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T Ishiwata

Publications and source records attributed to T Ishiwata.

At least 55 records · Page 3Linked to original sources
PubMed1998

Enhanced fibroblast growth factor 5 expression in stromal and exocrine elements of the pancreas in chronic pancreatitis.

BACKGROUND: Fibroblast growth factor 5 (FGF-5) belongs to a group of mitogenic and angiogenic heparin binding growth factors but its potential role in chronic inflammatory conditions is not known. AIMS: To compare FGF-5 expression in the normal pancreas and in the pancreas of patients with chronic pancreatitis (CP) and to characterise FGF-5 expression and secretion in TAKA-1 cells, an immortalised Syrian hamster pancreatic duct cell line. METHODS AND RESULTS: Northern blotting revealed the presence of a 4.0 kb FGF-5 mRNA transcript in both normal and CP tissue samples. Densitometric analysis indicated that the transcript levels were increased by a factor of 1.44 in CP tissue samples compared with normal tissue samples (p = 0.039). By immunohistochemisty and in situ hybridisation, FGF-5 was faintly expressed in ductal and islet cells in the normal pancreas. In contrast, in CP tissue samples, there was abundant expression of FGF-5 in ductal, acinar, and islet cells, as well as in periductal fibroblasts. FGF-5 was also expressed in TAKA-1 cells as determined by Northern blotting. By immunoblotting of heparinsepharose precipitates, TAKA-1 cells were shown to secrete FGF-5 into the medium. CONCLUSION: Exocrine and stromal derived FGF-5 has the potential to participate in autocrine and paracrine pathways that may contribute to the pathobiology of chronic pancreatitis.

Adult↗
PubMed1998

Deposition of advanced glycation end products (AGE) and expression of the receptor for AGE in cardiovascular tissue of the diabetic rat.

Advanced glycation end products (AGE) in tissues are important for the central pathological features of diabetic complication. Although AGE bind to several cell-surface sites, resulting in altered cellular functions, receptor for AGE (RAGE) appears to have a central role. We examined AGE accumulation and RAGE expression in the aorta and heart of rats with streptozotocin (STZ)-induced diabetes, 0, 4, 8, 12, 16 and 24 weeks after STZ administration. Early atherosclerotic findings in the intima and medial thinning were observed in the aorta after 16 weeks of STZ-Induced diabetes. Immunohistochemistry and microscope spectrophotometry showed that AGE deposition increased significantly in the aorta and vessels of the myocardium, depending on the period of hyperglycaemia. RAGE was expressed in the endothelial cells and vascular smooth muscle cells of all animals. The number of smooth muscle cells with RAGE immunoreactivity increased until 12 weeks after STZ injection, and then decreased in rats with diabetes between 16 and 24 weeks. On the other hand, total RAGE mRNA levels in the aorta and heart continued to increase with the duration of hyperglycaemia. Furthermore, AGE-BSA induced RAGE mRNA expression of human umbilical vein endothelial cells in vitro. Taken together, the AGE accumulation might initiate diabetic macroangiopathy through RAGE, and the increase of RAGE expression by endothelial cells could be a reason that diabetes mellitus accelerates atherosclerosis rapidly.

Animals↗
PubMed1997

Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions.

Fibroblast growth factor (FGF)-1 and -2 are overexpressed in human pancreatic cancer. In this study the role of FGF-5 in human pancreatic cancer was investigated, as FGF-5 has a classical signal sequence for secretion not found in FGF-1 or -2. Northern blot analysis with a 306 bp FGF-5 cDNA revealed the presence of 4.0 kb and 1.6 kb FGF-5 mRNA transcripts in both normal and cancerous pancreatic tissues. Densitometric analysis indicated that 4.0 kb and 1.6 kb FGF-5 mRNA transcripts levels were increased 2.4- and 2.7-fold in the cancers by comparison with normal tissues, respectively (P < 0.002, P < 0.0001). Immunohistochemistry and in situ hybridization demonstrated that FGF-5 localized in the cancer cells, stromal fibroblast and inflitrating macrophages. FGF-5 mRNA was also detected in COLO-357 human pancreatic cancer cells. Furthermore, secreted FGF-5 protein was present in conditioned medium of COLO-357 cells. Exogeneous FGF-5 (0.37 nM) increased the growth of COLO-357 cells by 48% (P < 0.0001) and increased mitogen-activated protein kinase activity. COLO-357 cells expressed the IIIc isoform of the type I FGF receptor, the preferred FGF receptor for FGF-5. These observations suggest that FGF-5 may participate in autocrine and paracrine pathways promoting pancreatic cancer cell growth in vivo.

Adenocarcinoma↗
PubMed1997

Amphiregulin antisense oligonucleotide inhibits the growth of T3M4 human pancreatic cancer cells and sensitizes the cells to EGF receptor-targeted therapy.

Human pancreatic cancers overexpress the epidermal growth factor (EGF) receptor (EGFR) and all 5 ligands that bind to this receptor, including amphiregulin. It is not known, however, whether amphiregulin contributes in an autocrine manner to enhance pancreatic cancer cell growth. Therefore, we used an amphiregulin antisense oligonucleotide (AR-AS) to suppress amphiregulin expression in T3M4 human pancreatic cancer cells. These cells express high levels of EGFR and amphiregulin. AR-AS abolished amphiregulin immunoreactivity in T3M4 cells, decreased amphiregulin release into the medium and inhibited cell growth in a dose-dependent manner. Exogenous amphiregulin reversed AR-AS-mediated growth inhibition. A random oligonucleotide (AR-R) did not alter either cell growth or cellular amphiregulin immunoreactivity. AR-AS also increased cellular EGFR protein levels and enhanced the growth-inhibitory actions of TP40, a chimeric protein consisting of transforming growth factor-alpha coupled to Pseudomonas exotoxin that internalizes into cells via EGFR. These findings indicate that there is an important EGFR/ amphiregulin autocrine loop in T3M4 cells and raise the possibility that modalities aimed at abrogating amphiregulin action may prove useful in pancreatic cancer, especially when used in conjunction with EGFR-targeted therapy.

Amino Acid Sequence↗
PubMed1997

Differential localization of transforming growth factor-beta isoforms in human gastric mucosa and overexpression in gastric carcinoma.

Transforming growth factor beta (TGF-beta) isoforms comprise a family of multifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. We examined TGF-beta expression in normal human gastric mucosa and carcinoma. The distribution and expression of TGF-beta isoforms in 4 normal mucosa samples from organ donors, in 12 normal mucosa samples adjacent to gastric cancer and in 12 gastric carcinomas were examined using immunohistochemistry and Northern blot analysis. Because TGF-beta s regulate collagen expression, collagen type I alpha1 mRNA amounts were also examined. Immunohistochemical analysis of normal human gastric tissue samples indicated that TGF-beta1 localized principally in parietal cells but also in some surface mucus cells, TGF-beta2 was present exclusively in chief cells and TGF-beta3 was present in parietal, chief and mucus cells. In the gastric cancers, strong colocalization of TGF-beta1, -beta2 and -beta3 was evident in the cancer cells. Northern blot analysis indicated that, compared to normal gastric tissue, gastric cancers showed a 4.8- and 6-fold increase in mRNA amounts encoding TGF-beta1 and TGF-beta3, respectively. In contrast, TGF-beta2 mRNA amounts were comparable in both groups. Northern blot analysis showed a 10-fold increase in human collagen type I alpha1 mRNA amounts compared to normal gastric tissue. These findings imply a role forTGF-beta s in normal human gastric mucosa function, and raise the possibility that the aberrant colocalization and overexpression of all 3 TGF-beta isoforms in human gastric cancer cells in vivo may contribute to the pathobiology of gastric carcinoma.

Adenocarcinoma↗
PubMed1997

Dissection of the promoter/operator region and evaluation of N-acylhomoserine lactone mediated transcriptional regulation of elastase expression in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, expression of the lasB gene which codes for the metalloprotease, elastase, depends on small diffusible N-acylhomoserine lactones. lasB expression is regulated through the interactions of N-3-oxododecanoyl-L-homoserine lactone and N-butanoyl-L-homoserine lactone with the transcriptional activators LasR and VsmR(RhlR), respectively. To investigate lasB expression further, we first located the transcriptional start site to a position 141 bp upstream from the translational start site. Using this information, we constructed a series of plasmids containing consecutive 5' deletions of the upstream region of lasB fused to a promoterless chloramphenicol acetyltransferase reporter gene. The results obtained indicate that three regions are required for efficient transcription of lasB; a 35 bp palindromic sequence located at +26 to +60 bp upstream from the translation start site, and two regions located upstream of the transcription start site, at -135 to -85 bp and -63 to -26 bp, respectively. Deletion of the latter region results in the loss of both N-butanoyl-L-homoserine lactone- and N-3-oxododecanoyl-L-homoserine lactone-mediated stimulation of lasB expression and provides further support for the role of this operator site as a target for either or both LasR and VsmR.

Bacterial Proteins↗
PubMed1997

Production of macrophage colony-stimulating factor by murine liver in vivo.

In previous reports, the authors demonstrated that M-CSF was produced by primary-cultured non-parenchymal (NPLC) and parenchymal (PLC) liver cells. In order to clarify the biological role of M-CSF produced by the liver, macrophage colony-stimulating factor (M-CSF)-producing cells in vivo were investigated using reverse transcriptase polymerase chain reaction (RT-PCR), dot blot analysis, in situ hybridization and immunohistochemistry. M-CSF mRNA was constantly identified by RT-PCR in the liver, NPLC and PLC, before and after partial hepatectomy. Dot blot analysis showed that fluctuations of M-CSF mRNA level after partial hepatectomy were not statistically significant. In situ hybridization revealed that M-CSF mRNA was expressed mainly in NPLC and vascular endothelial cells (VEC). In addition, a small number of PLC also expressed M-CSF mRNA. Neither the distribution nor the frequency of M-CSF mRNA positive cells in regenerative livers differed significantly from normal livers. M-CSF immunoreactivity was present in NPLC and VEC at all the times before and after partial hepatectomy, while PLC exhibited M-CSF immunostaining 0.5 days after partial hepatectomy. As normal liver expressed M-CSF mRNA to the same degree as regenerative liver, hepatic M-CSF mRNA production in vivo may be related to the physiological function of the liver. However, transient expression of M-CSF protein in PLC at an early stage after partial hepatectomy may be associated with liver regeneration.

Animals↗
PubMed1997

Altered expression of insulin-like growth factor II receptor in human pancreatic cancer.

The insulin-like growth factor-II (IGF-II) receptor (IGF-IIR) is a single-chain transmembrane protein identical to the mannose-6-phosphate receptor. In the present study we examined IGF-IIR expression in normal and cancerous human pancreatic tissues. In the normal pancreas, moderately strong IGF-IIR immunoreactivity was present in the cytoplasm of islet cells, and mild cytoplasmic immunoreactivity was evident occasionally in ductal and acinar cells. Some ductal cells also exhibited nuclear IGF-IIR immunoreactivity. In the pancreatic cancers, regions of strong IGF-IIR immunoreactivity were present in the duct-like cancer cells within the tumor mass, often exhibiting nuclear localization. Expression of IGF-IIR mRNA in the cancer cells was confirmed by in situ hybridization. By comparison with normal pancreatic tissues, 7 of 12 pancreatic cancers exhibited a 5.6-fold increase in IGF-IIR mRNA levels, whereas in 3 cancers the IGF-IIR transcript was below the level of detection. Furthermore, all six tested cultured human pancreatic cancer cell lines expressed the IGF-IIR mRNA transcript. Our data indicate that IGF-IIR is overexpressed in a significant number of human pancreatic cancers, where it has a tendency to localize in the nucleus, and raise the possibility that IGF-IIR may contribute to the pathobiology of pancreatic cancer.

Adolescent↗
PubMed1997

Differential distribution of ecNOS and iNOS mRNA in rat heart after endotoxin administration.

To evaluate the relationship between cardiovascular injury and the pathological significance of endothelial constitutive nitric oxide synthase (ecNOS) and inducible nitric oxide synthase (iNOS) in endotoxic shock, Wistar rats were injected intraperitoneally with 10 mg/kg Escherichia coli endotoxin and the resulting cardiovascular changes observed using immunohistochemistry, immunoelectron microscopy, the reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization at 4, 6, 8 and 10 h after endotoxin administration. Immunohistochemical and electron microscopic observations showed that ecNOS was localized in the cytoplasmic vesicles and rough endoplasmic reticulum of the endothelium of coronary arteries and intermyocardial capillaries in both control and endotoxin-treated rats. iNOS was localized in the cytoplasmic vesicles and endoplasmic reticulum of vascular endothelial cells, vascular smooth muscle cells and cardiomyocytes after endotoxin administration. The RT-PCR study confirmed the expression of ecNOS and iNOS mRNA in the heart tissues of all animals including controls. In situ hybridization showed that ecNOS mRNA was expressed in the cytoplasm of vascular endothelial cells in control and endotoxin-treated rats. After endotoxin administration, iNOS mRNA was strongly expressed in vascular endothelial cells, vascular smooth muscle cells, cardiomyocytes and a small number of macrophages. Bacterial lipopoly-saccharide induces rapid release of nitric oxide in the microvasculature and cardiomyocytes resulting in the depression of cardiomyocyte contraction. These findings may describe the cardiac response after endotoxin treatment.

Animals↗
PubMed1997

Enhanced expression of vascular endothelial growth factor in human pancreatic cancer correlates with local disease progression.

Vascular endothelial growth factor (VEGF) is an angiogenic polypeptide that has been implicated in cancer growth. In the present study, we characterized VEGF expression in cultured human pancreatic cancer cell lines and determined whether the presence VEGF in human pancreatic cancers is associated with enhanced neovascularization or altered clinicopathological characteristics. VEGF mRNA transcripts were present in all six tested cell lines (ASPC-1, CAPAN-1, MIA-PaCa-2, PANC-1, COLO-357, and T3M4). Immunoblotting with a highly specific anti-VEGF antibody revealed the presence of VEGF protein in all of the cell lines. Northern blot analysis of total RNA revealed a 5.2-fold increase in VEGF mRNA transcript in the cancer samples in comparison with the normal pancreas. Immunohistochemical and in situ hybridization analysis confirmed the expression of VEGF in the cancer cells within the tumor mass. Immunohistochemical analysis of 75 pancreatic cancer tissues revealed the presence of strong VEGF immunoreactivity in the cancer cells in 64% of the cancer tissues. The presence of VEGF in these cells was associated with increased blood vessel number, larger tumor size, and enhanced local spread but not with decreased patient survival. These findings indicate that VEGF is commonly overexpressed in human pancreatic cancers and that this factor may contribute to the angiogenic process and tumor growth in this disorder.

Adenocarcinoma↗
PubMed1996

Purification and characterization of LasR as a DNA-binding protein.

In Pseudomonas aeruginosa, the activator protein LasR and a cognate autoinducer (AI) are required for expression of the elastase gene (lasB). In the present study, we investigated the binding properties of the P. aeruginosa lasR gene product. The LasR protein was overexpressed and purified as a glutathione S-transferase (GST) fusion protein. Using gel retardation and UV cross-linking analysis, we demonstrated that the GST-LasR could bind to a separate site in the lasB upstream operator regions 1 and 3 in the presence of the autoinducer. Regions 1 and 3 are located at 105 and 42 base pairs upstream, respectively, from the lasB transcriptional start site. Our present results clearly demonstrate that LasR is a specific DNA-binding protein that regulates the transcription of the lasB gene in the presence of an autoinducer.

4-Butyrolactone↗
PubMed1995

[A molecular pathological study on the localization of the human papillomavirus using nested PCR and in situ hybridization methods in paraffin embedded tissues].

This study was designed to use nested polymerase chain reaction (nested PCR), in situ hybridization (ISH) and immunohistochemical techniques to identify human papillomavirus (HPV) in tissues fixed in formalin and embedded in paraffin. Eighty cases including 17 cases of condyloma acuminatum, 10 cases of cervical dysplasia, 6 cases of carcinoma in situ, 16 cases of squamous cell carcinoma of the uterine cervix, 14 cases of nasal and paranasal papilloma, 1 case of transitional cell papilloma and 16 cases of esophageal squamous cell carcinoma were examined. With the nested PCR method, the positive reaction rate of HPV was higher than with the single step PCR method. The detection rate was about ten-fold higher in condyloma acuminatum and two times higher in cervical dysplasia with the nested PCR method. In four cases of condyloma acuminatum, ISH was positive. Three of those cases were type 18 HPV and the another was type 11 HPV. Immunohistochemically, HPV was detected only in the cases of condyloma acuminatum. The nuclei of superficial epithelial cells, especially kilocytes reacted positively both by ISH and immunohistochemical methods. Nested PCR is not a very complicated technique, and the detection rate is higher than that of commonly used pathological techniques. These results suggest that nested PCR will be a useful test for HPV routine pathological diagnosis.

Base Sequence↗
PubMed1995

[Two autopsy cases of malignant mixed müllerian tumor of the uterus with metastasis to alimentary tract and liver].

Two autopsy cases (69 and 87-year-old women) of malignant mixed müllerian tumor (MMMT) following radiation therapy for uterine cervical cancer sixteen and twenty years ago respectively were reported. They were admitted due to abdominal pain and diagnosed as ileus. In the first case, CT examination revealed a tumor measuring about 8 x 10 cm in size in the uterine posterior wall. Recurrence of the uterine cancer was suspected and hysterooophorectomy combined with sigmoidectomy was performed. In the second case, artificial anus formation was performed because of sigmoid stricture by the invasion of the tumor. Histologically, tumors in both cases were composed of carcinomatous and sarcomatous components including heterologous elements such as cartilage. The patients died of extensive spreads and metastasis in the liver and alimentary tract.

Aged↗
PubMed1995

Role of vascular endothelial growth factor in angiogenesis of rheumatoid arthritis.

OBJECTIVE: To examine the localization and role of vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen, in rheumatoid arthritis (RA). METHODS: Immunohistochemical staining, reverse transcription polymerase chain reaction (RT-PCR) analysis and in situ hybridization for VEGF were performed on synovial tissues from 10 patients with RA, 5 patients with osteoarthritis (OA) and 3 autopsy cases. RESULTS: In RA, the proliferative ratio of synovial lining cells and vascular endothelial cells was significantly higher than that in OA and normal synovial tissues. Immunohistochemical staining demonstrated that VEGF polypeptide was strongly expressed in subsynovial macrophages, fibroblasts surrounding microvessels, vascular smooth muscle cells, and synovial lining cells, but not in vascular endothelial cells of patients with RA. On the other hand, in patients with OA and normal cases, VEGF polypeptide was slightly expressed in synovial lining cells and fibroblasts. RT-PCR showed a positive reaction for VEGF messenger RNA in RA, OA, and normal synovial tissues. A large number of subsynovial macrophages, fibroblasts, vascular smooth muscle cells, and synovial lining cells from patients with RA expressed VEGF mRNA using in situ hybridization. CONCLUSION: In RA, VEGF is synthesized and released by a large number of subsynovial macrophages, fibroblasts surrounding microvessels, vascular smooth muscle cells, and synovial lining cells and may stimulate endothelial proliferation in a paracrine manner via VEGF receptors.

Arthritis, Rheumatoid↗
PubMed1994

Intracellular receptor-type transcription factor, LasR, contains a highly conserved amphipathic region which precedes the putative helix-turn-helix DNA binding motif.

We have cloned and sequenced the lasR gene, which is involved in the transcriptional activation of several pathogenic factors, from Pseudomonas aeruginosa IFO3455 and PA103. These clones were predicted to be an open reading frame of 239 amino acids as reported for the PAO1 strain. There is only a single base change resulting in an amino acid exchange from M145 (PAO1) to I (IFO3455). PA103 DNA differs with PAO1 DNA in two bases resulting in only a single amino acid substitution from R179 to W. When the IFO3455 LasR was expressed in a PA103 strain which is known to be LasR defective, proteinase gene activation was detected, however, when PA103 LasR was expressed, no enhancement was measurable. From these results, it appears that the amino acid substitution of R179 to W inactivated LasR activity. This substitution is located in the highly conserved sequence found in many transcription factors, including sigma factors, and may disrupt amphipathic alpha-helix, predicted for the 176 to 189 region, which precedes the putative helix-turn-helix DNA binding motif. We presumed that these three helices may contribute to specific DNA binding.

Amino Acid Sequence↗
PubMed1994

Elastase gene expression in non-elastase-producing Pseudomonas aeruginosa strains using novel shuttle vector systems.

In order to determine whether non-elastase-producing strains of Pseudomonas aeruginosa such as N-10, PA103 and IFO3080 can express foreign elastase genes, we introduced elastase genes from P. aeruginosa IFO3455 (elastase-producing) as well as from PA103 and N-10 into non-elastase-producing P. aeruginosa strains. Results suggested that gene expression, secretion, and precursor processing systems of elastase were essentially normal in P. aeruginosa N-10 and IFO3080. Our studies using various elastase genes showed that both the elastase structural gene and 5'-upstream regions of P. aeruginosa PA103 were also normal. This was confirmed by the finding that P. aeruginosa N-10 and IFO3080 which carry the PA103 elastase gene produced elastase. Several deleted or chimeric genes were constructed using the 5'-upstream regions of elastase genes from P. aeruginosa N-10 or PA103 and studies of expression revealed that two individual DNA bases seem to be important in suppressing P. aeruginosa N-10 elastase gene expression. Possible reasons for the lack of elastase expression in these non-elastase-producing strains are discussed.

Base Sequence↗