Biomedical subjects

M Osaki

Publications and source records attributed to M Osaki.

At least 55 records · Page 3Linked to original sources

PubMed1998

Demonstration of the third antigenically distinct outer membrane lipoprotein (OmlA) in Actinobacillus pleuropneumoniae serotype 7.

The gene encoding an outer membrane lipoprotein (OmlA) of Actinobacillus pleuropneumoniae strain WF83 (serotype 7 reference strain), designated omlA7, was sequenced. The amino acid sequence of OmlA7 showed 64.5 and 71.6% identity to that of OmlA from serotypes 1 (OmlA1) and 5 (OmlA5), respectively. The first 134 amino acids of OmlA7 were identical to those of OmlA5. A Southern blot analysis revealed the presence of a gene highly homologous to the omlA7 in the reference strains of serotypes 3, 4, 6, and 7. A Western blot analysis using a specific antiserum against a recombinant OmlA7 detected expression of the homologous proteins in the serotypes 4, 6, and 7 reference strains and a serotype 3 field strain, but not in a serotype 3 reference strain. The data demonstrate the third antigenically distinct OmlA is expressed in A. pleuropneumoniae.

Actinobacillus Infections↗

PubMed1998

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene.

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon-gamma enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirus vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.

Journal Article↗

PubMed1998

Endoscopic osteosynthesis for frontal bone fracture.

Endoscopic surgery to repair a frontal bone fracture is described. This operation is less invasive than the conventional open technique, which is performed under direct vision through a bicoronal or eyebrow incision. Endoscopic surgery leaves no conspicuous scars in the forehead and is free from complications such as nerve or vascular injuries, and appears to be of significant clinical value.

Adult↗

PubMed1998

Pretreatment with restriction enzyme or bovine serum albumin for effective PCR amplification of Epstein-Barr virus DNA in DNA extracted from paraffin-embedded gastric carcinoma tissue.

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.

Animals↗

PubMed1998

Respiratory syncytial virus infection of neonatal monocytes stimulates synthesis of interferon regulatory factor 1 and interleukin-1beta (IL-1beta)-converting enzyme and secretion of IL-1beta.

Interleukin-1beta (IL-1beta) production in response to respiratory syncytial virus (RSV) was investigated in normal neonate monocytes. Intracellular or culture supernatant IL-1beta protein levels were measured by enzyme immunoassay. The expression of mRNAs for interferon regulatory factor 1 (IRF-1), IL-1beta-converting enzyme (ICE), and IL-1beta in the cells was analyzed semiquantitatively by reverse transcriptase-PCR. Before RSV exposure, some IRF-1, ICE, and IL-1beta transcripts were already expressed in the monocytes. The levels of these transcripts increased significantly 2 h after RSV exposure compared with those in mock-infected cells. At that time, significantly higher intracellular IL-1beta protein levels were observed in RSV-exposed cells. After 20 h of RSV exposure, quantities of soluble IL-1beta secreted from RSV-exposed cells were moderately higher than those from noninfected cells. These observations suggest that RSV infection of neonatal monocytes triggers enhanced transcription and increased translation of the IL-1beta gene and increased secretion of the soluble protein. The later phase of these processes may be promoted by ICE activity, which was upregulated by increased IRF-1. The increase in IRF-1 activity may also result from RSV infection.

Apoptosis↗

PubMed1998

Respiratory syncytial virus infection of human alveolar epithelial cells enhances interferon regulatory factor 1 and interleukin-1beta-converting enzyme gene expression but does not cause apoptosis.

The induction kinetics of the transcriptional activities of interferon regulatory factor 1 (IRF-1), interleukin-1beta-converting enzyme (ICE), and CPP32 by respiratory syncytial virus (RSV) infection of human type II alveolar epithelial cells (A549 cells) were analyzed semiquantitatively by reverse transcriptase PCR. The appearance of ICE and CPP32 protein in cell lysate was examined by Western blotting analysis. The induction of apoptosis by RSV infection was examined by the appearance of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. RSV moderately enhanced IRF-1 mRNA as early as 4 h after infection, and this enhancement lasted several hours. Following induction of the IRF-1 gene, ICE gene expression increased significantly, and an increase of ICE protein was observed in the RSV-infected cell lysate. These increments were observed in cells treated with live RSV but not in cells treated with inactivated RSV or control antigen. However, no infection-specific increase of CPP32 gene expression or the protein was observed. No nucleosomal fragmentation was observed in RSV-infected cells during the whole course of infection, despite the appearance of extensive cytopathic change and cell death. These observations suggest that RSV infection of human alveolar epithelial cells induces the ICE gene and its protein as a result of increased IRF-1 induction but that the increased ICE was insufficient to cause apoptosis in the RSV-infected cells. ICE might not be able to activate CPP32, which is thought to be the more important protease for apoptosis.

Apoptosis↗

PubMed1998

Improved electroporation of Rhodococcus equi.

The condition of an electroporation method was re-evaluated for the introduction of foreign plasmid DNA into Rhodococcus equi. The method is based on an electroporation of the bacteria made competent by culturing in a broth containing glycine and by heat shock at 50 degrees C. Transformation of R. equi could be achieved with a chloramphenicol-resistant shuttle vector originating from Rhodococcus fascians at an efficiency of about 10(4) transformants/microgram DNA. The bacteria were also shown to become competent when they were incubated with a chemical transformation buffer prior to washing with an electroporation buffer.

Culture Media↗

PubMed1997

Expression of p21 (waf1/cip1/sdi1), but not p53 protein, is a factor in the survival of patients with advanced gastric carcinoma.

BACKGROUND: The authors examined whether expression of p21 (waf1/cip1/sdi1) and p53 protein was related to survival, rates in patients with advanced gastric carcinoma. METHODS: The expression of p21 and p53 protein was analyzed by immunohistochemistry in 93 patients with advanced gastric carcinoma with serosal invasion and lymph node metastasis. All patients underwent curative surgery. The probability of survival was calculated by the Kaplan-Meier method and compared by the generalized Wilcoxon test. RESULTS: Various levels of p21 and p53 immunoreactivities in carcinoma cells were detected in 30 (32%) and 60 (65%), respectively, of 93 samples. There was no correlation between p21 and p53 expression. The 5-year survival rate of patients with p21 expression was 69.4%, which was significantly better than that of patients without p21 expression (38.1%; P < 0.05). However, p53 protein expression did not correlate with patient survival. CONCLUSIONS: Expression of p21 protein may be a better prognostic factor than p53 protein expression in patients with advanced gastric carcinoma.

Adenocarcinoma↗

PubMed1997

Expression of parathyroid hormone-related peptide in human osteoarthritis.

To evaluate the involvement of the expression of parathyroid hormone-related peptide gene in human articular cartilage pathology, we performed immunohistochemical staining and in situ hybridization on specimens of femoral head cartilage obtained from 15 patients with osteoarthritis, 11 with rheumatoid arthritis, and 12 control subjects. Parathyroid hormone-related peptide-positive chondrocytes were observed predominantly in degenerated lesions of osteoarthritic tissue and were less evident in rheumatoid arthritic samples, while the normal cartilage expressed little parathyroid hormone-related peptide. In addition, the level of parathyroid hormone-related peptide expression was clearly dependent on the degree of cartilage degeneration; cartilage tissues with moderate degenerative changes contained more positive chondrocytes compared with mildly or severely degenerated cartilage. In situ hybridization confirmed the localization of parathyroid hormone-related peptide protein and demonstrated intense expression of mRNA of the peptide in osteoarthritic samples. This is the first demonstration of parathyroid hormone-related peptide expression in chondrocytes from pathologic articular cartilage of humans. Our results suggest that parathyroid hormone-related peptide may be involved in the pathophysiology of osteoarthritis.

Aged↗

PubMed1997

Systemic and local immune responses of four cases with lower respiratory tract illness due to reinfection with respiratory syncytial virus.

Four infants and children who had tracheobronchitis or bronchiolitis by respiratory syncytial virus (RSV) reinfection were presented. Their clinical features, systemic and local immune reactions to RSV were compared with those of eleven age-matched patients who had lower respiratory tract illness with primary RSV infection. Moderate antibody activities in their nasopharyngeal secretions and sera which were comparable to those of convalescent phase of primary infection were observed within several days after onset of illness; however, their clinical symptoms, such as fever or wheezing, lasted almost as long as in primary RSV infection. These observations suggest that the higher serum and secretory antibody activity to RSV during acute phase does not always bring about clinical amelioration in RSV reinfection. The contribution of immunopathological reaction between RSV and RSV-specific antibodies might also be considered as a cause of inflammation during the acute phase of RSV reinfection.

Antibody Formation↗

PubMed1997

Flow injection analysis for measurement of activity of matrix metalloproteinase-7 (MMP-7).

A simple and convenient method for measuring the activity of a recombinant human matrix metalloproteinase 7 (MMP-7, matrilysin) was developed by flow injection analysis (FIA). For this method, purified recombinant MMP-7 zymogen expressed in E. coli and the substrate peptide (MOCAc-Pro-Leu-Gly-Leu-A2pr(DNP)-Ala-Arg-NH2) were used. Following the incubation of substrate peptide with activated r-proMMP-7, the resulting fluorescent product peptide (MOCAc-Pro-Leu-GLY) was monitored with a fluorescence detector (lambda ex 328 mm, lambda em 393 mm) without chromatographic separation. In this FIA system, the analysis time is 2 min and the standard curve is linear from 5 to 100 pmol of the product peptide injected. In order to use this FIA system as a method for screening inhibitors against MMP-7, the effects of CaCl2, EDTA and of the tissue inhibitor of metalloproteinase-1, and -2, were tested. A synthetic PRCGXPD-containing peptide (BS-10) was also observed to inhibit MMP-7 activity, with an IC50 value of 104 microM. Thus, it was concluded that the activity of r-MMP-7 can be reliably measured by the proposed system. Furthermore, to confirm the utility of this FIA system as a screening method, the inhibitory activity of the MMP-related substance in Joro spider (Nephilia clavata) venom was measured by this method. This inhibitory activity was observed in an extract of a venom diluted 1000-fold. Thus, the FIA method is not only simple and quick, but also sensitive enough to screen and analyze the inhibitory properties of a large number of test compounds.

Enzyme Inhibitors↗

PubMed1997

5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene.

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 microM 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 microM 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 microM 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.

Journal Article↗

PubMed1997

Expression of Fas antigen and its mediation of apoptosis in human gastric cancer cell lines.

Fas, a member of the tumor necrosis factor receptor/nerve growth factor receptor family, induces apoptosis by crosslinking with Fas ligand or anti-Fas antibody in a variety of cultured cells. We examined the expression of Fas antigen and its mediation of apoptosis in six human gastric carcinoma cell lines. Flow cytometric analysis and western blotting revealed relatively high expression of Fas antigen in MKN-74 (wild-type p53 gene) and MKN-45 (wild-type), followed by MKN-1 (mutated), MKN-7 (mutated) and KATO-III (deleted). MKN-28 (mutated) showed minimal expression of the antigen. The expression was apparently enhanced by interferon-gamma, except for MKN-1 and MKN-28. Anti-Fas antibody (100 ng/ml) induced nuclear fragmentation characteristic of apoptosis. Apoptosis occurred in a delayed fashion and the apoptotic index at 72 h was approximately 60% in MKN-74, 35% in MKN-45, and 20% in MKN-1 and KATO-III. A DNA ladder was noted in MKN-74 at 72 h. Expression levels of P53 and P21Waf1 did not change for up to 48 h in MKN-74. The biological effects did not correlate with endogenous Bcl-2 expression. These results indicated that a) Fas antigen is variably expressed in human cultured gastric carcinoma cells, b) the protein transduces an apoptotic signal which leads to delayed cell death, and c) susceptibility to the antibody correlates well with the expression level of Fas antigen.

Apoptosis↗

PubMed1997

Genetic diversity of the genes encoding the outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae.

An allelic variation of the genes encoding the protective outer membrane lipoprotein (omlA) in Actinobacillus pleuropneumoniae was investigated with polymerase chain reaction (PCR)-restriction fragment length polymorphisms. Primers for PCR were selected from a conserved sequence compared between the omlA genes of A. pleuropneumoniae serotypes 1 and 5a. A DNA fragment of 970 bp was amplified from the genomic DNA of all 12 serotypes of A. pleuropneumoniae. The amplified DNA sequence specifically hybridized under a low stringent condition to the cloned omlA gene of A. pleuropneumoniae. Digestion of the amplified DNA with the enzymes either HinfI or VspI yielded specific polymorphic patterns, allowing discrimination of all serotypes into five distinct groups.

Actinobacillus pleuropneumoniae↗

PubMed1997

Detection of mycobacterial DNA in formalin-fixed, paraffin-embedded tissue specimens by duplex polymerase chain reaction: application to histopathologic diagnosis.

Granuloma is a chronic inflammatory process associated with noninfectious agents or infectious diseases such as tuberculosis. Determination of the causative agent might be occasionally difficult in histopathologic sections. In this study, we examined 60 specimens of granuloma or inflammatory lesions that were originally diagnosed as 51 cases of granulomatous inflammation, 6 of leprosy, and 3 of atypical mycobacteriosis. The diagnoses in the last two categories were made both histologically and clinically. All of the sections and DNA were prepared from formalin-fixed, paraffin-embedded blocks. Histopathologic and immunohistochemical findings were compared with the results of duplex polymerase chain reaction (PCR) using two primers to amplify mycobacterial-common 383-base pair (bp) DNA and Mycobacterium tuberculosis-complex-specific 240-bp DNA. Six samples of leprosy and three of atypical mycobacteriosis showed the 383-bp but not the 240-bp band. Among the 51 specimens of granulomatous inflammations, nine showed no band of even the beta-globin, the cases being excluded from this analysis. The 42 specimens of granulomatous inflammation were subdivided into three categories by PCR: (1) 383- and 240-bp positive; (2) 383-bp positive and 240-bp negative; and (3) both negative. Category 1 included 32 specimens (76.2%), being considered as tuberculosis. One specimen was classified into Category 2, indicating possible atypical mycobacterium. Category 3 included nine specimens, composed of five of sarcoidosis and four other agent-induced granulomas, when compared with histologic and clinical findings. These findings indicate that the PCR assay using DNA extracted from paraffin-embedded materials provides useful information to differentiate tuberculosis from other type of granulomas.

DNA Primers↗

PubMed1996

Low frequency of apoptosis in Epstein-Barr virus-associated gastric carcinoma with lymphoid stroma.

In order to understand the character of Epstein-Barr virus (EBV)-associated gastric carcinoma with lymphoid stroma (GCLS), we examined cell proliferation and death in comparison with gastric carcinoma without prominent lymphoid stroma (conventional gastric carcinoma). The EBV-encoded small RNA I (EBER-1) and apoptotic cells were examined by in situ hybridization (ISH) and by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL), respectively, on formalin-fixed paraffin-embedded surgical specimens from 22 GCLSs and 23 conventional gastric carcinomas. Immunostaining was performed for the detection of Ki-67 antigen (Ki-67), P53 protein (P53) and bcl-2 oncoprotein (BCL-2). The percentages of apoptotic and Ki-67-positive cells were expressed as apoptotic indices (AI) and Ki-67 labeling indices (KI), respectively. EBER-1 was detected in 19 (86%) GCLSs in contrast to none in conventional carcinomas. Mean AI was 1.8 +/- 0.6 in the EBER-1-positive GCLSs and 3.3 +/- 1.7 in the conventional gastric carcinomas, the value being significantly lower in the former (p < 0.01). Mean KI was 40.0 +/- 10.8 in the GCLSs and 48.5 +/- 9.1 in the conventional gastric carcinomas, the value being significantly lower in the former (p < 0.05). P53 expression showed no significant difference between the 2 categories of carcinoma.

Adult↗

PubMed1996

Numerical aberration and point mutation of p53 gene in human gastric intestinal metaplasia and well-differentiated adenocarcinoma: analysis by fluorescence in situ hybridization (FISH) and PCR-SSCP.

This study examined p53 gene alterations in human gastric mucosa, intestinal metaplasia and well-differentiated (cohesive type) adenocarcinomas to clarify to the role of the p53 gene in gastric tumorigenesis by means of fluorescence in situ hybridization (FISH), polymerase-chain-reaction-single-strand-conformation polymorphism (PCR-SSCP) and immunohistochemistry. Gene alterations were compared with numerical changes of chromosome 17. Samples were obtained from 31 surgically resected stomachs affected with gastric cancer. There was no nuclear p53 protein in cells from normal gastric mucosa. Among 23 specimens of intestinal metaplasia, cells with p53 protein were variably detected in 5 incomplete metaplasias (colonic type). A histological study revealed a mildly dysplastic appearance. PCR-SSCP identified p53-gene mutation in exons 5 and 8 in 2 of the 5 samples. Numerical aberrations of chromosome 17 and the p53 gene were undetectable both in normal mucosa and in intestinal metaplasia. Variable numbers of tumor cells contained p53 protein in 13 (54%) of 24 carcinomas, and PCR-SSCP revealed abnormal bands in 5 of them. Mutations were detected in exons 5, 7, 7, 7 and 8. FISH analysis demonstrated that 6 carcinomas emitted 3 or 4 signals for chromosome 17, and 7 gave one signal for the p53 gene in over 20% of the cells. Ten (77%) of 13 carcinomas examined by FISH appeared to show a p53-gene deletion.

Adenocarcinoma↗

PubMed1996

Parathyroid hormone-related peptide in synovial fluid and disease activity of rheumatoid arthritis.

To understand the clinical role of parathyroid hormone-related peptide (PTHrP) in rheumatoid arthritis (RA), we analysed the circulatory and synovial fluid (SF) concentrations of the N- and C-terminal regions of PTHrP (N- and C-PTHrP) in RA (n = 38), osteoarthritis (OA, n = 45) and control (n = 11) subjects. The SF level of C-PTHrP was markedly higher in RA compared with control and OA groups, while no differences in circulatory C-PTHrP were present among the three groups. In contrast, the SF level of N-PTHrP was marginally higher in OA patients. C-PTHrP levels in SF correlated significantly with CRP, ESR and SF IL-1 receptor antagonist. To identify the mechanism of elevated PTHrP levels in SF, immunohistochemistry and in situ hybridization of synovial membrane (SM) were performed in each subject. Overexpression of PTHrP was identified in the sublining cells within papillary proliferated SM of RA patients only. Our results indicate that C-PTHrP produced from SM into SF reflects the disease activity in RA.

Adult↗