Biomedical subjects
J Kishi
Publications and source records attributed to J Kishi.
Degradation of T-kininogen by cathepsin D and matrix metalloproteinases.
Cathepsin D, matrix metalloproteinase (MMP)-2, MMP-3 (stromelysin), and MMP-9 were isolated from rat granulomatous tissues. HT1080 human fibrosarcoma cells and rheumatoid synovial cell CM. At acidic conditions, cathepsin D cleaved T-kininogen into small peptides and released Met-T-kinin-Leu (kinin precursor), but failed to release kinin. MMP-3 cleaved T-kininogen into a 57 kDa fragment as measured by SDS-PAGE and Western blot analysis using anti-T-kininogen antiserum. On the other hand, no degradation of T-kininogen occurred during incubation with MMP-2 or MMP-9100/1) at pH 7.5 for 7 h.
Establishment of a new human urinary bladder mixed carcinoma cell line UMK-1 and its cell-density-dependent secretion of gelatinases and tissue inhibitor of metalloproteinase-1.
A new human carcinoma cell line, UMK-1, was established from primary urinary bladder carcinoma with histological diagnosis of transitional cell carcinoma grade 2, with squamous and adenocarcinomatous differentiation. These histologic features were maintained in the cultured cells and in xenografted tumors in nude mice. Ultrastructurally, the cultured cells were characterized by formation of glandular structure with well-formed junctional complexes and microvilli, intracytoplasmic lumina and tonofilament bundles. Upon continuous propagation in serum-free medium, UMK-1 cells secreted gelatinases and tissue inhibitor of metalloproteinase (TIMP)-1 into the conditioned medium. The gelatinolytic activities which were inhibited by EDTA, were composed of a major band at 95 kD and a minor band at 68 kD on gelatin zymography. The measurement of TIMP-1 in the conditioned medium was performed by the one-step sandwich enzyme immunoassay system. The production of gelatinases and TIMP-1 was observed during the late log phase of the cell growth and those productions showed similar kinetics. UMK-1 cells can be used to study the interaction of tumor cell-derived gelatinolytic proteinases and TIMP-1 in cancer invasion and metastasis.
Stimulation of TIMP-1 and metalloproteinase production in co-cultures of human tumor cells and human fibroblasts.
The co-cultures of five different human tumor cell lines with human normal fibroblasts significantly stimulated the production of tissue inhibitors of metalloproteinases-1 (TIMP-1) when compared to cultures of individual cells. In the co-culture of T24 human urinary bladder carcinoma cells and CCD18 human fibroblasts, production of both TIMP-1 and metalloproteinases was stimulated, and the stimulatory effects were dependent on the cellular ratio between the fibroblasts and carcinoma cells. On day 6 of culture, collagenase and stromelysin were stimulated at a ratio of CCD18 fibroblasts to T24 cells of 1:0.1, while the maximum TIMP-1 production occurred at a ratio of 1:1. Thus, the cellular ratio in the interaction of carcinoma cells with host fibroblasts affects the production of TIMP-1 and metalloproteinases and hence modulates the balance between them.
Gelatinases and metalloproteinase inhibitor secreted by murine colonic carcinoma cells with differing metastatic potential.
Gelatinase activity and inhibitory activity against collagenase were measured in serum-free medium conditioned by murine colonic carcinoma cells with different spontaneous metastatic potentials to the lung. The medium conditioned with poorly metastatic NM11 cells gave higher inhibitory activity than that conditioned with highly metastatic LuM1 cells, while the level of secreted gelatinases in the same medium was lower in NM11 medium than in LuM1 case. Northern analysis showed the higher gene expression of both tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in NM11 cells than in LuM1 cells, suggesting that both TIMPs are responsible for the increase of inhibitory activity in NM11 conditioned medium. Examination of the balance of gelatinases and inhibitor revealed that the amount of inhibitor exceeded that of gelatinases in the medium conditioned with NM11 cells. In contrast, the medium conditioned with LuM1 cells contained excess amounts of gelatinases. The results indicated a close correlation between the balance of gelatinases and inhibitors and the metastatic behavior of murine tumor cells.
Developmental blockage of mouse embryos caused by fatty acids.
PURPOSE: It has been shown that lipid peroxides derived from polyunsaturated fatty acids (PUFAs) inhibit the proliferation of various cells. In the meantime, it has been suggested that oxidative stress is closely related to the developmental blockage of mammalian embryos cultured in vitro. In this study, we investigated the effects by various fatty acids on mouse embryo development in vitro, and the reversal of these effects by various antioxidants such as superoxide dismutase, ascorbic acid, alpha-tocopherol, uric acid, and ethylenediaminetetraacetic acid. METHODS: Pronuclear and two-cell stage mouse (ICR) embryos were cultured in Biggers-Whitten-Whittingham medium with 0.3% bovine serum albumin alone or complexed with one of the following fatty acids: palmitic, stearic, oleic, linoleic, linolenic, or arachidonic acid. We also measured the fluorescence emission of embryos in media containing various fatty acids in order to investigate the involvement of H2O2 or lipid peroxidation in embryo development. RESULTS: Palmitic acid and PUFAs including linoleic acid inhibited the embryo development. The inhibitory effect of PUFAs was attenuated by adding antioxidants into the media, while the inhibitory effect of palmitic acid was not. Both pronuclear and two-cell stage embryos with PUFAs showed markedly more intensive emissions than those under other conditions. CONCLUSIONS: These results suggest that lipid radicals can easily be generated in early stage embryos and that blastomeres are among the cells vulnerable to the damage by lipid peroxidation.
TPA-enhanced invasion of Matrigel associated with augmentation of cell motility but not metalloproteinase activity in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1.
We previously found that the enhanced activity to invade Matrigel upon stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) was one of the major properties of a highly metastatic variant (L-10) of a human rectal adenocarcinoma cell line RCM-1. To clarify the mechanism of this enhancement, we examined the effect of TPA on 2 major biological factors involved in tumor cell invasion: cell motility and matrix-degrading metalloproteinase activity. The enhanced invasiveness was inhibited by protein-kinase-C inhibitors. TPA markedly enhanced both haptotactic response to type-IV collagen and motility on tissue-culture glass substrate of L-10 cells in a dose-response manner quite similar to that of TPA-enhanced invasion of Matrigel. On the other hand, TPA showed little enhancement of metalloproteinase production, which was assessed by gelatin- and casein-zymography, and of type-IV collagenolytic activity. Addition of TIMP (tissue inhibitors of metalloproteinase)-I inhibited TPA-enhanced invasion of Matrigel by only up to 13%. Thus, TPA treatment of L-10 cells enhanced invasion of Matrigel in association with augmentation of cell motility but did not enhance metalloproteinase activity.
The fate of embryos transferred into the uterus.
PURPOSE: The purpose of this study is to elucidate the fate of embryos transferred into the uterus. METHODS: Implantation rates were compared between synchronous and asynchronous intrauterine embryo transfers in mice. Then transferred embryos were recovered from the uterus and examined morphologically 24 hr after transfer. Moreover, the same transfer-recovery experiments were performed after ligation of the uterus two-thirds from the uterotubal junction immediately before transfer. RESULTS: The implantation rate was high (58.4% per embryo, 87.5% per recipient) when blastocysts were transferred into the uteri of recipients of pseudopregnant Day 4, but it was very low (0-2.4% per embryo, 0-8.3% per recipient) when blastocysts were transferred to recipients of pseudopregnant Day 2, or two-cell embryos into recipients of pseudopregnant Days 2 and 4. When transferred embryos were recovered from the uterus 24 hr after transfer, few embryos (1.4%) were recovered in asynchronous transfers, while 66.3% of the embryos were recovered in synchronous transfers. However, from recipients in which the lower portions of uteri were ligated, embryos at more advanced stages were recovered even in asynchronous transfers. CONCLUSIONS: These results suggest that discharge from the uterus is one of the major causes of the low implantation rate for asynchronous transfer.
Cloning and sequencing of the cDNA encoding a mouse tissue inhibitor of metalloproteinase-2.
A mouse cDNA encoding a tissue inhibitor of metalloproteinases (mTIMP-2) was cloned and the 1695-bp sequence was determined. While high homology was observed with the sequences encoding the human (hTIMP-2) and bovine (bTIMP-2) genes, mTIMP-2 contained a long 3'-nontranslated region not observed in the hTIMP-2 or bTIMP-2 genes.
Salivary gland morphogenesis: possible involvement of collagenase.
When 12-day rudiments of mouse submandibular glands were cultured, they formed an average two clefts within 24 h. The average number of the clefts, however, increased up to five in the presence of bovine tissue inhibitor of metalloproteinases (TIMP). Contrary to this, either bacterial or bovine interstitial collagenase in the medium completely inhibited the cleft formation of the glands. Electron microscopic observations revealed that the amounts of collagen bundles at the cleft points significantly increased with TIMP, but decreased with bacterial collagenase. These results strongly support the idea that endogenous collagenase regulates the cleft formation through the modulation of fibrillar architectures containing collagen in the extracellular matrix. Recently, our immunohistochemical studies clarified that collagen III, known to be rich in embryonic tissue, accumulated preferentially at the cleft points of the epithelium, and may play a crucial role in submandibular gland morphogenesis.
Involvement of superoxide radicals in the mouse two-cell block.
The effect of oxygen toxicity on the development of mammalian embryos was assessed by the use of superoxide dismutase (SOD), a potent scavenger of superoxide radicals. Mouse pronuclear embryos recovered 17 h after human chorionic gonadotropin (hCG) were cultured in medium BWW at 37 degrees C under an atmosphere of 5% CO2 in air. Culture of mouse pronuclear embryos in the presence of Cu.Zn-SOD (500 micrograms/ml) significantly increased the blastulation rate (44.6%) when compared with the control culture system (4.2%). Essentially the same effects were observed in SOD containing either Mn or Fe in the catalytic center. Heat treatment of the SOD preparation, and the addition of anti-SOD antibodies to the culture medium, significantly reduced the attenuation of the two-cell block by SOD, indicating that this effect is SOD dependent. SOD activity was detected in rabbit oviduct fluid (3.675 +/- 3.084 mIU/mg protein) by electron spin resonance. These results suggest that active oxygen is involved in the two-cell block phenomenon in mouse embryos exposed to air and that SOD in the oviduct may play an important role in the protection of embryos from superoxide radicals.
Purification and characterization of a new tissue inhibitor of metalloproteinases (TIMP-2) from mouse colon 26 tumor cells.
Mouse colon 26 tumor cells were shown to produce collagenase inhibitor in culture. The inhibitor was purified more than 2,000-fold from the culture medium by passage through DE-52 cellulose, CM-52 cellulose, Ultrogel AcA 54, Con A-Sepharose, and Sephadex G-50 Superfine columns. The inhibitor did not bind to Con A-Sepharose as do most other collagenase inhibitors. The inhibitor showed a single band (Mr = 20.5 k) on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and inhibitory activity against interstitial collagenases and gelatinases, except for bacterial collagenase. Double-immunodiffusion analysis using monospecific anti-serum against tissue inhibitor of metalloproteinases (TIMP) from bovine dental pulp showed that colon 26 inhibitor did not cross-react immunologically with the pulp inhibitor. NH2-Terminal protein sequence data were obtained for the first 36 residues of the colon 26 inhibitor, and the first 20 of them exhibited a sequence almost identical with that of a new TIMP recently designated as TIMP-2.
Block to development in cultured rat 1-cell embryos is overcome using medium HECM-1.
Rat pronuclear embryos were cultured in hamster embryo culture medium-1 (HECM-1) or a modified Krebs-Ringer bicarbonate solution (mKRB). Embryo cultures in HECM-1 were also challenged with low oxygen concentrations. In HECM-1, 57.9% (70/121) of the pronuclear embryos developed into the 4-cell stage after 48 h of culture. The rates of 8-cell, morula and blastocyst formation were 32.2% (39/121), 17.4% (21/121) and 9.9% (12/121), respectively. On the other hand, in mKRB, rat pronuclear embryos showed developmental blockages at the 2-cell and 4-cell stages, and never developed beyond the 4-cell stage. The rate of blastocyst formation under a low oxygen concentration was 20.1% (43/214), showing a significant difference from the value of 5.5% (11/201) obtained under a standard oxygen concentration (P less than 0.005). This is the first report of successful culture of rat pronuclear embryos to the blastocyst stage. Furthermore, it is suggested that protection from oxidation stress is a prerequisite for rat embryo development in vitro.
Correction: mouse colon 26 inhibitor.
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Induction of angiogenesis in chick yolk-sac membrane by polyamines and its inhibition by tissue inhibitors of metalloproteinases (TIMP and TIMP-2).
Treatment of yolk-sac membranes of 4-day-old chick embryos with spermine or spermidine resulted in angiogenesis in the membranes. The angiogenic activity of spermine was stronger than that of spermidine. Putrescine, polylysine and histamine did not induce angiogenesis in the membranes. Administration of putrescine, spermidine and spermine increased their respective levels in yolk-sac membranes, but no interconversion of these amines was observed. The increases in spermidine and spermine levels in yolk-sac membranes preceded induction of angiogenesis. The angiogenesis induced by spermine was inhibited by tissue inhibitors of metalloproteinases, that is, TIMP and TIMP-2. These findings suggest that spermine and spermidine are angiogenesis factors in yolk-sac membranes of chick embryos and that matrix metalloproteinases represented by collagenase are involved in their action.
Mutations that substitute serine for glycine alpha 1-598 and glycine alpha 1-631 in type I procollagen. The effects on thermal unfolding of the triple helix are position-specific and demonstrate that the protein unfolds through a series of cooperative blocks.
Cultured skin fibroblasts from two probands with lethal variants of osteogenesis imperfecta synthesized type I procollagen that was posttranslationally over-modified. Analysis of cDNAs and genomic DNAs from the two probands demonstrated that proband I had a single-base mutation that converted the codon for glycine alpha 1-631 to a codon for serine, and proband II had a single-base mutation that converted the codon for glycine alpha 1-598 to a codon for serine. Although the two serine-for-glycine substitutions were separated by only 35 residues, they had markedly different effects on the thermal unfolding of the collagen triple helix as assayed by brief protease digestion. The type I procollagen from proband I (serine alpha 1-631) had an essentially normal temperature for thermal unfolding. In contrast, type I procollagen from proband II (serine alpha 1-598) was cleaved to readily identifiable intermediate fragments of about 630 residues at 20 degrees C. With procollagens from both probands, collagenase A fragments containing the first 775 amino acids of the alpha chain domains had a lowered temperature for thermal unfolding as assayed by brief protease digestion. The collagenase A fragments from proband I were cleaved to intermediates of about 600 amino acids at 36 degrees C and to fragments of about 510 residues at 37 degrees C. The collagenase A fragments from proband II were cleaved to intermediates of about 630 residues at 32 degrees C, to fragments of about 600 residues at 36 degrees C, and to fragments of about 510 at 37 degrees C. The fragments of about 510 residues from both mutated procollagens were more stable to protease digestion than the collagenase A fragments of 775 residues from normal type I collagen. The results demonstrate that the effects of glycine substitutions on the thermal unfolding of type I collagen are highly position-specific. They also provide direct evidence for previous indications that the triple helix of the protein undergoes micro-unfolding of a series of relatively independent "cooperative blocks" in the predenaturation range of temperatures.
A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells.
A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.
Tissue inhibitor of metalloproteinases from human bone marrow stromal cell line KM 102 has erythroid-potentiating activity, suggesting its possibly bifunctional role in the hematopoietic microenvironment.
In this study, we demonstrated that tissue inhibitor of metalloproteinases (TIMP) produced by human bone marrow stromal cell line KM-102 had erythroid-potentiating activity (EPA) which stimulates the proliferation of erythroid progenitor cells. We, then, propose a scheme for the bifunctional role of TIMP/EPA in hematopoietic microenvironment, that is, the maintenance of the integrity of bone marrow matrix and the proliferation of erythroid progenitor cells proceeding on the matrix.