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C Hanski

Publications and source records attributed to C Hanski.

71 records · Page 4Linked to original sources
PubMed1987

Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane.

Dipeptidyl peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns. The specific activity of the final preparation was 49.4 mumol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-polyacrylamide gel electrophoresis of the arginine-Sepharose eluate showed a single protein band with a molecular weight of 105,000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.

Animals↗
PubMed1987

Increased expression of a high molecular weight matrix component in rat hepatocellular carcinoma.

The urea extract of the glycoproteins from the extracellular matrix of rat liver has been compared with that of Morris hepatoma 7777. A high molecular weight glycoprotein present in Morris hepatoma 7777 was not found in the extract of liver matrix. Under reducing conditions in SDS-gel electrophoresis this component gave two glycoprotein bands with Mr 53 k and 56 k. The indirect immunofluorescence staining with a monospecific antiserum directed against the component showed its abundant presence in Morris hepatoma 7777 as well as in the less malignant Morris hepatoma 9121 in form of extracellular network structures. The antigen also densely filled some cumuli of cells. In contrast the liver tissue showed only very weak staining of the extracellular areas. The overall distribution of the component could be correlated with the distribution of several hydrolases in the tumor matrix, notably beta-D-glucuronidase.

Animals↗
PubMed1987

Proteases in the human full-term placenta.

Aminopeptidase A, not yet defined aminopeptidases and endopeptidases, dipeptidyl peptidase I, II and IV, gamma-glutamyl transferase and oxytocinase were investigated in the normal human full-term placenta using qualitative (catalytic) cytochemistry, isoelectric focusing, immunocytochemistry and kinetic fluorometry. Aminopeptidase A could be visualized cytochemically in the smooth muscle cells of the chorionic plate, stem villi and basal plate blood vessels. Aminopeptidases were found in connective tissue fibres of the chorionic plate, villous stroma, basal plate and paraplacenta. Dipeptidyl peptidase IV was detected at the same sites as the aminopeptidases and, in addition, in amniotic epithelial cells, fibroblasts of the villous stroma, endothelium of chorionic plate and villous blood vessels as well as in the basophilic cytotrophoblast cells (x-cells) of the basal plate and paraplacenta, and it possibly also occurred in some domains of the plasma membrane of the syncytiotrophoblast and cytotrophoblast cells. The x-cells surrounded the fetus in the form of a dipeptidyl peptidase IV-positive shell at the border to the mother. The enzyme represented the first specific marker for x-cells. Dipeptidyl peptidase I and II were primarily found in Hofbauer cells (macrophages) of the villous stroma, but also in the syncytiotrophoblast, other villous stromal cells and cells of the chorionic and basal plate. gamma-Glutamyl transferase was present in some connective tissue elements of the chorionic plate. Oxytocinase and endopeptidases were not detected. Isoelectric focusing of proteases revealed different molecular forms of dipeptidyl peptidase IV in the paraplacenta and villous tree, while the aminopeptidases shared the same pattern in both regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopeptidases↗
PubMed1986

Identification of a 110-kDa glycoprotein involved in cell-substratum adhesion.

Plasma membrane glycoproteins are involved in cell-matrix interactions. For identification of such glycoproteins a recently developed cell system was used. Two cell populations differing in their adhesion properties were selected from Morris hepatoma 7777. One population was able to grow as a monolayer, while the other proliferated in suspension. From both cell lines spontaneous revertants were selected. By using antibodies raised against plasma membranes of the hepatoma and of rat liver for sequential immunoprecipitation a glycoprotein of Mr 110000 was identified. This glycoprotein was only expressed in adherent hepatoma cells as well as in normal rat liver, but was absent in non-adherent hepatoma cells and in the in vivo growing Morris hepatoma 7777. This suggests that the glycoprotein is involved in cell-substratum adhesion of hepatocytes and adherent hepatoma cells.

Animals↗
PubMed1986

Increased activity of dipeptidyl peptidase IV in serum of hepatoma-bearing rats coincides with the loss of the enzyme from the hepatoma plasma membrane.

The specific activity of dipeptidyl peptidase IV (DPP IV E.C. 3.4.14.-) in the plasma membrane of Morris hepatoma 9121 or hepatoma 7777 was 3.5% and 2.9%, respectively, of that in the plasma membrane of rat liver. The enzyme activity in the serum of hepatoma-bearing rats was 141% (hepatoma 91219) and 162% (hepatoma 7777) of the normal value. Cytochemical investigation showed that the DPP IV activity was almost completely absent from the hepatoma cell plasma membrane and was not sequestered within these cells. Indirect immunofluorescence staining with a polyclonal antibody directed against DPP IV indicated that the loss of activity was due to the absence of DPP IV molecules in the plasma membrane. The possibility that the enzyme is transferred from the membrane into the serum as a result of structural alterations is discussed.

Animals↗
PubMed1986

Quantitative study of cell interaction with collagen and fibronectin.

The interaction of BHK-fibroblasts with collagen or fibronectin-collagen complex was investigated quantitatively. For that purpose an improved method for production of defined cell substrata was developed. The method permitted reproducible coupling of different ligands to glass via an amino or carboxyl group. BHK-cells grown on collagen required a minimum density of 15-20 ng collagen/cm2 for spreading. When grown on fibronectin adsorbed on collagen the cells were found to remove fibronectin from the substratum at a rate of 0.15 pg/(cell X h).

Animals↗
PubMed1985

1-Methylguanine and 7-methylguanine increase cell agglutinability.

1-Methylguanine and 7-methylguanine, both metabolic products of tRNA degradation, are known to induce transformation of Chinese hamster fibroblasts in culture. The effects of these compounds on the cell membrane have been studied by the method of Concanavalin A-mediated hemadsorption. 1-Methylguanine or 7-methylguanine induced a 50% increase of Con A-mediated hemadsorption within 20 hours of exposure of the cells to the agent at a concentration of 10(-5) M. This alteration was reversed within 13 days when the cells were grown in the control medium. Prolonged treatment with 1-methylguanine or 7-methylguanine resulted in changes which were only slowly reversed during growth of the cells in the control medium. The effect of the methylated purines on the cell membrane could be completely inhibited by simultaneous addition of dibutyryl-cAMP at a concentration of 10(-5) M. The possible mechanism of cell membrane alteration by methylated purines and its relevance to transformation in vitro are discussed.

Agglutination↗
PubMed1985

Urinary excretion of 3-methyladenine and 1-methylnicotinamide by rats, following administration of [methyl-14C]methyl methanesulphonate and comparison with administration of [14C]methionine or formate.

Following i.p. injection of [methyl-14C]methyl methanesulphonate (MMS) into rats (100 mg/kg) 3-[14C]methyladenine was identified as a urinary product excreted mainly up to 24 h after treatment, the amount over this period being about 0.02 mumol 3-methyladenine. When [14C]MMS and L-[methyl-3H]methionine were injected together no methyl-3H-label was detected in 3-methyladenine, nor was this product detected following injection of [methyl-14C]methionine alone or of [14C]formate. Isotopically labelled 1-methylnicotinamide (1-meNmd) was detected following all the treatments listed, and as previously found by Chu and Lawley, 1-meNmd excretion was enhanced by MMS treatment as judged by increased excretion of 1-[3H]meNmd when [14C]MMS and [3H]methionine were given together. The extent of labelling of 1-meNmd was much lower following injection of [14C] formate, than that from methionine or MMS. The results showed that 3-methyladenine derived only from direct chemical methylation by MMS. They also support the previous suggestion that [methyl-14C]meNmd can result from direct methylation, with a maximal amount of about 3% of excreted meNmd deriving from this route. The possible utility of the methods described for monitoring in vivo alkylation is discussed.

Adenine↗
PubMed1985

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen.

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.

Animals↗
PubMed1984

Turnover of the protein and carbohydrate moieties of a 105kD glycoprotein of plasma membranes from mouse liver determined by immunoprecipitation.

The turnover of a mouse liver plasma membrane glycoprotein (Mr 105 k) has been studied by means of immunoprecipitation and radioactive labeling. The method of immunoprecipitation proved to be a fast and efficient way of isolating a single protein for measurement of its specific radioactivity. The half-life of the carbohydrate moiety determined using 3H-galactose was 41 h and using 3H-mannose was 23 h, whereas that of the protein moiety determined using 3H-leucine was 63 h. The implications of this differential degradation of the protein and carbohydrate moieties are discussed in terms of the recycling of glycoproteins between the plasma membrane and intracellular membranes.

Animals↗
PubMed1980

Increased concentration of 7-methylguanine and 1-methylhypoxanthine in urine of rats bearing Yoshida tumour.

Sprague-Dawley rats bearing Yoshida Tumour showed an increased excretion of 7-methylguanine and 1-methylhypoxanthine with their urine as compared to control animals. The concentration of these methylated purines, which belonged to the non-specific cancer markers, was already significantly higher 2 days after tumour injection and more than 2-fold after 3 additional days. This was proved by daily L-[14CH3]methionine supply. Separation and identification of these compounds was done by liquid chromatography and mass spectrometry. By RNA analysis it became evident that tumour RNA contained more methyl groups than liver RNA of control rats. This suggested that the increased excretion of 7-methylguanine and 1-methylhypoxanthine should not be only ascribed to an increased turnover of tumour RNA but also to its higher degree of methylation.

Animals↗
PubMedDate not supplied

Novel adapter protein AP162 connects a sialyl-Le(x)-positive mucin with an apoptotic signal transduction pathway.

Glycoproteins modified with a sialyl-Le(x)-moiety are important sensors for extracellular signals regulating cellular recognition, adhesion and migration. The transduction pathways and signals mediated by these glycoproteins within the cell are largely unknown. In search of novel glycoproteins modified with sialyl-Le(x)-moiety, we screened a human colonic cDNA expression library with a rabbit antiserum produced against sialyl-Le(x)-positive mucins. The antiserum detected a new protein, named B2, which was cloned and characterised in detail. The analysis of the B2 gene revealed a 5.7 kb RNA transcript detectable in all investigated tissues and a complete coding sequence of 2778 bp. The B2 protein exhibited two putative PH (pleckstrin homology) domains and a leucine zipper motif but no homology to any known proteins. Monospecific antibodies against the B2-protein precipitated from the solubilised membrane fraction of the colon carcinoma cell line LS 174T a protein with an apparent Mr = 162 kDa and, additionally, a mucin-like glycoprotein with an apparent Mr = 220 kDa. Protein fractionation on a CsCl gradient, Western blots and sandwich ELISA showed that the 220 kDa mucin carries the sialyl-Le(x) moiety and is tightly bound to the 162 kDa protein. The expression of the recombinant B2-protein enhanced staurosporine-induced apoptosis in epithelial cancer cell lines. These data indicate that B2 is a novel, ubiquitously expressed protein with a putative adapter function. The protein has been named AP162 (adapter protein 162). In colon carcinoma cells B2-protein is tightly associated with a sialyl-Le(x)-positive mucin and has a potential for involvement in sialyl-Le(x)-mediated transduction of apoptotic signals.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

De novo expression of the Muc2 gene in pancreas carcinoma cells is triggered by promoter demethylation.

It has been established that mucin-producing variants of different subtypes of pancreatic carcinomas, including the intraductal papillary and ductal mucinous tumors, have usually a more favorable prognosis. Intraductal papillary and ductal mucinous tumors have also been shown to ectopically express the intestinal mucin gene MUC2. The mechanism of the de novo expression of this gene in tumors may have potential implications for the modulation of its behavior. We studied, therefore, the mechanism of the de novo expression of MUC2 in pancreas carcinoma cells in vitro. The MUC2 gene promoter is methylated in the nonexpressing pancreatic cell line PANC-1 and is not methylated in the expressing cell line BxPC-3. The promoter is silenced by methylation as shown by reporter expression assays. De novo expression of MUC2 in PANC-1 cells is triggered by treating the cells with a pharmacological inhibitor of DNA methylation (5-aza-2'-deoxycytidine). There was no decrease or loss of expression of the methyltransferase DNMT1 in the MUC2-producing cells. These data show that the de novo expression of the MUC2 gene in pancreas carcinoma cells is associated with promoter demethylation. They warrant further investigations on the relationship between MUC2 promoter demethylation in pancreatic cancer and the prognosis of carcinoma patients.

Azacitidine↗
PubMedDate not supplied

Further evidence for different isoenzymic forms of a cell surface protease, guanidinobenzoatase, associated with tumours.

Normal colonic epithelial cells possess a cell surface protease referred to as guanidinobenzoatase (GB) and a corresponding cytoplasmic protein inhibitor of GB. Colonic carcinoma cells possess two isoenzymic forms of GB, the normal and the carcinoma specific form, each of which is recognised by the corresponding inhibitors present in the cytoplasm of colonic carcinoma cells. An affinity-purified inhibitor preparation obtained from the cytoplasm of cultured colonic carcinoma cells inhibited these two colonic carcinoma isoenzymic forms of GB but not the GB associated with other forms of tumour. The data suggest that each cell type possessing isoenzymic forms of cell surface GB also possesses the corresponding cell-specific inhibitors of GB.

Carboxylic Ester Hydrolases↗