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T H Watts

Publications and source records attributed to T H Watts.

46 records · Page 3Linked to original sources
PubMed1985

T-cell activation by peptide antigen: effect of peptide sequence and method of antigen presentation.

A series of synthetic peptide analogues of a determinant recognized by the ovalbumin-specific, I-Ad-restricted, T-cell hybridoma 3DO-54.8 were synthesized. The resulting peptides were tested for activation of 3DO-54.8 cells by using glutaraldehyde-fixed cells as well as reconstituted membranes as antigen-presenting surfaces. The results show that the minimum epitope for activation of this T cell is between 7 and 11 amino acids in length. This region includes two important histidine residues. The order of preference of the various peptide analogues was the same regardless of the method of antigen presentation. However, the amount of peptide required for T-cell activation was considerably higher when reconstituted membranes, rather than fixed cells, were used as antigen-presenting surfaces.

Animals↗
PubMed1984

Antigen presentation by supported planar membranes containing affinity-purified I-Ad.

I-Ad, purified from A20-1.11 cells by affinity chromatography, was incorporated into supported planar membranes by incubation of I-Ad-containing phospholipid vesicles with clean glass coverslips. Such planar membranes present a peptide digest of ovalbumin to the ovalbumin-specific, I-Ad-restricted T-cell hybridoma 3DO-54.8, resulting in the antigen-specific release of interleukin 2. However, when the same material was provided in the form of small unilamellar vesicles, no response was obtained. Antigen presentation by the I-Ad-containing planar membranes was inhibited by the monoclonal antibody MKD6 (anti-I-Ad) but not by the antibody 10-2.16 (anti-I-Ak). The antibody GK1.5, which recognizes the T-cell surface antigen L3T4, was also inhibitory. In contrast to the results with purified I-Ad, crude membrane preparations from A20-1.11 cells were effective in antigen presentation in both planar and vesicular forms.

Animals↗
PubMed1983

Spectral properties of three quaternary arrangements of Pseudomonas pilin.

Pseudomonas aeruginosa possess multisubunit, filamentous appendages called pili which are involved in adhesion, twitching motility, and bacteriophage adsorption. The spectral properties of three forms of pili have been compared. These are native pili, pilin dimers in octyl glycoside, and an in vitro assembled form of pilin which we call reassembled pilin filaments. Alkaline pH titrations, solvent perturbation, quenching of tryptophan fluorescence with acrylamide, and circular dichroism were used to demonstrate that tyrosines-24 and -27 are at a dimer/dimer interface in both native pili and in the reassembled pilin filaments. Dissociation of pili by octyl glucoside results in exposure of the two tyrosines and in partial exposure of a least one tryptophan in pilin.

Amino Acid Sequence↗
PubMed1983

Mapping of the antigenic determinants of Pseudomonas aeruginosa PAK polar pili.

The polar pili of Pseudomonas aeruginosa are flexible filaments 5.2 nm in diameter and 2.5 microns in average length. They consist of a single subunit, pilin, which is a 144-residue polypeptide containing a hydrophobic N-terminal region (residues 1 to 30) and eight hydrophilic regions distributed throughout the remainder of the molecule. To delineate the antigenic regions of pilin, we cleaved the protein at Arg30, Arg53, and Arg120 to produce peptides TCI (residues 1 to 30), TCII (31 to 53), TCIII (54 to 120), and TCIV (121 to 144). TCIII and TCIV were further cleaved into several subfragments. The purified peptides were coupled to bovine serum albumin by using the N-hydroxysuccinimide ester of 4-azidobenzoic acid and were then subjected to immunological analysis, using the enzyme-linked immunosorbent assay and immunoblot procedures with polyclonal antiserum. Four antigenic regions were identified; one in TCI was found to be common to both PAK and PAO pilin. The remaining three were found to be specific to PAK pilin. Two of these were subfragments of TCIII, whereas the third was located close to the C-terminus of the molecule, most likely between Cys129 and Cys142. Modification of these cysteines by reduction and carboxymethylation of the disulfide linkage did not abolish the antigenicity of the C-terminal type-specific antigenic determinant.

Antigens, Bacterial↗
PubMed1982

Formation of 9-nm filaments from pilin monomers obtained by octyl-glucoside dissociation of Pseudomonas aeruginosa pili.

Pili isolated from Pseudomonas aeruginosa PAK were solubilized in the detergent octyl-glucoside. Subsequent removal of the detergent by dialysis resulted in the formation of short rods of about 9 nm in diameter and various lengths. The aggregation process was followed by ultracentrifugation, viscometry, and electron microscopy to show that the aggregates produced in this way are distinct from pili produced by the bacteria, both in diameter, as measured by electron micrographs, and in their inability to compete in a biological assay for pili.

Adsorption↗
PubMed1982

Identification of pilin pools in the membranes of Pseudomonas aeruginosa.

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.

Antibodies↗
PubMed1982

Dissociation and characterization of pilin isolated from Pseudomonas aeruginosa strains PAK and PAO.

Pili isolated from Pseudomonas aeruginosa strains PAK and PAO have been dissociated into subunits using the detergent octyl glucoside. Circular dichroism studies indicated that no change in protein conformation occurs as a result of this treatment. Ultracentrifugation measurements showed that both pilins have a Stokes radius of 21 A (1 A = 0.1 nm) corresponding to an axial ratio of between 3:1 and 5:1, when approximated as prolate or oblate ellipsoids. Sedimentation equilibrium measurements show that even at low protein concentrations the pilin-detergent complex exists as a mixture of monomers and dimers.

Bacterial Proteins↗
PubMed1979

Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO.

Pseudomonas aeruginosa strains PAO and PAK bear polar pili which are flexible filaments having a diameter of 6 nm and an average length of 2500 nm. Both types of pili are retractile and promote infection by a number of bacteriophages. The present communication describes the partial biochemical characterization of PAO pili isolated from a multipiliated nonretractile mutant of PAO. The observed properties are compared to those of PAK pili which were characterized previously. PAO pili were found to contain a single polypeptide subunit of 18 700 daltons. This is similar to PAK pili which contain a single polypeptide of 18 100 daltons. The amino acid composition of PAO pilin was also similar to that of PAK pilin. Neither protein contained phosphate or carbohydrate residues and both were found to contain N-methylphenylalanine at the amino terminus. Sequencing of 20 amino acid residues at the amino terminal end of PAO pilin revealed the sequence to be identical with that of PAK pilin, while tryptic peptide analyses of PAO and PAK pilin indicated that the two proteins probably contain a number of homologous regions within the polypeptide. It was concluded that PAO and PAK pili were closely related structures.

Amino Acid Sequence↗
PubMedDate not supplied

T-cell-mediated association of peptide antigen and major histocompatibility complex protein detected by energy transfer in an evanescent wave-field.

Helper T cells recognize foreign antigen displayed on antigenpresenting cells which also express self-molecules of the major histocompatibility complex (MHC). A single T-cell receptor mediates recognition of both MHC and foreign antigen. A proposed ternary complex between T-cell receptor, foreign antigen and MHC antigen has not yet been demonstrated (see ref. 1 for review). Here, we show that a fluorescein-labelled synthetic peptide, together with Texas red-labelled class II MHC antigen, I-Ad, stimulates the production of interleukin-2 by a peptide-specific I-Ad-restricted T-cell hybridoma when reconstituted in a lipid membrane on a glass substrate. Under the same conditions, resonance-energy transfer from donor peptide to acceptor I-A can be stimulated in an evanescent wave-field only in the presence of the specific T-hybrid. Our results show that the T cell stabilizes an association between peptide antigen and class II MHC protein to within a distance of about 40 A.

Animals↗