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Biomedical subjects

Takashi Kanno

Publications and source records attributed to Takashi Kanno.

At least 19 recordsLinked to original sources
PubMed2004

Three-dimensional microarray compared with PCR-single-strand conformation polymorphism analysis/DNA sequencing for mutation analysis of K-ras codons 12 and 13.

BACKGROUND: We developed a rapid, precise, and accurate microarray-based method that uses a three-dimensional platform for detection of mutations. METHODS: We used the PamChip microarray to detect mutations in codons 12 and 13 of K-ras in 15 cell lines and 81 gastric or colorectal cancer tissues. Fluorescein isothiocyanate-labeled PCR products were analyzed with the microarray. We confirmed the microarray results with PCR-single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. RESULTS: We could correctly identify wild-type, heterozygous, and homozygous mutant genotypes with the PamChip microarray in <3.5 h. The array data were consistent with those of PCR-SSCP analysis and DNA sequencing. All 15 cell lines and 80 of 81 clinical cancer specimens (98.8%; 95% confidence interval, 96.4-100%) were genotyped accurately with the microarray, a rate better than that of direct DNA sequencing (38.9%) or SSCP (93.8%). Only one clinical specimen was misdiagnosed as homozygous for the wild-type allele. Densitometric analysis of SSCP bands indicated that the content of the mutant allele in the specimen was approximately 16%. The PamChip microarray could detect mutant alleles representing more than 25% of the SSCP band proportions. Therefore, the limit for detection of mutant alleles by the PamChip microarray was estimated to be 16-25% of the total DNA. CONCLUSIONS: The PamChip microarray is a novel three-dimensional microarray system and can be used to analyze K-ras mutations quickly and accurately. The mutation detection rate was nearly 100% and was similar to that of PCR-SSCP together with sequencing analysis, but the microarray analysis was faster and easier.

Cell Line, Tumor↗
PubMed2004

Influence of fibrate treatment on malondialdehyde-modified LDL concentration.

BACKGROUND: Drug therapy is considered essential to the clinical prevention of atherosclerotic lesions in patients with diabetes mellitus (DM). METHODS: To confirm the effects of fibrate therapy, we determined low-density lipoprotein (LDL) size by gradient gel electrophoresis and malondialdehyde-modified LDL (MDA-LDL) concentrations by enzyme-linked immunosolvent assay (ELISA) and clarified the association between apolipoprotein B (apo B) and MDA-LDL during the fibrate therapy. RESULTS: Mean MDA-LDL concentrations were higher in healthy men than in healthy women. There were no significant differences in mean MDA-LDL concentrations between age groups for males or females. According to the regression equation (y = 0.063x + 10.9) obtained for apo B and MDA-LDL concentrations with fibrate treatment, the apo B concentration in those may need to be decreased to 1260 mg/l to restore the MDA-LDL concentration to the control concentration (65 +/- 25 units/l). This slope of the apoB/MDA-LDL regression line was approximately half of that with no-drug treatment (y = 0.109x - 10.8). CONCLUSIONS: Fibrate therapy had an effect on reducing serum MDA-LDL concentration in diabetic patients.

Adult↗
PubMed2004

High frequency of anti-ribosomal P antibody in patients with systemic lupus erythematosus-associated hepatitis.

Hepatic manifestations are a common phenomenon in patients with systemic lupus erythematosus (SLE). However, their cause may be difficult clinically to determine. A significantly increased frequency of anti-ribosomal P antibody has recently been found in patients with SLE-associated hepatitis. Thus, we examined the prevalence of anti-ribosomal P antibody and clinical differences between anti-ribosomal P antibody positive and negative SLE patients with liver dysfunction using ELISA kits against recombinant ribosomal P0 protein. Sera of 61 patients with SLE and 20 patients with autoimmune hepatitis (AIH) were assayed. Of 34 SLE patients with liver dysfunction, anti-ribosomal P antibody was detected in 15 (44.1%), consisting of 11 (68.8%) of 16 patients with SLE-associated hepatitis, 2 (28.6%) of 7 patients with fatty liver, 1 (16.7%) of 6 patients with drug-induced hepatitis, and 1 (20.0%) of 5 patients with SLE complicated by AIH. This antibody was not detected in patients with AIH. Except for those with SLE-associated hepatitis, anti-ribosomal P antibody positive patients were complicated by renal dysfunction and CNS lupus. The positive rate of anti-ribosomal P antibody was significantly higher in patients with SLE-associated hepatitis (68.8%) than in patients with SLE complicated by AIH (20%) ( [Formula: see text] ) and AIH (0%) ( [Formula: see text] ). These findings suggest that anti-ribosomal P antibody may be a useful marker of SLE-associated hepatitis to differentiate it from AIH and other liver dysfunctions in SLE patients without renal dysfunction or CNS lupus.

Journal Article↗
PubMed2003

Effects of mupirocin at subinhibitory concentrations on flagella formation in Pseudomonas aeruginosa and Proteus mirabilis.

Colonization of Pseudomonas aeruginosa at trachea, nares and oropharynx can cause ventilator-acquired pneumonia. To identify beneficial effects of antibiotics on expression of virulence factors related to colonization by such pathogens, we evaluated the effect of mupirocin on flagella formation in P. aeruginosa and on motility and flagella formation in Proteus mirabilis. In P. aeruginosa, subinhibitory concentrations of mupirocin inhibited flagella formation, which was associated with reduced flagellin expression. In P. mirabilis, subinhibitory concentrations of mupirocin dose-dependently suppressed bacterial motility and flagella formation, again with reduced flagellin expression. Our results indicate that subinhibitory concentrations of mupirocin can suppress expression of virulence factors in P. aeruginosa and P. mirabilis.

Anti-Bacterial Agents↗
PubMed2003

Detection of mutations in quinolone resistance-determining regions in levofloxacin- and methicillin-resistant Staphylococcus aureus: effects of the mutations on fluoroquinolone MICs.

The minimum inhibitory concentrations (MICs) of 18 antibiotics were determined for 66 clinical isolates of staphylococci. Genotypes, mutations in the quinolone resistance-determining regions (QRDRs), and effect of efflux were determined in the 18 levofloxacin-resistant isolates, for which the MICs of levofloxacin were high (> or =8 microg/ml). The increased levofloxacin resistance mainly resulted from some combinations of mutations in the QRDRs, although NorA-mediated efflux may play a minor role in resistance. A combination of mutations in GrlA (Ser80Phe), GrlB (Pro451Ser), and GyrA (Ser84Leu) was found in 4 methicillin-resistant Staphylococcus aureus (MRSA) isolates that were unrelated genotypically. The mutations in grlA QRDR varied in the isolates classified as being in an identical pulsed-field gel electrophoresis (PFGE) group, although the grlB, gyrA, and gyrB QRDRs were the same. These results suggest that the patterns of amino acid mutations in the QRDRs can provide distinct epidemiologic information from PFGE genotypes in fluoroquinolone-resistant MRSA. A combination of at least three mutations in GrlA, GrlB, and/or GyrA is required to increase the MICs of fluoroquinolones, although all of the levofloxacin-resistant MRSA retained the MICs of sitafloxacin in the range of 1 to 2 microg/ml.

Anti-Infective Agents↗
PubMed2003

Effect of basic amino acids on susceptibility to carbapenems in clinical Pseudomonas aeruginosa isolates.

We evaluated effects of medium composition, including basic amino acid content and pH, on susceptibility to carbapenems such as imipenem, panipenem and meropenem, in clinical isolates of Pseudomonas aeruginosa. Susceptibility to carbapenems was reduced by basic amino acids in the medium, while susceptibilities to ceftazidime and aztreonam were not. Among carbapenems, susceptibility to panipenem was most sharply reduced by addition of basic amino acids to 1:16 Mueller-Hinton agar (MHA). In 174 of 175 clinical isolates, MICs for carbapenems were affected to different degrees by medium composition. One isolate, in which MICs for carbapenems did not differ between MHA and 1:16 MHA, showed reduced production of porin (OprD). Our results suggest that susceptibility to individual carbapenems, especially panipenem, is difficult to evaluate based on MICs for other carbapenems determined on MHA. For a better prediction of antibiotic efficacy, it may be important to evaluate the susceptibility for each carbapenem individually.

Amino Acids, Basic↗
PubMed2003

Detection of macrolide resistance in Streptococcus pneumoniae.

We determined the susceptibilities of recent clinical isolates of Streptococcus pneumoniae to 19 antibiotics. The frequency of erythromycin nonsusceptibility was high, i.e. 8/13 (61.5%), 10/14 (71.4%) and 11/11 isolates (100%) from 13 penicillin-susceptible, 14 penicillin-intermediate and 11 penicillin-resistant S. pneumoniae, respectively. Macrolide resistance was detected by polymerase chain reaction (PCR) and disk diffusion methods. Of these erythromycin-nonsusceptible pneumococcal isolates, 13/29 (44.8%) isolates contained genomic copies of MEFA and showed non-'D'-shaped zones of inhibition observed around rokitamycin and/or clindamycin disks. Sixteen out of 29 isolates (55.2%) contained copies of ERMB and showed typical 'D'-shaped zones of inhibition, except one isolate. Although the macrolide resistance determinants, MEFA and ERMB, could be identified by PCR and disk diffusion methods, PCR methods were more reliable in elucidating these determinants. The susceptibility pattern to 14-, 15- and 16-membered macrolides and clindamycin differed between the MEFA+ and ERMB+ isolates. All isolates were susceptible to levofloxacin, sparfloxacin and vancomycin. The MICs of sitafloxacin were lowest among the fluoroquinolones examined for 38 isolates.

Anti-Bacterial Agents↗
PubMed2003

A common truncated variant of lipoprotein lipase in the Japanese population is characterized by pattern B phenotype.

To investigate the relationship between the lipoprotein lipase Ser447Ter (S447X) mutation, lipid profiles and risk of atherosclerotic disease, we studied two groups of Japanese subjects. These groups consisted of a dyslipidemic group (triglyceride (TG) > 1.69 mmol/l and high-density lipoprotein-cholesterol (HDL-C) < or = 0.91 mmol/l; n = 106) and a control group (TG < or = 1.69 mmol/l and HDL-C > or = 1.16 mmol/l; n = 106). All subjects in the control group were confirmed to the pattern A phenotype (normal low-density lipoprotein (LDL) pattern), and 21 individuals in this group were heterozygous for the S447X mutation, but not homozygous. In the patient group, pattern A was shown by 44 of 106 patients. The rest were of the pattern B phenotype, which is associated with an increased risk of coronary heart disease. Homozygosity was concentrated in the patient group (p < 0.05) and in those with the pattern B phenotype (p < 0.05). In contrast, heterozygosity between both groups was not statistically significant. In conclusion, heterozygous and homozygous status with respect to the LPL S447X mutation appears to have different meanings with respect to biochemical and clinical phenotypes of atherosclerosis.

Adolescent↗
PubMed2003

A case of polyarteritis nodosa limited to lower legs with a high titer of MPO-ANCA under precedence of idiopathic pulmonary fibrosis.

A 58-year-old man with a 15-year history of idiopathic pulmonary fibrosis was hospitalized for rapid progression of muscle weakness to bilateral foot drop. Although laboratory data revealed high titers of myeloperoxidase anti-neutrophil cytoplasmic antibody (489 EU), the patient was diagnosed as polyarteritis nodosa limited to the lower portions of the legs. Despite of the treatment with large doses of corticosteroids and cyclosporin A, his symptoms barely improved during the following two months.

Adrenal Cortex Hormones↗
PubMed2003

Relationship between high-density lipoprotein-cholesterol and malondialdehyde-modified low-density lipoprotein concentrations.

Several reports have suggested that HDL has anti-oxidative actions. We investigated the relationship between HDL-cholesterol (HDL-C) and malondialdehyde-modified LDL (MDA-LDL) concentrations using enzyme linked immunosolvent assay. We divided our study subjects into four groups on the basis of concentrations of triglyceride (TG) and HDL-C by the following lipid profiles: serum TG < or = 1.69 mmol/L and HDL-C > or = 1.16 mmol/L (control group, n = 26); TG >1.69 and HDL-C < or = 1.16 (high TG group, n = 22); TG >1.69 and HDL-C < or = 0.91 (high TG & low HDL group, n = 67); TG < or = 1.69 and HDL-C < or = 0.91 (low HDL group, n = 21). MDA-LDL concentrations, MDA-LDL/apolipoprotein B (apo B) ratio, and LDL size were different between subjects in high TG & low HDL and control groups. MDA-LDL concentrations in both high TG and low HDL groups did not differ significantly from those in the control. However, MDA-LDL/apo B ratio in low HDL group was significantly higher than that in the control (P < 0.05). The MDA-LDL/apo B ratio reflects the extent of MDA modification of apo B in LDL. Therefore, our data suggest that as HDL-C concentrations fall, the extent of MDA modification per one LDL particle increases. Moreover, accompanied by high TG concentration, LDL size in subjects with lower HDL-C concentrations became smaller.

Adult↗
PubMed2003

[Basic studies on mutation analysis of K-ras codon 12 by use of three-dimensional microarray system].

A next-generation DNA microarray system, FD10 has been developed. It is based around the PamChip, a custom-made microarray, which consists of a solid three-dimensional structure that facilitated the incorporation of probe molecules. We applied this microarray system on a detection of K-ras mutation at codon 12 in some cancer cell lines. The PCR products amplified by use of FITC labeled primers were applied onto probe-absorbed microarray. After hybridization, the signal was imaged by CCD camera and analyzed by the exclusive software. We confirmed the microarray results by PCR-SSCP and sequencing analyses. Ten, two and three out of 15 cell lines were homozygous for wild type allele, heterozygous for wild and mutant allele, and homozygous for mutant alleles, respectively. Signals hybridized with antisense probes were stronger than those with sense probes, without PSN1 cell line. The system had a good reproducibility. Essentially, the microarray results were consistent with PCR-SSCP and sequencing results. In conclusion, the FD10 microarray system was easy to operate and short to get results. It might be useful for a focused array applicable for specific purposes. The K-ras mutation detection system worked well and will be applied to clinical specimens soon.

Codon↗
PubMed2002

Size distribution measurement of vesicles by atomic force microscopy.

Vesicles have been utilized as nanoscale vehicles for reagents including potential drug delivery systems. When used to deliver drugs, vesicle size and the size distribution are important factors in the determination of the dosage, cell specificity, and rate of clearance from the body. Current size measurement techniques for vesicles are electron microscopy and dynamic light scattering, but their results are not equal. Therefore atomic force microscopy was attempted as another size measurement technique. After adsorption of the vesicles from a low-concentration solution of vesicles on mica substrate, each vesicle is generally found as a flattened structure. The diameters of vesicles in these solutions and their distribution have been successfully estimated from the surface area of the flattened structure of each vesicle. At higher concentrations, we have found a monolayer crammed with dome-shaped vesicles on the substrate. The diameters of vesicles in these solutions have also been successfully estimated from the surface area of the dome-shaped structure of each vesicle. Diameters of vesicles in solution estimated from two different vesicle concentrations are not close to those reported by electron microscope studies but are close to those reported by dynamic light scattering studies.

Adsorption↗
PubMed2002

Increased circulating malondialdehyde-modified LDL levels in patients with coronary artery diseases and their association with peak sizes of LDL particles.

Recent establishment of a sensitive ELISA system using antibodies against malondialdehyde-modified low density lipoprotein (MDA-LDL) made it possible to determine the circulating oxidized lipoprotein levels. Here, we investigated the serum levels of MDA-LDL in 62 patients with coronary artery disease (CAD) compared with the levels in 42 patients without CAD [groups CAD(+) and CAD(-), respectively], which are adjusted for age, serum total cholesterol, LDL and high density lipoprotein cholesterol, and triglyceride levels. Serum MDA-LDL levels were 113.4+/-49.1 IU/L in CAD(+), which were significantly higher than the levels in CAD(-) (85.2+/-22.5 IU/L, P<0.0005). The ratio of MDA-LDL/LDL cholesterol was 0.95+/-0.32 in CAD(+), indicating a significant increase compared with the ratio in CAD(-) (0.68+/-0.19, P<0.0005). The positive correlation of MDA-LDL level and the ratio of MDA-LDL/LDL cholesterol with intima-media thickness in carotid arteries was observed. Age was not clearly associated with the MDA-LDL level (P=0.865). The serum MDA level was positively correlated with LDL cholesterol (P<0.0001) and with triglycerides (P<0.001) and negatively correlated with high density lipoprotein cholesterol (P<0.05). Furthermore, the MDA-LDL level was negatively correlated with the peak size of the LDL particle (P<0.01). The LDL subclasses that were identified by using the sera collected from the subjects by sequential ultracentrifugation showed that the ratios of MDA-LDL/apolipoprotein B in LDL3 and LDL4 were nearly 3-fold higher than those in LDL1 and LDL2 for CAD(+) and CAD(-). These results indicate that the circulating MDA-LDL level is increased in CAD(+), independent of the serum LDL cholesterol level but in association with the peak size of LDL particles. The measurement of serum MDA-LDL level may be useful for the identification of patients with advanced atherosclerosis.

Aged↗
PubMed2002

Clinicolaboratory characteristics of patients with primary biliary cirrhosis associated with CREST symptoms.

Aims: Patients with primary biliary cirrhosis (PBC) occasionally suffer complications from other autoimmune diseases. When PBC was associated with calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasias (CREST) symptoms, it has been proposed that it is a distinct clinical entity. This study aimed to investigate whether PBC associated with CREST symptoms is a distinct disease complex. Method: Clinicolaboratory data, HLA type of leukocytes and disease prognosis were compared between 31 patients with PBC associated with CREST symptoms and 68 patients with PBC alone. Results: The characteristic findings and significant differences observed in patients with PBC associated with CREST symptoms compared with PBC alone are as follows: all women with older age with milder clinical features of both PBC (asymptomatic PBC in 84%) and CREST syndrome (incomplete CREST in 81%), more frequent occurrence of esophageal varices (28.6 vs. 9.3%), better prognosis (87.5 vs. 45.5% in 10 years survival), lower serum levels of AST (39.8 vs. 63.6 IU/l) and IgM (460 vs. 676 mg/dl), higher prevalence of discrete speckled pattern of antinuclear antibodies (93.5 vs. 12.3%), higher median titers of anti-CENP-B antibodies (1.22 vs. 0.31), lower median titers of antimitochondrial antibody (1:80 vs. 1:160), and a higher prevalence of HLA-DR9 (54.5 vs. 24.3%). Conclusion: These findings support the presence of a subgroup in PBC as PBC associated with CREST symptoms.

Journal Article↗
PubMed2002

Antibacterial activities of beta-lactamase inhibitors associated with morphological changes of cell wall in Helicobacter pylori.

BACKGROUND: Recent study has demonstrated that beta-lactamase inhibitors including clavulanate, sulbactam and tazobactam have an vitro antibacterial effect on Helicobacter pylori. Here we describe the relationship between viability and cell profiles of H. pylori exposed to beta-lactamase inhibitors and some antibiotics in a short-time course. MATERIALS AND METHODS: The antibacterial effects of beta-lactamase inhibitors including clavulanate, sulbactam and tazobactam on the bacterial viability of and morphological changes in H. pylori ATCC43504 were examined. RESULTS: The beta-lactamase inhibitors such as clavulanate and sulbactam alone decreased the viable counts of H. pylori, depending on the antibiotic concentrations. Exposure to these beta-lactamase inhibitors resulted in morphological changes of cell shape, cell-wall disintegration and cell lysis. Among these beta-lactamase inhibitors, clavulanate was the most active, causing a decrease in viable counts and morphological changes such as short filamentous to sphaeroplast formation and lysis. One x minimum inhibitory concentration (MIC) of amoxicillin plus 1 x MIC of clavulanate decreased viable counts effectively compared with 1 x MIC of amoxicillin or 1 x MIC of clavulanate alone, and induced morphological changes of cell shape and cell wall. CONCLUSION: Our results suggest that the beta-lactamase inhibitors alone have concentration-dependent antibacterial activities against H. pylori and affect the morphology of the cell shape and the cell wall in vitro.

Anti-Bacterial Agents↗