Search PubMed⌕ Search

Biomedical subjects

N Koshikawa

Publications and source records attributed to N Koshikawa.

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

cDNA cloning of a novel trypsin inhibitor with similarity to pathogenesis-related proteins, and its frequent expression in human brain cancer cells.

A novel trypsin inhibitor (P25TI) with an apparent molecular size of 25 kDa has previously been purified from the culture medium of human glioblastoma cells. In this study, the cDNA encoding P25TI was isolated by the polymerase chain reaction (PCR) screening system, and its complete amino acid sequence was determined. The cDNA consisted of 1440 nucleotides and encoded a sequence of 258 amino acids. The deduced structure of P25TI seemed to consist of a putative signal peptide sequence (residues 1-25), a propeptide sequence (26-60) and a mature protein (residues 61-258). The P25TI sequence has no homology to other proteinase inhibitors, but has similarity to insect venom allergens, mammalian testis-specific proteins and plant pathogenesis-related proteins. P25TI mRNA was frequently expressed in human neuroblastoma and glioblastoma cell lines. Although Northern blotting analysis failed to detect P25TI mRNA in various human tissues, PCR analysis showed its expression in the brain, placenta and lymphocytes.

Amino Acid Sequence↗
PubMed1998

Sister chromatid exchange analysis of human cells carrying retroviral DNA.

Chromosomal abnormalities have been seen in human cells following infection with human T lymphotropic virus type I (HTLV-I). Effects on sister chromatid exchange (SCE) in cells carrying HTLV-I related DNA were examined by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Peripheral blood specimens were obtained from 16 donors. Cells were processed for SCE analysis and sera were tested for HTLV-I antibodies by Western blot analysis. DNA from fresh lymphoid cells was analyzed by PCR-SSCP for HTLV-I genes. Of 16 donors, eight were HTLV-I seropositive and eight seronegative. Five HTLV-I infected lymphocyte cultures were also analyzed for SCE. Cells were stained immunocytochemically with anti-p19 for the viral expression. By MNNG treatment, SCE was significantly enhanced in cells derived from two seropositive donors with HTLV-I genes. In cells of six seropositive and eight seronegative donors with or without HTLV-I related DNA, SCE frequency was influenced insignificantly. Cells from all persons were negative for the p19 expression. SCE was greatly enhanced by MNNG treatment in cells from three productively infected cultures when compared with SCE in two nonproductively infected cultures.

Adult↗
PubMed1998

Differential expression of trypsin in human ovarian carcinomas and low-malignant-potential tumors.

It is widely recognized that matrix metalloproteinases and serine proteinases play an important role in cancer invasion and metastasis. We have reported that trypsin is synthesized in ovarian carcinomas as well as in some other types of cancers. In general, ovarian cancers easily tend to invade, metastasize, and spread widely into the peritoneal cavity. However, low-malignant-potential (LMP, borderline tumor) ovarian tumors are known to have limited malignant potential for progression, although microinvasion and distant metastasis have been reported among them. To analyze the relationship between varied degrees of trypsin expression and malignant behavior of ovarian tumors, immunohistochemical studies with monoclonal antibodies to human trypsin and clinicopathologic analysis were performed in human ovarian carcinomas, low-malignant-potential tumors, and benign cystadenomas. Thirteen (44.8%) cases of 29 ovarian carcinomas showed prominent trypsin expression, while only 2 (18.2%) cases of 11 LMP ovarian tumors demonstrated low levels of expression. Benign tumors and normal ovaries did not show any positivity for trypsin. These data suggest that tumor-derived heterotropic trypsin may be associated with ovarian tumors in parallel with malignant potential or behavior such as invasiveness or metastasis. At least in some ovarian carcinomas, prominent stromal invasion or metastasis might require the acquisition of or association with tumor-derived trypsin production.

Female↗
PubMed1998

Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse.

It has long been believed that trypsin is normally synthesized only in the pancreas. In the present study, expression of trypsin in human and mouse nonpancreatic tissues was examined. Northern blot analysis of normal human tissues indicated that the trypsin gene is expressed at high levels in the pancreas and spleen and considerably in the small intestine. However, in situ hybridization and immunohistochemistry demonstrated that trypsin is widely expressed in epithelial cells of the skin, esophagus, stomach, small intestine, lung, kidney, liver, and extrahepatic bile duct, as well as splenic and neuronal cells. In the spleen, trypsin message was detected in macrophages, monocytes, and lymphocytes in the white pulp. In the brain, it was detected in the nerve cells of the hippocampus and cerebral cortex. Analysis by gelatin zymography confirmed the presence of a latent or an active form of trypsin in various normal mouse tissues. Reverse transcription-polymerase chain reaction analysis also confirmed the expression of trypsin genes in the spleen, liver, kidney, and brain of normal mice. Such a broad distribution of trypsin suggests its general roles in the maintenance of normal epithelial cell functions, the immune defense system, and the central nervous system.

Animals↗
PubMed1998

Clonidine reduces dopamine and increases GABA in the nucleus accumbens: an in vivo microdialysis study.

The effects of clonidine, an alpha2 adrenoceptor agonist, on extracellular concentrations of dopamine and gamma-aminobutyric acid (GABA) in the nucleus accumbens of rats were studied by using in vivo brain microdialysis. Clonidine (5 microg/kg i.v.) significantly decreased the brain microdialysate concentration of dopamine in the nucleus accumbens up to a maximum of 16% at its peak effect. This effect was inhibited by a dose of idazoxan (10 microg/kg i.v.), an alpha2-adrenoceptor antagonist. which itself did not affect the efflux of dopamine. A smaller dose of clonidine (1 microg/kg i.v.), which had no significant effect on dopamine efflux per se, decreased the dopamine efflux (21% reduction) when given together with an ineffective dose of midazolam (0.075 mg/kg i.v.), a benzodiazepine receptor agonist. The effect of clonidine (5 microg/kg i.v.) on mesolimbic dopamine efflux was abolished by bicuculline (1 mg/kg i.v.), a GABA(A) receptor antagonist, counteracted by beta-carboline-3-carboxylate ethyl ester (beta-CCE, 3 mg/kg i.p.), a benzodiazepine receptor inverse agonist, but not affected by flumazenil (6 microg/kg i.v.), a benzodiazepine receptor antagonist. Clonidine (5 microg/kg i.v.) increased the dialysate concentration of GABA in the nucleus accumbens up to a maximum of 250% at its peak effect, but not in the ventral tegmental area. It is hypothesized that GABA(A) binding sites in the nucleus accumbens form part of the sequence of events that is triggered by clonidine in an alpha2-adrenergic-specific manner and that ultimately results in a decreased release of dopamine in the nucleus accumbens.

Adrenergic alpha-Agonists↗
PubMed1998

Production of trypsins by human gastric cancer cells correlates with their malignant phenotype.

Proteolytic degradation of extracellular matrix is a critical step in tumour invasion and metastasis. To examine the role of trypsin in tumour dissemination, we cloned two variants (S4 and R3 cells) from STKM-1, a trypsinogen 1-producing diffuse gastric cancer cell line. Western blot analysis with antitrypsin antibody showed that 26 and 24 kDa proteins were highly detected in S4 conditioned medium (CM) in comparison to R3 CM. In addition to the 26 and 24 kDa proteins, 25 and 23 kDa bands, which correspond to enterokinase-activated trypsin, were found only in S4 CM. When the CMs of the two clones were treated with enterokinase, the 25 and 23 kDa trypsin activities in S4 CM were effectively increased as compared with R3 CM. When the two clones were inoculated intraperitoneally (i.p.) into nude mice, S4 cells strongly invaded the liver, pancreas and peritoneum and killed the hosts more rapidly than R3 cells: the 50% survival time was 50 days for S4 and 82 days for R3 cells. These results suggest that trypsin production is associated with the invasive growth of STKM-1 gastric cancer cells.

Animals↗
PubMed1998

Stimulation of cellular growth and adhesion to fibronectin and vitronectin in culture and tumorigenicity in nude mice by overexpression of trypsinogen in human gastric cancer cells.

It has previously been reported that the trypsinogen gene is expressed in various human cancers. To investigate the possible role of trypsin in tumor malignancy, trypsinogen-1 cDNA was introduced into the human gastric carcinoma cell line MKN-1. The overexpression of trypsinogen-1 in MKN-1 cells stimulated cellular growth and adhesion to fibronectin and vitronectin when the trypsinogen activator enterokinase was added into the culture. Enterokinase treatment of the conditioned medium of the MKN-1 transfectants partially converted the proforms of gelatinases B and A to their apparent active forms. When the MKN-1 transfectants expressing trypsinogen-1 were intraperitoneally transplanted into nude mice, the mice frequently produced tumors in the colon, spleen and liver. However, the mice implanted with control MKN-1 cells produced no tumors. These results strongly suggest that tumor-derived trypsin contributes to the disseminated growth of some types of cancer cells including gastric cancer.

Adenocarcinoma↗
PubMed1998

Wide distribution of laminin-5 gamma 2 chain in basement membranes of various human tissues.

Laminin 5 (LN5), a heterotrimer of laminin alpha 3. beta 3. and gamma 2 chains. is a laminin isoform which strongly promotes adhesion. migration. and scattering of cells through binding to integrins alpha 3 beta 1, alpha 6 beta 1 and alpha 6 beta 4. To get an insight into the physiological functions of LN5, we prepared a mouse monoclonal antibody to human laminin gamma 2 chain and used it for immunohistochemical analysis of laminin gamma 2 chain in normal human tissues. The basement membranes of various epithelial tissues, such as the skin, lung, small intestine, stomach, kidney and prostate, were immunostained with the anti-laminin gamma 2 chain monoclonal antibody. In addition, the basement membrane of the surface germinal epithelium in the ovary was also positive for laminin gamma 2 chain. These results suggest general roles of LN5 in the anchorage of various types of epithelial cells to the underlying basement membrane and in the expression of their cellular functions. Moreover, deposition of laminin gamma 2 chain around small arteries and veins was observed in the thymus and spleen. This lymphatic organ-specific expression of vascular LN5 might provide a novel function of LN5 in immune responses.

Adult↗
PubMed1998

Effects of propofol and fentanyl on extracellular levels of gamma-aminobutyric acid in the rat nucleus accumbens: an in vivo microdialysis study.

The effects of propofol, a hydrophobic intravenous anaesthetic, and fentanyl, an opiate analgesic, on extracellular concentrations of gamma-aminobutyric acid (GABA) in the nucleus accumbens of rats were studied using in vivo brain microdialysis. The concentrations of GABA in the microdialysate of the nucleus accumbens reached a stable baseline value approximately 24 h after probe insertion and were not affected over the ensuing 250 min by intravenous injection of vehicle (1.0 ml/kg) or perfusion of tetrodotoxin (2 microM) into the nucleus accumbens via the dialysis membrane. Propofol (2.5 mg/kg and 10.0 mg/kg i.v.) did not significantly affect the accumbens microdialysate concentration of GABA over the 250 min of quantification. Fentanyl (100 microM) infused for 25 min into the nucleus accumbens via the dialysis membrane produced a marked and transient increase in accumbens GABA concentration to a maximum of approximately 1200% at its peak effect that occurred at 25 min after cessation of the fentanyl infusion.

Analgesics, Opioid↗
PubMed1998

Inhibitory effect of matrilysin antisense oligonucleotides on human colon cancer cell invasion in vitro.

In colorectal cancer, matrilysin (matrix metalloproteinase-7) is mainly produced by the tumor cells themselves and is thought to play an important role in tumor invasion and metastasis. In the study reported here, we examined the effects of matrilysin antisense phosphorothioate oligonucleotides on both the expression of matrilysin and the invasive potential of the human colon cancer cell line CaR-1 in vitro. To select the most specific and potent oligonucleotide sequence, we performed extensive analyses of the binding specificities of all antisense candidates in the GenBank database by using a computer program we developed. As a result, a 15-mer matrilysin-specific antisense oligonucleotide that hybridizes to the coding region of matrilysin mRNA (AS-1) and a random control oligonucleotide (CL-1) were designed. Reverse transcription-polymerase chain reaction and western blot analysis demonstrated that 10 microM AS-1 suppressed matrilysin expression at both the mRNA level (92%) and protein level (64%). In vitro invasion assays demonstrated that this same concentration of AS-1 inhibited the ability of cells to invade a reconstituted basement membrane by 50% as compared with the ability of untreated cells to do so. On the other hand, CL-1, which had the same length and GC content as AS-1, did not show any inhibitory effect. These results demonstrate that the antisense oligonucleotide AS-1 inhibits matrilysin activities in a sequence-specific manner and suggest that AS-1 has the potential to be used as an anti-metastatic agent in an in vivo experimental model of colon cancer.

Antineoplastic Agents↗
PubMed1997

Expression of matrilysin in vascular endothelial cells adjacent to matrilysin-producing tumors.

Matrilysin is believed to play important roles in tumor progression and metastasis. In the present study, we analyzed matrilysin-producing cells in various human cancer tissues by immunohistochemistry and in situ hybridization. Tumor cells in colorectal carcinomas, pancreatic carcinomas, transitional-cell carcinomas of the kidney and small-cell lung carcinomas were frequently positive for matrilysin. In addition, we found that endothelial cells of arterioles and venules adjacent to matrilysin-positive tumors expressed matrilysin mRNA and protein. The endothelial cells adjacent to matrilysin-negative tumors and those in normal tissues were negative for matrilysin. Furthermore, analyses by casein zymography, Western blotting and RT-PCR showed that matrilysin was weakly expressed by cultured human umbilical vein endothelial cells. Our results suggest that the expression of matrilysin in vascular endothelial cells and in tumor cells may be regulated by common soluble factors, and that endothelial cell-derived matrilysin may contribute to tumor angiogenesis and tumor metastasis.

Animals↗
PubMed1997

Expression of trypsin in vascular endothelial cells.

Proteinases produced by vascular endothelial cells are expected to play important roles in many biological processes. Here we report that human vascular endothelial cells express trypsinogen-2 mRNA and its protein product in culture. The trypsinogen production was stimulated by a tumor promoter and associated with cell growth. In situ hybridization analysis showed that the trypsinogen gene was significantly expressed in vascular endothelial cells around gastric tumors and in patients with disseminated intravascular coagulation (DIC). These results suggest the possible roles of endothelial cell-derived trypsin in tumor angiogenesis and abnormal blood coagulation.

Cells, Cultured↗
PubMed1997

Assignment of human membrane-type matrix metalloproteinase-2 (MT2-MMP) gene to 16q12 by FISH and PCR-based human/rodent cell hybrid mapping panel analysis.

A new group of matrix metalloproteinase with a potential membrane domain was reported as membrane-type matrix metalloproteinases (MT-MMPs), and the gene coding for one of them, MT2-MMP was recently cloned from a human lung cDNA library. To predict its physiological functions by the relation to the genetic disorders mapped on the chromosomes, the chromosomal location of the human MT2-MMP gene was examined by fluorescence in situ hybridization (FISH) and PCR-based analysis with human/rodent hybrid cell mapping panels, and it was assigned to 16q12.

Animals↗
PubMed1997

Expression of gelatinase A, tissue inhibitor of metalloproteinases-2, matrilysin, and trypsin(ogen) in lung neoplasms: an immunohistochemical study.

Lung cancer is a heterogeneous tumor in terms of clinical and biological behavior, and its aggressiveness depends on its invasive and metastatic properties. Matrix metalloproteinases and serine proteinases are believed to play a crucial role in invasion and metastasis of malignant tumor cells. In the present study, the authors evaluated immunohistochemically the expression of gelatinase A; tissue inhibitor of metalloproteinases-2 (TIMP-2), an inhibitor of gelatinase A; matrilysin; and trypsin(ogen) in 67 lung tumors from a variety of histological types including 17 squamous cell carcinomas, 16 adenocarcinomas, 15 small cell carcinomas, and 12 carcinoids. Interestingly, normal bronchial, bronchiolar, and alveolar epithelial cells expressed gelatinase A, TIMP-2, matrilysin, and trypsin(ogen) at varying frequencies and intensities. Bronchial smooth muscle cells and cartilage cells expressed gelatinase A alone, whereas endothelial cells, fibroblasts, and macrophages expressed gelatinase A and TIMP-2. Gelatinase A was expressed at high levels in most lung tumors examined (47% to 80%). TIMP-2 was also expressed at high levels except in the small cell carcinomas, which showed TIMP-2 expression at a lower frequency (60%) compared with other types of lung tumors (80% to 100%). Although matrilysin was expressed by tumor cells of all the histological types at various frequencies (13% to 63%), its expression was most common in adenocarcinomas. Expression of trypsin(ogen) was observed almost exclusively in adenocarcinomas (56%); other types of lung tumors expressed trypsin(ogen) far less frequently (0% to 12%). The present results, taken together with those of previous studies, suggest that gelatinase A is associated with malignant behavior of all the types of lung tumors, whereas its activity may be controlled by the endogenous inhibitor TIMP-2. The aggressive clinical behavior of small cell carcinoma may be attributable, at least in part, to a loss of the inhibitory effect of TIMP-2, as a significant proportion of these tumors showed negative or low levels of TIMP-2 expression. Matrilysin and trypsin(ogen) expressions are unlikely to be correlated with the aggressiveness of lung tumors. The expression of trypsin (ogen) may rather reflect the differentiation of adenocarcinoma cells toward normal airway epithelial cells.

Adult↗
PubMed1997

Effects of YM-14673, a thyrotropin-releasing hormone analogue, injected into the shell and the core of the nucleus accumbens on production of repetitive jaw movements in rats: comparison with the effects of a dopamine D1 and D2 receptor agonist combination.

The present study examined whether the shell and the core of the nucleus accumbens play a differential role in the display of YM-14673-induced jaw movements in rats. For that purpose the effects of YM-14673 were compared to those of a SKF 82958 and quinpirole combination, a dopamine D1 and a D2 receptor agonist respectively, that is known to functionally differentiate these two subregions of the nucleus. Consistent with the previous report, bilateral injections of a mixture of SKF 82958 (5 micrograms) and quinpirole (10 micrograms) into the shell of the nucleus accumbens produced repetitive jaw movements, whereas similar injections of the mixture into the core did not induce such an effect. In contrast, there was no regional difference in the effects of YM-14673 on the production of repetitive jaw movements. Thus, both bilateral injections of YM-14673 (0.1 or 1.0 microgram) into the shell or the core produced similar repetitive jaw movements in a dose-related manner. Moreover, the pattern of oral movements induced by YM-14673 differed from that induced by the mixture of SKF 82958 and quinpirole; frequent tongue protrusions were evident in rats treated with the mixture but were not seen in YM-14673-treated rats. It therefore appears that, unlike the effects of the mixture of dopamine D1 and D2 receptor agonists, the effects of YM-14673 in the shell on the production of rat jaw movements do not differ from the effects of the compound in the core.

Animals↗
PubMed1997

Chromosome changes associated with growth potential of HTLV-I infected human lymphocytes.

The association between chromosomal changes and cellular growth potential was investigated in HTLV-I infected human lymphocytes. Cell lines studied include Coculture-5 (HTLV-I infected immortalized cells dependent of IL-2), Coculture-15 and -18 (semi-transformed Coculture-5 cells following cocultivation with transformed Coculture-5 cells), UV-5 (Coculture-5 cells transformed following UV irradiation), and MNNG-1 (Coculture-5 cells transformed following MNNG treatment). Immortalized cells were grown in IL-2 medium (IL-2+PHA+TPA), whereas semi-transformed and transformed cells were grown in RPMI medium. By G-band karyotyping, double band formation was seen in 3-33% of spreads at the centromeric region of chromosomes 1 and 7 to which structural abnormalities were found to cluster. The double band formation was also seen by FISH using alpha satellite DNA probe in Coculture-5 and -15 thus far examined. The ploidy of immortalized, semi-transformed, and transformed cell lines, was 4n, 4n to 2n, and 2n range, respectively. These findings suggest that chromosomal rearrangements cause abnormalities in cell division kinetics resulting in numerical and structural abnormalities of chromosomes, and render host cells growth advantage.

Cell Division↗
PubMed1997

Cytokine mediated growth inhibition of HTLV-I infected transformed human lymphocytes.

The interaction between HTLV-I infected immortalized cells and transformed cells was studied. HTLV-I infected immortalized human lymphocytes (Coculture-5) dependent of IL-2 medium (IL-2+PHA+TPA) and transformed cells (UV-5) derived from Coculture-5 following UV irradiation were cocultured in equal numbers in IL-2 medium. UV-5 cells were suppressed and disappeared about 1 month in cocultivation. UV-5 cells were not suppressed in IL-2 medium. Coculture-5 cells and UV-5 cells were seeded in Transwell dishes that were separated by membrane with pores 0.4 micron in diameter and cultured in IL-2 medium. UV-5 cells were suppressed indicating that cell-cell contact was not required for the observed suppression. UV-5 cells were markedly suppressed in IL-2 medium harvested from Coculture-5 cells when recombinant interferon alpha (1000 U/ml) was added to the medium. These findings demonstrated that growth of HTLV-I infected transformed cells could be suppressed by cytokine(s) released by HTLV-I carrier lymphocytes.

Cell Division↗