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Biomedical subjects

Johbu Itoh

Publications and source records attributed to Johbu Itoh.

18 recordsLinked to original sources
PubMed2005

Establishment of stable GH3 cell line expressing enhanced yellow fluorescein protein-growth hormone fusion protein.

To investigate, in real time, the transport and secretion of pituitary hormone, we have developed an experimental pituitary cell line, GH3 cell, which has secretory granules of growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes secretory granules of GH linked to EYFP on stimulation by Ca2+ influx or Ca2 release from storage. This GH3 cell will be useful for the real-time visualization of the intracellular transport and secretion of GH.

Animals↗
PubMed2005

Cell type-specific intervention of transforming growth factor beta/Smad signaling suppresses collagen gene expression and hepatic fibrosis in mice.

BACKGROUND & AIMS: Transforming growth factor beta and its intracellular mediators, Smad proteins, play important roles in stimulating collagen gene transcription and, thus, could be the targets for treating hepatic fibrosis. However, intervention of transforming growth factor beta/Smad signaling affects physiological signal transduction as well and may cause serious adverse effects on clinical application. Here we have attempted to suppress hepatic fibrosis by expressing a transforming growth factor beta/Smad antagonist selectively in collagen-producing cells only in the fibrotic liver. METHODS: Recombinant adenoviruses expressing either green fluorescent protein or a transforming growth factor beta/Smad signal repressor, YB-1, were injected into mice untreated or treated with carbon tetrachloride. Green fluorescent protein expression was analyzed under a confocal laser scanning microscope. Antifibrotic effects of YB-1 overexpression were examined by luciferase assays and histological examination with transgenic reporter mice. RESULTS: When the CAG expression unit was used as a control, green fluorescent protein was strongly expressed in a large number of hepatocytes in both normal and carbon tetrachloride-treated liver. In contrast, green fluorescent protein expression driven by a tissue-specific enhancer of the mouse alpha2(I) collagen gene ( COL1A2 ) was detected in activated hepatic stellate cells in carbon tetrachloride-induced fibrotic liver, but not in untreated normal liver. No green fluorescent protein fluorescence was observed in any other organs when the COL1A2 enhancer was used. Adenovirus-mediated YB-1 expression under the control of the COL1A2 enhancer significantly decreased COL1A2 promoter activity after carbon tetrachloride injection and subsequently suppressed the progression of hepatic fibrosis. CONCLUSIONS: These results validate a new concept of the therapy for hepatic fibrosis to achieve cell type-specific gene expression only in the fibrotic liver, with little damage to other organs.

Adenoviridae↗
PubMed2005

Case of uterine cervical carcinosarcoma.

Carcinosarcoma (CS) is a rare neoplasm that is called a mixed epithelial and mesenchymal malignancy. CS of the uterine cervix is much less common than its counterparts in the uterine corpus. A 61-year-old, gravida 2, para 2 woman, who had undergone menopause 16 years prior to the presentation, was diagnosed with CS of the uterine cervix. A semiradical hysterectomy was carried out on the diagnosis of stage Ib1 cervical cancer. The patient underwent whole pelvic 45 Gy radiation as a postoperative additional treatment, but she died from multiple organ failure by metastasis 17 months after the operation. The tumor protruded from the cervix to the vagina and measured 4.5 x 3.0 cm. Histologically, the tumor was characterized as a squamous cell carcinoma and mesenchymal malignancy, represented by osteosarcomatous components. The stroma was largely composed of atypical spindle-shaped cells, which were immunohistochemically demonstrated to be of epithelial origin. Uterine cervical CS is one of the aggressive malignancies, and squamous cell carcinomas are common epithelial counterparts of cervical CS as well as adenocarcinomas.

Carcinosarcoma↗
PubMed2005

Three-dimensional imaging of the intracellular localization of growth hormone and prolactin and their mRNA using nanocrystal (Quantum dot) and confocal laser scanning microscopy techniques.

Semiconductor nanocrystals (Quantum dots, Qdots) have recently been used in biological research, because they do not fade on exposure to light, and they enable us to obtain multicolor imaging because of a narrow emission peak that can be excited via a single wavelength of light. There have been no reports of simultaneous localization of mRNA and protein using Qdots. We successfully applied these advantages of Qdot and confocal laser scanning microscopy (CLSM) to three-dimensional images of the intracellular localization of growth hormone and prolactin and to their mRNA. In situ hybridization and immunohistochemistry using Qdots combined with CLSM can optimally illustrate the relationship between protein and mRNA simultaneously in three dimensions. Such an approach enables us to visualize functional images of proteins in relation with mRNA synthesis and localization.

Animals↗
PubMed2005

Association of neuroendocrine differentiation with neoadjuvant hormone therapy effects in prostatic cancer.

Histological therapeutic effects of neoadjuvant hormone therapy (NHT) in prostatic cancer were examined, focusing on the association with neuroendocrine differentiation (NED), using 69 radical prostatectomy cases. The effects of NHT were classified into 3 grades based on the extent of tumor degeneration as observed with hematoxylin and eosin staining. NED cells in the cancer were semi-quantified into 4 grades (negative, 1+, 2+, and 3+) by immunohistochemical staining of chromogranin A (CgA). According to the therapeutic effects, the cases are divided as follows: good response in 26 patients, intermediate in 20, poor in 23. The histological therapeutic effects were significantly weaker in the CgA-positive group than the CgA-negative group (p=0.02). A close relationship between the extent of CgA expression and the histological response was also demonstrated (p=0.007). In the biopsy specimens before NHT, CgA was positive in 46% (32/69) and there was no significant difference in histological therapeutic effects between the positive and negative groups. However, the therapeutic effects were significantly weaker in 22 CgA-positive cases for both biopsy and prostatectomy specimens than in 18 CgA-negative cases for both specimens (p=0.001). In conclusion, although it seems difficult to predict the therapeutic effects of NHT using the biopsy specimens of prostatic cancer, we believe that NED is negatively associated with histological response of prostatic cancer to NHT.

Adenocarcinoma↗
PubMed2004

Post-ischemic delayed expression of hepatocyte growth factor and c-Met in mouse brain following focal cerebral ischemia.

We investigated long-term changes in the expression of protein and mRNA of hepatocyte growth factor (HGF) and its receptor c-Met in mouse brain after permanent occlusion of the middle cerebral artery, by using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. HGF-immunopositive cells were observed in the periinfarct region from 4 days after occlusion, peaking at 14-28 days. The area containing HGF-immunopositive cells continued to expand until 28 days after occlusion. c-Met-immunopositive cells were observed exclusively at the periinfarct region at 7 and 14 days after occlusion. At 28 days after occlusion, there were many c-Met-immunopositive cells in the widespread periinfarct region. Triple immunohistochemical staining by using confocal laser scanning microscopy (CLSM) demonstrated that most of the HGF-immunopositive cells were localized to reactive astrocytes. The c-Met-immunopositive cells were also localized to reactive astrocytes. HGF mRNA was upregulated exclusively in the periinfarct region at 14 days. c-Met mRNA was upregulated in the periinfarct region from as late as 28 days after occlusion. Thus, HGF and c-Met show delayed expression in the periinfarct region at both protein and mRNA levels after induction of ischemia. Because HGF was recently shown to play critical roles in angiogenesis and neurotrophic activities, the temporal profiles of their expression may imply the involvement of HGF in the process of post-ischemic brain tissue repair.

Animals↗
PubMed2004

Immunohistochemical evaluation of hormone receptors in breast cancer: which scoring system is suitable for highly sensitive procedures?

To evaluate hormone receptors immunohistochemically, standardized staining procedures and scoring systems are required. The authors previously reported that highly sensitive procedures affected basic factors for technical validation. The aim of the present study was to show the characteristics of scoring systems for highly sensitive procedures. To examine how highly sensitive procedures enhance the staining intensity and increase the positive cell population, two different staining methods were compared. To evaluate scoring systems, three systems--cell counting score, modified immunoreactive score, and H score--were compared using the same samples stained by an autostaining system. It was found that the highly sensitive procedure increased the positive cell population, especially in breast cancers with a low enzyme immunoassay (EIA) level of less than 100 fmol/mg, and strengthened the staining intensity. This enhancement led to a correlation in a logarithmic curve rather than a linear correlation by all three scoring systems. The results showed that scoring systems including a factor of staining intensity did not have an absolute advantage because boosted staining intensity by highly sensitive procedure did not reflect EIA value or protein contents accurately. To the authors' knowledge, there is no report discussing the nonlinear correlation between biochemical and immunohistochemical assay by highly sensitive procedures; however, it is important to select a scoring system and threshold based on nonlinear correlation.

Breast Neoplasms↗
PubMed2004

Malignant transformation of atypical endometrial hyperplasia after progesterone therapy showing germ-cell tumor-like differentiation.

A 31-year-old woman was treated for atypical endometrial hyperplasia (AEH) with high-dose medroxyprogesterone acetate (MPA) therapy to preserve fertility. The AEH was found by repeated cytologic and histologic examinations to have completely disappeared with the therapy, but 3 years after her last follow up she required emergency surgery to treat severe genital bleeding. The hysterectomied uterus consisted mostly of poorly differentiated adenocarcinoma, G3 endometrioid type. Minor AEH was present in the exophytic area, in which some glands were cystically dilated. Part of the AEH had transformed into other histologic features with germ-cell-like differentiation, demonstrated by immunohistochemical positive reaction of placental alkaline phosphatase, alpha-fetoprotein, and human chorionic gonadotrophin. Recurrent AEH had undergone malignant transformation, resulting in the development of well- and poorly differentiated adenocarcinoma and tumor exhibiting germ-cell-like differentiation. The patient died of a massive tumor extension 7 months after surgery. The AEH before MPA therapy and the recurrent tumors had genetically different characteristics based on evidence of a loss of heterozygosity, detected at D8S1132 (chromosomal locus, 8q22.1) in the latter but not in the former, by analysis of genetic alterations using microsatellite markers.

Adenocarcinoma↗
PubMed2003

Dynamics of subcellular organelles, growth hormone, Rab3B, SNAP-25, and syntaxin in rat pituitary cells caused by growth hormone releasing hormone and somatostatin.

Rab3B is involved in the exocytosis of synaptic vesicles and secretory granules in the central nervous system and the anterior pituitary cells. The aim of this study was to elucidate both the role of rab3B in GH secretion and the mutual relationship of rab3B and SNARE proteins. Adult male rats were injected intravenously with 10 microg of growth hormone releasing hormone (GHRH) or 10 microg of somatostatin (SRIF). Untreated rats were used as controls, and their pituitary glands were sectioned for histochemical examination. Rab3B is localized on the limiting membrane of the secretory granules and the cytosol. Confocal laser scanning microscopic observation of immunohistochemical double staining of rab3B and GH revealed that immunoreactivity of rab3B increased in GHRH-treated rats and decreased in SRIF-treated rats. Confocal laser scanning microscopic observation of immunohistochemical double staining of SNAP-25, syntaxin, and rab3B revealed the co-localization of rab3B and these SNARE proteins in GHRH-treated rats, and their dissociation in SRIF-treated rats. These results suggest that rab3B plays a principal role in GH secretion in the anterior pituitary cells and that SNAP-25 and syntaxin act as co-workers with rab3B in the functional regulation of GH secretion.

Animals↗
PubMed2003

Vascular networks and endothelial cells in the rat experimental pituitary glands and in the human pituitary adenomas.

There has been considerable interest in the relationship between hormone-secreting endocrine cells (HSEC) and their microvessels (MVN) in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). Therefore, this study was designed to visualize and to reveal the relationship between hormone secreting endocrine cells and their microvessel environment including vascular endothelial cells in 3D using rat pituitary glands under various experimental conditions by confocal laser scanning microscopy (CLSM). By CLSM, the 3D distributions of MVN were visualized and revealed a relationship between HSEC and MVN in experimental pituitary glands and human pituitary adenomas. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways.

Adenoma↗
PubMed2003

In vivo and in vitro differentiation of myocytes from human bone marrow-derived multipotent progenitor cells.

OBJECTIVE: Recent studies have shown that bone marrow (BM) contains cells capable of differentiating into myocytes in vivo. However, addition of demethylation drugs has been necessary to induce myocyte differentiation from BM cells in vitro, and precise mechanisms of BM cells' conversion to myocytes and the origin of those cells have not been established. We investigated the expression of myogenic markers during differentiation and maturation of myocytes from BM-derived multipotent adult progenitor cells (MAPC) under physiological culture condition. MATERIALS AND METHODS: Frozen BM samples from 21 healthy donors were used as a source of MAPC. To induce myocyte differentiation MAPC was cultured in the presence of 5% FCS, VEGF, bFGF, and IGF-1, and the expressions of myocyte markers were examined at various time points. We also investigated engraftment and differentiation of MAPC-derived myocytes in vivo. RESULTS: Frozen BM-derived MAPC, cultured under the physiological myogenic condition, demonstrated spatial expression patterns of several myocyte markers similar to that of authentic myocyte differentiation. When injected into murine muscles, MAPC treated with the myogenic condition engrafted and differentiated into myocyte marker-positive cells and myotubes in vivo. CONCLUSION: For the first time, we were able to induce myocyte formation from BM cells under the physiological condition in vitro and demonstrated that treating cells with this condition prior to intramuscular injection increased efficiency of engraftment and differentiation in vivo.

Adolescent↗
PubMed2003

Rapid and quantitative detection of human septin family Bradeion as a practical diagnostic method of colorectal and urologic cancers.

BACKGROUND: Malignant tumor progression is a complex and multi-gene event which can not be easily detected or predicted. The detection of malignant cells using marker genes is hampered by the fact that these markers are only expressed by certain malignancies or lack sensitivity and/or specificity. We have reported a human septin family gene Bradeion, which shows strong cancer-specific expression in colorectal and urologic cancers as a result of carcinogenesis. MATERIAL/METHODS: Diagnostic efficacy and validity of Bradeion gene expression were tested by two independent systems, one is a protein detection method using monoclonal antibody based immuno-chromatographic membrane strip tests (a nitrocellulose test strip assay), and another is a gene expression detection method, quantitative RT-PCR. The technology has been established using Bradeion fusion proteins, in vitro cultivated human cancer cell lines, and also patients' test samples with controls. RESULTS: Bradeion test strip by combination with two monoclonal antibodies are valid for the detection of 1 ng/ml Bradeion, and successfully applied for patient urine samples with no false-positive results. Positive detection rates were over 70% of the patient urine samples so far tested (prostate cancer, renal cell carcinoma, and bladder cancer) in 15 to 30 minutes. Quantitative RT-PCR resulted in significantly high copy numbers of 0.4-3.0-3.0 x 10(5) per microg total RNA in patients' tissue samples, whereas those from normal tissue or other cancers found negative. CONCLUSIONS: The present study introduces the practical diagnostic methods using a disease-specific molecular marker, which provides safe, economical, and rapid clinical screening of cancer.

Animals↗
PubMed2002

Impaired expression of a human septin family gene Bradeion inhibits the growth and tumorigenesis of colorectal cancer in vitro and in vivo.

We have identified a novel human septin family gene Bradeion, which is specifically expressed in human colorectal cancer and malignant melanoma. In order to analyze the implications of tumor-specific gene expression, ribozymes and its derivatives were specifically designed and transfected into various colorectal adenocarcinoma cell lines for Bradeion inactivation. We constructed ribozyme expression plasmids controlled by a human tRNA(Val) promoter, and both hammerhead ribozyme and its allosteric derivative maxizyme were used for two different forms of Bradeion mRNA. The sequence-specific cleavage of Bradeion mRNA resulted in significant growth inhibition and G2 arrest in human cancer cell lines, detected by flow cytometry analysis. In addition, in vivo mice studies demonstrated marked tumor growth suppression by the Bradeion-specific ribozymes. Thus, the tumor-specific and selective marker Bradeion also provides valuable tools as a potential target for colorectal cancer therapy.

Animals↗
PubMed2002

Evaluation of Na+ active transport and morphological changes for bioartificial renal tubule cell device using Madin-Darby canine kidney cells.

The function of current hemodialysis as an artificial kidney is insufficient because of the lack of reabsorptive function. In this study, we intend to develop a bioartificial renal tubule cell device (RTD) using tubular epithelial cells and artificial membranes and to evaluate the reabsorptive function of the confluent layers. Madin-Darby canine kidney (MDCK) cells were cultured on a nucleopore polycarbonate membrane for up to 4 weeks after confluence to examine the influence of the culture period on their ability to transport Na+ actively using Na+/K+ATPase (NKA). The results were (1) active Na+ transport of the cells averaged 24.8 mM/m(2) x 24 h during the initial 2 weeks after confluence and then decreased to about 4.2 mM/m(2) x 24 h during the next 2 weeks; (2) NKA localized on the basal-lateral sides of the cells during the initial 2 weeks, whereas it also localized on the apical side of the cells during the next 2 weeks; (3) long-term culture resulted in an increased number of upheaving cell mass, increased fatty droplets in the cells, and necrosis; and (4) scanning electron microscopy showed fewer microvilli 3-four weeks after confluence. It is concluded that the culture period is critical for developing RTD using cultured renal tubular epithelial cells.

Animals↗
PubMed1999

Electron Microscopic and Confocal Laser Scanning Microscopic Observation of Subcellular Organelles and Pituitary Hormone mRNA: Application of Ultrastructural In Situ Hybridization and Immunohistochemistry to the Pathophysiological Studies of Pituitary Cells.

Nonradioisotopic electron microscopic (EM) in situ hybridization (ISH) (EM-SH) with biotinylated oligonucleotide probes is utilized for the ultrastructural visualization of pituitary hormone mRNA in rat pituitary cells. EMISH is an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cell using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex can provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein. Another focus of this review is the utilization of a recently developed imaging system of confocal laser scanning microscopy (CLSM). The combination of CLSM and image analysis system (lAS) enables us to visualize an individual dimensional image of the intracellular distribution of mRNA and subcellular organelles successfully at any optional cross sections of light microscopic ISH studies, and can be another useful tool for the ultrastructural ISH study of mRNA.

Journal Article↗
PubMedDate not supplied

Transcellular water transport and stability of expression in aquaporin 1-transfected LLC-PK1 cells in the development of a portable bioartificial renal tubule device.

We investigated a portable bioartificial renal tubule device (BRTD) consisting of renal tubule cells and hollow fibers, to improve the quality of life of patients. It is necessary for a BRTD system to be compact. A compact portable BRTB requires transfection of an appropriate water channel or electrical pump genes in tubular epithelial cells, which should be based on physiological similarities to human kidney function. LLC-PK(1) cells, into which rat kidney aquaporin 1 (AQP1) cDNA was stably transfected, were evaluated for water transport ability. The expression and localization of water AQP1 were examined by Western blotting, RT-PCR, and immunofluorescence. To measure transcellular water permeation, a simple method was applied, using phenol red as a cell-impermeant marker of concentration. In contrast to wild-type LLC-PK(1) cells, rat AQP1-transfected cells had high transcellular osmotic water permeability. The expression of rat AQP1 mRNA (ratio of AQP1 to beta-actin mRNA) and protein bands (a 28-kDa band and a broad, 35- to 45-kDa band) was confirmed to be stably maintained until a population doubling level of 24. In AQP1-transfected LLCPK(1) cells, the protein was localized mainly to the basolateral side, but also the apical side, of the plasma membrane. Wild-type LLC-PK(1) cells were not stained at the plasma membrane. It is possible that enough AQP1-transfected tubule epithelial cells were supplied for a bioartificial renal tubule device.

Animals↗