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Biomedical subjects

N Kato

Publications and source records attributed to N Kato.

At least 325 records · Page 18Linked to original sources
PubMed1998

Detection of negative-stranded hepatitis C virus RNA using a novel strand-specific reverse transcription-polymerase chain reaction.

We developed a novel single-tube reverse transcription-polymerase chain reaction (RT-PCR) for the specific detection of negative-stranded hepatitis C virus (HCV) RNA. By using in vitro synthesized positive- and negative-stranded HCV RNAs, it was demonstrated that as few as 50 copies of negative-stranded HCV RNA could be specifically detected with a set of primers that amplify a 232-base pair sequence unique to the 5'-non-coding region of HCV RNA, while 10(8) copies of positive-stranded HCV RNA were not detected. In addition, we demonstrated that this method allows the detection as few as 100 copies of negative-stranded HCV RNA even with the coexistence of a 100-fold excess of positive-stranded HCV RNA. Furthermore, with this method, negative-stranded HCV RNA was detected in RNAs from liver biopsy specimens obtained from patients with chronic hepatitis C, but not in RNAs from HCV-positive sera.

Hepacivirus↗
PubMed1998

Human hepatocyte clonal cell lines that support persistent replication of hepatitis C virus.

We previously found that a human T-cell leukemia virus type I infected T-cell line, MT-2, was susceptible to hepatitis C virus (HCV) infection, and that cloned MT-2C cells could support HCV replication more persistently than the parental MT-2 cells. Recently we found that a non-neoplastic hepatocyte line, PH5CH, showed good susceptibility to HCV infection. In this study, we cloned PH5CH cells to obtain cells that supported more persistent HCV replication, and consequently three clones (PH5CH1, PH5CH7 and PH5CH8) in which intracellular HCV RNA could be detected at least 25 days postinoculation (p.i.) were obtained. Semi-quantitative analysis of HCV RNA indicated that HCV replicated in these cloned PH5CH cells was released into the culture medium. Semi-quantitative analysis of internalized HCV RNA after treatment of cloned PH5CH cells and parental PH5CH cells with proteinase K immediately after virus inoculation revealed that PH5CH1, PH5CH7 and PH5CH8 cells contained 10-fold higher levels of HCV RNA than low susceptible cloned PH5CH or parental PH5CH cells. Furthermore, we demonstrated that HCV replication was maintained for 70-100 days in these three clonal lines when the temperature of cell culture after virus inoculation was reduced from 37 to 32 degrees C. Moreover, we demonstrated that interferon alpha had antiviral effect on HCV-infected PH5CH8 cells. The three PH5CH clones obtained in this study will provide a useful tool for the study of HCV replication and proliferation, and for development of an assay system for antiviral agents.

Antiviral Agents↗
PubMed1998

T cell responses to 53-kDa outer membrane protein of Porphyromonas gingivalis in humans with early-onset periodontitis.

Patients with early-onset periodontitis (EOP) are susceptible to infection with periodontopathic bacteria, such as Porphyromonas gingivalis. Ag53, 53-kDa outer membrane protein of P. gingivalis, evokes strong humoral immune responses in EOP patients. In a first step to clarify how host immune cells recognize Ag53, we established Ag53-specific short-term T cell lines from 22 subjects including 6 EOP patients and 16 healthy donors, using overlapping peptides based on Ag53 amino acid sequences. All T cell lines from active EOP patients recognized a common region (p141-181, especially p141-161) on Ag53, while those from healthy donors showed heterogeneous specificity. p141-181 was not recognized by T cell lines established from EOP patients following therapy. A monoclonal antibody to HLA-DRB 1 inhibited Ag53-induced proliferation of most of the T cell lines. Our observations suggest that, although antigen-presenting molecules are common in EOP patients and in healthy individuals, p141-161 includes a major T cell epitope(s) on Ag53 for active EOP patients but not for healthy individuals or inactive EOP patients.

Adult↗
PubMed1998

An Escherichia coli protein that exhibits phosphohistidine phosphatase activity towards the HPt domain of the ArcB sensor involved in the multistep His-Asp phosphorelay.

The Escherichia coli sensory kinase, ArcB, possesses a histidine-containing phosphotransfer (HPt) domain, which is implicated in the His-Asp multistep phosphorelay. We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity. Using in vivo screening, we first identified a gene that appeared to interfere with the His-Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid. The cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine-histidine-glycine (RHG) signature, at its N-terminus. Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose-2,6-bisphosphatase, E. coli periplasmic phosphatase and E. coli glucose-1-phosphate phosphatase, and ubiquitous phosphoglycerate mutase. Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA. It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not. Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling. These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His-Asp phosphorelay through regulating the phosphorylation state of the HPt domain.

Amino Acid Sequence↗
PubMed1998

Involvement of the tongue by lymphomatoid papulosis.

We report on a case of lymphomatoid papulosis (LyP) with involvement of the tongue. The patient was a 34-year-old Japanese man. Three reddish, centrally depressed, slightly elevated nodules were evident on the dorsal tongue, along with lesions elsewhere on the skin. One of them was biopsied and exhibited a superficial and deep, perivascular and interstitial mixed cellular infiltrate including atypical lymphoid cells, lymphocytes, neutrophils, and histiocytes. The patient also showed rhythmical recurrence of reddish papules and ulcerated nodules on the trunk, extremities, and anogenital area. Histologically, these papules showed a dense, wedge-shaped mixed cellular infiltrate in the dermis, which included medium and large atypical lymphoid cells, lymphocytes, neutrophils, and histiocytes. Immunoperoxidase staining for CD30 was positive in the cell membrane and cytoplasm of the atypical cells. We could not find other reports of LyP involving the tongue. Systemic treatment with interferon (INF)-alpha2a was dramatically effective in inhibiting recurrence of the eruption.

Adult↗
PubMed1998

Primary cutaneous adenoid cystic carcinoma with lymph node metastasis.

We report on a case of primary cutaneous adenoid cystic carcinoma with local recurrence and lymph node metastasis. The patient was a 52-year-old Japanese woman. The initial cutaneous lesion was a skin-colored, centrally elevated, thumb-sized nodule with hair loss on the scalp in the right side of the parietal region. Three and half years after the initial combined treatment consisting of surgery and irradiation, a recurrent nodule on the left side of the parietal region of the scalp and a lymph node metastasis with perinodal tissue involvement in the left retroauricular area were discovered. In the literature on primary cutaneous adenoid cystic carcinoma, the average age in the 37 cases reported, including the present case, is 58.1 years, and 23 of the patients are women. Thirty-two percent of the carcinomas (12 cases) occur on the scalp and 16% (6 cases) on the breast. The average size of the tumors in the 24 documented cases is 3.2 cm. The local recurrence rate is 51% (19 cases). Distant metastasis to the lungs and pleura are recorded in three cases. Lymph node metastasis is recorded only in two cases including the present case. Multiple modalities including surgery, irradiation, and chemotherapy should be used for this locally aggressive and potentially metastasizing carcinoma.

Adolescent↗
PubMed1998

Structural analysis and evaluation of the 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) gene in human essential hypertension.

AIM: Mutations of the 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) gene cause the syndrome of apparent mineralocorticoid excess, a rare autosomal recessive form of hypertension. We therefore investigated the question of whether variants of the 11beta-HSD2 gene can contribute to genetic susceptibility to essential hypertension. SUBJECTS AND METHODS: We performed a linkage study in 162 French hypertensive sibships using the affected sib-pair method on 347 sibling pairs and a polymorphic microsatellite marker that we identified in a 30 kb cosmid clone containing the 11beta-HSD2 gene. The coding sequence, introns 2-4 and 350 bp of the 5'-flanking region of the 11beta-HSD2 gene were screened for polymorphisms by polymerase chain reaction/single-strand conformation polymorphism, and a single polymorphism, Glu178/Glu (G534A), was identified in exon 3, which did not change the encoded amino acid sequence. A case-control study was conducted on 370 hypertensive subjects with a positive family history of hypertension and 783 French subjects with hypertension with or without a family history of hypertension, compared with 313 normotensive control subjects, all of whom were analyzed for the newly identified bi-allelic polymorphism. RESULTS: Statistical analyses using the affected sib-pair method did not show significant linkage between the 11beta-HSD2 microsatellite marker and hypertension. Furthermore, no positive association with hypertension was found with the Glu178/Glu (G534A) polymorphism. CONCLUSION: Our data do not suggest that variants of the 11beta-HSD2 gene contribute substantially to essential hypertension in Caucasians.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1998

Hepatitis C virus population dynamics in human lymphocytes and hepatocytes infected in vitro.

We previously found two cell lines (MT-2 and PH5CH) that were susceptible to hepatitis C virus (HCV) infection. Analysis of the infectivity of sera from HCV-positive blood donors for MT-2 and PH5CH cells suggested the cell tropism of HCV. To investigate further the cell tropism of HCV, the dynamics of HCV populations during culture were examined using three MT-2 clones and three PH5CH clones, infected with inoculum 1B-2. To type HCV populations in these infected cells, the HCV hypervariable region 1 (HVR1) in these cloned cells was characterized by sequence analysis and HpaII digestion analysis, which could distinguish three major HVR1 types (I, II and III) derived from the inoculum 1B-2. It was found that genomes containing HVR1 type I became predominant in MT-2 clones, and genomes containing HVR1 type II became predominant in PH5CH clones during culture after inoculation. These results suggest that inoculum 1B-2 contains both lymphotropic and hepatotropic HCV species, which can be distinguished by HVR1 type. To search for cell type-specific sequences in regions other than HVR1, three HCV cDNA clones (3.4 kb of the 5' noncoding region to the nonstructural 2 region) containing HVR1 type I obtained from HCV-infected MT-2C cells, and three HCV cDNA clones containing HVR1 type II obtained from HCV-infected PH5CH7 cells were sequenced. Following a comparison of the sequences, 11 amino acids were identified as candidates for determinants of the cell tropism of HCV.

Amino Acid Sequence↗
PubMed1998

Multiple cutaneous epithelioid hemangioendothelioma: a case with spindle cells.

A case of multiple cutaneous epithelioid hemangioendothelioma (EH) in a Japanese male is reported. The patient was a 52-year-old Japanese bureaucrat. Clinically, about 10 purplish-red, elastic soft or hard papules and nodules had appeared on both lower extremities and the left buttock over the past 20 years. They were excised, but some of them recurred. Histologically, the papules and nodules were well-circumscribed tumors located in the superficial and deep dermis. They appeared to arise from medium-sized vessels and were composed of proliferations of characteristic epithelioid endothelial cells with intracytoplasmic vacuolization and spindle cells. No other extracutaneous tumors were found. We diagnosed the present case as multiple cutaneous EH with spindle cells. The presence of epithelioid endothelial cells and absence of pleomorphism and mitoses of nuclei of spindle cells made differential diagnosis from Kaposi's sarcoma possible. The protracted clinical course seems characteristic of EH in various organs including skin.

Blood Vessels↗
PubMed1998

Single-step reverse transcription-polymerase chain reaction for the detection of hepatitis C virus RNA.

We developed a novel single-step reverse transcription-polymerase chain reaction (RT-PCR), which is equal in sensitivity and specificity to RT-nested PCR, based on both reverse transcriptase and Taq DNA polymerase working efficiently under single buffer reaction conditions. Using in vitro synthesized hepatitis C virus (HCV) RNA, it was demonstrated that 10-100 copies of HCV RNA could be detected with a set of primers that amplify a 144 base-pair sequence unique to the 5'-noncoding region of HCV RNA. Furthermore, this method was successfully performed on serum and liver biopsy specimens obtained from patients with chronic hepatitis C. In addition, HCV RNA from in vitro HCV-infected MT-2C cells, which supported HCV replication, was also detected by this method. The method is anticipated to improve the detection of small amounts of RNA, such as that of HCV, promoting both labor savings and the prevention of carry-over contamination.

Hepacivirus↗
PubMed1998

New monoclonal antibodies against a recombinant second envelope protein of Hepatitis C virus.

To study the immunological features of the hepatitis C virus (HCV) envelope protein (E2 protein), new specific monoclonal antibodies (mAbs) were generated. WKA/H rats were immunized with syngeneic cells infected with a vaccinia virus expressing the E2 protein and with soluble E2 protein obtained from Chinese hamster ovary cells with a plasmid-based expression system. By screening hybridoma cells obtained from spleen cells of the immunized rats, three specific mAbs were obtained. One mAb was reactive to a peptide corresponding to the hypervariable region 1 (HVR1) in E2 protein, while the others reacted to regions outside HVR1. The significance of these antibodies for the diagnosis of HCV infection as well as for analysis of the structure of the HCV E2 protein will be discussed.

Amino Acid Sequence↗
PubMed1998

Power Doppler sonography for hepatocellular carcinoma: factors affecting the power Doppler signals of the tumors.

AIMS/BACKGROUND: A recent advancement in Doppler ultrasonography (US) is power Doppler for detecting low-velocity blood flow at the microvascular level with angle independence. The present study was performed to characterize the factors contributing to the power Doppler signals of hepatocellular carcinoma (HCC). METHOD: Correlation of Doppler signals of HCC in 114 patients with 178 HCC nodules was analyzed in relation to the findings of CT and angiography, tumor characteristics (size, echo pattern, and histological differentiation of tumor), viral markers and severity of liver disease. RESULTS: The sensitivity of power Doppler US was superior to that of CT and angiography (each p<0.05; McNemar's test). The detection rate of power Doppler signal was significantly higher in tumors with diameter > or =2 cm (vs <2 cm in diameter), and with low/mixed echo pattern (vs high echo appearance), and with moderately/poorly differentiated HCC (vs well-differentiated HCC). Univariate analysis revealed that echo pattern, tumor size and histological differentiation of HCC in addition to CT and angiographic findings were significant. Multivariate analysis showed that tumor size and differentiation were significant. CONCLUSION: These results indicate that tumor characteristics play an important role in power Doppler signals and that these could be assessed by the presence or absence of power Doppler signals.

Angiography↗
PubMed1998

[Retropharyngeal abscess in an adult and an elderly woman].

Retropharyngeal abscess is reported to be decreasing in frequency in recent years. We report two cases of retropharyngeal abscess that were diagnosed within four years in Department of Otorhinolaryngology, Gifu University Hospital. Case 1 was a 36-year-old male who was suggested to have an abscess as primary infection and case 2 was a 71-year-old female whose abscess seemed to be a secondary infection following unknown primary infection. Endoscopic or open neck drainage as well as antibacterial chemotherapy mainly with combinations of flomoxef and clindamycin in the case 1 and piperacillin and clindamycin in the case 2 was successfully carried out. They were discharged an 15-day and 24-day after operation in the cases 1 and 2, respectively. A 30 min. culture after sample collection on operation demonstrated aerobe-anaerobe mixed infection in both cases; three aerobes and four anaerobes in the case 1 and three aerobes and two anaerobes in the case 2. These results suggest that retropharyngeal abscess may be a complicated infection involving more bacterial species than has been commonly believed. Measuring susceptibility of isolates to antimicrobial agents, a strain of Capnocytophaga sp. and a strain of anaerobic gram-negative rod were highly resistant to piperacillin, cefroxine and ofloxacin. It is important for adequate antibacterial chemotherapy to grasp the bacteriology of retropharyngeal abscess and analyze susceptibility of antimicrobial agents.

Adult↗
PubMed1998

Regulation and physiological role of the DAS1 gene, encoding dihydroxyacetone synthase, in the methylotrophic yeast Candida boidinii.

The physiological role of dihydroxyacetone synthase (DHAS) in Candida boidinii was evaluated at the molecular level. The DAS1 gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed. Western and Northern analyses revealed that DAS1 expression was regulated mainly at the mRNA level. The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase. The DAS1 gene was disrupted in one step in the host genome (das1Delta strain), and the growth of the das1Delta strain in various carbon and nitrogen sources was compared with that of the wild-type strain. The das1Delta strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y. Sakai et al., J. Bacteriol. 179:4480-4485, 1997). These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the das1Delta strain on media containing methylamine or choline as a nitrogen source) suggested that DAS1 is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.

Aldehyde-Ketone Transferases↗
PubMed1998

Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR.

Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A-, toxin B-) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A-, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A-, toxin B-strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A-, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A-, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A-, toxin B+ strains from toxin A+, toxin B+ and toxin A-, toxin B- strains and will contribute to elucidation of the precise role of toxin A-, toxin B+ strains in intestinal diseases.

Adolescent↗