphe nuclei.

Immunohistochemical localization of the substance P receptor (SPR) was examined in the developing rat medulla oblongata, with special reference to the development of substance P (SP)-immunoreactive neurons which form the medullary raphe nuclei. During development, SPR immunoreactivity was detected in cells lying lateral to the medullary midline from embryonic day 13 (E13) to postnatal day 5 (P5). The SPR-positive cell bodies were located close to the fourth ventricle, and bore long processes extending to the ventral pial surface. This SPR immunoreactivity co-localized with staining for monoclonal antibody 1D11, a specific marker of immature astrocytes. Substance P (SP)-immunoreactive neurons were first detected at E14 in the ventrolateral part of the medulla. By E16 their number had increased and they were arrayed in two rows closely parallel to the SPR-immunoreactive processes of non-neuronal cells. By P1, two separate SP-immunoreactive cell clusters could be recognized at the midline, representing dorsally the nascent raphe pallidus and ventrally the raphe obscurus. In addition, many SP-immunoreactive fibers traveled rostrocaudally in the medulla oblongata, juxtaposed to the midline sheets of SPR-immunoreactive long processes. SPR-immunoreactive processes at the midline were also immunoreactive for S-100, a glia-specific calcium-binding protein that is known to promote axonal growth of raphe neurons. These results suggest that SPR-expressing immature glial cells at the medullary midline are involved in the development of SP-immunoreactive raphe neurons, both in the formation of the medullary raphe nuclei and in axon guidance and growth.

Animals↗

PubMed2000

Adenoviral vector-mediated GDNF gene transfer prevents death of adult facial motoneurons.

We examined neuroprotective effects of an adenoviral vector encoding glial cell line-derived neurotrophic factor (AxCAhGDNF) on the lesioned adult rat facial motoneurons. After facial nerve avulsion, animals locally injected into the facial canal with AxCALacZ (adenovirus encoding beta-galactosidase gene) or AxCAhGDNF showed expression of beta-galactosidase activity or intense immunolabeling for GDNF in lesioned facial motoneurons, respectively. The treatment with AxCAhGDNF after avulsion significantly prevented the loss of lesioned facial motoneurons, ameliorated choline acetyltransferase immunoreactivity, and suppressed the activity of nitric oxide synthase in these neurons. These results indicate that the adenovirus-mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with peripheral nerve injury and motor neuron diseases.

Adenoviridae↗

PubMed2000

Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect cranial and spinal motoneurons, that suggests potential uses of GDNF in the treatment of spinal cord injury and motor neuron diseases. We examined neuroprotective effect of human GDNF encoded by an adenovirus vector (AxCAhGDNF) on the death of lesioned adult rat spinal motoneurons. The seventh cervical segment (C7) ventral and dorsal roots and dorsal root ganglia of adult Fisher 344 rats were avulsed, and AxCAhGDNF, AxCALacZ (adenovirus encoding beta-galactosidase gene) or PBS was inoculated in C7 vertebral foramen. One week after the avulsion and treatment with AxCALacZ, the animals showed expression of beta-galactosidase activity in lesioned spinal motoneurons. Animals avulsed and treated with AxCAhGDNF showed intense immunolabeling for GDNF in lesioned spinal motoneurons and expression of virus-induced human GDNF mRNA transcripts in the lesioned spinal cord tissue. Nissl-stained cell counts revealed that the treatment with AxCAhGDNF significantly prevented the loss of lesioned ventral horn motoneurons 2 to 8 weeks after avulsion, as compared to AxCALacZ or PBS treatment. Furthermore, the AxCAhGDNF treatment ameliorated choline acetyltransferase immunoreactivity in the lesioned motoneurons after avulsion. These results indicate that the adenovirus-mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with spinal cord injury and motor neuron diseases.

Adenoviridae↗

PubMed2000

Antibody against a novel, myriocin (ISP-I)-based immunosuppressant, FTY720.

An antibody was prepared by immunizing rabbits with an ovalbumin conjugate of 2-amino-2-(2-(4-(4-mercaptobutyl)phenyl)ethyl)propane-1,3-diol HCl (AMPD-4), which contains the essential structure of the novel immunosuppressant FTY720. As the antibody reacted to not only AMPD-4, but also FTY720, it should be useful for immunoassay of FTY720 in body fluids, tissues and cells.

Animals↗

PubMed2000

Localization of a novel tumor metastasis suppressor region on the short arm of human chromosome 2.

Much of the lethality of malignant neoplasms is attributable directly to their ability to develop secondary growths in organs at a distance from the primary tumor mass, whereas few patients die from their primary neoplasm. Little is known about the molecular mechanism of tumor metastasis, however, which is controlled by a variety of positive and negative factors. In the search for metastasis suppressor genes, we have used the microcell-mediated chromosome transfer method and a rat prostate tumor model in SCID mice. When human chromosome 2 was introduced into the highly metastatic rat prostatic tumor cell, AT6.1, the metastatic ability of this cell was significantly (>99%) decreased in animals. An STS-based PCR analysis for 8 hybrid clones indicates that the suppressor activity is located in the p25-22 region of the chromosome. Furthermore, the AT6.1 cell with human chromosome 2 showed a reduced ability to invade Matrigel, suggesting that the suppressor activity is involved in the step of tumor invasion during the progression of prostate cancer. We have also examined the status of the suppressor region on chromosome 2 in human prostate cancer specimens and found that this region was often lost in high-grade tumors. These results suggest that the putative suppressor gene on chromosome 2 is functionally involved in the progression of human prostate cancer. Genes Chromosomes Cancer 28:285-293, 2000.

Aged↗

PubMed2000

Synthesis and characterization of the spore proteins of Bacillus subtilis YdhD, YkuD, and YkvP, which carry a motif conserved among cell wall binding proteins.

We have previously reported that YaaH and YrbA are spore proteins of Bacillus subtilis that are required for spore resistance and/or germination and that they have a motif conserved among so-called cell wall binding proteins [Kodama et al. (1999) J. Bacteriol. 181, 4584-4591, Takamatsu et al. (1999) J. Bacteriol. 181, 4986-4994]. In this study, we analyzed the expression of ydhD, ykuD, and ykvP genes, which encode putative proteins containing the same motif. Transcription of ydhD was dependent on SigE, and the mRNA was detectable from 2 h after the cessation of logarithmic growth (T(2) of sporulation). ykuD was transcribed by SigK RNA polymerase from T(4) of sporulation. Both SigK and GerE were essential for ykvP expression, and this gene was transcribed from T(5) of sporulation. Inactivation of these genes by insertion of an erythromycin resistance gene did not affect vegetative growth, spore resistance to heat, chloroform, and lysozyme, or spore germination in the presence of L-alanine or in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride. The His tag fusions of YdhD, YkuD, and YkvP downstream of their natural promoter regions were introduced into a multicopy plasmid. These fusion proteins were produced during sporulation in B. subtilis transformants and were detected in mature spores, indicating that YdhD, YkuD, and YkvP are all proteins intrinsic to spores. Excessive YkuD and YkvP in the sporulating cells did not affect spore resistance or germination. The cells producing excessive YdhD also did not show impaired spore resistance, but their germination properties were changed: the spores revealed reduced response to L-alanine and some of them germinated even without germinants. Escherichia coli b-lactamase, whose signal sequence had been genetically replaced by the cell wall binding motif of YaaH, was produced in sporulating cells, and Western blot analysis indicated that the fused protein was assembled into spores. We speculate that the conserved motif functions as a kind of signal sequence involved in assembly of these proteins on forespores.

Amino Acid Motifs↗

PubMed2000

Annexin VII as a novel marker for invasive phenotype of malignant melanoma.

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. While examining the genetic difference between the two sublines, we found a marked reduction of annexin VII expression in BL6 cells. In addition, fusion cell clones of both sublines were as poorly metastatic as F10 cells after subcutaneous injection, and contained the annexin VII message as abundantly as F10 cells. Hence, we examined whether the annexin VII expression was correlated with the less malignant phenotype of clinical cases by immunohistochemistry. Immunoreactivities to anti-annexin VII antibody in melanoma cells were evaluated quantitatively by using skin mast cells as an internal positive control. Eighteen patients with malignant melanoma were divided into two groups: lymph node metastasis-negative and positive groups. The ratio of numbers of patients positive versus negative to the antibody was significantly larger in the former than in the latter group. These results not only indicated that annexin VII serves as a marker for less invasive phenotype of malignant melanoma, but also suggested a possible role of annexin VII in tumor suppression.

Adolescent↗

PubMed2000

The Bacillus subtilis yabG gene is transcribed by SigK RNA polymerase during sporulation, and yabG mutant spores have altered coat protein composition.

The expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T(4)) in wild-type cells and in a gerE36 (GerE(-)) mutant but not in spoIIAC (SigF(-)), spoIIGAB (SigE(-)), spoIIIG (SigG(-)), and spoIVCB (SigK(-)) mutants. The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yabG spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B. subtilis.

Alanine↗

PubMed1999

A new member of the GTPase superfamily that is upregulated in highly metastatic cells.

Two sublines of B16 melanoma cells, F10 and BL6, are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found a new member of the GTPase superfamily, namely TIB929, which displayed an induction of expression in BL6 cells. It conserved three consensus sequences for GTP-binding site motifs and showed a significant homology to the yeast Gtr2 gene throughout the coding sequence. TIB929 was expressed ubiquitously in human tumor cells, with a marked expression in highly metastatic cells. TIB929 was mapped on mouse chromosome 4D, syntenic to human chromosome 1p. The results suggested an involvement of TIB929 in malignant progression.

Amino Acid Sequence↗

PubMed1999

Assembly of the CotSA coat protein into spores requires CotS in Bacillus subtilis.

The CotSA protein, encoded by cotSA (ytxN) of Bacillus subtilis, was detected from the cells at 5 h after the onset of sporulation (T5) and in the spore coat of wild-type cells, but not in cotE, cotS, gerE, or cotSA mutant spores. CotSA was also detected in the sporangium at T5 to T7 but not in the sporangium at T18 of cotS mutant cells, while the incorporation of CotS into the coat was not dependent upon CotSA. These results suggested that CotSA was synthesized simultaneously with CotS during T5 to T7 of sporulation and assembled into the coat dependent upon CotS.

Bacillus subtilis↗

PubMed1999

Assessment of mucosal immune response in genitourinary tract using urine.

A novel method to assess mucosal immune response in the genitourinary mucosa after immunization with a mucosal vaccine has been developed. In this method, secretory IgA antibody is measured by a highly sensitive enzyme immunoassay (immune-complex transfer enzyme immunoassay) using urine as a specimen. The urinary IgA antibody response could be detected by the immune-complex transfer enzyme immunoassay. In contrast, a conventional enzyme immunoassay (enzyme-linked immunosorbent assay (ELISA)) could not detect this response because of its low sensitivity. Because urine samples can be collected easily and nontraumatically, not only from experimental animals but also from humans, both males and females, the present method may be applicable for assessing the protective efficacy of candidates for mucosal vaccines against sexually transmitted microorganisms, such as human immunodeficiency virus. Furthermore, the usefulness of this method for novel mucosal vaccine formulae was shown for a model in which vaccine antigen and Bordetella pertussis adjuvant were adsorbed onto CaCO, and enclosed in enteric coated capsules.

Adjuvants, Immunologic↗

PubMed1999

The Bacillus subtilis yaaH gene is transcribed by SigE RNA polymerase during sporulation, and its product is involved in germination of spores.

The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T(2)) of sporulation in wild-type cells and in spoIIIG (SigG(-)) and spoIVCB (SigK(-)) mutants but not in spoIIAC (SigF(-)) and spoIIGAB (SigE(-)) mutants. The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced from T(2) of sporulation in a B. subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein. Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yaaH mutant spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination. These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T(2) of sporulation and that it is required for the L-alanine-stimulated germination pathway.

Amino Acid Sequence↗