Biomedical subjects

L Jaso-Friedmann

Publications and source records attributed to L Jaso-Friedmann.

57 records · Page 4Linked to original sources

PubMedDate not supplied

NCCRP-1: a novel receptor protein sequenced from teleost nonspecific cytotoxic cells.

The recognition structure responsible for binding to conventional antigen on target cells has not previously been described for nonspecific cytotoxic cells (NCC) or for mammalian natural killer (NK) cells. Although several biochemical pathways may be available for initiation of the lytic cycle in NCC, evidence presented indicates that initial contact with a tumor cell or protozoan parasite is facilitated by recognition of a target antigen by a membrane protein of Mr 34,000 on NCC (NCC receptor protein, NCCRP-1). Binding to NCCRP-1 by monoclonal antibody 5C6, by target cell antigen or by cognate synthetic peptide initiates a signalling response leading to increased cytotoxicity. In the present study, three 20-mer microsequences were obtained from tryptic digests of purified NCCRP-1. Degenerate primers were synthesized (based on each peptide sequence) and were used for RT-PCR with mRNA purified from homogeneous NCC populations. An NCCRP-1 specific cDNA sequence was used to synthesize nondegenerate primers. These primers were used in a 5'/3' RACE PCR to obtain the entire NCCRP-1 specific cDNA. A deduced aa sequence consisted of 235 aa with a derived molecular weight of 30,628 Da. NCCRP-1 is proline rich (9%), has two glycosylation sites and 18% of all amino acids are potential phosphorylation sites (serine, threonine, tyrosine). The identity of the protein was confirmed by finding the previously microsequenced peptides in the derived sequence. Homology searches revealed that NCCRP-1 is a novel protein. Northern blot analysis of mRNA content from teleost NCC, B-cells and T-cells revealed only one band in NCC preparations. Functional studies demonstrated a decrease in membrane NCCRP-1 expression and inhibition of NCC cytotoxicity following treatment with NCCRP-1 anti-sense oligonucleotides. Treatment of NCC with sense oligonucleotides had no inhibitory effects on cytotoxicity. An algorithm predicting the membrane conformation of NCCRP-1 suggests one extracellular proline-rich domain, a transmembrane portion of 15 18 aa and a cytoplasmic tail composed of a high frequency of phosphorylation sites. Current studies suggest that NCC and NCCRP-1 may participate in innate resistance functions in teleost fish.

Amino Acid Sequence↗

PubMedDate not supplied

Role of function-associated molecules in target cell lysis: analysis of rat adherent lymphokine-activated killer cells.

Monoclonal antibodies (MAbs) against fish nonspecific cytotoxic cells (NCC) inhibit the lysis of target cells by NCC and by human and rat natural killer (NK) cells. We now show that the anti-function-associated molecule (anti-FAM) MAb 5C6 also inhibits the cytolytic activity of rat (F344) nonadherent lymphokine-activated killer (LAK) cells and adherent LAK (ALAK) cells. Rat nylon-wool-nonadherent (NWNA) cells were cultured for 48 h in recombinant human interleukin-2 (IL-2). Nonadherent cells were then removed and the plastic-adherent (ALAK) cells were recultured for 9 days. Cells were daily analyzed by cytometry (using MAb 5C6 and the rat NK cell MAb 3.2.3) and cytotoxicity. Approximately 40% of the initially cultured NWNA cells, 50-60% of the LAK cells and 87-98% of the ALAK cells were positive for MAb 5C6. Tissue culture supernatants containing MAb 5C6 caused a dose-dependent inhibition of cytotoxicity against P815 and YAC-1 targets. Approximately 50% inhibition was obtained at MAb concentrations of 1.25-2.5 micrograms/ml. Maximum inhibition of ALAK cytotoxicity was 86% on day 4 of culture. Two-dimensional SDS-PAGE and Western blot analysis of acetone-precipitated cell membrane preparations identified a 55-kD molecule (pI 6.1-6.3) on NWNA and ALAK cells and an additional slightly more acidic 45-kD protein on ALAK cells. These data indicate that rat ALAK cells may initiate lysis of target cells via receptor binding.

Animals↗

PubMedDate not supplied

Human interleukin-2-activated adherent natural killer cells recognize a conserved antigen found on tumor cells and protozoan parasites.

Plastic adherent interleukin-2-activated human natural killer (NK) cells (ALAK) lyse many different histological types of tumor target cells. In order to effect their function as cytotoxic mediators of innate immunity, ALAKs may 'recognize' antigen(s) of protozoan parasites, select virus-infected cells and they may release certain cytokines in response to bacterial antigens. In the present study, we demonstrate that CD3-/CD56dim/CD16dim/monoclonal antibody 5C6bright human ALAKs bind to an antigenic determinant on tumor cells independent of target cell H-2 allotype expression. The conserved antigen was originally obtained from the protozoan Tetrahymena pyriformis, however it is also located on the membranes of many ALAK-sensitive tumor cells. The sequence of this protein, i.e. NK target antigen/NKTag, was previously deduced from cDNA. One ALAK cognate determinant of NKTag was identified by inhibition of cytotoxicity using NKTag-derived synthetic peptides. Biotinylated synthetic peptide [amino acids (aa) 58-74] bound to ALAKs, and synthetic peptides corresponding to this sequence inhibited ALAK lysis of U937 target cells. Inhibition effects of peptide binding were nonreversible. To determine the requirements for recognition by ALAKs of this antigenic determinant, the cognate peptide aa 55-74 was truncated to 17-, 14-, 10-, 7- and 6-mer lengths and tested for inhibition of cytotoxicity. All inhibited except the 6-mer. A possible mechanism of peptide inhibition of cytotoxicity following ALAK binding to an antigenic determinant was a requirement for recognition of one anchor peptide (arginine) and receptor occupancy by a minimum of five to six additional amino acids. In antibody-dependent cell-mediated cytotoxicity experiments, synthetic peptide (aa 68-74) inhibited ALAK killing of anti-H-2d-sensitized P815 targets. This same peptide also inhibited conventional lysis of nonsensitized P815 and IM-9 targets. However, the cognate synthetic peptide (aa 58-74) did not inhibit conjugate formation between ALAKs and U937 target cells. These data demonstrate that ALAK binding to a soluble monomeric peptide inhibited cytotoxicity. Peptide binding appeared to negatively regulate cytotoxicity, and the inhibitory effects following peptide binding were nonreversible. Effector:target cell conjugate formation was not affected by peptide binding, however, recognition was required because inhibition was specific for the amino acid sequence of the synthetic peptide.

Adjuvants, Immunologic↗