Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies.

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium.

The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2.

A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein.

Cytoskeletal reorganization induced by engagement of the NG2 proteoglycan leads to cell spreading and migration.

Cells expressing the NG2 proteoglycan can attach, spread, and migrate on surfaces coated with NG2 mAbs, demonstrating that engagement of NG2 can trigger the cytoskeletal rearrangements necessary for changes in cell morphology and motility. Engagement of different epitopes of the proteoglycan results in distinct forms of actin reorganization. On mAb D120, the cells contain radial actin spikes characteristic of filopodial extension, whereas on mAb N143, the cells contain cortical actin bundles characteristic of lamellipodia.

PDGF (alpha)-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from the NG2 knockout mouse.

A line of null mice has been produced which fails to express the transmembrane chondroitin sulfate proteoglycan NG2. Homozygous NG2 null mice do not exhibit gross phenotypic differences from wild-type mice, suggesting that detailed analyses are required to detect subtle alterations caused by the absence of NG2. Accordingly, dissociated cultures of aortic smooth muscle cells from null mice were compared to parallel cultures from wild-type mice for their ability to proliferate and migrate in response to specific growth factors.

Expression of CD45 isoforms lacking exons 7, 8 and 10.

The CD45 exon usage pattern of various CD8+ and CD4+ T cell lines was studied. By using the reverse transcription-polymerase chain reaction (RT PCR) and Southern analysis with exon specific or exon junction probes, we showed that all of the cytotoxic T cell lines and the majority of the helper T cells expressed the 789 isoform as a major splice variant. Expression of the splice product lacking exons 4-7 (isoform 89) was not as ubiquitous.

Murine mast cells and monocytes express distinctive sets of CD45 isoforms.

CD45 isoform expression patterns were examined in various mast and monocyte cell populations. The reverse transcription-polymerase chain reaction (RT/PCR) and Southern analysis showed these myeloid cells express characteristic sets of CD45 isoforms. Mast cells produce mRNA for two splice variants, one containing exons 5, 7 and 8 of the alternatively expressed exons (therefore lacking exons 4 and 6) and another containing variable exons 7 and 8.

The role of Tyr13 and Lys15 of interleukin-8 in the high affinity interaction with the interleukin-8 receptor type A.

Interleukin-8 (IL-8) has at least two binding regions for both the A and the B type IL-8 receptors. This study defines an important region between Cys7 and Cys50 that, together with the Glu4-Leu5-Arg6 sequence of the NH2 terminus, accounts for the high affinity binding of IL-8 to the IL-8 A receptor on leukocytes. Utilizing rabbit IL-8 that shares 82% sequence identity with human IL-8, but has 200-fold lower binding affinity for the IL-8 A receptor, residues of the human homologue were sequentially exchanged into the rabbit molecule.

Multiple sites on IL-8 responsible for binding to alpha and beta IL-8 receptors.

To define the structural features important for IL-8 binding to its two known receptors, mutants of IL-8 and melanoma growth-stimulating activity (MGSA) and chimerae consisting of segments of these two chemokines were constructed and purified from the pGEX 2T Escherichia coli expression vector. IL-8 alpha and beta receptors were expressed stably and individually in 293 kidney epithelial cells and HL60 human leukemia cells. The Kd for IL-8 itself and copy numbers for both receptors in transfected cells were comparable.

Analysis of Ly5 chromosome 1 position using allelic differences and recombinant inbred mice.

Differences between the mouse Ly5a and Ly5b alleles can be distinguished on the basis of polymerase chain reaction (PCR)-restriction enzyme analysis and differential monoclonal antibody reactivities. To more precisely map the Ly5 gene on the mouse chromosome 1, analytical DNA and protein tests were performed on recombinant inbred strains of mice prepared from SJL/J (Ly5a) and BALB/cke (Ly5b) progenitor strains. Each recombinant inbred strain was characterized to determine whether it carried the Ly5a or Ly5b allele.

Comparison of mouse Ly5a and Ly5b leucocyte common antigen alleles.

The family of leucocyte common antigen (LCA) transmembrane glycoproteins is expressed in most hematopoietic cells. Molecular isoforms of the LCA molecule are generated by alternative splicing of a single gene encoded on the murine chromosome 1. Three LCA alleles with different antigenic reactivities have been identified in inbred mouse strains.

Characterization of the neisserial lipid-modified azurin bearing the H.8 epitope.

The pathogenic Neisseria have multiple genes encoding proteins that bind monoclonal antibody (MAb) H.8. We previously reported the cloning and sequencing of a meningococcal gene (laz) encoding an H.

Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence.

Expression of outer membrane protein II (P.II) of Neisseria gonorrhoeae is subject to reversible phase variation at a rate of 10(-3)-10(-4)/cell/generation. The signal peptide coding regions of P.

Recombination among protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the protein II family.

Expression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.

Reversible phase variation of expression of Neisseria meningitidis class 5 outer membrane proteins and their relationship to gonococcal proteins II.

Neisseria meningitidis class 5 proteins are major outer membrane proteins that share many properties with the proteins II (P.II) of Neisseria gonorrhoeae. We generated two bactericidal monoclonal antibodies, each of which bound specifically to one of the two identified class 5 proteins produced by N.

Antigenic and structural differences among six proteins II expressed by a single strain of Neisseria gonorrhoeae.

Gonococci express a family of related outer membrane proteins designated protein II (P.II), which undergo both phase and antigenic variation. Six P.

Resistance to meningococcemia apparently conferred by anti-H.8 monoclonal antibody is due to contaminating endotoxin and not to specific immunoprotection.

We evaluated the ability of a monoclonal antibody directed against the common H.8 antigen of pathogenic Neisseria sp. to confer passive protection against meningococcal disease in mice.

Genetic and biochemical analyses of protein II.

We compared the structure of P.II proteins of gonococcal strain FA1090 by N-terminal sequence analysis of purified proteins and by DNA sequencing of cloned P.II genes.

The H8 antigen of pathogenic Neisseriae.

We cloned and sequenced the H8 gene from N. meningitidis FAM18. The predicted amino acid sequence included a consensus lipoprotein signal sequence processing site, consistent with lipid modification that could account for the unusual electrophoretic and solubilization properties of H8.

Abrupt withdrawal of atenolol in patients with severe angina. Comparison with the effects of treatment.

The effects of abrupt withdrawal of atenolol, a long acting cardioselective beta blocker, were studied in 20 patients with severe stable angina pectoris admitted to hospital for coronary arteriography. During the 144 hour postwithdrawal period no serious coronary events occurred. Mean and maximal daily heart rates rose steadily for at least 120 hours.

Prophylaxis against ventricular arrhythmias in suspected acute myocardial infarction: a comparison of tocainide and disopyramide.

Five hundred and seventy one patients admitted to a coronary care unit with suspected acute myocardial infarction were considered for entry into a double-blind study. Two hundred and eighty-three patients were excluded, mainly because of recent treatment with beta-adrenoceptor blocking agents, life threatening arrhythmias requiring specific treatment and left ventricular failure presenting with hypotension or pulmonary oedema. Two hundred and eighty-eight entered the trial of whom 202 were subsequently confirmed to have had myocardial infarction.