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V Miggiano

Publications and source records attributed to V Miggiano.

At least 19 recordsLinked to original sources
PubMed1991

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver.

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.

Amino Acid Sequence↗
PubMed1991

Human catechol-O-methyltransferase: cloning and expression of the membrane-associated form.

A cDNA clone for human catechol-O-methyltransferase (hCOMT; S-adenosyl-L-methionine:catechol O-methyltransferase; EC 2.1.1.6) was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (COMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The endogenous COMT activity, which was approximately 9.98 units per mg of protein in the untransfected cells, increased to 206 units per mg of protein upon transfection with a plasmid containing the COMT cDNA. The COMT activity of recombinant protein was inhibited competitively (IC50 = 700 nM) by the selective COMT inhibitor Ro 40-7592. An anti-COMT monoclonal antibody recognized, on immunoblots, a major polypeptide with apparent molecular mass of 29 kDa, in reasonable agreement with the predicted molecular mass. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A)+ RNA.

Amino Acid Sequence↗
PubMed1990

Monoclonal antibodies recognizing both soluble and membrane bound catechol-O-methyltransferase.

Both cytosolic, soluble and membrane-bound catechol-O-methyl-transferase (COMT) from pig and rat liver or kidney were recognized by mouse monoclonal antibodies (MAbs) raised against soluble COMT isolated from pig liver. In ELISA, the MAbs Co 16 and Co 54 reacted better with the pig than with the rat enzyme. The MAb Co 60 showed good reactivity with both pig and rat COMT. In addition, all three MAbs recognize the soluble (23 kDa) as well as the membrane-bound (26 kDa) forms of the COMT enzyme.

Animals↗
PubMed1985

Monoclonal antibodies against antigens on breast cancer cells.

Of 360 mAb obtained in a cell fusion experiment with the spleen cells of a mouse immunized with a mixture of different human breast carcinoma cell lines, 30 mAb were selected which reacted more strongly with tumor cells than with (noncancerous) fibroblasts. These mAb were tested for reactivity with additional types of cancerous and noncancerous tissues. Two mAb showed high tumor selectivity, but the corresponding epitopes on individual tumor cells were heterogeneously expressed. The mAb will be evaluated for in vivo applications.

Animals↗
PubMed1985

Immunohistochemical characterization of an anti-epithelial monoclonal antibody (mAB lu-5).

A mouse monoclonal antibody (mAB lu-5) was prepared using a lung cancer cell line as an antigen. The selected clone produces an IgG with a gamma-1 heavy chain and a kappa-light-chain. Immunohistochemical testing of mAB lu-5 on 117 normal tissue biopsies and 474 tumours revealed reactivity with an intracytoplasmic, formaldehyderesistant antigen present in most epithelial and mesothelial cells, but absent in mesenchymal cells. The antibody can therefore be used as a first order, pan-epithelial marker. It proved also useful for fast tumour diagnosis on frozen sections.

Animals↗
PubMed1984

A rapid and highly sensitive solid-phase enzyme immunoassay specific for human fibronectin using a characterized monoclonal antibody.

A solid-phase enzyme immunoassay (EIA) was developed for the quantitation of human fibronectin in body fluids and cell culture media. In the assay a human fibronectin-specific murine monoclonal IgG1 (f-33) was used as capture antibody and polyclonal rabbit anti-fibronectin as detector antibody. The antibody showed no reactivity to purified monkey, dog, rabbit, horse, sheep, mouse, bovine or chicken fibronectins. The determinant of the monoclonal antibody was mapped to the cell-binding region of the fibronectin molecule. This localization was based on the use of purified fragments of fibronectin, immunoblotting, EIA and inhibition of fibroblast adhesion and spreading by the antibody. The detection limit of the fibronectin assay was 2 ng/ml. The assay was used for the quantitation of fibronectin in human plasma, urine and cerebrospinal fluid specimens and culture media of human cells.

Animals↗
PubMed1983

Monoclonal antibody against the N-terminal end of human plasma fibronectin.

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.

Antibodies, Monoclonal↗
PubMed1983

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them.

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.

Antibodies, Monoclonal↗
PubMed1983

Human fibroblasts X mouse cell hybrids, containing a human 11/X translocation, do not express human fibronectin.

Using a monoclonal antibody specific for human fibronectin (FN), we screened hybrid clones derived from the fusion of FN+ human fibroblasts, carrying a 11/X translocation, and FN-, HPRT- mouse cells for the production of this glycoprotein. Since no hybrid clone retaining the human der 11 chromosome was found to produce any human fibronectin, the segment of chromosome 11 included in the rearranged chromosome (11qter leads to 11p13) probably does not carry the structural locus for fibronectin.

Animals↗
PubMed1981

Production of hybridomas secreting monoclonal antibodies to the human leukocyte interferons.

Thirteen monoclonal antibodies to human leukocyte interferon have been obtained. They exhibit different patterns of binding to purified leukocyte interferon species that are consistent with the structural multiplicity of the human leukocyte interferons. These antibodies will be useful as probes into the structure of the human leukocyte interferons, for their purification, and for rapid assay of leukocyte interferon.

Animals↗
PubMed1980

High frequencies of antigen-specific hybridomas: dependence on immunization parameters and prediction by spleen cell analysis.

Hybridomas producing antibodies against soluble antigens have in most cases been difficult to establish. After fusion of myeloma cells with spleen cells obtained from mice immunized with a soluble protein, hybridomas secreting specific antibodies have been observed to occur very rarely among non-specific hybridomas. We found that the frequency of specific hybridomas correlates directly with the increase over background of the frequency of blast and/or plasma cells in the spleen (measured by cell size analysis) after antigenic stimulation. High yields of specific hybridomas were obtained simply by following a novel immunization technique consisting of several conventional preimmunization courses followed by 4 very high doses of antigen in saline on each of the last 4 days before fusion.

Animals↗
PubMed1975

I-region-associated determinants: expression on mitogen-stimulated lymphocytes and detection by cytotoxic T cells.

We have studied the expression of Ia-antigens, controlled by genes in the I-region of the H-2 complex, on phytohemagglutinin (PHA)-stimulated lymph node cells and on lipopolysaccharide (LPS)-stimulated spleen cells, and have compared these two types of cell populations as targets for killer cells in the cell mediated lympholysis (CML) assay. PHA targets are almost completely insensitive to complement-mediated lysis by anti-Ia sera while the majority of LPS targets are killed. T cell-mediated lysis against I-region determinants was also detected, and these determinants, in contrast to H-2K and H-2D CML determinants, seem to be much more strongly expressed on LPS-stimulated cells. No differences in the kinetics of the response to K- or I-region CML determinants were observed. Lysis by killer cells can also be obtained against incompatibilities which do not give rise to strong skin graft rejection and against determinants which are most probably controlled by genes outside the K-D interval.

Animals↗