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Biomedical subjects

R Tsuboi

Publications and source records attributed to R Tsuboi.

At least 73 records · Page 4Linked to original sources
PubMed1992

Primary cutaneous plasmacytoma: treatment with intralesional tumour necrosis factor-alpha.

We report a case of primary multiple cutaneous plasmacytomas, without underlying multiple myeloma, treated with intralesional tumour necrosis factor-alpha (TNF-alpha). A marked reduction in tumour size, with no significant adverse effects, suggests that intralesional TNF-alpha is an effective treatment for non-solid malignant tumours. Extramedullary plasmacytoma, especially involving the skin as a primary site, has rarely been reported in the English literature. We describe the ninth reported case of multiple cutaneous plasmacytomas without underlying multiple myeloma. Intralesional tumour necrosis factor-alpha (TNF-alpha) was used to treat this disease and its anti-tumour effect quantitatively evaluated by comparing the tumour size and histological change before and after treatment.

Aged↗
PubMed1990

Performance of wide-pore silica- and polymer-based packing materials in polypeptide separation: effect of pore size and alkyl chain length.

The effects of pore size and alkyl chain length of silica- and polymer-based packing materials in the elution of polypeptides with an acetonitrile gradient in the presence of trifluoroacetic acid were studied. Considerable differences were found in the performance of alkylsilylated phases prepared from various wide-pore silica particles assumed to have 30-50-nm pores. The pore size of such silica gels was found to be the critical factor in determining the efficiency for high-molecular-weight polypeptides. Silica C18 phases having small pore volumes below 20 nm pore diameter showed comparable performances to C4 and C8 phases for polypeptides with molecular weights of up to 80,000, and were more stable. Polymer-based packing materials with adequate pore size provided excellent column efficiencies and recoveries for polypeptides with higher chemical stabilities than silica-based materials.

Acetonitriles↗
PubMed1990

Bimodal relationship between invasion of the amniotic membrane and plasminogen activator activity.

Three human tumor cell lines, Bowes' melanoma, HT1080 and Osmond cells, were characterized for their ability to invade the amniotic membrane and their production of plasminogen activator. Bowes' melanoma cells, which release large amounts of tissue plasminogen activator (tPA), were poorly invasive on the amniotic membrane. The addition of plasmin inhibitors, anti-tPA antibody or tissue inhibitor of metalloproteinase (TIMP) to the amnion assay enhanced invasiveness. The depletion of plasminogen from the growth medium also enhanced the degree of invasiveness. Similarly, HT1080 cells, which produce high levels of urokinase-type plasminogen activator (uPA), were poorly invasive under standard conditions but invasion was enhanced by plasmin inhibitors or anti-uPA antibodies. Conversely, Osmond cells, which produce low levels of uPA, were very invasive on the amniotic membrane. Invasion by these cells was blocked by the addition of plasmin inhibitors or anti-uPA antibodies to the amnion assay. These results suggest that invasion requires only a minimum level of PA activity and that, as PA production exceeds this optimal level, the degree of invasion decreases. We propose that high levels of plasmin, generated by the tPA or uPA secreted by the cells, may cause uncontrolled matrix degradation and interrupt the interaction of cells and matrix in the early stages of invasion. The inhibition of excessive plasmin activity may stabilize and increase cell matrix contacts and result in an enhancement of invasion.

Amnion↗
PubMed1990

Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice.

The stimulatory effect of recombinant basic fibroblast growth factor (bFGF) on wound healing was assessed using healing-impaired (db/db) mice. Full-thickness wounds were made in female diabetic C57BL/KsJ db/db mice, and their normal (db/+) littermates with a punch biopsy instrument. Recombinant bFGF was applied locally to the open wound once a day. The mice were later killed and histological sections of the wounds were prepared. The degree of wound healing was evaluated using several histological parameters such as degree of reepithelialization, granulation tissue thickness, matrix density, number of infiltrated cells, and number of capillaries. Wounds from normal mice displayed good reepithelialization rates and granulation tissue formation, while wounds from db/db mice had poor responses, especially in the dermal parameters. Although the application of bFGF to wounds in the normal (db/+) mice had little effect, application of bFGF to wounds in db/db mice induced significant responses in all of the dermal parameters compared with nontreated db/db mice (p less than 0.001). In the presence of bFGF, these parameters approximated those observed in nontreated littermates. A minimum of 0.5 microgram bFGF in either single or multiple applications was required for a significant effect. bFGF that was either boiled or pretreated with neutralizing antibody had little stimulatory effect. Time-course experiments indicated that the granulation response in bFGF-treated mice peaked between 8 and 12 d, and decreased after 12 d, while matrix density continued to increase until the 18th day (p less than 0.05). The breaking strength of healed linear wounds in db/db mice was also decreased when compared with heterozygous littermates. This parameter was also improved by the administration of bFGF to the wounds (p less than 0.05).

Animals↗
PubMed1990

Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells.

The levels of endogenous basic fibroblast growth factor (bFGF) in seven clones of cultured bovine capillary endothelial (BCE) cells were assayed, and their relation to cell morphology, bFGF receptor number, cell migration, amniotic membrane invasivity, and proteinase levels were studied. Immunoblotting experiments with anti-bFGF IgG demonstrated that cells from these clones contained different amounts of bFGF. The cells containing high levels of bFGF had a spindle or elongated appearance at confluence and a low number of high affinity receptors for bFGF. The cells containing low levels of bFGF had a cobblestone-like appearance and a higher number of high affinity receptors. When exposed to 10 ng/ml bFGF, cells containing a low level of bFGF took on an elongated appearance with a crisscross pattern similar to that seen with the high producer bFGF cells. The endogenous bFGF levels of the BCE cell clones correlated with the extent of cell migration after wounding of a monolayer and the degree of invasion of the human amniotic membrane. Cells from the clone with the highest endogenous bFGF level migrated well, invaded the amnion membrane without the addition of exogenous bFGF, and were relatively unaffected by the addition of bFGF. Cells from the clone containing the lowest level of bFGF did not migrate or invade under normal conditions. However, the addition of bFGF to the culture medium strongly enhanced both of these processes. The inclusion of anti-bFGF IgG in the media suppressed cell migration and invasion. The plasminogen activator (PA) activities of cell lysates of the clones, assayed by the 125I-fibrin plate technique, indicated that the PA levels did not correlate with the bFGF levels. Metalloproteinase activities in the conditioned medium, assayed by gelatin zymography, correlated with the endogenous bFGF levels, suggesting that the degree of expression of metalloproteinases might be critical for cell migration and invasion. These data suggest that endogenous bFGF may have an important role for migration and invasion of BCE cells during neovascularization via the induction and/or activation of specific metalloproteinases.

Animals↗
PubMed1990

Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system.

The conversion of latent transforming growth factor beta (LTGF-beta) to the active species, transforming growth factor beta (TGF-beta), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-beta in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-beta as determined by this assay. The concentration of TGF-beta in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-beta 1 and TGF-beta 2, while SMCs produced primarily TGF-beta 1. No change in the expression of these two forms of TGF-beta was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-beta indicated that most of the active TGF-beta was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-beta in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-beta in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI-1 subsequent to TGF-beta formation blocked the activation of the protease required for conversion of LTGF-beta to TGF-beta as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-beta. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-beta appears to be a self-regulating system.

Animals↗
PubMed1990

Purification and characterization of an extracellular proteinase from Hendersonula toruloidea.

An extracellular proteinase from a fast-growing strain of Hendersonula toruloidea was demonstrated when it was cultivated in liquid medium containing bovine serum albumin as the sole nitrogen source. Purification to homogeneity of the proteinase was performed by carboxymethyl cellulose, CM Sephadex G-50, and Sephacryl S-200 column chromatographies. The purified enzyme was a chymotrypsinlike serine proteinase, as indicated by the strong inhibitory activities of diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, L-1-tosylamido-2-phenylethylchloromethyl ketone, and chymostatin and good kinetic constants for a synthetic substrate, Suc-Ala-Ala-Pro-Phe-MCA. The enzyme had a pI of 8.4, a pH optimum of 9.0, and a molecular weight of 34,000. Skin constituents such as stratum corneum and nail, but not hair, were easily digested by this enzyme. Thus, this extracellular proteinase may play a role in the invasion of thickly keratinized skin and nail by this organism.

Amino Acid Sequence↗
PubMed1990

[Relation between movement of acid or alkaline proteinase from Trichophyton rubrum and the growth of fungus, pH in medium].

We have previously reported that T. rubrum produced a new type of extracellular proteinase in liquid medium containing bovine serum albumin. This enzyme had an optimal pH 4.5 for azocoll. In this paper, we studied about the relation between movement of acid or alkaline proteinase and environmental pH. In the beginning of cultivation, acid proteolytic activity increased radically, and 3 or 4 weeks later, it was decreased. In place of acid proteinase, alkaline proteinase which had an optimal pH 8.0 was increased gradually. During incubation of T. rubrum in bovine serum albumin medium which was adjusted to pH 6.0, the pH of medium was gradually increased to about pH 7.0 toward 4 weeks. The growth of T. rubrum increased until pH in medium was fixed to pH 7.0.

Aspartic Acid Endopeptidases↗
PubMed1989

Correlation between culture medium pH, extracellular proteinase activity, and cell growth of Candida albicans in insoluble stratum corneum-supplemented media.

Candida albicans produces a major extracellular proteinase whose activities are observed only in weakly acidic pH. However, in affected lesions, a variety of pH conditions exist, including neutral pH. To verify the pathological importance of the extracellular proteinase, the correlation between culture medium pH, extracellular proteinase activity, and cell growth of C. albicans was followed for 3 weeks with unbuffered and insoluble stratum corneum-supplemented liquid media. Each medium pH, initially adjusted within a range of pH 3-7 by the addition of sodium hydroxide or hydrochloric acid solution, was acidified, and a subsequent high proteolytic activity and rapid fungal growth were observed. After full fungal growth, neutralization of each medium to pH 7 and reduction of proteinase activity occurred. Results from a glucose addition experiment suggest that acidification of each medium was produced by the acid formation from glucose and neutralization by the exhaustion of glucose and increase of ammonia from denatured stratum corneum. These data suggest that extracellular proteinase from C. albicans could act as a virulence factor under a wide range of pH conditions by the acidification of the environmental pH close to the organism.

Ammonia↗
PubMed1989

Differences in induction of lysosomal protease activity by protease inhibitors in B16 melanoma cell lines.

1. The effects of potent protease inhibitors in vitro (leupeptin, pepstatin and E-64[N-[L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine]) on intracellular cathepsin B (EC 3.4.22.1), hemoglobin (Hb)-hydrolase and acid phosphatase (EC 3.1.3.2) from cultured B16 melanoma variants (B16-F1, F10 and BL6) were studied. 2. E-64 induced all the cultured B16 melanoma variants to decrease the activity of intracellular cathepsin B but did not have this effect with Hb-hydrolase or acid phosphatase. Furthermore, E-64 decreased the activity of cathepsin B in both the lysosomal and cytosol fractions. 3. Leupeptin induced all the cultured B16 melanoma variants to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. An increase in the level of cathepsin B activity was most significant in B16-BL6 followed by F10 and then F1 variants. 4. Leupeptin induced all the cultured B16 melanoma variants to increase the cathepsin B activity in the lysosomal fraction. Our data differed from the results of Tanaka et al. (1981) in that leupeptin induced rat cultured hepatocytes to inhibit the activity of intracellular cathepsin B and increase the Hb-hydrolase activity, especially in the cytosol fraction.

Acid Phosphatase↗
PubMed1989

In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases.

The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis.

Amnion↗
PubMed1989

Isolation of a keratinolytic proteinase from Trichophyton mentagrophytes with enzymatic activity at acidic pH.

A keratinolytic proteinase with enzyme activity at acidic pH was isolated from culture filtrates of Trichophyton mentagrophytes, a major pathogenic fungus of dermatophytosis. The molecular weight of the proteinase was estimated to be 41,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 38,000 by gel filtration. The isoelectric point was determined to be 3.9. The proteinase had a pH optimum of 4.5 for keratin and 5.5 for hemoglobin. This enzyme hydrolyzed the synthetic chymotrypsin substrate Suc-Ala-Ala-Pro-Phe-MCA (Km, 0.59 mM), and its activity was strongly inhibited by chymostatin. Previously reported proteinases from dermatophytes have had enzyme activities in neutral or alkaline pH; however, healthy skin has a weakly acidic pH. Thus, the purified proteinase which has an optimal activity at acidic pH and hydrolyzes skin constituents could be an important virulence factor in dermatophytosis.

Cations, Divalent↗