PubMed Health⌕ Search

Biomedical subjects

Masaaki Kobayashi

Publications and source records attributed to Masaaki Kobayashi.

At least 19 recordsLinked to original sources
PubMed2006

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications.

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (muTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the "initial start-up" in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with muTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.

DNA↗
PubMed2005

The inhibitory effect of disease-modifying anti-rheumatic drugs and steroids on gliostatin/platelet-derived endothelial cell growth factor production in human fibroblast-like synoviocytes.

Gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) is known to have both angiogenic and arthritogenic activities. The purpose of this study was to investigate whether disease-modifying anti-rheumatic drugs (DMARDs) and steroids are involved in the regulation of GLS expression. Fibroblast-like synoviocytes (FLSs) obtained from patients with rheumatoid arthritis (RA) were cultured and stimulated by interleukin (IL)-1beta with or without DMARDs and steroids. The expression levels of GLS were determined using the reverse transcription-polymerase chain reaction and an ELISA. In cultured rheumatoid FLSs, the expression of GLS mRNA was significantly increased by stimulation with IL-1beta. By contrast, GLS mRNA levels in IL-1beta-stimulated FLSs were reduced by treatment with aurothioglucose (AuTG) and dexamethasone (DEX). These findings indicate that AuTG and DEX have anti-rheumatic activity, which is mediated via the suppression of GLS production. Neither methotrexate (MTX) nor sulfasalazine (SSZ) had a significant influence on GLS levels in our study.

Adrenal Cortex Hormones↗
PubMed2005

Establishment of a simple and quantitative immunospot assay for detecting anti-type II collagen antibody using an infrared fluorescence imaging system (IFIS).

Antibodies to type II collagen (col II) have been detected in patients with rheumatoid arthritis and in animal models of collagen induced arthritis. Here, we describe a novel method to detect anti-col II antibodies using an immunospot assay with an infrared fluorescence imaging system. This method showed very high sensitivity and specificity, and was simple, with low background levels. It also showed higher reproducibility and linearity, with a dynamic range of approximately 500-fold, than the conventional immunospot assay with enhanced chemiluminescence detection. Using this method we were able to demonstrate the antibody affinity maturation process in mice immunized with col II. In these immunized mice, although cross-reactive antibodies reacting with other collagen species were detected in earlier stages of immunization, the titers of cross-reactive antibodies rapidly diminished after the antigen boost, concomitantly with the elevation of the anti-col II antibody. The method and its possible applications are discussed.

Animals↗
PubMed2005

Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes.

A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.

Biosensing Techniques↗
PubMed2005

Hippocalcin protects against caspase-12-induced and age-dependent neuronal degeneration.

Hippocalcin is a neuronal calcium binding protein, but its physiological function in brain is unknown. We show here that hippocampal neurons from hippocalcin-deficient mice are more vulnerable to degeneration, particularly using thapsigargin, elevating intracellular calcium. Caspase-12 was activated in neurons lacking hippocalcin, while calpain was unchanged. Neuronal viability was accompanied by endoplasmic reticulum (ER) stress and a change in the relative induction of the ER chaperone, BiP/GRP78. Neuronal apoptosis inhibitor protein (NAIP), known to interact with hippocalcin, was not altered, but hippocampal neurons from gene-deleted mice were more sensitive to excitotoxicity caused by kainic acid. In addition, an age-dependent increase in neurodegeneration occurred in the gene-deleted mice, showing that hippocalcin contributes to neuronal viability during aging.

Aging↗
PubMed2004

On-chip nanoliter-volume multiplex TaqMan polymerase chain reaction from a single copy based on counting fluorescence released microchambers.

A novel method for multiplex TaqMan PCR in nanoliter volumes on a highly integrated silicon microchamber array is described. Three different gene targets, related to beta-actin, sex-determining region Y (SRY), and Rhesus D (RhD) were amplified and detected simultaneously on the same chip by using three different types of human genomic DNA as the templates. The lack of cross-contamination and carryover was shown using alternate dispensing of mineral oil-coated microchambers containing template and those without template. To confirm the specificity of our system to beta-actin, SRY, and RhD genes, we employed the larger volume PCR samples to a commercial real-time PCR system, SmartCycler. The samples were cycled with the same sustaining temperatures as with the microchamber array. Instead of the conventional method of DNA quantification, counting the number of the fluorescence released microchambers in consequence to TaqMan PCR was employed to our chip. This simple method of observing the end point signal had provided a dynamic quantitative range. Stochastic amplification of 0.4 copies/reaction chamber was achieved. The microfabricated PCR chip demonstrated a rapid and highly sensitive response for simultaneous multiple-target detection, which is a promising step toward the development of a fully integrated device for the "lab-on-a-chip" DNA analysis.

Actins↗
PubMed2004

Reduced expression of liver-intestine cadherin is associated with progression and lymph node metastasis of human colorectal carcinoma.

Liver-intestine cadherin (LI-cadherin) is a recently identified member of the cadherin superfamily. We examined LI-cadherin expression in four human colorectal carcinoma cell lines and 45 human primary colorectal carcinomas using a monoclonal antibody against LI-cadherin. We also investigated the correlation between LI-cadherin expression and clinicopathologic parameters. Among the cell lines, LI-cadherin expression was detected in a differentiated phenotype, but not in dedifferentiated phenotypes. Among the 45 tumor samples, LI-cadherin expression was preserved in 28 (62%) and reduced in 17 (38%). Reduced LI-cadherin expression was significantly associated with a high tumor grade (P = 0.015), lymphatic invasion (P = 0.033), lymph node metastasis (P = 0.015), and an advanced pTNM stage (P = 0.033). These results suggest that analysis of LI-cadherin expression may help to indicate the biological aggressiveness of this malignancy.

Aged↗
PubMed2004

Application of on-chip cell cultures for the detection of allergic response.

In this report, the development of a microfluidic cell chip for monitoring allergic response is described. A rat basophilic leukemia cell line (RBL-2H3), a tumor analog of rat mucosal mast cells, has been used as a model to observe its allergic response upon antigen stimulus. The cells were cultivated on a poly(dimethylsiloxane) (PDMS) chip, the surface of which was modified by several methods. The PDMS chip, which comprised a cell cultivation chamber and microfluidic channels, was fabricated by conventional molding methods. In order to detect the allergic response, a fluorescent dye, quinacrine, was introduced inside the cell compartment that included histamine. The cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with anti-DNP IgE. When exocytosis events occurred, the microfluidic system detected the fluorescent signal of quinacrine, which was released from RBL-2H3 cells by using a photomultiplier tube (PMT) fitted onto a microscope.

Animals↗
PubMed2004

Effects of bilateral lingual and inferior alveolar nerve anesthesia effects on masticatory function and early swallowing.

OBJECTIVE: To determine whether bilateral lingual and inferior alveolar nerve anesthesia affects the onset of swallowing including masticatory function. STUDY DESIGN: Twenty young male volunteers were asked to chew and swallow corned beef and then a mixture of corned beef and liquid in their usual manner before and after local anesthesia. The oral and pharyngeal swallowing was investigated using videofluoroscopic examination in a lateral plane. Eight objective indicators including oral containment time, swallowing threshold, and pharynx-to-swallow interval were measured. RESULTS: Oral containment time and total sequence duration, total number of chews, and total number of swallows for the 2 test foods varied significantly between those before and after anesthesia. Individual pharynx-to-swallow intervals varied with both foods without remarkable increases or decreases in the swallow duration and cricopharyngeal opening time. CONCLUSION: Bilateral lingual and inferior alveolar nerve anesthesia may affect early swallowing; changes in the onset of swallowing varied among the participants.

Adult↗
PubMed2004

A picoliter chamber array for cell-free protein synthesis.

The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.

Cell-Free System↗
PubMed2004

DNA condensation monitoring after interaction with hoechst 33258 by atomic force microscopy and fluorescence spectroscopy.

DNA condensation was only observed after the addition of Hoechst 33258 (H33258) among various types of DNA binding molecules. The morphological structural change of DNA was found to depend on the H33258 concentration. On comparison of fluorescence spectrum measurements with AFM observation, it was found that fluorescence quenching of DNA-H33258 complexes occurred after DNA condensation. Additionally, we showed that DNA condensation by H33258 was independent of sequence selectivity or binding style using two types of polynucleotides, i.e. poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Moreover, it was concluded that the condensation was caused by a strong hydrophobic interaction, because the dissolution of condensed DNA into its native form on dimethyl sulfoxide (DMSO) treatment was observed. This study is the first report, which defines the DNA condensation mechanism of H33258, showing the correlation between the single molecule scale morphology seen on AFM observation and the bulky scale morphology observed on fluorescence spectroscopy.

Bisbenzimidazole↗
PubMed2004

Listening difficulty as a subjective measure for evaluation of speech transmission performance in public spaces.

Listening difficulty ratings, using words with high word familiarity, are proposed as a new subjective measure for the evaluation of speech transmission in public spaces to provide realistic and objective results. Two listening tests were performed to examine their validity, compared with intelligibility scores. The tests included a reverberant signal and noise as detrimental sounds. The subject was asked to repeat each word and simultaneously to rate the listening difficulty into one of four categories: (1) not difficult, (2) a little difficult, (3) fairly difficult, and (4) extremely difficult. After the tests, the four categories were reclassified into, not difficult [response (1)] and some level of difficulty, (the other 3 responses). Listening difficulty is defined as the percentage of the total number of responses indicating some level of difficulty [i.e. not (1)]. The results of two listening tests demonstrated that listening difficulty ratings can evaluate speech transmission performance more accurately and sensitively than intelligibility scores for sound fields with higher speech transmission performance.

Adult↗
PubMed2004

Anthropometric, lifestyle and biomarker assessment of Japanese non-professional ultra-marathon runners.

BACKGROUND: Anthropometric characteristics, lifestyle, and baseline biological markers of Japanese non-professional ultra-marathon runners have not been fully assessed. METHODS: We evaluated anthropometric characteristics, lifestyle, and baseline biological markers of 180 Japanese amateur ultra-marathon runners (144 males [mean age: 50.5 +/- 9.4 (standard deviation) years] and 36 females [48.9 +/- 6.9]), and compared them with those of participants in a community heath check-up program and with the figures in the literature. We furthermore evaluated baseline blood indices according to monthly running distance with analysis of variance adjusted for age, body mass index, smoking and alcohol drinking habits. RESULTS: The ultra-marathon runners demonstrated more favorable values for body mass index and bone density, and the proportion of smoking, and undertaking physical activity (for both sexes), eating breakfast (for males), and having daily bowel movements (for females), while greater proportion of alcohol drinking habit (for both sexes), than the comparison group. Average monthly running distances and standard deviations (km) were 257.2 +/- 128.9 for males and 209.0 +/- 86.2 for females. Male runners possessed beneficial markers, including lowered triglyceride and elevated high-density lipoprotein cholesterol, and their values showed hockey-stick (or inverse hockey-stick) patterns depending on their monthly running distance. Some subjects running more than 300 km/month exhibited signs of an over-reaching/training syndrome, including somewhat lowered hemoglobin, ferritin and white blood cell count, and elevated creatine kinase and lactate dehydrogenase. CONCLUSIONS: Together with a desirable lifestyle, Japanese non-professional ultra-marathon runners with vigorous exercise habit demonstrated a preferable health status according to biological indices.

Adult↗
PubMed2003

Induction of Notch signaling by tumor necrosis factor in rheumatoid synovial fibroblasts.

Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with abberrant proliferation of synoviocytes. In order to explore the characteristics of rheumatoid synovial fibroblasts (RSF), we performed the comparative gene expression profile analysis between RSF and normal synovial fibroblasts (NSF) upon tumor necrosis factor (TNF) stimulation. As an initial screening for the genes preferentially induced by TNF in RSF compared with NSF, we have adopted a cDNA array containing well-defined sets of genes responsible for cell growth, cell fate determination, and cellular invasiveness. Differentially expressed genes of interest were confirmed using real-time RT-PCR. We found that TNF induced the expression of Notch-1, Notch-4, and Jagged-2 in RSF. The expression of these proteins was detected in the RA synovial tissues. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 and Notch-4 antibody. TNF induced the nuclear translocation of Notch intracellular domain in RSF, indicating the elicitation of the Notch signaling. Notch-1, Notch-4, and Jagged-2 proteins were also detected in the developing synovium of mouse embryo. Thus, RSF may have re-acquired the primordial phenotype, accounting for the hyperproliferation and aggressive invasiveness, exhibiting tumor-like phenotype.

Active Transport, Cell Nucleus↗
PubMed2003

Prophylactic clip application does not decrease delayed bleeding after colonoscopic polypectomy.

BACKGROUND: The most common major complication of colonoscopic polypectomy is postpolypectomy hemorrhage. Although several factors have been implicated in the occurrence of hemorrhage, accurate prediction of delayed bleeding remains difficult. This randomized controlled trial evaluated the efficacy of prophylactic clip application for prevention of delayed postpolypectomy bleeding. METHODS: Postpolypectomy ulcers created by colonoscopic removal of polyps (mean size 7.8 [4.0] mm) with the endoscopic mucosal resection technique were randomly assigned to prophylactic clip placement (n = 205) or no clip (n = 208). Baseline characteristics of the patients and polyps excised were comparable between the groups. Delayed bleeding was defined as the postprocedure passage of bloody stool or massive hematochezia. The site of delayed bleeding was identified at emergent colonoscopy. RESULTS: Delayed bleeding was identified from 2 ulcers in each group from 1 to 4 days after resection (mean 2.3 days). Delayed bleeding occurred from 0.98% of ulcers in the clip group and 0.96% in the non-clip group (p > 0.9999). No patient with delayed bleeding required transfusion or surgery. CONCLUSIONS: Prophylactic clip placement did not decrease the occurrence of delayed bleeding after colonoscopic polypectomy.

Adenocarcinoma↗
PubMed2002

Gene expression profile analysis of rheumatoid synovial fibroblast cultures revealing the overexpression of genes responsible for tumor-like growth of rheumatoid synovium.

To elucidate the aberrant growth properties of rheumatoid synoviocytes, we have examined the gene expression profile of rheumatoid synovial fibroblasts (RSFs) and compared with that of normal synovial fibroblasts (NSF). Gene expression profile analysis was conducted with synoviocyte cultures obtained from five rheumatoid arthritis (RA) patients and five control cases using a commercial cDNA array containing the defined 588 cancer-related genes. The results were confirmed by real-time RT-PCR. Gene expression levels for the platelet-derived growth factor receptor alpha (PDGFRalpha), plasminogen activator inhibitor-1 (PAI-1), and stromal cell derived factor 1A (SDF1A) are constitutively augmented in RSF compared with NSF. The mRNA levels of PDGFRalpha, PAI-1, and SDF1A in RSF over NSF were 4.6-, 14-, and 2.8-fold, respectively, by real-time RT-PCR. In fact, we found that RSFs showed greater sensitivity to the cell proliferative effect of PDGF. T his aberrant gene expression profile suggests that RSF may have retained the premature phenotype of primordial synoviocytes.

Adult↗