Biomedical subjects

Megumi Nakamura

Publications and source records attributed to Megumi Nakamura.

13 recordsLinked to original sources

PubMed2006

Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress.

Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.

Blotting, Western↗

PubMed2006

Application of ion attachment mass spectrometry to evolved gas analysis for in situ monitoring of porous ceramic processing.

Ion attachment mass spectrometry was applied to evolved gas analysis-mass spectrometry (EGA-MS), generally known as thermogravimetry-mass spectrometry. Characteristic species arising from poly(vinyl alcohol) (PVA) and poly(methyl methacrylate) (PMMA) resin, used as a binder and a porogen, respectively, in the starting materials for porous ceramics, were detected in the mass spectra. The EGA curves of the characteristic mass peaks from PVA and PMMA, when plotted against the programmed temperature, successfully showed the individual pyrolysis behavior of each polymer during the firing process.

Journal Article↗

PubMed2006

Living-related intestinal transplantation for a patient with hypoganglionosis.

A 14-yr-old boy with total parenteral nutrition-dependent short-bowel syndrome associated with hypoganglionosis underwent the LR-IT by using a 150 cm segment of distal ileum taken from a healthy donor. The graft vessels were connected to infrarenal aorta and inferior vena cava. The immunosuppressive regimen consisted of daclizumab, tacrolimus, and steroid. The graft surveillance for ACR was accomplished using zoom endoscopy and mucosal biopsy. The blood trough level of tacrolimus was maintained between 20 and 25 ng/mL for the first 2 months, followed by 15-20 ng/mL thereafter. The 50 mg of daclizumab was administered on the day of operation, and same dosage was repeated at 2-wk intervals. The first ACR occurred on POD-9 and was progressive, and required a 14-day course of OKT-3 injection. After the treatment with OKT-3, the graft recovered from the ACR, and began to function well enough to discontinue the intravenous nutrition on POD-55. No infectious complication has occurred. The patient was discharged in POD-112, and currently tolerates full oral intake without requiring intravenous nutritional or fluid supplementation. The donor was discharged without any complications. The LR-IT could successfully be performed with minimal risk to the donor, and it can be a treatment of choice for patients with short-gut syndrome associated with hypoganglionosis.

Adolescent↗

PubMed2006

Nurses' perception on information privacy in Japan.

This study explores how nurses perceive patients' privacy and with whom they share the patients' information. A descriptive study was conducted on 710 nurses from three hospitals. The results show 1) nurses think of patients' privacy more importantly than their own privacy, 2) nurses hesitated somewhat to share information on "family structure" and "elimination habit" with other nurses, 3) nurses shared more limited information with the staff on the hospital food service unit than with other health professionals. Compared to the previous study conducted three years ago, this study showed nurses have become more sensitive to patient privacy in Japan.

Confidentiality↗

PubMed2005

Expression of versican and ADAMTS1, 4, and 5 during bone development in the rat mandible and hind limb.

Extracellular matrix (ECM) remodeling is achieved by both production and degradation of ECM molecules during bone development. ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motifs) constitutes a family of extracellular proteases which are implicated in cleaving the protein versican. The present study was designed to investigate the expression of versican and ADAMTS1, 4, and 5 mRNA during bone development in rat mandibles and hind limbs by RT-PCR and in situ hybridization. Versican was localized by immunohistochemistry. The process of bone development from day 14 postcoitum through week 6 postnatum was divided into the beginning of osteogenesis, woven bone, and lamellar bone stages. Versican protein was abundant in the woven bone matrix, but decreased in the lamellar bone matrix. Versican mRNA was prominent in some osteoblasts with corresponding localization of the cognate protein. The temporal and spatial mRNA expression pattern of ADAMTS1, 4, and 5 was comparable to that of versican. These results suggest that woven bone rich in versican alters into lamellar bone containing little versican during bone development in both mandibles and hind limbs, where some osteoblasts may be involved in production as well as degradation of versican by secreting ADAMTS1, 4, and 5.

ADAM Proteins↗

PubMed2005

Expression of caveolin-1 and its correlation with cisplatin sensitivity in oral squamous cell carcinoma.

PURPOSE: Cisplatin (CDDP) is widely used for chemotherapy of oral squamous cell carcinoma (OSCC). However, the mechanism of resistance to CDDP is unclear. Recently, caveolin-1 was identified as being associated with both metastasis and multidrug resistance. In the present study, we showed that caveolin-1 expression is significantly related to chemosensitivity in OSCC. METHODS: We established a CDDP-resistant cell line, H-1R, from the parental OSCC cell line, H-1. Caveolin-1 expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) in both cell lines. We analyzed expression of caveolin-1 in 30 OSCC biopsy specimens and investigated the relationship between expression of caveolin-1 and patients' clinicopathological parameters and chemotherapeutic responses. RESULTS: The 3-(3,4-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that H-1R has a ten-times greater resistance to CDDP than H-1 has. The level of caveolin-1 expression in H-1R was significantly decreased in comparison with that in H-1 by real-time RT-PCR analysis. Positive caveolin-1 immunostaining correlated positively with a complete response (16/20, 80.0%). However, negative immunostaining was found in 6/7 (85.7%) cases with no response. Positive immunohistochemical staining of caveolin-1 correlated positively with chemosensitivity to CDDP-based combination chemotherapy (P=0.02). CONCLUSIONS: These results suggest that overexpression of the caveolin-1 gene may provide novel diagnostic markers associated with CDDP sensitivity in OSCC.

Aged↗

PubMed2005

Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin.

Insulin/IGF-I-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism, and autophagy in various cells, including hematopoietic cells. Although the early protein kinase activation cascade has been intensively studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells 1 h after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 and glutathione S-transferase-pi. Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, upregulation of CLIC1 was not observed in one-dimensional Western blotting, unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus insulin induces the proteasome-dependent degradation of SRp20 as well as the subnuclear relocalization of CLIC1.

Amino Acid Sequence↗

PubMed2005

Expression of versican and ADAMTS during rat tooth eruption.

A disintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS) is a family of extracellular proteases and implicated in cleaving proteoglycans, such as aggrecan, versican and brevican. No information is available about expression or localization of these ADAMTSs in teeth. Versican is a large chondroitin sulfate proteoglycan that is present in a variety of connective tissue including dental pulp, dentin, cementum and periodontal ligaments. The present study was designed to investigate expression of ADAMTSs and versican during rat tooth eruption. Rat maxillary first molars in weeks 1, 2, 3, 4 and 6 were examined. The mRNA expression of ADAMTS1, ADAMTS4, ADAMTS5 and versican was localized using in situ hybridization. ADAMTS1, ADAMTS4, ADAMTS5 and versican were expressed in dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes. The temporal and spatial expression pattern in these cellular phenotypes was comparable among ADAMTSs and versican. The present study suggests that dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes may be involved in both production and degradation of versican with secreting ADAMTS1, ADAMTS4 and ADAMTS5.

ADAM Proteins↗

PubMed2005

Thoracoscopic removal of neurogenic mediastinal tumors in children.

PURPOSE: The aim of this study was to evaluate the feasibility and advantages of thoracoscopic removal of neurogenic mediastinal tumors (NMTs) in children. METHODS: From January 1998 to December 2001, 6 patients, ages 1.1 to 6.8 years (mean, 3.5 years), underwent thoracoscopic removal of NMT. A retrospective study was done to assess the type of anesthesia, conversions to standard thoracotomy, operative time, complications, and the current status of each patient. RESULTS: General anesthesia using a Fogarty catheter as an ipsilateral bronchial blocker was utilized. Four 5-mm ports were placed in all patients. All NMTs were successfully removed without a need to convert to standard thoracotomy. The tumor was captured in an extraction bag, fragmented, and then removed through the trocar site, which had been enlarged up to 2.0-2.5 cm in each case. Pathologic diagnosis was neuroblastoma in 1 patient, ganglioneuroblastoma in 2, and ganglioneuroma in 3 patients. The mean operative time was 230 minutes. There was no need for transfusion and no serious complication related to this approach. All patients are alive at the time of writing, without any evidence of tumor recurrence. CONCLUSION: All tumors were successfully removed. Thoracoscopic surgery is a feasible, safe, and effective technique in the treatment for children with NMT.

Child↗

PubMed2005

Establishment and gene analysis of a cisplatin-resistant cell line, Sa-3R, derived from oral squamous cell carcinoma.

Cisplatin (CDDP) is widely used for chemotherapy of many malignancies, especially of oral squamous cell carcinoma (SCC). However, because the mechanism of resistance to CDDP is unclear, we established a CDDP-resistant cell line, Sa-3R, from a CDDP-sensitive cell line, Sa-3, which was derived from moderately differentiated SCC of the lower gingiva. The 3-(3,4-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide assay indicated that Sa-3R has 7.5-fold greater resistance to CDDP than Sa-3. Comparing gene expression levels in the cell lines using an in-house cDNA microarray, which represented 2,201 oral disease origin genes, many differentially expressed genes were identified. The ATP-binding cassette transporter genes (MDR-1, MRP-1, and MRP-2), and FANCONI, GRP58, FLJ12089, and SPINT-2 were up-regulated, whereas FOSL1, MRPS27, and PGK-1 were down-regulated. These results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction. The Sa-3/Sa-3R cell lines could be useful to identify the candidates responsible for the mechanism of CDDP-resistance and the up- or down-regulated genes identified by the gene expression profiles in the Sa-3R cell line may be, in part, associated with the mechanism.

Biopsy↗

PubMed2005

Establishment and characterization of a cisplatin-resistant oral squamous cell carcinoma cell line, H-1R.

Cisplatin (CDDP) is a widely used potent chemotherapeutic agent for many malignancies. However, the mechanism of resistance to CDDP remains unclear. To investigate the molecular mechanism, we established a CDDP-resistant cell line (H-1R) from a CDDP-sensitive cell line (H-1) which was derived from moderately differentiated squamous cell carcinoma of the lower gingiva. The 3-(3,4-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay indicated that H-1R had a 10-fold greater resistance to CDDP than H-1. When we compared gene expression levels in the cell lines using an in-house cDNA microarray, which represented 2,201 genes originating from normal oral tissue, primary oral cancer, and oral cancer cell lines, 12 genes showing elevated mRNA expression in H-1R compared with H-1 were identified. Among them, the up-regulated expression of ATP-binding cassette transporter genes (MDR1, MRP1, and MRP2), CD55, and PGK1 and down-regulated expression of Caveolin 1 were further confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (PCR) or real-time PCR. Our results suggest that H-1 and H-1R cell lines could be useful for elucidating the candidate genes responsible for CDDP resistance, including the genes found in this study.

ATP-Binding Cassette Transporters↗

PubMed2004

Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts.

Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O (theta-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BCtheta) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BCtheta can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BCtheta, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BCtheta. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.

Journal Article↗

PubMed2003

Cellular aging-dependent decrease in cholesterol in membrane microdomains of human diploid fibroblasts.

Recently it has been shown that cholesterol plays indispensable roles in the function of cholesterol-rich microdomains (rafts), such as in ligand-mediated signal transduction. Using a perfringolysin O derivative (BCtheta) that binds selectively to cholesterol in rafts without causing membrane damage (Proc. Natl. Acad. Sci. USA 98 (2001) 4926), we have investigated the effect of in vitro replicative aging of human diploid fibroblasts, TIG-1, on the distribution of plasma membrane cholesterol. The amount of BCtheta-labeled membrane cholesterol decreased during replicative aging of TIG-1 cells, whereas total cholesterol increased somewhat. The relationship was confirmed by double staining with BCtheta and senescence-associated-beta-galactosidase, a biomarker of senescent cells. Cell fractionation experiments revealed decreases in both cholesterol in rafts and a raft marker, flotillin, during replicative aging. In addition, hydroxyurea-induced prematurely senescent cells also showed a lower level of BCtheta-labeled cholesterol than untreated cells, despite maintaining the total amount of cholesterol. When TIG-1 cells were cultured in cholesterol-deficient medium, BCtheta labeling was first diminished and then premature senescence was induced. Taken together with the reduced signaling capacity of aged cells, these results suggest that plasma membrane cholesterol in raft microdomains is more sensitive to senescence than total cholesterol and is a primary target in aging.

Apoptosis↗