Abstract.A membrane-bound adhesive protein that promotes neurite outgrowth in brain neurons has been isolated from rat brain (Rauvala, H., and R. Pihlaskari. 1987. J. Biol. Chem. 262:16625-16635). The protein is an immunochemically distinct molecule with a subunit size of ~ 30 kD (p30). p30 is an abundant protein in perinatal rat brain, but its content decreases rapidly after birth.In the present study the amino-terminal sequence of p30 was determined by automated Edman degradations. A single amino-terminal sequence was found, which is not present in previously studied adhesive molecules. This unique sequence has a cluster of five positive charges within the first 11 amino acid residues: GlyLys-Gly-Asp-Pro-Lys-Lys-Pro-Arg-Gly-Lys. Antisynthetic peptide antibodies that recognize this sequence were produced in a rabbit, purified with a peptide affinity column, and shown to bind specifically to p30.The antipeptide antibodies were used, together with anti-p30 antibodies, to study the localization of p30 in brain cells and in neuroblastoma cells as follows. (a) Immunofluorescence and immunoelectron microscopy indicated that p30 is a component of neurons in mixed cultures of brain cells. The neurons and the neuroblastoma cells expressed p30 at their surface in the cell bodies and the neurites. In the neurites p30 was found especially in the adhesive distal tips of the processes. In addition the protein was detected in ribosomal particles and in intracellular membranes in a proportion of cells. (b) The antibodies immobilized on microtiter wells enhanced adhesion and neurite growth indicating that p30 is surface exposed in adhering neural cells.(c) Immunoblotting showed that p30 is extracted from suspended cells by heparin suggesting that a heparinlike structure is required for the binding of p30 to the neuronal cell surface. A model summarizing the suggested interactions of p30 in cell adhesion and neurite growth is presented. , 1975). In an attempt to identify adhesive molecules that might play a role in such phenomena, we have fractionated solubilized membranes from young rat brain and monitored the fractionations with brain neurons Rauvala and Pihlaskari, 1987). These studies have resulted in the isolation of an adhesive molecule that has a subunit size of • 30 kD (p30). Due to its neuron-binding and neurite outgrowth-promoting properties and the developmental regulation of its content in brain tissue, p30 has been suggested to play a regulatory role in neuronal growth (Rauvala and Pihlaskari, 1987).
HF.S]Or~ of neurons to other cells or to extracellular materials is thought to play an important role in the outgrowth and guidance of neurites (LetourneauTo further characterize the structural and functional properties of p30 and its possible relationship to other adhesive and neurite-promoting factors, we have analyzed the aminoterminal amino acid sequence of p30 and produced antipeptide antibodies that specifically detect this unique sequence. The affinity-purified antipeptide and anti-p30 antibodies have been used ...
An enzyme immunoassay (EIA) for serum antibodies to human immunodeficiency virus type 1 (HIV-1), based on the synthetic pentadecapeptide SGKLICT-TAVPWNAS, a segment of the transmembrane glycoprotein (gp41) of the virus, was developed and tested for sensitivity and specificity. Sera of 152 individuals at various stages of HIV-1 infection, including two prospectively and six retrospectively studied patients exposed to HIV-1 but seronegative on initial testing in whole-virus EIA and immunoblotting, were screened with the gp41 peptide antibody EIA. The reference population consisted of 1,000 healthy HIV-1 antibody-negative blood donors. In addition, five individuals with antibodies to HIV-2 were studied. Antibodies to the synthetic peptide were detected in 100% of those with asymptomatic infection. Only one patient with LAS failed to react in the peptide EIA. Patients with HIV-2 infection did not react in this test. The peptide antibodies appeared rapidly after infection, were detectable at the time when seroconversion was observed by immunoblotting, and preceded reactivity in whole-virus EIA. Sera of seven patients with verified HIV-1 infection did not react with gp41 in immunoblotting, although antibodies were readily detectable in the gp41 peptide EIA.
Antigenicity of rubella virus E1 polypeptide was analyzed using synthetic peptides with predicted amino acid sequences. Overlapping solid-phase bound peptides were used to define antibody binding domains and a panel of free peptides to study T-cell responsiveness. Several antibody-binding areas including those earlier described to contain major neutralizing epitopes were recognized by human sera positive for rubella antibodies. T-cell lines specific for rubella virus were established from 14 rubella immune subjects. All cell lines responded to rubella virion-derived antigen but only eight (57%) responded to one or more of the synthetic peptides. Individual patterns of peptide recognition were found but peptide 8 representing amino acids 402-422 was most often stimulatory to T-cells lines, either alone (3 subjects) or in combination with peptide 3 (amino acids 245-269) or 3 and 4 (amino acids 269-287). The response was HLA restricted but no single DR specificity for this restriction was identified.
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