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C Miles

Publications and source records attributed to C Miles.

At least 55 records · Page 3Linked to original sources
PubMed1990

DNA sequence analysis of gamma radiation-induced deletions and insertions at the APRT locus of hamster cells.

Gamma radiation-induced gene rearrangements at the Chinese hamster ovary cell locus coding for the purine salvage enzyme adenine phosphoribosyl transferase (APRT) consist of both simple deletions and more complex alterations that are presumably the result of multiple strand breaks. To characterize these mutations at the DNA sequence level, fragments altered by deletion and insertion mutations were obtained by cloning in lambda phage vectors or by using the polymerase chain reaction. The radiation-induced deletions characterized here eliminate 3-4 kb and have at least one breakpoint in an AT-rich region or near short direct or inverted repeats. Insertions involve small fragments (102 and 456 bp) of repetitive DNA that appear to be related to B2 (short interspersed repetitive) and long interspersed repeat families. The novel fragments bear little resemblance to each other or to sequences at the integration sites, and their introduction is accompanied by a small target site deletion.

Adenine Phosphoribosyltransferase↗
PubMed1989

Structural requirements for the interaction between peptide antigens and I-Ed molecules.

We have analyzed the structural characteristics of the interaction between I-Ed molecules and their peptide ligands. It was found that unrelated good I-Ed binders share structurally similar "core" regions that were experimentally demonstrated to be crucial for binding to I-Ed molecules. Single amino acid substitution analogues of one good I-Ed binder, hen egg lysozyme 107-116, were analyzed for their capacity to bind to I-Ed molecules and to activate two different I-Ed-restricted T cell hybridomas. The results illustrate the great permissiveness of I-Ed-peptide interaction and the great specificity of T cell recognition. It was concluded from these analyses that basic residues on the peptide molecule play a crucial role in binding to I-Ed. This contrasts with the structural requirements for binding to the other Iad isotype, I-Ad, the crucial hydrophobic residues. Thus, different class II molecules of the same MHC haplotype may have rather distinct peptide binding specificities, thereby expanding the repertoire of possible immunogenic peptides presented for T cell recognition.

Amino Acid Sequence↗
PubMed1989

G-repeats: a novel hamster sine family.

A fragment of a hamster repetitive element inserted into the aprt locus of a radiation-induced mutant is a member of a novel interspersed repetitive (SINE) family constituting approximately 0.3 to 0.5% of the hamster genome (30 to 50,000 family members). Since this family was first detected in a gene rearranged after exposure to gamma irradiation, we have called these G-repeats. In common with other repetitive elements, members of this family are about 300 bp in length, are highly divergent (an average of 30% from the consensus), have an A + T rich sequence flanking one side, and can be found in short polydisperse circular (SPC) DNA. In contrast to some other families, G-repeats are not flanked by short direct repeats and lack sequences corresponding to the RNA polymerase III consensus promoter.

Animals↗
PubMed1989

Induction of class I MHC-restricted, peptide-specific cytolytic T lymphocytes by peptide priming in vivo.

The present study investigated the possibility that protein Ag fragments in the form of peptides could serve as the priming Ag in the generation of a MHC class I-restricted immune response. Trypsin-digested chicken ovalbumin (OVA-TD) fragments were used as the model Ag. The results demonstrate the peptides within OVA-TD, when injected into C57BL/6 mice, could prime T cells which lysed H-2b Ia-EL4 target cells in an OVA-TD-specific manner. In contrast to priming with OVA-TD, immunization of mice with intact OVA did not lead to generation of CTL against OVA-TD or OVA. Furthermore, target cells sensitized with intact OVA failed to be recognized by OVA-peptide-specific CTL indicating that the target cells serving as APC were unable to generate the relevant peptide determinants recognized by the T cells. These results support the idea that the processing pathway within APC for class I-restricted T cells may differ from that used for class II-restricted T cells. Using OVA-TD-specific CTL clones (phenotypically Thy 1+, CD8+, CD4-, Pgp-1+) isolated from primed animals to screen OVA-TD fractions separated by HPLC, two T cell peptide determinants were identified corresponding to OVA sequences 111-122 and 370-381. Both determinants were recognized by CTL clones in the context of the H-2Db molecule.

Animals↗
PubMed1989

Effect of conformational propensity of peptide antigens in their interaction with MHC class II molecules. Failure to document the importance of regular secondary structures.

In an attempt to define some of the conformational requirements for binding of the antigenic peptide OVA 323-336 to purified IAd molecules, three distinct experimental approaches were applied. First, the effect of introducing proline or glycine residues within the region of OVA 323-336 crucial for its IAd binding capacity was analyzed. In most instances these substitutions had little or no effect, suggesting that neither alpha-helical nor beta-sheet regular structures may be strictly required for productive interaction with MHC molecules. Some of the same substitutions were also found to have no effect on the capacity of the peptide to stimulate OVA 323-336 specific T cell hybridomas, suggesting that regular structures such as alpha-helices or beta-sheets may not be strictly required for T cell stimulation, either. Second, we introduced, within the OVA 323-336 molecule, structural modifications predicted to alter its dipole characteristics and stabilize helical structures. No improvement of the IAd binding capacity was detected following these structural alterations. Surprisingly, some but not others of these analogs displayed increased antigenicity for OVA 323-336 specific T cell hybridomas. Third, a panel of analogs of OVA 323-336 were synthesized in which the crucial IAd binding core region was linked to non-native sequences of differing conformational propensities. When 22 such analogs were tested for IAd binding, it was found that these non-native sequences could drastically influence the binding capacity, but no correlation was found between their effect and their alpha-helical, beta-sheet, or beta-turn conformational propensity as calculated by the Chou and Fasman algorithm. In summary, all the data presented herein suggest that, at least in the case of OVA 323-336 and IAd, the propensity of the antigen molecule to form secondary structures such as alpha-helices, beta-sheets, or beta-turns does not correlate with its capacity to bind MHC molecules.

Amino Acid Sequence↗
PubMed1989

Structural requirements and biological significance of interactions between peptides and the major histocompatibility complex.

Previous studies indicate that T cells recognize a complex between the major histocompatibility complex (MHC) restriction-element and peptide-antigen fragments. Two aspects of this complex formation are considered in this paper: (1) what is the nature of the specificity of the interactions that allows a few MHC molecules to serve as restriction elements for a large universe of antigens; and (2) what is the relative contribution of determinant selection (i.e. antigen-MHC complex formation) and T-cell repertoire in determining the capacity of an individual to respond to an antigen? By analysing single amino acid substitution analogues of a peptide antigen (Ova 325-335) as well as by analysing the structural similarities between unrelated peptides capable of binding to the same MHC molecule, we have been able to document the very permissive nature of the antigen-MHC interaction. Despite this permissiveness of binding, it is possible to define certain structural features of peptides that are associated with the capacity to bind to a particular MHC specificity. With respect to the question of the relative role of 'determinant selection' and 'holes in the T-cell repertoire' in determining immune responsiveness, we present data that suggest both mechanisms operate in concert with one another. Thus only about 30% of a collection of peptides that in sum represent the sequence of a protein molecule were found to bind to Ia. Although immunogenicity was restricted to those peptides that were capable of binding to Ia (i.e. determinant selection was operative), we found that about 40% of Ia-binding peptides were not immunogenic (i.e. there were also 'holes in the T-cell repertoire').

Amino Acid Sequence↗
PubMed1989

Structural analysis of peptides capable of binding to more than one Ia antigen.

The Ia binding regions were analyzed for three unrelated peptide Ag (sperm whale myoglobin 106-118, influenza hemagglutinin 130-142, and lambda repressor protein 12-26) for which binding to more than one Ia molecule has previously been demonstrated. By determining the binding profile of three separate series of truncated synthetic peptides, it was found that in all three cases the different Ia reactivities mapped to largely overlapping regions of the peptides; although, for two of the peptides, the regions involved in binding the different Ia specificities were distinct. Moreover, subtle differences were found to dramatically influence some, but not other, Ia reactivities. Using a large panel of synthetic peptides it was found that a significant correlation exists between the capacity of peptides to interact with different alleles of the same molecule (i.e., IAd and IAk), but no correlation was found with the capacity of peptides to interact with different isotypes within the same haplotype (i.e., IAd and IEd). These data suggest that different alleles of the same MHC molecule may actually recognize closely related structures, whereas different isotypes may recognize unrelated, albeit non-mutually exclusive, structures on an Ag molecule.

Amino Acid Sequence↗
PubMed1989

DNA sequence determination of gamma-radiation-induced mutations of the hamster aprt locus.

From a collection of 85 independent gamma-radiation hamster aprt- mutants, 27 having no major structural alterations were analysed at the nucleotide level by using the polymerase chain reaction to amplify mutant exons and then directly sequencing the double-stranded products. The majority of these mutations were simple base substitutions of all types, particularly transversions (11/27). Frameshifts and small deletions were also induced. The 'spectrum' of mutations produced by gamma-radiation was not significantly different from that occurring spontaneously at this locus. Differences with respect to the target and structure of frameshifts and small deletions occurring in the two collections were apparent.

Adenine Phosphoribosyltransferase↗
PubMed1989

Prediction of major histocompatibility complex binding regions of protein antigens by sequence pattern analysis.

We have previously experimentally analyzed the structural requirements for interaction between peptide antigens and mouse major histocompatibility complex (MHC) molecules of the d haplotype. We describe here two procedures devised to predict specifically the capacity of peptide molecules to interact with these MHC class II molecules (IAd and IEd). The accuracy of these procedures has been tested on a large panel of synthetic peptides of eukaryotic, prokaryotic, and viral origin, and also on a set of overlapping peptides encompassing the entire staphylococcal nuclease molecule. For both sets of peptides, IAd and IEd binding was successfully predicted in approximately 75% of the cases. This suggests that definition of such sequence "motifs" could be of general use in predicting potentially immunogenic peptide regions within proteins.

Amino Acid Sequence↗
PubMed1989

Sensitivity of methods for calculating energy expenditure by use of doubly labeled water.

Attempts to estimate human energy expenditure by use of doubly labeled water have produced three methods currently used for calculating carbon dioxide production from isotope disappearance data: 1) the two-point method, 2) the regression method, and 3) the integration method. An ideal data set was used to determine the error produced in the calculated energy expenditure for each method when specific variables were perturbed. The analysis indicates that some of the calculation methods are more susceptible to perturbations in certain variables than others. Results from an experiment on one adult human subject are used to illustrate the potential for error in actual data. Samples of second void urine, 24-h urine, and breath collected every other day for 21 days are used to calculate the average daily energy expenditure by three calculation methods. The difference between calculated energy expenditure and metabolizable energy on a weight-maintenance diet is used to estimate the error associated with the doubly labeled water method.

Adult↗
PubMed1989

Cross-modal, auditory-visual Stroop interference: a reply to Cowan and Barron (1987).

This series of experiments was performed to verify Cowan and Barron's (1987) reported effect of auditory color-word interference on a visual Stroop task. Extant theory predicts effects of irrelevant speech on visual memory tasks involving immediate, ordered recall of the items. Interference between the two sources of information (visual and auditory) is assumed to be located at the phonological store component of the articulatory loop. Perceptual tasks such as the Stroop task, which do not require use of the articulatory loop for rehearsal purposes, should not be similarly susceptible. The present data fail to replicate Cowan and Barron's findings, and are thus consistent with contemporary theory.

Adult↗
PubMed1988

I-Ad-binding peptides derived from unrelated protein antigens share a common structural motif.

Purified Ia molecules can specifically bind many unrelated peptide Ag, and such binding appears to be a necessary, albeit not sufficient, prerequisite for the immunogenicity of the proteins from which such peptides are derived. We have recently analyzed the affect of single amino acid substitutions on the I-Ad binding of the immunogenic peptide OVA 323-339. The results obtained demonstrated the very permissive nature of Ag-Ia interaction. We also showed that unrelated peptides that are good I-Ad binders share a common structural motif and speculated that recognition of such motifs could represent a mechanism to achieve a very permissive type of interaction that yet retained some degree of specificity. In the present set of experiments we analyzed the I-Ad binding pattern of a series of overlapping peptides derived from sperm whale myoglobin (residues 102-125) and influenza hemagglutinin (residues 121-146) to determine whether the peptide regions predicted on the basis of structural similarity to be involved in I-Ad binding were in fact involved. In both cases, the I-Ad-interacting determinants were found to contain the sequence motif postulated to be important for I-Ad binding. These data support the hypothesis that I-Ad molecules recognize a large library of Ag by virtue of common structural motifs present in peptides derived from phylogenetically unrelated proteins.

Amino Acid Sequence↗
PubMed1988

Insertion of unique and repetitive DNA fragments into the aprt locus of hamster cells.

Two insertion mutations occurring at the aprt locus of Chinese hamster ovary cells were analyzed at the nucleotide level by cloning and sequencing the mutant genes. The insertions are similar with respect to events at the target site in that both are accompanied by small deletions. The nature of the DNA introduced, on the other hand, is very different, a unique fragment in a spontaneously occurring mutant and a highly dispersed, repetitive fragment in a gamma radiation-induced strain. The inserts are small (285 and 58 base-pairs) and have none of the structural features or sequences related to putative mobile sequences in mammalian cells. The mechanism of transposition was further examined by cloning the unique donor fragment of the spontaneous mutant. These analyses revealed that the insert in the mutant gene was a precise duplicate of the donor DNA fragment.

Animals↗
PubMed1988

Continuity of care in hypertension. An important correlate of blood pressure control among aware hypertensives.

Hypertensive patients frequently discontinue follow-up care. In a population-based survey of 4688 adults, we examined the impact of nonattendance on blood pressure control in aware hypertensives. Nonattendance was defined as a failure to visit a physician for hypertensive care within a six-month interval and was reported by 29% of 907 aware hypertensives. Nonattenders had a higher prevalence of diastolic blood pressure above 90 mm Hg (67% vs 30% for attenders). The nonattender profile was male, young, active in the work force, and without coexisting chronic diseases. Poor blood pressure control among nonattenders was associated with a lower treatment rate. Uncontrolled diastolic hypertension and less adherence to medications in nonattenders warrants concern by clinicians.

Adult↗
PubMed1987

The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides.

The capacity of purified I-Ad, I-Ed, I-Ak, and I-Ek to bind to protein derived peptides that have been previously reported to be T cell immunogens has been examined. For each of the 12 peptides studied strong binding to the relevant Ia restriction element was observed. All the peptides bound more than one Ia molecule; however, for 11 of 12 peptides, the dominant binding was to the restriction element, whereas in one instance the dominant binding was to a nonrestriction element. When the peptides were used to inhibit the presentation of antigen by prefixed accessory cells to T cells, an excellent correlation was found between the capacity of a peptide to inhibit the binding of an antigen to purified Ia and the capacity of the peptide to inhibit accessory cell presentation of the antigen. Thus, the binding of peptide to purified Ia is immunologically relevant, and Ia seems to be the only saturable molecule on the surface of the accessory cell involved in antigen presentation. Inhibition analysis also indicated that all peptides restricted to a particular Ia molecule competitively inhibited one another, suggesting that each Ia restriction element has a single binding site for antigen. Cross-linking of labeled peptides to Ia followed by electrophoretic analysis and autoradiography suggested that this single binding site is made up of portions of both alpha and beta chains of Ia.

Animals↗