Biomedical subjects

Yukio Nakamura

Publications and source records attributed to Yukio Nakamura.

At least 19 recordsLinked to original sources

PubMed2007

Efficient production of recombinant human (pro)renin utilizing a decahistidine tag attached at the C-terminus.

Human prorenin attached by a decahistidine tag at the C-terminus was produced in Chinese hamster ovary cells. The tagged protein secreted into the culture medium was in the inactive prorenin form, and was activated to mature renin by proteolytic removal of its prosegment by trypsin in the same manner as native prorenin. The tagged (pro)renin was efficiently purified by metal-chelate affinity chromatography. The enzymatic properties of mature renin carrying the tag were similar to native renin. These results indicate that the introduction of a decahistidine tag at the C-terminus does not interfere with either the correct folding of prorenin or the catalytic activity of mature renin.

Amino Acid Sequence↗

PubMed2007

[A case of stage IV gastric cancer treated sequentially for over two years by TS-1, paclitaxel, and CPT-11].

We report a patient with advanced stage IV gastric cancer treated by chemotherapy for over two years. The patient was a 69-year-old man with paraaortic lymph node metastasis of gastric cancer. He underwent a distal gastrectomy in non-curative resection. After surgery, chemotherapy with TS-1 (100 mg/body/day) was performed. At 7 months after surgery, progression of lymph node metastasis in porta hepatis was recognized, and paclitaxel was administered at a weekly dose of 80 mg/m(2) for 3 weeks followed by one week rest. He remained stable for 12 months under paclitaxel treatment. At 26 months after surgery, progression of lymph node metastasis in porta hepatis was recognized again, and CPT-11 was administered at a bi-weekly dose of 80 mg/m(2). Although the patient died two years seven months after surgery, the chemotherapy with sequential administration of TS-1, paclitaxel and CPT-11 was thought to be effective for advanced gastric cancer.

Adenocarcinoma↗

PubMed2006

An assessment following root canal preparation by Er,Cr: YSGG laser irradiation in straight and curved roots, in vitro.

In the present study, the effectiveness of Er,Cr:YSGG laser in straight and curved root canal preparation was compared with that of the conventional canal preparation technique, in vitro. The degree of root curvature of 40 root canals was determined, and then 20 canals were prepared by an Er,Cr:YSGG laser of 2 W by using the crown-down technique, while the other 20 canals were shaped by K-file (control). The achievement degree of root canal preparation and debris score was investigated morphologically. The results indicated that straight root canals could be successfully prepared by Er,Cr:YSGG laser irradiation; a significant decrease of smear layer or debris was also recognized (P<0.01). However, canal preparation by laser device in curve root often leads to a ledge or zipped formation, perforation or over-instrumentation. The results demonstrated that further development in laser device and technique are required to ensure its success in root canal preparation, especially in curve root.

Dental Pulp Cavity↗

PubMed2006

Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells.

Erythroblast enucleation is thought to be largely dependent on signals mediated by other cells, such as macrophages. In an attempt to improve the in vitro production of red blood cells (RBCs) from immature hematopoietic progenitor cells, we have developed a method to produce enucleated RBCs efficiently in the absence of feeder cells. Our method may represent an efficient way to produce transfusable RBCs on a large scale from hematopoietic progenitors.

Antigens, CD↗

PubMed2006

A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation.

Human embryonic stem cells (hESCs) exhibit pluripotency and indefinite proliferation and are a potential source of cells for transplantation therapies and drug discovery. These applications will require large amounts of hESCs. However, hESCs are difficult to culture and maintain at larger scales, in part because of their low resistance to dissociation during passaging. To circumvent this, we developed a simple and easy method for establishing hESC sublines tolerant of complete dissociation. These cells exhibit high replating efficiency and also high cloning efficiency, and they maintain their ability to differentiate into the three germ layers. Several sublines have no detectable abnormalities in their karyotypes, and they retained their characteristics under feeder-free culture conditions and after freeze-thawing. Thus, these hESC sublines would be valuable for hESC applications.

Biomarkers↗

PubMed2006

Long-lasting in vitro hematopoiesis derived from primate embryonic stem cells.

OBJECTIVE: Induction of hematopoietic cells from human embryonic stem (ES) cells has been reported recently. However, before cells derived from human ES cells can be used in the clinic, preclinical studies using these cells in experimental primates will be necessary. Therefore, we attempted to establish a method to induce hematopoietic cells robustly and abundantly from primate ES cells. METHODS: A primate ES cell line, CMK-6, derived from the cynomolgus monkey was used in this study. We adapted a method to induce hematopoiesis from CMK-6 cells on feeder cells, and tested the effectiveness of three kinds of feeder cell lines (OP9, C2C12, and C3H10T1/2). In addition, we tested the effect of vascular endothelial growth factor (VEGF) and insulin-like growth factor-II (IGF-II) on hematopoiesis induction from CMK-6 cells. RESULTS: VEGF and IGF-II showed an extremely strong synergistic effect to induce hematopoiesis from CMK-6 cells. C3H10T1/2 cells proved to be very useful for the induction of hematopoiesis from CMK-6 cells, and the production of blood cells on C3H10T1/2 cells has been maintained as long as 5 months. During this long period, ES cell derivatives continuously produced mature blood cells, including terminally differentiated cells. CONCLUSION: We have developed an original method to produce enriched blood cells abundantly from primate ES cells for an extremely long period. This method may represent a good in vitro model for studying primate hematopoiesis and related diseases. Furthermore, our method may be useful for preclinical studies of transfusion therapy using blood cells derived from ES cells in experimental primate systems.

Animals↗

PubMed2006

Coordinate expression of BMP-2, BMP receptors and Noggin in normal mouse spine.

The purpose of this study was to determine the localization of bone morphogenetic protein-2 (BMP-2), BMP receptors (BMPRs) and Noggin in mouse spinal tissues. The coordinate expression of these positive and negative regulators of BMP signaling may elucidate regulatory mechanisms for bone induction in the spine. Whole spines from 3-week-old mice were used and the spatial expression profiles of BMP-2, BMPR-1a, -1b, -2 and Noggin were examined using in situ hybridization. BMP-2, BMPR-1b and -2 were observed in bone marrow cells in the vertebrae, chondrocytes, hyaline cartilage cells and fibrous cells in the intervertebral discs and neurons of the spinal cord in the entire spine. BMPR-1a was also observed in these cells, but only in the cervical spine. Noggin was expressed in bone marrow cells in the vertebrae, chondrocytes and hyaline cartilage cells and fibrous cells in the intervertebral discs in the entire spine and in neurons in the spinal cord in the cervical and thoracic regions. Noggin was also expressed in the anterior longitudinal, posterior longitudinal and yellow ligaments in the cervical spine, and in the fibrous cells in the anterior longitudinal and yellow ligaments of the lumbar spine.

Animals↗

PubMed2006

Basic study of morphological changes and surface roughness of cavities prepared by TEA CO2 laser irradiation.

OBJECTIVE: The purpose of this study was to investigate the morphological change of dental hard tissue and surface roughness of cavities prepared by transversely excited, atmospheric pressure (TEA) CO2 laser irradiation. BACKGROUND DATA: It has been reported that dental hard tissues and bone can be removed by a long-pulse of TEA CO2 laser irradiation with minimal thermal damage. However, there are few reports on the surface roughness of lased teeth. METHODS: The TEA CO2 laser was irradiated on the enamel and dentin surfaces of extracted human teeth under the following conditions: wavelength, 10.6 microm; output, 95 mJ/pulse; pulse repetition rate, 1 Hz; irradiation time, 7.5 microsec/shot; and energy density, 7.9 J/cm2. Morphological studies were performed by histological and scanning electron microscopic (SEM) examination. Surface roughness of prepared cavities was measured by three-dimensional laser microscopy. RESULTS: Irradiated dentin produced a deeper defect (705 +/- 11 microm) than the enamel (501 +/- 10 microm). Histological appearance showed a basophilic line at the margin of lased dentin. SEM observation noted that the surfaces of the enamel cavity seem to be melted, and dentinal tubules were sealed. The surface roughness of the enamel cavity wall and dentin floor were 175 +/- 5 microm and 170 +/- 6 microm, respectively. CONCLUSION: These findings suggest that it is possible to remove carious dental hard tissue or cavity preparation with the TEA CO2 laser irradiation. Lased dental hard tissue can facilitate caries prevention, and surface roughness of the cavities might improve the bond strength of restorative dental materials.

Dental Caries↗

PubMed2006

Refinement of cytokine use in the in vitro expansion of erythroid cells.

Blood transfusion is indispensable for many clinical applications. However, the supply of transfusable material is insufficient in many countries. Human cord blood contains many hematopoietic stem and progenitor cells, providing a promising resource for the production of transfusable material in vitro. In this study, we have refined a protocol to produce abundant red blood cells (RBC) from human cord blood in an in vitro culture system. We found that erythropoietin and interleukin-3 were most effective when they were added to the culture medium sequentially rather than simultaneously. Although insulin-like growth factor-I (IGF-1) has been reported to function as a positive regulator of RBC production in some in vitro culture systems, we found that IGF-1 had a negative effect upon RBC production. However, IGF-II appeared to function as a positive regulator of RBC production. Finally, stem cell factor functioned to both expand and accelerate the differentiation of immature erythroid cells.

Animals↗

PubMed2006

Essential role for gene profiling analysis in the authentication of human cell lines.

Cross-contamination between cultured cell lines can result in the generation of erroneous scientific data. Hence, it is very important to eliminate cell lines that are of an origin different from that being claimed. Inter-species contamination can be detected by various established methods, such as karyotype and isozyme analyses. However, it has been impossible to detect intraspecies cross-contamination prior to the development of technology to detect differences between cell lines at the molecular level. Recently, profiling of short tandem repeat (STR) polymorphisms has been established as a method for the analyses of gene polymorphism. Gene profiling by STR polymorphism (STR profiling) is a simple and reliable method to identify individual cell lines. Each human cell line currently provided by the Cell Engineering Division of the RIKEN BioResource Center was analyzed by STR profiling to authenticate its identity. We found that more than 10 human cell lines out of approximately 400 were in fact identical to a different cell line deposited in the collection, and therefore had been misidentified. We conclude that STR profiling is a useful and powerful method for eliminating cell lines that have been misidentified by cross-contamination or by other causes. Hence, STR profiling of human cell lines used in published research will likely be a prerequisite for publication in the future, so that the problem of misidentification of cell lines can be eliminated.

Cell Line↗

PubMed2005

Lipocalin 2 functions as a negative regulator of red blood cell production in an autocrine fashion.

Members of the lipocalin protein family are typically small, secreted proteins that possess a variety of functions. Although the physiological role of lipocalin 2 remains to be fully elucidated, a few pivotal functions have recently been reported, e.g., regulation of the apoptosis of leukocytes. Unexpectedly, lipocalin 2 is abundantly expressed in erythroid progenitor cells. An in vitro culture experiment demonstrated that lipocalin 2 induces apoptosis and inhibits differentiation of erythroid progenitor cells. During acute anemia the expression of lipocalin 2 was reduced in erythroid cells by a feedback system. Furthermore, injection of recombinant lipocalin 2 into mice suffering from acute anemia retarded the recovery of red blood cell (RBC) numbers, suggesting the importance of reduced expression of lipocalin 2 for the efficient recovery of RBC numbers. These results indicate that lipocalin 2 suppresses RBC production in an autocrine fashion. Hence, anemia arising from pathological conditions, such as chronic inflammation, might be partly due to increased levels of lipocalin 2 secreted from expanded leukocytes and/or macrophages. Also, anemia arising from malignancies might be partly due to the abundant secretion of lipocalin 2 from tumor cells. Thus, lipocalin 2 may represent an attractive therapeutic target for anemia under certain pathological conditions.

Acute-Phase Proteins↗

PubMed2005

Angiotensin II and III upregulate body fluid volume of the clam worm Perinereis sp. via angiotensin II receptors in different manners.

Angiotensin III (Ang III) as well as angiotensin II (Ang II) suppressed body weight loss of the clam worm Perinereis sp. under a hyper-osmotic condition, and enhanced body weight gain under a hypo-osmotic condition. Under a drying condition where the water inflow from outside the body was eliminated, Ang II suppressed body weight loss, but Ang III did not. Under these conditions, angiotensins I, IV, and (1-7) had no effect, and saralasin blocked the effects of Ang II and Ang III. It is concluded that Ang II and Ang III upregulate body fluid volume of the clam worm via Ang II receptors in different ways.

Angiotensin II↗

PubMed2005

Use of bone morphogenetic protein 2 and diffusion chambers to engineer cartilage tissue for the repair of defects in articular cartilage.

OBJECTIVE: To examine the ability of cartilage-like tissue, generated ectopically in a diffusion chamber using recombinant human bone morphogenetic protein 2 (rHuBMP-2), to repair cartilage defects in rats. METHODS: Muscle-derived mesenchymal cells were prepared by dissecting thigh muscles of 19-day postcoital rat embryos. Cells were propagated in vitro in monolayer culture for 10 days and packed within diffusion chambers (10(6)/chamber) together with type I collagen (CI) and 0, 1, or 10 microg rHuBMP-2, and implanted into abdominal subfascial pockets of adult rats. Tissue pellets were harvested from the diffusion chambers at 2 days to 6 weeks after implantation, and examined by histology, by reverse transcription-polymerase chain reaction (PCR) for aggrecan, CII, CIX, CX, and CXI, MyoD1, and core binding factor a1/runt-related gene 2, and by real-time PCR for CII. Tissue pellets generated in the chamber 5 weeks after implantation were transplanted into a full-thickness cartilage defect made in the patellar groove of the same strain of adult rat. RESULTS: In the presence of 10 microg rHuBMP-2, muscle-derived mesenchymal cells expressed CII messenger RNA at 4 days after transplantation, and a mature cartilage mass was formed 5 weeks after transplantation in the diffusion chamber. Cartilage was not formed in the presence of 1 microg rHuBMP-2 or in the absence of rHuBMP-2. Defects receiving cartilage engineered with 10 microg rHuBMP-2 were repaired and restored to normal morphologic condition within 6 months after transplantation. CONCLUSION: This method of tissue engineering for repair of articular defects may preclude the need to harvest cartilage tissue prior to mosaic arthroplasty or autologous chondrocyte implantation. Further studies in large animals will be necessary to validate this technique for application in clinical practice.

Animals↗

PubMed2005

Microbubble-enhanced ultrasound exposure promotes uptake of methotrexate into synovial cells and enhanced antiinflammatory effects in the knees of rabbits with antigen-induced arthritis.

OBJECTIVE: To evaluate whether microbubble-enhanced ultrasound (US) treatment promotes the delivery of methotrexate (MTX) into synovial cells and the enhanced antiinflammatory effects of intraarticular MTX therapy in a rabbit arthritis model. METHODS: Arthritis was induced in both knees of 53 rabbits by immunization with ovalbumin. MTX including a microbubble agent was then injected into the left and right knee joints, and the right knees were exposed to US (MTX+/US+ group), while the left knees were not (MTX+/US- group). The knee joints were evaluated histologically in 7 rabbits at 5 time points up to day 56. Quantitative gene expression of interleukin-1beta (IL-1beta) in synovial tissue was measured on days 7 and 28. Eight rabbits were used for the measurement of MTX concentration in synovial tissue 12 hours after treatment. To evaluate the effect of microbubble-enhanced US treatment in the absence of MTX, only the microbubble agent was injected into the left and right knee joints of 10 rabbits with or without US exposure, and these animals were evaluated histologically on days 7 and 28. RESULTS: The MTX concentration in synovial tissue was significantly higher in the MTX+/US+ group than in the MTX+/US- group. Synovial inflammation was less prominent in the MTX+/US+ group compared with the MTX+/US- group, judging from the results of the histologic evaluation and the gene expression levels of IL-1beta in synovial tissue. It also appeared that microbubble-enhanced US exposure itself did not affect inflammation. CONCLUSION: Microbubble-enhanced US exposure promoted the uptake of MTX into synovial cells, which resulted in enhancement of the antiinflammatory effects of the intraarticular MTX injection. These results suggest that application of this technique may have clinical benefit.

Animals↗

PubMed2005

Usefulness of noise adaptive non-linear gaussian filter in FDG-PET study.

OBJECTIVE: In positron emission tomography (PET) studies, shortening transmission (TR) scan time can improve patient comfort and increase scanner throughput. However, PET images from short TR scans may be degraded due to the statistical noise included in the TR image. The purpose of this study was to apply non-linear Gaussian (NLG) and noise adaptive NLG (ANLG) filters to TR images, and to evaluate the extent of noise reduction by the ANLG filter in comparison with that by the NLG filter using phantom and clinical studies. METHODS: In phantom studies, pool phantoms of various diameters and injected doses of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) were used and the coefficients of variation (CVs) of the counts in the TR images processed with the NLG and ANLG filters were compared. In clinical studies, two normal volunteers and 13 patients with tumors were studied. In volunteer studies, the CV values in the liver were compared. In patient studies, the standardized uptake values (SUVs) of tumors in the emission images were obtained after processing the TR images using the NLG and ANLG filters. RESULTS: In phantom studies, the CV values in the TR images processed with the ANLG filter were smaller than those in the images processed with the NLG filter. When using the ANLG filter, their dependency on the phantom size, injected dose of FDG and TR scan time was smaller than when using the NLG filter. In volunteer studies, the CV values in the images processed with the ANLG filter were smaller than those in the images processed with the NLG filter, and were almost constant regardless of the TR scan time. In patient studies, there was an excellent correlation between the SUVs obtained from the images with a TR scan time of 7 min processed with the NLG filter (x) and those obtained from the images with a TR scan time of 4 min processed with the ANLG filter (y) (r = 0.995, y = 1.034x - 0.075). CONCLUSIONS: Our results suggest that the ANLG filter is effective and useful for noise reduction in TR images and shortening TR scan time while maintaining the quantitative accuracy of FDG-PET studies.

Adult↗

PubMed2005

The effects of heat on the biological activity of recombinant human bone morphogenetic protein-2.

This study was designed to investigate effects of heat on the bone-inducing activity of recombinant human bone morphogenetic protein (rhBMP)-2. rhBMP-2 samples were heated at 50, 70, 90, or 100 degrees C for 15 min, or 1, 2, 4, or 8 h, or autoclaved at 120 degrees C for 15 min. The bone-inducing activity of the rhBMP-2 before and after heating was assayed in in vivo and in vitro systems. For the in vivo assay, 5 microg rhBMP-2 samples were impregnated into porous collagen disks (6 mm in diameter, 1 mm thickness), freeze dried, and implanted into the back muscles of ddY mice. Three weeks later, the implant was harvested from the host and examined for ectopic new bone tissue by radiography. The new bone mass was quantified by single-energy X-ray absorptiometry. The in vitro activity of the rhBMP-2 was assayed by adding the BMP sample at a concentration of 100 ng/ml to cultures of MC3T3-E1 cells. After 48 h, the alkaline phosphatase activity was measured. After heating at 50 degrees or 70 degrees C, no significant reduction in bone-inducing activity was noted in either in vivo or in vitro assay systems unless the protein was exposed to sustained heat at 70 degrees C for 8 h, based on in vitro assay data. However, heating above 90 degrees C and for longer periods led to a decrease in the biological activity of the rhBMP-2 in a time- and temperature-dependent manner. rhBMP-2 was rendered inactive when exposed to temperatures at or in excess of 120 degrees C.

Alkaline Phosphatase↗

PubMed2005

Expression profiles of BMP-related molecules induced by BMP-2 or -4 in muscle-derived primary culture cells.

The formation of ectopic bone in muscle following the implantation of decalcified bone matrix led to the search and eventual discovery of bone morphogenetic proteins (BMPs) in bone matrix. The precise sequence of molecular events that underpin the cellular transformation of undifferentiated mesenchymal cells into bone has not been established, and is the subject of this study. Northern and Western blot analyses were used to examine changes in gene expression of cells treated with BMP-2 or -4. The molecules, which included BMP receptors (BMPRs), Noggin (a BMP-specific antagonist), osteocalcin (OC), Smad-4, and MyoD, were examined at messenger RNA (mRNA) and protein levels. The changes in expression of these molecules were followed in mouse muscle-derived primary culture cells, and osteoblastic or nonosteoblastic embryonic cell lines. We show the early up-regulation of BMPR-1A, -2, Noggin, OC, and Smad-4 in muscle-derived primary culture cells in a dose-dependent manner in response to BMP-2 or -4. MyoD expression was not detected after BMP stimulation. The differential expression of these positive and negative regulators of BMP signaling points to a potential regulatory mechanism for bone induction in mesenchymal cells.

Blotting, Western↗

PubMed2005

Hypoxia correlates with angiogenesis in cervical cancers.

BACKGROUND: Tissue hypoxia stimulates the induction of the angiogenic substances vascular endothelial growth factor and erythropoietin in the locus of the tissue. We have previously demonstrated that erythropoietin promotes angiogenesis by binding to its receptor in the endothelial cells of uterine and ovarian malignancies. In the present study, we examined whether malignant uterine cervix tissue showed hypoxia and whether hypoxia correlated with high vascular density through vascular endothelial growth factor. METHODS: To detect tissue hypoxia, we estimated the content of ATP in squamous cell carcinoma of the uterine cervix and in the normal cervix, using liquid chromatography columns. Surgically resected samples were fixed in Zamboni solution and processed for immunohistochemical microscopy to identify the endothelial cells and the location of vascular endothelial growth factor, with the use of anti-factor VIII and anti-vascular endothelial growth factor165 antibody, respectively. The microvessels in a definite area were counted in sections of each specimen. RESULTS: Significantly lower ATP levels and significantly higher vascular density were seen in squamous cell carcinoma than in the controls (P<0.05). The microvessel number in relation to ATP content was significantly higher in squamous cell carcinoma than in the controls (P<0.001). Moreover vascular endothelial growth factor, the hyperplastic epithelium of the squamous cell carcinoma contained the immunoreactivity, with characteristic histopathological features suggesting retention of tissue fluid. CONCLUSION: Squamous cell carcinoma of the uterine cervix showed hypoxia which correlated with abundant vascularity. Vascular endothelial growth factor expressed in the hyperplastic epithelium appears to promote angiogenesis in squamous cell carcinoma of the uterine cervix.

Adenosine Triphosphate↗