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R C Seid

Publications and source records attributed to R C Seid.

11 recordsLinked to original sources
PubMed1989

Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus.

We describe the covalent attachment of palmitate to the fusion glycoprotein of respiratory syncytial virus and the identification of the attachment site. Labeling of respiratory syncytial virus-infected Vero cells with [3H]palmitate, followed by the purification and subsequent analysis of the fusion glycoprotein in conjunction with polyacrylamide gel electrophoresis, demonstrated that the fatty acid is covalently attached to the F1 subunit of the fusion glycoprotein. The bound palmitate was sensitive to 1 M hydroxylamine at neutral pH. In addition, the release of palmitate label by reduction with sodium borohydride showed that the palmitate is linked to the protein through a thioester bond. Isolation of a radiolabeled peptide from a tryptic digest of the protein and subsequent amino-terminal sequence analysis revealed that the cysteine residue (amino acid residue 550) within the anchor sequence, located at the carboxyl terminus of the F1 subunit, is the covalent attachment site for palmitate.

Acylation↗
PubMed1989

Synthetic peptide representing a T-cell epitope of CRM197 substitutes as carrier molecule in a Haemophilus influenzae type B (Hib) conjugate vaccine.

The cross-reactive material (CRM197) of diphtheria toxin is considered to be advantageous as a carrier molecule in the formulation of a Haemophilus influenzae type b conjugate vaccine. In order to more precisely understand the function of the CRM197 in the vaccine, we have begun mapping the T-cell epitopes of the protein. A peptide which represents a segment of the primary sequence of CRM197 has been identified and found to stimulate diphtheria toxoid or CRM197-primed murine T-lymphocytes. In addition, the peptide is capable of priming T-cells in vivo for a subsequent in vitro T-cell response to itself or to the intact CRM197 molecule. The ability of the peptide to replace CRM197 as a carrier molecule was examined by immunizing mice with PRP, PRP-CRM197 conjugate, or PRP covalently coupled to the peptide. Antibodies to PRP were only detected in the PRP-CRM197 or PRP-peptide immunized groups. Both conjugates elicited primary and secondary antibody responses. Thus, a synthetic peptide representing a defined T-cell epitope of CRM197 has been functionally demonstrated based on its ability to act as a carrier molecule in a PRP conjugate vaccine.

Animals↗
PubMed1989

Chemical evidence for covalent linkages of a semi-synthetic glycoconjugate vaccine for Haemophilus influenzae type B disease.

We have defined the nature of the covalent linkages in a Haemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative, N epsilon-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed between H. influenzae type b oligosaccharides (HbO) and the lysyl epsilon-amino groups of CRM197 (a non-toxic, cross-reactive variant of diphtheria toxin), followed by acid hydrolysis of HbOC. Quantification of OHEt-Lys per CRM197 molecule allowed the determination of a covalency ratio, a useful parameter for evaluating the stoichiometry and consistency of HbOC preparations. Covalent association between HbO and CRM197 was also demonstrated by the coincidence of immunoreactivity of gel-electrophoresed HbOC on a Western blot probed with anti-CRM197 and anti-saccharide antisera.

Amino Acids↗
PubMed1989

Purification, cyanogen bromide cleavage, and amino terminus sequencing of class 1 and class 3 outer membrane proteins of meningococci.

Meningococcal class 1 and 3 outer membrane proteins (OMPs) were subjected to cyanogen bromide treatment. The class 3 OMP was found to be resistant to cyanogen bromide, while the class 1 OMP was cut into two main fragments of 25 and 17 kilodaltons. The N-terminal sequences were determined for class 1 and class 3 proteins, which exhibit similarities to one another and to OMP I of gonococci. The C-terminal class 1 OMP fragment bound the bactericidal monoclonal antibodies tested.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of a peptidoglycan-associated lipoprotein from Haemophilus influenzae.

We have purified to homogeneity a peptidoglycan-associated protein from Haemophilus influenzae. Our purification process used differential extraction of cell envelopes with nondenaturing detergents. Solubilization of this protein was accomplished by heating a peptidoglycan-enriched subcellular fraction in the presence of one of several nondenaturing detergents at 55-60 degrees C. The purified protein migrated as a single band, with a Mr approximately 15,000, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein contains covalently linked fatty acids, is rich in tyrosine, but lacks methionine and tryptophan. Amino acid analysis also revealed the presence of glycerylcysteine, which has been shown to be the site of fatty acylation in other bacterial lipoproteins. Over 87% of the primary structure has been determined by sequencing high pressure liquid chromatography purified fragments derived from several endoproteinase digests. This protein belongs to a family of proteins, known as peptidoglycan associated lipoproteins, which appear to be components of the outer membranes of most Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1988

Peptides bound to proteosomes via hydrophobic feet become highly immunogenic without adjuvants.

Addition of either a lauroyl or a pentapeptide (FLLAV) hydrophobic foot to the NH2 terminus of a small, synthetic peptide allowed the peptide to hydrophobically complex to meningococcal outer membrane protein proteosomes by simple dialysis. Both conventional and LPS-hyporesponsive mice immunized with these complexes without any adjuvants developed high-titered and persistent anti-peptide IgG. Since proteosomes have been safely given to many people and since important antigenic determinants are generally hydrophilic, this system should be widely applicable to the development of peptide vaccines for human use.

Adjuvants, Immunologic↗
PubMed1986

Identification of ester-linked fatty acids of bacterial endotoxins by negative ion fast atom bombardment mass spectrometry.

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.

Amides↗
PubMed1981

Preparation and characterization of detoxified lipopolysaccharide-protein conjugates.

Alkaline treatment of Pseudomonas aeruginosa type 5 lipopolysaccharide (LPS) resulted in reduced toxicity as measured by both the Limulus amoebocyte assay and the rabbit pyrogenicity test. Chemical analysis of the deacylated LPS (D-LPS) revealed that ester-linked fatty acids were removed while the amide-linked fatty acids remained intact. The neutral and amino sugar compositions for native LPS and D-LPS were identical within experimental error. Antigenic determinants for complement-dependent human opsonic antibody were retained under these deacylation conditions. To enhance its immunogenicity, D-LPS was covalently coupled to Pseudomonas pili and the 1,4-diaminobutyl derivatives of Pseudomonas exotoxin A and tetanus toxoid. Quantitative amino sugar analyses revealed that 2.6 and 3.2 mol of D-LPS were covalently bound to aminobutyl Pseudomonas exotoxin A and aminobutyl tetanus toxoid, respectively. Gel electrophoresis data indicated at least 1 mol of D-LPS covalently bound per pilus subunit protein. Initial immunologic data indicated that antibody against D-LPS could be induced when the D-LPS is covalently attached to protein carriers.

Amino Acids↗
PubMed1977

Studies on Limulus amoebocyte lysate II. Purification of the coagulogen and the mechanism of clotting.

A coagulogen in Limulus lysate has been purified to apparent homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purified coagulogen identified by its ability to clot with either trypsin or the purified Limulus clotting enzyme (Tai, J.Y., and Liu, T. Y. (1976) Fed. Proc. 35, 1486) has a molecular weight of 24,500. It consists of a single polypeptide chain of about 220 amino acids with glycine and serine as its NH2- and COOH-terminal residues; respectively. When acted upon by the Limulus clotting enzyme, the coagulogen releases a soluble C-peptide of about 45 amino acids and an insoluble coagulin of about 170 amino acids. The latter interacts in a noncovalent fashion to form the clot. Amino acid analyses together with the results of NH2- and COOH-terminal analyses suggest that the clot formation involves the cleavage of an--Arg--Lys--bond. Trypsin acts on the coagulogen to cause clotting by splitting the same peptide bond.

Amino Acids↗