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Biomedical subjects

Y Okumura

Publications and source records attributed to Y Okumura.

At least 19 recordsLinked to original sources
PubMed2003

[Adhesion or organization during surgery due to mediastinoscopy before induction therapy for non-small cell lung cancer].

We conducted a retrospective study to investigate amounts of adhesion or organization during surgery due to mediastinoscopy before induction therapy for non-small cell lung cancer (NSCLC). A case series of 13 patients (c-stage IIIA in 11 and IIIB in 2) with right-side NSCLC who underwent induction therapy were assessed. They were between 39 and 71 years old and 8 males and 5 females. Induction therapy was chemotherapy alone in 4 cases and chemo-radiotherapy in 9 cases. The mediastinocopy was used in 4 cases. Mediastinal lymph node resection was incomplete in 3 of the 4 (75%) cases among the mediastinoscopy group but the no-mediastioscopy group had no incomplete resection (p = 0.01), though 2 of the 9 (22%) cases among the chemo-radiation group and 1 of the 4 (25%) among the chemo alone group (p > 0.9). Severe adhesion or organization can occur after the mediastinoscopy and induction therapy, which might be a course of incomplete mediastinal lymph node resection.

Adult↗
PubMed2002

Induction of DNA double strand breaks in scid cells by carbon ions.

The DNA double strand breaks (DSBs) induced by X ray and carbon ion beam irradiation in scid cells were analysed using pulsed-field gel electrophoresis. Scid cells and hybrid cells were ideal to study the DNA DSB repair mechanisms, because their genetic backgrounds were identical except DNA-PK activity. Induction of DNA DSBs was determined after exposure to X rays and carbon beams. DNA DSB repair was by biphasic kinetics with a fast and a slow component. For scid cells only a slow component was observed, whereas the kinetics of DSBs repair was biphasic with a fast and a slow component. It was concluded from the experimental data that the induced DSB rejoining in scid cells was due to the lack of DNA-PK activity.

Carbon Radioisotopes↗
PubMed2002

Ambroxol suppresses influenza-virus proliferation in the mouse airway by increasing antiviral factor levels.

The protective effect of ambroxol, a mucolytic agent which has antioxidant properties and stimulates the release of pulmonary surfactant, against influenza-virus proliferation in the airway was investigated in mice. Ambroxol or the vehicle was administered intraperitoneally twice a day for 5-7 days to mice shortly after intranasal infection with a lethal dose of influenza A/Aichi/68 (H3N2) virus, and the survival rate, virus titre and levels of factors regulating virus proliferation in the airway fluid were analysed. Ambroxol significantly suppressed virus multiplication and improved the survival rate of mice. The effect of ambroxol reached a peak at 10 mg x kg(-1) x day(-1), higher doses being less effective. Ambroxol stimulated the release of suppressors of influenza-virus multiplication, such as pulmonary surfactant, mucus protease inhibitor, immunoglobulin (Ig)-A and IgG, although it stimulated the release of a trypsin-type protease that potentiates virus proliferation. In addition, ambroxol transiently suppressed release of the cytokines, tumour necrosis factor-alpha, interferon-gamma and interleukin-12, into airway fluid. Although ambroxol had several negative effects on the host defence system, overall it strikingly increased the concentrations of suppressors of influenza-virus multiplication in the airway.

Ambroxol↗
PubMed2001

Picometer-scale dynamical x-ray imaging of single DNA molecules.

Time-resolved dynamical x-ray imaging of individual DNA molecules with picometer-scale precision is demonstrated for the first time. Diffracted x-ray tracking (DXT), a single-molecule experiment with x rays, monitors the rotating motions, rather than the translational motions, of a labeled nanocrystal. DXT can obtain information about the dynamics of single molecules through a quantitative analysis, since the signals from DXT are independent of the chemical conditions.

DNA↗
PubMed2001

The growth and cyst formation of a toxic dinoflagellate, Alexandrium tamarense, at low water temperatures in northeastern Japan.

A field survey was carried out in early spring to investigate the growth physiology and efficiency of cyst formation of Alexandrium tamarense in low water temperatures. A bloom of A. tamarense occurred in a stratified water column, formed by river inflow. The in situ growth rate estimated from daily cell abundance was high, 0.33 divisions day(-1), at 7.5-9 degrees C. New cysts began to be observed during the late growth phase. Maximum cyst flux (600 cysts cm(-2) day(-1)) was observed just after maximum cell abundance occurred. PO(4)-P Chl a(-1) gradually decreased and reached extremely low levels beyond the mid-growth phase of A. tamarense. As sinking cysts were also recognized at that time, it suggests cyst formation may have been induced by depletion of phosphorus source. The incidence of cyst formation (C.I) was 30%. The number of C.I was the same as reported previously for batch cultures under conditions suitable for vegetative growth. These results indicate that A. tamarense grows with considerably higher growth rate and transforms to cysts in high numbers, in low water temperatures in the field.

Journal Article↗
PubMed2001

Detection of hepatic metastasis from medullary thyroid cancer with Tc-99m-MIBI scintigraphy in a patient with Sipple's syndrome.

We report a case of Sipple's syndrome with a hepatic tumor. A 28-year-old male with medullary thyroid cancer and bilateral pheochromocytomas was diagnosed as having Sipple's syndrome. The hepatic tumor was found to be hypervascular by means of a dynamic abdominal CT scan. An I-131-MIBG scan showed no increased accumulation in the tumor. A whole body Tc-99m-MIBI scan had shown, however, increased accumulation in the tumor by 10 minutes after i.v. Tc-99m-MIBI. The hepatic tumor was diagnosed histologically as a metastasis from the medullary thyroid cancer. To our knowledge, there have been no previous reports of increased Tc-99m-MIBI accumulation in hepatic metastases from medullary thyroid cancer.

Adrenal Gland Neoplasms↗
PubMed2001

Influence of organic solvents on the growth of marine microalgae.

Ninety-six-hour toxicity tests of five organic solvents (methanol, ethanol, acetone, N,N-dimethylformamide [DMF], and dimethyl sulfoxide [DMSO]) on nine species of marine microalgae (Bacillariophyceae; Skeletonema costatum, and Chaetoceros calcitrans, Prasinophyceae; Tetraselmis tetrathele, Haptophyceae; Isochrysis galbana and Pavlova lutheri, Dinophyceae; Prorocentrum minimum, Raphidophyceae; Heterosigma akashiwo, Euglenophyceae; Eutreptiella sp., Chlorophyceae; Dunaliella tertiolecta) were conducted. For D. tertiolecta, T. tetrathele, I. galbana, and P. lutheri, methanol was the least toxic solvent. For S. costatum, C. calcitrans and Eutreptiella sp., DMSO was the least toxic solvent. For Heterosigma akashiwo, DMF was the least toxic solvent. In general, solvent toxicity was ethanol > methanol or acetone > DMF > DMSO. In relation to sensitivity of the microalgae to solvents, D. tertiolecta and T. tetrathele were lower in sensitivity, and Eutreptiella sp. and H. akashiwo were higher in sensitivity than the other species. The no observed effect concentration (NOEC) of acetone, DMF, and DMSO were higher than for ethanol and methanol. Acetone, DMF, and DMSO were suitable for carrier solvents used in toxicity test of water-insoluble chemicals. Excellent regression between logEC(50) and logNOEC was obtained (logNOEC = 1.09 x logEC(50) - 0.84, R(2) = 0.91). This equation indicated that higher toxicity was higher application factor (EC(50)/NOEC) than lower toxicity. All application factors were higher than 0.1.

Dose-Response Relationship, Drug↗
PubMed2001

Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding.

BACKGROUND: Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in-door allergens. Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen-specific immunotherapy (AIT). Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed. OBJECTIVE: Our purpose is to establish an efficient system to prepare recombinant Der f 1(rDer f 1), identical in quality to native Der f 1. METHODS: The preproforms of Der f 1 and a mutant N53Q, whose consensus motif for N-glycosylation was disrupted, were expressed in yeast Pichia pastoris. Cysteine protease activity and IgE reactivity were analysed using synthetic substrates and by RAST-EIA, respectively. RESULTS: The proforms of the two rDer f 1 molecules were efficiently secreted into culture medium. Their prosequences were removed autocatalytically by dialysis against acidic buffer. Although the wild-type rDer f 1 was more highly glycosylated than native Der f 1, N53Q had almost the same apparent molecular weight as native Der f 1 on SDS-PAGE. Both the protease and IgE binding activities of the mature rDer f 1 molecules were the same as those of native Der f 1, whereas the proforms had no or markedly reduced activities. CONCLUSION: The efficient system to prepare active rDer f 1s established in this study is useful for diagnosis and standardized AIT for house dust mite allergy. Furthermore, the system would be a tool for analysis of IgE epitopes, determination of tertiary structure, allergen engineering for safer and more effective AIT, resolving the relation between the enzymatic activity and pathogenesis, and the development of therapeutic inhibitors.

Animals↗
PubMed2001

Mini-plasmin found in the epithelial cells of bronchioles triggers infection by broad-spectrum influenza A viruses and Sendai virus.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. Here we report a protease present in the airway that, like tryptase Clara, can process influenza A virus haemagglutinin and Sendai virus envelope fusion glycoprotein. This protease was extracted from the membrane fraction of rat lungs, purified and then identified as a mini-plasmin. Mini-plasmin was distributed predominantly in the epithelial cells of the upward divisions of bronchioles and potentiated the replication of broad-spectrum influenza A viruses and Sendai virus, even that of the plasmin-insensitive influenza A virus strain. In comparison with plasmin, its increased hydrophobicity, leading to its higher local concentrations on membranes, and decreased molecular mass may enable mini-plasmin to gain ready access to the cleavage sites of various haemagglutinins and fusion glycoproteins after expression of these viral proteins on the cell surface. These findings suggest that mini-plasmin in the airway may play a pivotal role in the spread of viruses and their pathogenicity.

Amino Acid Sequence↗
PubMed2001

Detection of occlusal caries under sealants by use of a laser fluorescence system.

OBJECTIVE: In recent years, various dental caries diagnosis systems, including digital radiography, light fluorescence, and lasers, have been developed. The aim of this study was to evaluate the new laser fluorescence system known as DIAGNOdent for its ability to detect occlusal caries under sealants. BACKGROUND DATA: Fluorescence induced by laser light for early diagnosis of enamel caries was introduced by Bjelkhagen et al. in 1982, but these systems were difficult to use in the oral cavity. Recently, DIAGNOdent has been shown to have a higher diagnostic validity for the detection and quantification of caries lesions than the electronic caries monitor and to have a higher reproducibility for measurements, according to work by Lussi et al. in 1999. The laser diagnosis system is able to indicate the value of reflected fluorescence on a digital display. This value is associated with the carious progression. METHODS: Thirty-two extracted permanent premolars and molars that had occlusal dental caries were used in this study. The reflected fluorescence was measured by use of DIAGNOdent at the occlusal surface of the tooth before and after chemical irrigation, acid etching, and application of a light-cured fluoride releasing sealant. The measurements were two or three points per experimental tooth. A total of 53 points on the experimental teeth was evaluated. RESULTS: The mean reflected fluorescence value was significantly decreased by chemical irrigation; however, no difference was noted between the value before and the salve after acid etching. These teeth were divided into three groups: clear, red, and white as tooth color sealants of the tooth. The reflected fluorescence value before and after sealants was tested for statistical significance. The diagnosis of caries under sealants was up to 10 of reflected fluorescence value in the study. Clear and red sealants were used to reveal caries under sealants using visual inspection, and all sealants groups achieved 90% of reflected fluorescence value after sealants application. However, the white sealants did not reveal caries through visual inspection. The detection ability of caries under white sealants was 53.5% (n = 23/42). CONCLUSIONS: This laser diagnosis system makes it easy to detect the existence of caries under a pit and fissure sealant during a routine check-up.

Dental Caries↗
PubMed2001

Expression of humanized Fab fragments that recognize the IgE-binding domain of human Fc(epsilon)RIalpha in COS and CHO cells.

Interfering with the binding of IgE to high-affinity IgE receptor alpha chain (Fc(epsilon)RIalpha) is a straightforward strategy for the specific prevention of the IgE-mediated allergic reaction specifically. A Fab fragment (Fab) of a humanized antibody against the membrane proximal IgE-binding domain of human Fc(epsilon)RIalpha inhibits the release of histamine from human basophils. We established an efficient expression system in which to produce directly the humanized anti-human Fc(epsilon)RIalpha Fabs without papain-digestion of the whole antibody. Four Fabs with different C-termini of CH1 were expressed directly in COS-7 cells transfected with expression vectors with or without the Fc gene downstream of a stop codon inserted within the hinge gene. The secretion of Fabs when transfected without the Fc gene was remarkably enhanced compared to that when transfected with the Fc gene. The ability of Fabs to inhibit IgE-Fc(epsilon)RIalpha binding when transfected without the Fc gene was equivalent to that of purified Fab prepared by papain-digestion of the whole antibody. No significant differences among the four Fabs were observed in secretion or activity. Clones of CHO-transfectant cells that secreted the Fabs constitutively were acclimatized to a serum-free medium. Analysis of the binding interface between the Fab and human Fc(epsilon)RIalpha will provide useful information for the design of therapeutic reagents for allergy and asthma.

Animals↗
PubMed2001

Effects of site-directed mutagenesis in the cysteine residues and the N-glycosylation motif in recombinant Der f 1 on secretion and protease activity.

BACKGROUND: The group 1 allergens from mite feces, which belong to the papain-like cysteine protease family, are the most significant in-door allergens. In this study, we analyzed the contribution of the cysteine residues and N-glycosylation in Der f 1, the group 1 allergen from Dermatophagoides farinae, to secretion and maturation by using systems for expression of recombinant Der f 1 (rDer f 1). METHODS: The rDer f 1 and its mutants were expressed in yeast Pichia pastoris and insect SF9 cells. Secretion of their proforms was checked by SDS-PAGE or immunoblotting. Protease activities of the secreted proform of a mutant and the mature form were compared with that of native Der f 1. RESULTS: The proform of a mutant Der f 1, pro-N53Q, whose consensus motif for N-glycosylation was disrupted, was not secreted in insect SF9 cells although secreted in P. pastoris. Indirect evidence was obtained to support the disulfide bond formation between Cys4 and Cys118, which were not conserved in papain. A mutant for Cys35 in the catalytic site of the cysteine protease, pro-C35S/N53Q, was secreted, but the other mutants for cysteines concerning intramolecular disulfide bonds were not secreted in P. pastoris. The prosequence of pro-C35S/N53Q was removed by an in vitro activation process. The mature C35S/N53Q showed low protease activity. CONCLUSION: N-glycosylation is essential for secretion in insect SF9 cells but not in P. pastoris. Disulfide bonds are essential for secretion in P. pastoris. A mutation in the catalytic site, C35S, is not completely critical to removal of the prosequence and protease activity. The findings are useful for future design of recombinant products for application in immunotherapy.

Amino Acid Motifs↗
PubMed2001

Production of humanized antibody against human high-affinity IgE receptor in a serum-free culture of CHO cells, and purification of the Fab fragments.

We describe the preparation of Fab fragments of a humanized anti-human high-affinity IgE receptor (Fc epsilonRIalpha) antibody potentially useful for treatment of IgE-mediated allergic diseases. IgE-binding capacities of sixteen combinations of light and heavy chains of four recombinant anti-Fc epsilonRIalpha antibodies, chimeric CRA2, humanized CRA2, chimeric CRA4, and humanized CRA4, were compared. A combination in which both chains were of humanized CRA2 had the highest activity. Stable transfectant clones of four kinds of host cells expressing recombinant antibodies were established. CHO-K1 cells were the most productive. Serum-free media suitable for culture of the stable CHO-transfectant clones were screened. The concentration of the humanized CRA2, which the most productive clone secreted into the chosen serum-free medium, was approximately 100 microg/ml. A procedure for the purification of the antibody, papain-digestion, and purification of Fab fragments was established. The highly purified humanized Fab fragments are suitable for use to examine their in vivo activity and immunogenicity in primates.

Animals↗
PubMed2001

Effects of double mutation at two distant IgE-binding sites in the three-dimensional structure of the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity.

Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.

Allergens↗
PubMed2001

Cloning and expression of cDNA encoding the complete prepro-form of an isoform of Der f 1, the major group 1 allergen from house dust mite Dermatophagoides farinae.

cDNA clones encoding a major house dust mite allergen, Der f 1, were isolated from a Dermatophagoides farinae cDNA library by plaque immunoscreening using rabbit anti-Der f 1 serum. The sequences cover the complete open reading frame encoding the prepro-form. The sequence is different from previously reported cDNA of Der f 1 in six bases and the encoded amino acid sequence is different in two residues. Pro-forms of Der f 1 and its mutant, in which the N-glycosylation motif was disrupted, expressed in Pichia pastoris were converted to the mature forms by an in vitro activation process and they showed significant IgE-binding. The biologically active rDer f 1 molecules would be useful for diagnostic testing and allergen-specific immunotherapy. In contrast, Der f 1 directly expressed in Escherichia coli without the prosequence had very low IgE binding. The hypoallergenic Der f 1 polypeptide could be useful for safer and more effective immunotherapy.

Allergens↗
PubMed2001

Reactivities of mutants of a major house dust mite allergen Der f 2 to mouse anti-Der f 2 monoclonal antibodies analyzed by immunoblotting.

A total of sixteen recombinant variants of a major house dust mite allergen Der f 2, the wild-type Der f 2, six cysteine mutants, six proline mutants, and three lysine mutants, were expressed in Escherichia coli. The cells were solubilized and run on SDS-PAGE under reducing conditions. Epitopes for five mouse anti-Der f 2 monoclonal antibodies, 1B2, 7C10, 13A4, 15E11, and 18G8, to the recombinant Der f 2 variants were characterized by immunoblot analysis.

Allergens↗