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Biomedical subjects

M Watabe

Publications and source records attributed to M Watabe.

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

Anti-Candida activity of a new platinum derivative.

A new platinum derivative of the form H[Pt(IV)(Hdigly)Cl(2)(OH)(2)] (Hdigly==glycylglycine) damaged the Candida albicans cell membrane and inhibited the growth of the cells. The cytotoxic activity of H[Pt(IV)(Hdigly)Cl(2)(OH)(2)] on mammalian cells was 10-fold lower than that of cis-diammine-dichloroplatinum (cisplatin). Substitution of platinum for peptides is effective for enhancement of antifungal activity and reduction of the toxicity to mammalian cells.

Antifungal Agents↗
PubMed1999

Requirement of protein kinase (Krs/MST) activation for MT-21-induced apoptosis.

Fas is a well characterized apoptosis-inducing factor. One of our synthetic compounds, MT-21, induced apoptosis in human leukemia HL-60 cells similar to Fas. MT-21 activated caspase-3, an important cysteine aspartic protease for apoptosis induction. MT-21 also activated c-Jun-NH2-terminal kinase (JNK), a member of mitogen activated protein kinase (MAPK) superfamily that is involved in the regulation of cell growth, differentiation and cell death. Moreover, MT-21 treatment resulted in the activation of a 36 kDa kinase which uses myelin basic protein (MBP) as a substrate. However, MAPK and p38 were not activated by treatment with MT-21. The 36 kDa MBP kinase was shown to be a proteolytic product derived from the Krs protein with a molecular weight of 60 kDa. The Krs protein is a Ser/Thr protein kinase whose activity is enhanced by digestion of its C-terminal regulatory domain by caspase-3. When a kinase-inactive mutant form of Krs protein was overexpressed in HL-60 cells, JNK activation and apoptosis induction by MT-21 were suppressed. Furthermore, overexpression of dominant negative c-Jun also suppressed apoptosis induction by MT-21. These findings indicate that MT-21 induces apoptosis by the activation of JNK via the Krs protein, which is activated by caspase cleavage.

Amino Acid Substitution↗
PubMed1999

Tiam1 is involved in the regulation of bufalin-induced apoptosis in human leukemia cells.

Bufalin, a component of the Chinese medicine chan'su, induces apoptosis in various lines of human tumor cells, such as leukemia HL60 and U937 cells, by altering the expression of apoptosis-related genes, for example, bcl-2 and c-myc. In this study, we characterized a gene that is involved in bufalin-induced apoptosis by the differential display (DD) technique. The partial nucleotide sequence of one of the differentially expressed clones obtained after treatment with bufalin was identical to that of the human gene for Tiam1. When U937 cells were treated with 10(-7) M bufalin, expression of both Tiam1 mRNA and the protein was induced 1 h after the start of the treatment. The increase of Tiam1 mRNA was transient but the level of Tiam1 protein continued to increase at least for 6 h. In addition, the activities of Rac1 and p21-activated kinase (PAK) were also stimulated by bufalin treatment. To evaluate the role of Tiam1 in the apoptotic process, we examined the effects of the expression of sense and antisense RNA for Tiam1 in U937 cells. Apoptosis was strongly induced by bufalin in cells that expressed sense RNA for Tiam1 as compared to apoptosis in control cells treated with bufalin only. Cells expressing antisense RNA for Tiaml were significantly more resistant than the control bufalin-treated cells to induction of DNA fragmentation in response to bufalin. Moreover, sense transformants had elevated activities of PAK and c-Jun NH2-terminal kinase (JNK). These results suggest that Tiaml might play a critical role in bufalin-induced apoptosis through the activation of Rac1, PAK, and JNK pathway.

Antineoplastic Agents↗
PubMed1999

[Preservation, thawing and infusion of hematopoietic stem cells].

Peripheral blood stem cells can be stored by the following 3 methods: liquid storage, non-rate controlled freezing and rate controlled freezing. Methods of processing of these cells including thawing, ex vivo purging and infusion are described in detail.

Cryopreservation↗
PubMed1998

Human chromosome 16 suppresses metastasis but not tumorigenesis in rat prostatic tumor cells.

Genomic aberrations at the chromosome 16q arm are one of the most consistent abnormalities observed by loss of heterozygosity and comparative genomic hybridization analyses in human prostate cancer, suggesting that there are tumor suppressor or metastasis suppressor genes encoded by this chromosomal region. To functionally identify such suppressor genes, we have conducted microcell-mediated chromosome transfer to introduce human chromosome 16 into the highly metastatic Dunning rat prostatic cancer cell line, AT6.1. The metastatic ability of the resultant microcell hybrid clones was then tested in a standard spontaneous metastasis assay using SCID mice. When the microcell-mediated chromosome transfer hybrid cells containing whole human chromosome 16 were injected, the number of metastatic lesions in the lung was significantly reduced as much as 99% on average. Therefore, chromosome 16 has a strong activity to suppress the metastatic ability of AT6.1 cells while it did not affect the tumorigenesis and tumor growth rate. A PCR analysis of various microcell hybrid clones with sequence-tagged site markers indicates that the metastasis suppressor activity is located in the q24.2 region of chromosome 16. Our results are consistent with the previous finding that the region of human chromosome 16q has frequent loss of heterozygosity in prostate cancer patients and suggest that there is a metastasis suppressor gene in this region that may play an important role in the progression of prostate cancer.

Animals↗
PubMed1998

The expression of the KAI1 gene, a tumor metastasis suppressor, is directly activated by p53.

KAI1 is a tumor metastasis suppressor gene that is capable of inhibiting the metastatic process in animals. The expression of the KAI1 gene also is found to be down-regulated during the tumor progression of prostate, breast, lung, bladder, and pancreatic cancers in humans, and this down-regulation appears to be at or posttranscription level. We have found that the tumor suppressor gene p53 can directly activate the KAI1 gene by interacting with the 5' upstream region. The p53 responding region is located at approximately 860 bases upstream of the transcriptional initiation site, and it contains a typical tandem repeat of the p53 consensus-binding sequence. A gel-shift mobility analysis showed that this sequence indeed had the ability to bind to the purified p53 protein. Mutations of this sequence abolished the responsiveness to p53 and also the binding ability to the p53 protein. Furthermore, immunohistochemical analysis of 177 samples of human prostate tumors revealed that the expression of the KAI1 gene was correlated strongly to that of the p53 gene and that the loss of these two markers resulted in poor survivals of patients. Our data indicate a direct relationship between p53 and KAI1 genes and suggest that the loss of p53 function, which is commonly observed in many types of cancer, leads to the down-regulation of the KAI1 gene, which may result in the progression of metastasis.

Base Sequence↗
PubMed1998

Activation of AP-1 is required for bufalin-induced apoptosis in human leukemia U937 cells.

In a previous study, we demonstrated that bufalin caused apoptosis in human leukemia U937 cells by the anomalous activation of mitogen-activated protein kinase (MAPK) via a signaling pathway that included Ras, Raf-1 and MAPK kinase-1. We report here the effect of bufalin on c-Jun N-terminal protein kinase (JNK), a member of the MAPK family, and on the signaling pathway downstream of MAPKs in U937 cells. When U937 cells were treated with 10(-8) M bufalin, the activity of JNK1 was markedly elevated 3 h after the start of treatment and remained so for 9 h. This activation of JNK and the induction of apoptosis by bufalin were suppressed by expression of antisense mRNA for MAPK kinase-1. c-Jun was translocated from the cytoplasm to the nucleus after treatment of U937 cells with bufalin. The transcriptional activity of AP-1 was transiently enhanced by the treatment with bufalin and this activation was suppressed by the expression of antisense mRNA for MAPK kinase-1. Both curcumin (1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione), an inhibitor of the biosynthesis of AP-1, and the expression of dominant negative c-Jun inhibited the activation of AP-1 and the induction of apoptosis by bufalin. Expression of a constitutively active mutant form of MAPK kinase-1 induced the activation of AP-1 and subsequent apoptosis in U937 cells. These results suggest that the activation of AP-1 via a MAPK cascade that includes JNK is required for the induction of apoptosis by bufalin in U937 cells.

Antineoplastic Agents↗
PubMed1997

Bcl-2 protein inhibits bufalin-induced apoptosis through inhibition of mitogen-activated protein kinase activation in human leukemia U937 cells.

In a previous study, we demonstrated that bufalin, which is an active principle of Chinese medicine, chan'su, caused apoptosis in human leukemia U937 cells by anomalous activation of mitogen-activated protein kinase (MAPK) via the signaling pathway of Ras, Raf-1, and MAPK kinase-1. Here, we report the effect of overexpression of bcl-2 in U937 cells on the signaling pathway of apoptosis that is induced by bufalin. The results indicated that the apoptosis induced by bufalin in U937 cells was significantly inhibited by overexpression of the Bcl-2 protein. No significant difference was detected in the activation of MAPK kinase-1 that is induced by bufalin in wild-type or Bcl-2-overexpressed U937 cells; however, the activation of MAPK by bufalin was significantly attenuated in the cells overexpressing Bcl-2. Bufalin treatment activated activator protein-1 transcriptional activity; however, this activation was decreased to 40% in bcl-2-overexpressed U937 cells. These results indicate that Bcl-2 acts downstream of MAPK kinase-1 but upstream of MAPK and suggest that, in the signaling pathway of the apoptotic process induced by bufalin, the transcriptional activity of activator protein-1 may be down-regulated through the inhibition of MAPK activity by Bcl-2.

Antineoplastic Agents↗
PubMed1997

Activation of protein kinase C prevents induction of apoptosis by geranylgeraniol in human leukemia HL60 cells.

In a previous study, we showed that geranylgeraniol (GGO) is a potent inducer of apoptosis in human leukemia cells, including HL60 promyelocytic leukemia cells. The present study describes the effects of activators of protein kinase C (PKC) on GGO-induced apoptosis in various lines of leukemia cells. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and diacylglycerol (DG) inhibited the GGO-induced morphological changes that are characteristic of apoptosis and the DNA fragmentation. Similar effects were observed with other lines of human and murine leukemia cells such as ML1, U937, M1 and P388. Flow cytometric analysis also revealed that both TPA and DG prevented GGO-induced DNA degradation in a dose-dependent manner. These inhibitory effects of TPA and DG were antagonized by inhibitors of PKC such as H-7 and staurosporin, and by amiloride, an inhibitor of Na+/H+ antiporter. In contrast to the inhibitory effects of TPA and DG on GGO-induced apoptosis, 4alpha-TPA, which is unable to activate PKC, failed to prevent GGO-induced DNA fragmentation. However, the selective activator of PKC-beta, 12-deoxyphorbol 13-phenylacetate 20-acetate, significantly inhibited GGO-induced DNA fragmentation. Our results suggest that PKC, and in particular the PKC-beta isoenzyme, might be involved in the process of GGO-induced apoptosis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Sequence and functional analysis of the 5'-flanking region of the mouse growth inhibitory factor gene.

1. The growth inhibitory factor (GIF) is a 68-amino acid protein which is capable of inhibiting the growth of neuronal cells in vitro. 2. We have cloned and sequenced the 5'-flanking region of the mouse GIF gene, which spans from the transcriptional initiation site to the -1854 nucleotide. 3. This region contains sequences homologous to hgcs, SPE, and the JCV silencer domain that functions in a glial cell specific manner. This region also contains two metal responding elements and putative binding sites for AP-1, AP-2, Sp-1, SP-2, NF-1, and CREB. 4. An analysis of the reporter plasmids containing the various regions of the 5'-flanking sequence revealed that the region indeed functioned in a tissue-specific manner in glial cells and that the region between -328 and 175 is responsible for suppression, while the region between -175 and -49 is involved in the activation of gene expression.

3T3 Cells↗
PubMed1997

Echographia as a symptom of interictal state in an epileptic patient: a case report.

Echographia is a phenomenon in which a patient continuously translates verbal stimuli into writing. We encountered a patient with epilepsy who developed visual echographia during interictal periods. In this case, echographia was observed during two different periods, namely the period of disturbed consciousness after the epileptic seizure and the period of clear consciousness after suppression of the seizures. Disinhibition due to disturbance of the consciousness is considered to have been the cause of echographia in the former period. In the latter period, it is considered that echographia was caused by the release of lower function from suppression of upper function by brain dysfunction, as the after effect of status epilepticus. As echographia can be observed in epileptic patients, attention and careful observation by epileptologists is needed.

Adolescent↗
PubMed1997

Treatment of U937 cells with bufalin induces the translocation of casein kinase 2 and modulates the activity of topoisomerase II prior to the induction of apoptosis.

The treatment of human leukemia U937 cells with 10(-8) M bufalin in the absence of serum resulted in the immediate translocation of casein kinase 2 (CK 2) from the cytoplasm to the nucleus, as determined by confocal laser-scanning microscopy. Concomitantly, the activity of topoisomerase (topo) II, as determined by monitoring activities specific to this enzyme such as DNA relaxation, DNA decatenation, and topo II-mediated DNA cleavage, was enhanced. The activity reached a maximum after 3 h and then decreased markedly after treatment with bufalin for 9 h. The amount of a complex of CK 2 and topo IIalpha in U937 cells was estimated by immunoprecipitation with antibodies raised against subunits of CK 2 and against topo IIalpha. The amount increased just after the start of treatment with bufalin and reached a maximum at 6 h. The results suggest that the topo IIalpha in the complex might have been phosphorylated by the translocated CK 2 and that the topo activity was stimulated by such phosphorylation. Apoptotic U937 cells with fragmented nuclei were observed between 9 and 12 h after the start of treatment using 10(-8) M bufalin. Therefore, it appears that the bufalin signal was transmitted to the nucleus by the translocation of CK 2, which formed a complex with topo IIalpha and modulated the activity of this enzyme, leading to the induction of apoptosis.

Apoptosis↗
PubMed1996

Antimicrobial activity of a 13 amino acid tryptophan-rich peptide derived from a putative porcine precursor protein of a novel family of antibacterial peptides.

It has long been speculated that porcine cathelin is an N-terminal fragment of a longer precursor protein which possesses antimicrobial activity. In an attempt to find such a precursor, a cDNA clone was recently isolated and sequenced by screening a cDNA library from porcine bone marrow. In order to identify the functional activity of the putative protein encoded by an open reading frame, we have synthesized various lengths of peptides that correspond to the C-terminal region of the protein and examined them for their antimicrobial activities. We found that a 13 amino acid tryptophan-rich region with the sequence of VRRFPWWWPFLRR had strong antimicrobial activity with a wide spectrum. It showed potency against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus epidermidis, Proteus mirabilis, and Streptococcus group D as well as Aspergillus fumigatus. The action of this peptide is bactericidal rather than bacteriostatic and this activity is completely inhibited by 2 mM MgCl2. Our results indicate that the previously identified putative precursor encoded by the isolated cDNA indeed possesses a potent antimicrobial activity and that this 13 amino acid synthetic peptide is considered to be a potentially effective drug against various infectious agents.

Amino Acid Sequence↗
PubMed1996

The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells.

Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells. When U937 cells were treated with 10(-8) M bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to bufalin. Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Effects of synthetic form of tracheal antimicrobial peptide on respiratory pathogens.

We have synthesized a C-terminal portion of tracheal antimicrobial peptide (TAP) with 38 amino acids and tested it for efficacy on various clinical isolates of Pseudomonas aeruginosa strains from patients with cystic fibrosis and also on Aspergillus fumigatus. Our results indicate that the synthetic TAP has both potent bactericidal and fungicidal activities and that a combination of TAP and amphotericin B showed strong additive effects of growth inhibition on A fumigatus. These results suggest that TAP is potentially an effective therapy for Aspergillus and multi-drug-resistant Pseudomonas, pathogens that are often a serious threat to patients with cystic fibrosis.

Anti-Bacterial Agents↗
PubMed1996

A synthetic form of tracheal antimicrobial peptide has both bactericidal and antifungal activities.

We have chemically synthesized tracheal antimicrobial peptide (TAP) with 38 amino acids and examined efficacy of the peptide on various organisms. The synthetic peptide showed potent bactericidal effect on both gram positive and negative bacteria. The action of bactericidal effect was relatively quick and 99.9% of E. coli cells were killed within 90 minutes at a concentration of 2.5 micrograms/ml of TAP. The peptide also showed antifungal activity against both mycelia (Aspergillus fumigatus) and yeast (Candida albicans) forms of fungi. Our domain analysis with a series of synthetic peptides of various lengths indicates that 17 amino acid residues of the C-terminal end is the minimum functional domain of the bactericidal activity.

Amino Acid Sequence↗
PubMed1995

Differential gene expression in experimental hepatocellular carcinoma induced by woodchuck hepatitis B virus.

Hepatitis B virus infection is closely linked to hepatocellular carcinoma (HCC), the pathological mechanism of hepatocarcinogenesis by this virus is not well understood. In order to gain further insight into the molecular mechanism of HCC, we constructed and screened a subtracted c-DNA library which was specific to HCC cells of a woodchuck infected with woodchuck hepatitis B virus. Among eight clones that were isolated based on their differential expressions, we determined nucleotide sequences of two genes whose expressions were most significantly stimulated in HCC. Our results indicate that these two genes appear to be woodchuck counterpart genes of hemopexin (HPX) and alpha-1 acid glycoprotein (AGP), suggesting that the expression of HPX and AGP genes are strongly augmented in tumor cells partly due to transcriptional regulation.

Amino Acid Sequence↗