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Biomedical subjects

R J Massey

Publications and source records attributed to R J Massey.

At least 19 recordsLinked to original sources
PubMed1994

Molecular cloning of a proteolytic antibody light chain.

The cDNA for an antibody light chain raised by immunization against vasoactive intestinal peptide (VIP) was cloned in a bacterial expression vector, and the recombinant light chain was purified to electrophoretic homogeneity. The light chain catalyzed the hydrolysis of VIP efficiently owing to its comparatively high affinity for the substrate. In control experiments, the catalytic activity was preserved at a constant level after further chromatography of the light chain on anion-exchange and gel-filtration fast protein liquid chromatography columns, and it was removed by immunoadsorption with immobilized anti-mouse light chain antibody. The amide bond linking methylcoumarinamide (MCA) and arginine in a tripeptide unrelated in sequence to VIP was cleaved by the light chain with lower affinity and kinetic efficiency (kcat/Km). Hydrolysis of the peptidyl-MCA conjugate was inhibited competitively by the alternate substrate, VIP. The Ki and Km values for VIP were in the same range, indicating that peptide-MCA and VIP hydrolysis occurs at a common catalytic site in the light chain. Molecular modeling suggested the presence of a serine protease-like site in the light chain. This was supported by inhibition of the hydrolytic activity by serine protease inhibitors, but not by inhibitors of other classes of proteases. These observations suggest a poorly discriminatory catalytic site, with specificity for VIP arising chiefly by means of the antigen recognition function of the light chain combining site.

Amino Acid Sequence↗
PubMed1992

Peptidolytic monoclonal antibody elicited by a neuropeptide.

We report evidence that a monoclonal antibody raised by immunization with a vasoactive intestinal peptide (VIP)-carrier protein conjugate selectively hydrolyzes VIP and a fluorescence quenched decapeptide (FQ14-22D), representing the region of VIP most susceptible to autoantibody-mediated cleavage (residues 14-22). A high affinity of the antibody for VIP and a lower affinity for FQ14-22D were revealed by kinetic studies and further substantiated by potent inhibition of FQ14-22D cleaving activity by full-length VIP. Sequencing of FQ14-22D hydrolysis products indicated selective cleavage at one peptide bond. These observations suggest that antibodies induced against naturally occurring polypeptide antigens can express peptidolytic activity targeted for specific sequences in the recognition epitope.

Amino Acid Sequence↗
PubMed1992

Rapid, non-separation electrochemiluminescent DNA hybridization assays for PCR products, using 3'-labelled oligonucleotide probes.

Described are rapid assays for the analysis of PCR products in a one step, non-separation assay based on the use of electrochemiluminescence generated from a tris-bipyridine ruthenium (II) label. The assay uses PCR incorporation of a biotinylated oligonucleotide as a primer, with the inclusion of a labelled oligonucleotide. Oligonucleotides were labelled with an N-hydroxy succinimide ester of tris-bipyridine ruthenium (II) dihexafluorophosphate (Origen-label) by modifying the 3' and 3' 5' ends of the oligonucleotide probes. The assay makes use of the inherent thermal stability and absence of polymerase activity on such probes to allow the PCR and probe hybridization to be completed automatically on the thermocycler. The assay is concluded by the addition of PCR samples to streptavidin beads on an electrochemiluminescence analyser for binding and analysis. Target genes evaluated were the HIV-1 gag gene, and cystic fibrosis delta F-508 deletion mutation. The results obtained from these assays demonstrated the detection of 10 copies of the HIV-1 gag gene, and cystic fibrosis delta F-508 mutation in 1 ng of human DNA within 15 min. This assay format allows a rapid and simple determination of specific amplified DNA sequences, reducing the contamination risks due to washes and multiple pipetting.

2,2'-Dipyridyl↗
PubMed1992

Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV-1 polymerase chain reaction products.

We describe the characterization and utility of a new electrochemiluminescent (ECL) label for oligonucleotides, utilizing phosphoramidite chemistry. This phosphoramidite of the tris(2,2-bipyridine)ruthenium(II) complex, bis(2,2-bipyridine)(4-[4-(2-cyanoethoxy-N,N-diisopropyl-amino) phosphinoxybutyl]4'-methyl)2,2-bipyridine ruthenium(II) dihexafluorophosphate or Origen phosphoramidite, enables the direct incorporation of the label during automated DNA synthesis. Efficiency of this automated synthesis allows the direct utilization of probes without further purification. Introduction of this labeling group is reproducible, and the ECL signal recovered is not influenced by hybridization. Furthermore, neither hybridization kinetics nor hybrid stability was affected by our label. We also demonstrate the utility of these labels for the development of rapid assays with oligonucleotides direct from automated synthesis. The clinical utility of these labeled oligonucleotides is shown with assays of total nucleic acid, extracted from peripheral blood lymphocytes of patients with acquired immunodeficiency syndrome (AIDS), to detect the human immunodeficiency virus (HIV-1). The results demonstrate the ability of the assay to quantify 30-2000 copies of HIV1 gag genes and to rapidly detect (less than 45 min) HIV-1 gag genes in a nonseparation assay. The application of this assay to clinical samples demonstrates the utility of these assays for rapid and quantitative analysis.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Cleavage of vasoactive intestinal peptide at multiple sites by autoantibodies.

Vasoactive intestinal peptide (VIP) fragments generated by autoantibodies purified from the blood of two human beings were separated and sequenced. Based on the identity of these fragments, seven peptide bonds cleaved by the antibodies were identified. Six of the seven scissile bonds are clustered in the region of VIP spanning residues 14-22 and were cleaved by antibodies from both human subjects. The seventh scissile bond is located at residues 7-8 and was cleaved by antibodies from one of the subjects. The scissile bonds link amino acid residues with different size, charge, and hydrophobicity. The hydrolytic activity of the antibodies was selective in that they failed to hydrolyze polypeptides unrelated in sequence to VIP (insulin and atrial natriuretic peptide). These observations demonstrate substrate specific hydrolysis by naturally occurring antibodies and expand the range of peptide bonds hydrolyzed by these antibodies.

Amino Acid Sequence↗
PubMed1990

Site specificity of a catalytic vasoactive intestinal peptide antibody. An inhibitory vasoactive intestinal peptide subsequence distant from the scissile peptide bond.

Ten fragments of vasoactive intestinal peptide (VIP) were tested for reactivity with a human catalytic autoantibody that cleaves full-length VIP(1-28) at the Gln16-Met17 peptide bond. A large COOH-terminal subsequence, VIP(15-28), was bound by the autoantibody with high affinity (Ki 1.25 nM), suggesting that it is the antibody binding epitope. VIP(22-28), a short subsequence distant from the scissile bond, inhibited the binding (Ki 242 microM) and hydrolysis (Ki 260 microM) of full-length VIP by the catalytic autoantibody in a competitive fashion. The autoantibody did not show detectable binding of short VIP subsequences that encompass the scissile bond (VIP(15-21), VIP(11-17), and VIP(13-20]. These data show that residues 22-28, located four amino acids distant from the scissile bond, contribute in recognition of VIP by the catalytic autoantibody.

Amino Acid Sequence↗
PubMed1989

Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody.

Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.

Amino Acid Sequence↗
PubMed1986

Transplantable primate tumors induced by Rous sarcoma virus. I. Induction of tumors transplantable into young marmosets.

The tumors induced in white-lipped marmosets (Saguinus fuscicollis, S. nigricollis) by Rous sarcoma virus (RSV) of chicken origin (RSV-SR) were not transplantable to allogeneic hosts. In contrast, RSV rescued from these tumors (RSV-M) induced sarcomas that were transplantable to young but not to adult marmosets. The tumors induced by RSV-M and the transplants rapidly enlarged, metastasized to various organs, and killed the recipients 29-59 days post inoculation. Cell lines were readily established from all transplantable sarcomas. No virus expression was detected in transplantable tumor cell lines by electron microscopy or by biochemical and biological assays. However, RSV of the same subgroup as RSV-SR was rescued from both short-term and long-term tumor cell cultures by cocultivation with chicken embryo fibroblasts (CEF). The rescued viruses transformed marmoset cells 100-fold more efficiently than CEF cells, although CEF cells remained permissive for virus replication. Cytogenetic studies revealed extensive chromosome abnormalities in tumor transplants but not in RSV-M-induced sarcomas. All cell lines were hyperploid and contained structurally abnormal, large metacentric and telocentric chromosomes. Immunologic studies failed to detect group-specific (gs) antigen of the avian sarcoma-leukemia complex in either RSV-M-induced, transformed cells or tumor transplants. By complement-dependent cytotoxicity assays, with the use of marmoset anti-gs serum, RSV-associated antigen could be detected on the surfaces of tumor cells. No differences in the expression of this antigen existed between transplantable and nontransplantable marmoset sarcomas. All transplantable cell lines contained abnormal amounts of lipids and glycogen in comparison to RSV-SR-induced tumors and normal marmoset cell lines. The glycogen was associated with unique cytoplasmic membrane complexes and was surrounded by either single- or double-membraned vesicles.

Animals↗
PubMed1985

A 3-dimensional tumor growth inhibition assay for testing monoclonal antibody cytotoxicity.

A 3-dimensional tumor growth inhibition assay [18] has been adapted to test the cytotoxic activity of a panel of monoclonal antibodies directed to various antigenic determinants on the surface of mouse mammary tumor cells. Target cells can be prepared from either cultured cells or from pieces of fresh tumor. Antibody and complement are added when cells are growing actively and cell growth can be measured, non destructively, over a 7-10-day period. Effective diffusion of antibody through collagen gel and binding to target cells embedded in the gel is demonstrated by indirect immunofluorescent staining. The specificity of monoclonal antibody AMT 101 cytotoxicity for mouse mammary tumor cells is the same in trypan blue exclusion assays of single-cell suspensions as in collagen gel assays, with complete killing seen in the collagen gel assay only. The collagen gel assay allows the testing of repeated treatments in vitro, as well as combined treatment with multiple antibodies. It also allows cell-cell interaction and preserves all cell components in the tumor. The collagen gel assay has potential as a method of predicting the outcome of monoclonal antibody treatment of solid tumors.

Animals↗
PubMed1985

In vivo interference between pathogenic and non-pathogenic viruses.

The interference among viruses is a well-documented biological phenomenon, both in animals and tissue culture systems. In two of our previous in vivo experiments and in four independent animal experiments, which are described in this presentation, interferences were successfully used to influence the outcomes of viral diseases by using non-pathogenic viruses. In this study, four pathogenic viruses were studied in their natural hosts, and against these viruses, in different combinations, 15 non-pathogenic viruses were tested. There was great variation in mutual effects among pathogenic and non-pathogenic viruses. In our four experiments, the viruses were either simultaneously inoculated or the non-pathogenic viruses were preinoculated. Newcastle disease vaccine (Strain H) had remarkable effects in the development of mouse ascites-associated lymphoma virus. The 50% mortality rate in mice caused by a vaccine strain of rabies virus was reduced to 15% using avian encephalitis virus. The clinical manifestations of rabbit myxoma virus effects were significantly delayed by Newcastle disease vaccine (Strain H). The 72% mortality rate due to Rous sarcoma virus in chickens was decreased to 33.3% when the animals were preinoculated with avian bursa virus vaccine.

Animals↗
PubMed1984

Interference between human hepatitis A virus and an attenuated apathogenic avian virus.

The effect of an attenuated apathogenic avian bursa virus on the course of human hepatitis A viral infection was studied in marmoset monkeys. The monkeys were infected with human hepatitis A virus, then superinfected with avian bursa virus one and three weeks after initial inoculation with human hepatitis A virus. The superinfected monkeys did not show the characteristic serum glutamic pyruvic transaminase (SGPT) elevation. Also their liver biopsies showed no pathologic changes. The virus control animals exhibited six times higher SGPT enzyme elevation than the superinfected groups, and hepatitis was detected by histopathology. This experiment, as known to us, is the first in which a definite interference was documented using a nonpathogenic virus against a highly pathogenic and clinically significant human virus. This should be considered a successful experiment demonstrating that the use of an apathogenic virus for the cure of a virus-induced disease is a realistic possibility.

Alanine Transaminase↗
PubMed1981

Viral epitopes and monoclonal antibodies: isolation of blocking antibodies that inhibit virus neutralization.

The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

Animals↗