Biomedical subjects

H Kotani

Publications and source records attributed to H Kotani.

At least 145 records · Page 8Linked to original sources

PubMed1989

Overproduction and crystallization of FokI restriction endonuclease.

To overproduce FokI endonuclease (R.FokI) in an Escherichia coli system, the coding region of R.FokI predicted from the nucleotide sequence was generated from the FokI operon and joined to the tac promoter of an expression vector, pKK223-3. By introduction of the plasmid into E. coli UT481 cells expressing the FokI methylase gene, the R.FokI activity was overproduced about 30-fold, from which R.FokI was purified in amounts sufficient for crystallization. The removal of a stem-loop structure immediately upstream of the R.FokI coding region was essential for overproduction.

Amino Acid Sequence↗

PubMed1989

The fokI restriction-modification system. I. Organization and nucleotide sequences of the restriction and modification genes.

A DNA fragment that carried the genes coding for FokI endonuclease and methylase was cloned from the chromosomal DNA of Flavobacterium okeanokoites, and the coding regions were assigned to the nucleotide sequence by deletion analysis. The methylase gene was 1,941 base pairs (bp) long, corresponding to a protein of 647 amino acid residues (Mr = 75,622), and the endonuclease gene was 1,749 bp long, corresponding to a protein of 583 amino acid residues (Mr = 66,216). The assignment of the methylase gene was further confirmed by analysis of the N-terminal amino acid sequence. The endonuclease gene was downstream from the methylase gene in the same orientation, separated by 69 bp. The promoter site, which could be recognized by Escherichia coli RNA polymerase, was upstream from the methylase gene, and the sequences adhering to the ribosome-binding sequence were identified in front of the respective genes. Analysis of the gene products expressed in E. coli cells by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weights of both enzymes coincided well with the values estimated from the nucleotide sequences, and that the monomeric forms were catalytically active. No significant similarity was found between the sequences of the two enzymes. Sequence comparison with other related enzymes indicated that FokI methylase contained two copies of a segment of tetra-amino acids which is characteristic of adenine-specific methylase.

Amino Acid Sequence↗

PubMed1989

Production and characterization of recombinant human neutrophil chemotactic factor.

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.

Amino Acid Sequence↗

PubMed1989

[Clinical survey on denture stomatitis. 1. Relation between denture plaque and denture stomatitis].

Denture stomatitis is a very frequent complication of the denture wearers. In this study, the relation between denture plaque and denture stomatitis was evaluated by a simplified culture (STOMASTAT). 583 patients wearing dentures (1981-1987) were selected, from 47 to 91 years old. The following results were obtained. 1. Degree of denture plaque in 583 patients was severe, moderate, and no detection: 30, 35 and 35%, respectively. 2. Grade of denture stomatitis in 583 patients was severe, moderate, and no symptom: 24, 36 and 40%, respectively. In this survey, the occurrence of denture stomatitis was about 60%. 3. The good correlation between degree of denture plaque and grade of denture stomatitis was recognized in this study. The present results suggest that denture plaque plays an etiologic role in denture stomatitis and, in daily practice, it is important to instruct denture plaque control for denture wearers.

Aged↗

PubMed1989

[Clinical studies on TMJ arthrosis in young people. A study of dental axis of lower molar teeth].

The subjects were 37 female patients with juvenile TMJ arthrosis (T group) and 27 healthy females (N group) who visited the Dept. of Oral Surgery, Nagoya University Hospital or the Dept. of Dent-Oral Surgery, Nagoya University Branch Hospital. Following evaluations were made: 1) L6 to L7 angle 2) L6 to occlusal plane 3) L7 to occlusal plane 4) L6 to mandibular plane 5) L7 to mandibular plane 6) occlusal plane angle 7) mandibular plane angle Following results were obtained: 1. Significant differences between T group and N group were not seen in the occlusal plane angle. 2. A majority in the T group showed a mesial inclination of the lower second molar. 3. Mandibular plane angle in the T group was larger than that in the N group.

Adolescent↗

PubMed1989

[Risk factors for peptic ulcers treated surgically].

To elucidate the risk factors of peptic ulcer which require surgical treatments, we examined the influences of following factors such as sex, age, smoking, and alcohol in 308 peptic ulcer patients. In the group with gastric ulcers, high age group was likely to be operated. On the contrary, young patients with duodenal ulcers were likely to be treated surgically. Sex, smoking and alcohol were not regarded as risk factors. The correlations between the degree of neurosis and the amounts of smoking and alcohol were analyzed in the cases with duodenal ulcers. In the cases treated surgically, the degree of neurosis showed no or little correlations. In the cases treated conservatively, however, the degree of neurosis showed positive correlation with the amounts of smoking and alcohol.

Adolescent↗

PubMed1988

Activation and function of an autoreactive T cell clone with dual immunoregulatory activity.

Previously it was demonstrated that the human autoreactive CD4+ T cell clone MTC-4 is bifunctional, having the capacity to augment differentiation of autologous B cells into Ig-secreting cells in the absence of PWM and the capacity to suppress such differentiation in the presence of PWM. In the present study it was shown that these two functions of MTC-4 are mediated by distinctly different mechanisms. In the presence of autologous class II MHC Ag, MTC-4 releases one or more non-MHC-restricted soluble factors which stimulate B cell differentiation. The helper factors are different from IL-2, and act on both resting (small) and activated (large) B cells. The suppressor function of MTC-4 cells is elicited when MTC-4 cells are co-cultured with autologous non-T cells preincubated with PWM for 4 h, but not with non-T cells preincubated with PWM for 24 h; thus, activated autologous non-T cells have a transient capacity to induce MTC-4 suppressor function. Induction of MTC-4 suppressor activity is not associated with increased proliferation of MTC-4 and is mediated by low numbers of these cells. Unlike helper function, MTC-4 suppression of Ig synthesis can occur late in B cell cultures, and MTC-4 suppresses Ig production by autologous B cells, but not by allogeneic B cells. Finally, in co-cultures with activated autologous non-T cells and allogeneic B cells, MTC-4 can simultaneously produce helper factors that augment Ig synthesis by allogeneic B cells and suppress Ig synthesis by autologous B cells. In summary, exposure of MTC-4 to autologous non-T cells causes release of non-MHC-restricted factors which augment Ig production by both resting and activated autologous B cells, whereas exposure of MTC-4 to recently activated B cells causes MTC-4 to express the additional function of directly suppressing Ig production by differentiated autologous B cells. Thus autoreactive T cells may be uniquely suited to regulate Ig production.

Adult↗

PubMed1988

[Localization of enolase in the cancer nest of digestive organs].

Gamma-enolase, otherwise called neuron specific enolase (NSE), is specific for neural and neuro-endocrine systems and is present in the tumors of these systems. Gamma-enolase is also reported to be present, however, in a localized form in the tissues of other nonneural systems in some cases of lung cancer in relatively large number of incidences. We examined immunohistologically specimens of digestive organ cancers. Two principal conclusions were obtained. First, gamma-enolase is not specific for neural and neuro-endocrine systems. Second, gamma-enolase is localized in the epithelium of cancers of some cases following the transformation into cancer.

Biomarkers, Tumor↗

PubMed1988

Isolation of mycoplasmas from house musk shrews (Suncus murinus).

Mycoplasmas were isolated from various sites of experimental and wild house musk shrews (Suncus murinus). The oral cavity was the most prominent site to harbor mycoplasmas (15/18; 83%), followed by the nasal cavity (9/18; 50%). All of the isolated strains required serum for their growth and all fermented glucose. They were found to be serologically homogeneous by growth inhibition test but did not cross-react with several type strains of mycoplasma or reference strains of murine, feline, canine, porcine, bovine and equine origins.

Animals↗

PubMed1987

Nucleotide sequence and expression of the cloned gene of bacteriophage SP6 RNA polymerase.

The coding region of the gene for bacteriophage SP6 RNA polymerase was cloned into pBR322, and its entire nucleotide sequence was deduced. The predicted amino acid sequence for the polymerase consists of 874 amino acid residues with a total molecular weight of 98,561 daltons. Comparison of the amino acid sequence with that of T7 RNA polymerase reveals that regions with partial homology are present along the sequence. The coding region of SP6 RNA polymerase was inserted into an E. coli expression vector. The polymerase gene was efficiently expressed in E. coli cells, and the enzymatic properties of the expressed polymerase were very similar to those of the enzyme synthesized in SP6 phage-infected Salmonella typhimurium cells.

Amino Acid Sequence↗

PubMed1987

Colonization with genital mycoplasmas in pregnant women and their neonates and birth weight.

The colonization by genital mycoplasmas of mothers and their newborn infants was examined in 114 pregnant women and their 84 neonates. Urine and cervical swabs were taken from the pregnant women in the last trimester, and urine from the neonates within six days after birth. Ureaplasma urealyticum was found in 73.7% of the pregnant women and in 17.9% of the neonates. Mycoplasma hominis was isolated in 8.8% of the material from the pregnant women and in 1.2% of that from the neonates. The isolation rate of U. urealyticum from newborn girls was significantly higher than that from newborn boys (p less than 0.01). There was no correlation between the germ density in the urine of the pregnant women and that of the neonates. The urine of the neonates harboured as many U. urealyticum as that of the adults. The frequency of colonization by mycoplasmas in the pregnant women or the neonates was not related to the duration of gestation or the babies' birth weight.

Adult↗

PubMed1987

Identification and isolation of mycoplasmas by immunobinding.

This laboratory has developed an immunobinding assay (IBA) to identify and detect mycoplasmas in a variety of specimens. The specimen is inoculated in volumes of 10 microliters onto nitrocellulose (NC) paper, which is then blocked, fixed, and incubated at room temperature. Specific antimycoplasma polyclonal or monoclonal antibody is first added, followed by peroxidase-labeled antibody directed toward the first immunoglobulin. Alternately, antimycoplasma IgG can be purified and conjugated to horseradish peroxidase for use in a direct assay. Addition of a developing solution results in the formation of purple color when mycoplasmas are present. Titers of rabbit antimycoplasma antisera range from 1:1,000 to 1:30,000. This assay can detect approximately 1 x 10(4) colony-forming units (CFU). This IBA has been used routinely to identify mycoplasmal isolates from 132 infected cell cultures. In addition, the procedure successfully detected Mycoplasma pneumoniae in throat swabs from patients with respiratory illness within 2 h. Perfect correlation was obtained with the IBA and microbiological culture of throat swabs for M. orale, M. salivarium and M. pneumoniae. The procedure was successfully used for other Mollicutes. It detected ureaplasmas in urogenital swabs and corn stunt spiroplasmas in infected corn plants and leafhoppers. A modification of the technique has been developed that identifies mycoplasma colonies on agar. It has also been used to assay for antimycoplasma antibodies in serum.

Animals↗

PubMed1986

An autoreactive T cell clone that can be activated to provide both helper and suppressor function.

To understand further the biologic significance of the autologous mixed lymphocyte reaction, we determined the functional properties of autoreactive T cell lines and clones. Initially, we found that cells in an uncloned autoreactive Leu-3+ T cell line helped immunoglobulin production when added to cultures containing fresh T and non-T cells in the absence of pokeweed mitogen (PWM) but suppressed immunoglobulin production in the same cultures in the presence of PWM. To explain this phenomenon, we studied the immunoregulatory potential of an autoreactive T cell clone termed MTC-4. This clone bore the phenotype Leu-3+, 2-, 8-, 11-, DR+ and underwent proliferation when co-cultured with autologous, but not allogeneic non-T cells. Of interest, the immunoregulatory potential of the MTC-4 cells varied according to how the cells were activated. When MTC-4 cells were cultured with autologous non-T cells in the absence of antigen or mitogen (unactivated non-T cells), polyclonal immunoglobulin production (detected by reverse PFC assay) was observed. This helper activity was MHC-restricted in that it was elicited only by autologous non-T cells or MHC-matched allogeneic non-T cells; however, once activated by autologous non-T cells, it could also help allogeneic non-T cells. In contrast, when MTC-4 cells were cultured with autologous non-T cells in the presence of PWM (activated non-T cells), immunoglobulin production was greatly suppressed. This suppression was also observed when MTC-4 cells were added to cultures containing exogenous T cell help (such as that provided by autologous fresh T cells) and was not due to a direct effect of PWM on the T cell clone, because preincubation of MTC-4 cells with PWM before culture with non-T cells did not result in suppression. On the basis of these data, we conclude that autoreactive T cells can have dual immunoregulatory function that is manifest, at least in part, at the single cell level. Moreover, these regulatory functions are differentially elicited depending on the state of activation of the stimulating autologous non-T cells: when stimulated by MHC antigens present on unactivated B cells, they provide helper activity; and when stimulated by MHC antigens present on activated B cells, they act as suppressor cells. Autoreactive T cells with dual regulatory potential appear to make up a substantial proportion of all autoreactive T cells and are cells that are uniquely adapted to maintain immunologic homeostasis.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1986

Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma, Spiroplasma mirum, strain SMCA.

A helical mycoplasma, Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T3 cells. The time of inoculation of S. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation.

Animals↗

PubMed1986

Comparative studies to determine the efficiency of 6 methylpurine deoxyriboside to detect cell culture mycoplasmas.

Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were: Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection with Spiroplasma mirum when this organism was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98.8, and 97.1%, respectively.

Bisbenzimidazole↗

PubMed1986

Detection of cell culture mycoplasmas by a genetic probe.

The utility of a genetic probe prepared against mycoplasmal ribosomal RNA was evaluated. The probe consisted of a tritiated DNA probe prepared with reverse transcriptase against Mycoplasma and Acholeplasma. Using a batch process without Southern blots and autoradiography, the probe assay detected mycoplasma infection in 27 of 52 cell cultures, an incidence of 51.9%. No false-positives were detected. Two false-negatives occurred; these were unusual mycoplasma infections of cell cultures grown in serum-free media. The sensitivity of the probe varied with mycoplasma species, from 9.3 X 10(3) for M. arginini to 2.6 X 10(6) for M. orale. The assay is rapid, taking only 2-3 h.

Acholeplasma↗

PubMed1986

Structure and gene organization of bovine neuromedin K precursor.

cDNA and genomic DNA clones for the precursor of a mammalian neuropeptide tachykinin, neuromedin K, have been isolated and characterized by molecular cloning and sequence analysis. The deduced amino acid sequence indicates that the bovine neuromedin K precursor (preprotachykinin B) consists of 126 amino acid residues including a putative signal peptide. There are two preprotachykinin B mRNAs that differ only at the 5' extremity of the untranslated regions. The major mRNA species is encoded by seven exon sequences, while the minor species includes two extra 5' exon sequences and lacks the 5' terminus of the first exon sequence for the major mRNA species. The above gene organization for the major preprotachykinin B mRNA closely resembles that for the preprotachykinin A mRNA encoding the precursor for substance P and substance K. This structural resemblance strongly suggests that the two preprotachykinin genes have evolved from a common ancestor gene. Furthermore, we have found that the preprotachykinin A and B mRNAs markedly differ in their major expression sites. The results described thus indicate that the diversity of the mammalian tachykinin system has been acquired through various cellular mechanisms including gene duplication and differential expression of duplicated genes.

Amino Acid Sequence↗

PubMed1986

Evaluation of cervical cord function in cervical spondylotic myelopathy and/or radiculopathy using both segmental conductive spinal-evoked potentials (SEP).

To evaluate the function of the cervical cord and to diagnose the level and severity of cervical cord lesions in myelopathy, both segmental and conductive spinal evoked potentials (SEP) were measured in 73 patients with cervical spondylotic myelopathy and/or radiculopathy. In normal subjects, segmental SEPs consisted of two waves (R and N waves). Ascending conductive SEPs also consisted of two waves (first and second waves). The function of the cervical cord, including roots, grey matter, and white matter, can be measured by the combined method using both segmental and conductive SEPs, and this allows differentiation among radiculopathy and various types of myelopathy.

Cervical Vertebrae↗