Perceptions of first year foundation dentists on oral health education and its role in general dental practice.

UNLABELLED: To explore the perceptions of first year foundation dentists (FD1s) regarding oral health education (OHE) and its role in general dental practice. DESIGN: Focus group discussions. SETTING: Postgraduate training venues and general dental practices utilised for foundation training in South Wales, UK. SUBJECTS (MATERIALS) AND METHODS: Nineteen FD1s accepted an invitation to take part in a series of focus groups. Focus groups were transcribed and data analysed using a constructive process of thematic content analysis to identify themes and theories relating to the FD1s' understanding of OHE and its role in the delivery of care as general dental practitioners. RESULTS: The data fell into three broad categories: the teaching of OHE delivery at undergraduate level; factors influencing the 'frequency and content' of OHE delivery; and barriers to 'effective and successful' OHE. The first category identified perceptions of the 'gold standard' of OHE following undergraduate experiences. The practicalities of the acquisition of technical skills had created a simplistic compartmentalised view of OHE which was not a priority in adult dental care. The second category covered triggers for delivering OHE; in general these were reactive rather than preventive. The last category dealt with successful OHE; unsuccessful OHE was attributed to the patient although communication barriers were recognised. CONCLUSION: The subtle but important difference between OHE and oral health promotion (OHP) in terms of its role in general dental practice is recognised theoretically but not as a reality in practice. OHE is often compartmentalised and a simplistic approach to its delivery is taken. Against a backdrop of commissioning to improve health this has implications in developing organisational processes within general dental practice and training in order to achieve this.

Generating MHC Class II+/Ii- phenotype after adenoviral delivery of both an expressible gene for MHC Class II inducer and an antisense Ii-RNA construct in tumor cells.

Tumor cells engineered by gene transduction to be MHC Class II+/Ii- are novel APCs capable of presenting endogenous tumor antigen epitopes to activate T helper cells. The MHC Class II+/Ii- tumor cell phenotype is created by transfecting genes for either CIITA or IFN-gamma, and inhibiting induced Ii mRNA by an Ii reverse gene construct (Ii-RGC). Adenoviral vectors are preferred for the delivery of such genes because of high infection efficiency and ubiquity of the adenoviral receptor on many cell types and tumors. Here we show that at 5 MOI (multiplicity of infection), recombinant adenoviruses with CIITA or IFN-gamma genes converted virtually all MC-38 colon adenocarcinoma cells and Renca renal carcinoma cells in culture to MHC Class II+/Ii+ cells. A single recombinant adenovirus with both genes for IFN-gamma and Ii-RGC (rAV/IFN-gamma/Ii-RGC) efficiently induced the MHC Class II+/Ii- phenotype. Injection of tumor nodules with rAV/Ii-RGC and rAV/CIITA/IFN-gamma combined with a suboptimal dose of rAV/IL-2 induced a potent antitumor immune response. The methods are adaptable for producing enhanced genetic vaccines, attenuated virus vaccines (eg, vaccinia), and ex vivo cell-based vaccines (dendritic and tumor cells).

MHC class II allosteric site drugs: new immunotherapeutics for malignant, infectious and autoimmune diseases.

The discovery of the interactions of the 'Ii-Key' segment of the Ii protein with the major histocmpatibility complex (MHC) Class II allosteric site, which is adjacent to the antigenic peptide-binding site, creates therapeutic opportunities by regulating the antigenic peptide binding to MHC class II molecules. The binding of Ii-Key to the MHC class II allosteric site loosens the hold of the MHC Class II 'clamshell' on antigenic peptides and leads to highly efficient antigenic peptide charging to or releasing from the MHC class II antigenic peptide-binding groove. Ii-Key peptide-induced spilling of bound antigenic peptide, or replacement with inert blockers, leads to 'inert immunosuppression'. Highly efficient replacement of ambient with vaccine peptides by Ii-Key permits 'active immunosuppression' for antigen-specific control of autoimmune diseases in the absence of cytokines or adjuvants. On the other hand, active immunization against cancer or infectious disease can result from epitope replacement mediated by Ii-Key and accompanied by cytokines or other adjuvants. Finally, linking the Ii-Key peptide through a simple polymethylene bridge to an antigenic sequence vastly increases the potency of MHC Class II peptide vaccines. In summary, the discovery of the MHC class II allosteric site allows one to increase the efficiency of MHC class II-related, antigenic epitope-specific therapy for malignant, infectious, and autoimmune diseases. The focus of this review is on the mechanism and potential clinical use of such novel allosteric site-directed, Ii-key drugs.

Increasing the potency of MHC class II-presented epitopes by linkage to Ii-Key peptide.

We previously found that peptide Ii77-92 from the immunoregulatory Ii protein significantly enhances the binding of antigenic peptides to MHC class II molecules. Now a series of hybrids have been constructed linking LRMK, the active core region of the Ii77-92 peptide, to an antigenic epitope of cytochrome C. In vitro T cell hybridoma stimulation by some of these hybrids is up to 250 times more potent than by the antigenic peptide. The biological activities of the hybrids were tested in terms of length and composition of the linker. Simple spacers containing a polymethylene bridge (-HN-CH(2)-CH(2)-CH(2)-CH(2)-CO(2)-) were fully active in these hybrids which can enhance vaccination with MHC class II-presented epitopes.

Genetic modulation of tumor antigen presentation.

An effective cancer-cell vaccine is created by expressing major-histocompatibility-complex (MHC) class II molecules without the invariant chain protein (Ii) that normally blocks the antigenic-peptide-binding site of MHC class II molecules at their synthesis in the endoplasmic reticulum. Such tumor-cell constructs are created either by the transfer of genes for MHC class IIalpha and beta chains, or by the induction of MHC class II molecules and Ii protein with a transacting factor, followed by Ii suppression using antisense methods. Preclinical validation of this approach is reviewed with the goal of using this immunotherapy for metastatic human cancers.

Cancer immunotherapy by antisense suppression of Ii protein in MHC-class-II-positive tumor cells.

This study was aimed at creating a more effective tumor cell vaccine by suppressing Ii protein in the presence of MHC class II molecules within a cancer cell. Absence of the Ii protein, which normally blocks the antigenic-peptide-binding site of MHC class II molecules at synthesis in the endoplasmic reticulum, presumably increases the range of cancer-related epitopes presented to CD4+ helper T cells. Effective suppression of Ii protein was achieved with an antisense, phosphorothioate oligonucleotide, which was selected on the basis of (1) the RNase H activation assay, (2) an assay for Ii protein suppression, and (3) a test for potency with respect to the extent of base sequence ("sequence walking"). The SaI murine sarcoma, which is MHC-class-I+ and MHC-class-II-, Ii-protein-, upon transfection with genes for either interferon gamma or the MHC class II transactivator, came to express MHC class II molecules and Ii protein. In each line of transfected tumor cells, the antisense oligonucleotide profoundly suppressed Ii protein in 35%-55% cells, without affecting expression of MHC class II molecules. Inoculation of mice with such Ii-protein-suppressed tumor vaccine cells, after either formaldehyde fixation or X-irradiation, led to much greater protection against challenge with the parental SaI sarcoma than did inoculation with untreated cells. This approach to cancer cell vaccination can be applied in a wide range of human tumors.

Studies on activities of invariant chain peptides on releasing or exchanging of antigenic peptides at human leukocyte antigen-DR1.

An invariant chain peptide (Ii77-92; YRMKLPKSAKPVSQMR; 'Ii-Key') enhances 10-50 times baseline levels, the presentation of synthetic antigenic peptides to murine T cell hybridomas, by an exchange mechanism at cell surface MHC class II molecules. Two differing activities, to promote the release of antigenic peptide in the presence or absence in solution of a second antigenic peptide, were characterized with truncation homologs through assays for release or binding of human myelin basic protein biotinylated (*) peptide 90-102 on purified HLA-DR1: 1) release of bound hMBP *peptide from DR1 in the presence or absence of free hMBP peptide in solution, 2) exchange of hMBP *peptide from solution with hMBP peptide on DR1, and 3) binding of hMBP *peptide to 'empty' DR1. Peptides such as Ii81-88, LPKSAKPV, released prebound hMBP *peptide from DR1 without free hMBP peptide in solution. They also exchanged hMBP *peptide from solution for prebound hMBP peptide. Peptides including hIi77-83, LRMKLPK, released hMBP *peptide only when free hMBP peptide was in solution. Nevertheless, hIi77-85, LRMKLPKPP, released hMBP peptide without hMBP peptide in solution. Either type of peptide accelerated hMBP *peptide binding to 'empty' DR1. Competitive binding assays with hMBP *peptide or several *Ii-Key truncation homologs, with respective not biotinylated forms, demonstrated that the Ii77-83, LRMKLPK, binding site was distinct from the HLA-DR1 antigenic peptide binding site.

Biological activity and therapeutic potential of homologs of an Ii peptide which regulates antigenic peptide binding to cell surface MHC class II molecules.

An invariant chain peptide (murine Ii76-92; Ii-Key) is known to produce a 10 to 50 times baseline enhancement of the presentation of specific antigenic peptides to murine T cell hybridomas by cell surface MHC class II molecules. In order to define structure-activity relationships in Ii-key, homologs were synthesized with the following systematic variations: 1) N- and C-terminal truncations, 2) N-terminal acetylation and C-terminal amidation, 3) substitutions with 13 natural amino acids in each position of the shortest, fully active peptide, and then with an additional 12 nonnatural amino acids at certain 'pharmacophore' positions, 4) substitutions with D-amino acids and N-methyl-leucine, and 5) cyclical forms. More than 160 homologs were tested for effects on antigen presentation by the murine MHC class II alleles: A(d), Ak, E(d), or Ek. For some compounds, allele specificity between E(d) and Ek exceeded 1:20. D-Amino acid and/or N-methyl-leucine substitutions were accepted at some residue positions, leading to peptides with relatively long half-lives in mouse serum and low toxicities in mice. An Ii-Key homolog inhibited in vitro presentation of an internally processed hen egg lysozyme determinant to a specific T hybridoma. The best compounds can be tested in vivo for therapeutic applications: 1) immunosuppression upon release of antigenic peptide, and 2) vaccination or immunomodulation upon co-administration of a second antigenic peptide.

Structural studies by 1H NMR of a prototypic alpha-helical peptide (LYQELQKLTQTLK) and homologs in trifluoroethanol/water and on sodium dodecyl sulfate micelles.

The 1H NMR-determined structure and dynamics of a synthetic, amphiphilic alpha-helical peptide, PH-1.0 (LYQELQKLTQTLK), and several homologs were compared in 50% trifluoroethanol-d2 (TFE-d2)/H20 and in sodium dodecyl-d25 sulfate (SDS-d25) micelles. The peptides were designed to test the influence on secondary structure of placement of favored and disfavored residues relative to a "longitudinal, hydrophobic strip-of-helix" defined by the repeating leucines. PH-1.0 was highly ordered as an alpha-helix in 50% TFE-d2/H20 and in SDS-d25 micelles. Homologs PH-1.1, in which L1 was replaced by T, and PH-1,4, in which L12 was replaced by T. were found to be partially helical in both media. Calculated structures in SDS-d25 revealed that the helix of PH-1.1 was slightly disordered at the N-terminus, but that of PH-1.4 was completely disordered at the C-terminus. Examination of distributions of hydrophobic residues in protein structures revealed that, when [symbol: see text] = LIVFM and [symbol: see text] = nonLIVFM, the pattern [symbol: see text] is favored and [symbol: see text] is disfavored in alpha-helices. Several analogs of PH-1.0 incorporating these patterns were studied. Peptide PH-1.12 (LYQELQKLLQTLK) retained alpha-helical structure in both 50% TFE-d2/H20 and in SDS-d25 micelles. However, although PH-1.13 (LYQELQKLTLTLK) was fully helical in 50% TFE, it was helical only through residue 6 in SDS micelles. Two homologs containing an additional loop of the helix and repeats of favored (PH-5.0, NYLQTLLETLKTLLQK) or suppressed LL patterns (PH-5.11, NYLQTLETLKLTQK) gave similar results, i.e. the latter peptide was helical only through residue 6 in SDS micelles. The degree of local order in these SDS micelle-adsorbed peptides correlates to placement of hydrophobic residues in motifs which are favored or disfavored in proteins in general and in alpha-helices specifically.

1H NMR studies of prototypical helical designer peptides. A comparative study of the amide chemical shift dependency on temperature and polypeptide sequence.

A series of designer alpha-helical peptides with hydrophobic residues located at different positions along the sequence (PH-1.0 = LYQELQKLTQTLK, PH-1.19 = LYQELQKLTQTFK, PH-1.12 = LYQELQKLLQTLK, PH-1.13 = LYQELQKLTLTLK, PH-1.4 = LYQELQKLTQTTK) were analyzed using one- and two-dimensional NMR methods (TOCSY and NOESY). The central feature of these designer peptides is the incorporation of a maximal hydrophobic strip which may play a role in antigen processing and the nucleation of alpha-helices in proteins (J. Immunol. 145, 899, 1990). Using the 2D-NMR, sequence specific assignments and NOE connectivities were determined in all peptides when dissolved in H2O/TFE mixtures. NOE connectivities indicated that all these peptides are helical in this medium. An unusually large number of NOEs was found for all these designer peptides. This is in accord with ultracentrifugation studies that showed that PH-1.0 forms a trimer in 50% H2O/TFE mixtures. Other peptides in the series behave in similar manner as PH-1.0. The structural differences among these peptides was addressed using the backbone amide chemical shift temperature coefficients, [symbol: see text], and the differences between the observed and random coil values, delta delta HN. The delta delta HN patterns along the peptide sequence are consistent with those expected for amphiphilic alpha-helices, where most delta delta HN values are below zero. However, no significant differences among the peptides in this series can be detected on the delta delta HN patterns, with the exception of PH-1.12. The [symbol: see text] values reveal differences among the peptides of the series. The patterns of [symbol: see text] along the peptide sequences are similar to that found for delta delta HN for PH-1.0, PH-1.19 and PH-1.4. The other peptides in the series, PH-1.12 and PH-1.13, showed different patterns for [symbol: see text]. The latter parameter was used to evaluate the helicity of this series of peptides. According to this parameter the relative helicity of this series is as follows: PH-1.12 > PH-1.0 > PH-1.4 > PH-1.19 > PH-1.13 The NMR data shown here correlated well with the helical propensities predicted for polypeptide sequences using statistical arguments (Proc. Natl. Acad. Sci. USA, 90, 9100, 1993).

Invariant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules.

Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine Ad, Ak, Ed, and Ek major histocompatibility complex (MHC) class II molecules. Naturally produced class II-associated invariant chain peptides human (h)Ii81-104/murine (m)Ii80-103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii-4 peptides hIi77-92/mIi76-91 enhanced presentation of antigenic peptides on I-E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigen-presenting cells (APC) with Ii-4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii-4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81-104 and mIi80-103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II-transfected L cells, as measured by flow cytometry, was inhibited by mIi80-103 and enhanced by mIi-4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.

Structural analysis of invariant chain subsets as a function of their association with MHC class II chains.

Respective subsets of human invariant chain (Ii), as identified with antibodies to two different epitopes, were characterized as a function of their associations with major histocompatibility complex (MHC) class II alpha,beta chains and intracellular processing. E1 antiserum to Ii(183-193) and VIC-Y1 monoclonal antibody to an N-terminal determinant identified Ii(E1) and Ii(VIC) populations, respectively. Ii proteins comprise several species which have been defined with either genomic or post-translational processes: Ii itself; IpN and IpO, which represent the glycosylated forms on asparagine or threonine/serine, respectively; gamma 2 and gamma 3, which originate from an alternative initiation site for transcription; and p41, which has a 64-amino-acid insert which originated from an additional exon placed after the sixth exon of Ii. Immunoprecipitates of detergent-solubilized protein complexes from [35S]methionine-labeled Raji cells showed that Ii(E1) consisted of Ii, p41, IpN, and immature alpha chain, while Ii(VIC) consisted of Ii, processed Ii with N- and O-linked glycosylation (IpN,IpO), p41, and associated MHC class II alpha,beta chains. In the MHC class II-deficient P3HR-1 cells, Ii(E1) and Ii(VIC) were virtually identical. Ii(E1) was resistant to cathepsin B digestion while Ii(VIC) was sensitive. In pulse-chase radiolabeling experiments with brefeldin A (BFA)-treated cells, Ii(VIC) progressively became resistant to endoglycosidase H (endo H) and had a longer half-life than that in cells not treated with BFA, but Ii(E1) remained susceptible to endo H and its half-life was unaffected by BFA. Since BFA redistributes Golgi enzymes to the endoplasmic reticulum, this observation suggests that Ii(E1) is protected from processing enzymes while Ii(VIC) is not. These studies define association of Ii with MHC class II molecules when certain epitopes on Ii are exposed or not. These differences relate to intracellular transport of Ii and to its release for the binding of antigenic peptides.

Cathepsin B cleavage and release of invariant chain from MHC class II molecules follow a staged pattern.

A staged pattern of cathepsin B cleavage of MHC class II alpha, beta-bound invariant (Ii) chain and release of fragments was defined. Charge-loss mutations in the Ii chain were created in three clusters of cathepsin B putative cleavage sites R78K80K83K86, K137K143, and R151K154. Products of HLA-DR1 alpha, beta and wild type (WT) or mutant Ii genes, co-transfected into COS1 cells, were cleaved by cathepsin B and immunoprecipitated by antibodies either to MHC class II chains or to different Ii epitopes. In WT Ii, cathepsin B digestion generated two forms of p21 Ii fragments: a p21 recognized by anti-C-terminus antibodies and a p21 recognized by an antibody to a determinant near the N-terminus. C-terminal p21 was released from MHC class II alpha, beta chains upon its formation while N-terminal p21 remained associated with MHC class II alpha, beta chains. Mutations at K137K143 inhibited the generation of N-terminal p21 by cathepsin B. Mutation at R78K80K83K86 led to an accumulation of MHC class II-bound N-terminal p21 without the appearance of MHC class II-bound p14, p10, and p6 fragments after cathepsin B digestion. These results indicate that cathepsin B cleaves wild type Ii first about K137K143 to produce a MHC class II-associated N-terminal p21, which is then cleaved about R78K80K83K86 to generate p14, p10 and finally p6 which still associates with MHC class II alpha, beta chains. This pattern of staged cleavage and release of Ii might be related to a concerted mechanism regulating the binding of antigenic peptides to MHC class II molecules.

More efficient peptide binding to MHC class II molecules during cathepsin B digestion of Ii than after Ii release.

The binding of a T cell-presented peptide to MHC class II alpha,beta chains occurs as a concurrent process with the release of the associated invariant chain (Ii) by cathepsin B. Ii was digested by cathepsin B from solubilized, MHC class II alpha,beta,Ii complexes in the presence of N-hydroxysuccinimidyl-4-azidobenzoate-conjugated, 125I-labeled, influenza virus matrix (18-29) peptide. The peptide was crosslinked where it became bound. This HLA-DR1-restricted peptide bound about three times more efficiently to class II alpha,beta chains of DR1-positive B cells when present during cathepsin B digestion of Ii than when added afterward, also at pH 5.0. Binding was competed by similarly DR-restricted peptides. Cathepsin D cleaved Ii but did not enhance peptide binding. However, a trace level of cathepsin D, added to the assay for peptide binding in the presence of cathepsin B, further enhanced peptide binding about three times. These experiments support an hypothesis for the staged release of Ii fragments by cathepsin D and cathepsin B, catalyzing at one point the insertion of a peptide into the antigen binding site formed by class II alpha and beta chains.

Assessment of homology with the helical mimicry algorithm.

Homologies based on structural motifs characterize conserved structures and mechanisms of maintaining function. An algorithm was developed to quantitate homology among segments of two proteins based upon structural characteristics of an amphipathic alpha-helix. This helical mimicry algorithm scored homology among sequences of two proteins in terms of: (i) presence of Leu, Ile, Val, Phe, or Met in a longitudinal, hydrophobic strip-of-helix at positions n, n + 4, n + 7, n + 11, etc. in the primary sequence, (ii) identity or chemical similarity of amino acids at intervening positions and (iii) exchanges of amino acids from positions n to n - 1, n + 3, n + 4, n + 1, n - 3, n - 4 around n (on the surface of a putative helix). While such exchanges of amino acids on the surfaces of homologous helices may conserve function, they did not maintain specific interactions of those residues with apposing groups.

Characterization of the invariant chain C-terminus (Glu183-Glu193) epitope which is obscured in processed Ii, MHC alpha,beta trimers.

The E1 serum was developed against invariant chain peptide Ii (183-193) in order to study the function of the Ii protein which associates with class II MHC alpha,beta chains from time of synthesis until cleavage and release, possibly regulating the binding of antigenic peptides. Subpopulations of Ii, Ii(VIC) and Ii(E1), respectively, were demonstrated by sequential immunodepletions and immunoprecipitations with: (1) VIC-Y1 monoclonal antibody to an N-terminal epitope of Ii, and (2) E1 rabbit antiserum to Ii(183-193). In 3 hr radiolabeled cells, VIC-Y1 recognized Ii, Ii and N- and O-linked glycosylation (IpN, IpO), p41 and co-precipitated class II alpha,beta chains, while E1 recognized Ii, IpN and immature Ii-alpha complex. In 15 min radiolabeled cells, each antibody recognized similar, immature Ii forms without alpha,beta. Urea denaturation of Ii(VIC) rendered the main Ii species but not IpO immunoprecipitable with E1. E1 recognized O-glycanase-treated Ii (VIC). We conclude that the Ii(183-193) epitope was obscured by interactions of Ii with class II alpha,beta chains and by the O-linked glycosylation of Thr187, which may in part regulate association of Ii to class II alpha and beta chains.

Favored and suppressed patterns of hydrophobic and nonhydrophobic amino acids in protein sequences.

Hydrophobic amino acids of the group Leu, Ile, Val, Phe, and Met (LIVFM) are distributed in favored or suppressed patterns within protein sequences. The frequencies of all five-position combinations of [symbol: see text] = LIVFM and [symbol: see text] = non-LIVFM residues were analyzed in 48 proteins of known crystallographic structure. Some motifs were strongly preferred or suppressed; e.g., [symbol: see text] was favored (z = 3.5), while [symbol: see text] was suppressed (z = -3.4). In longer patterns, [symbol: see text] followed by [symbol: see text] and one [symbol: see text] was favored ([symbol: see text], z = 5.1), while conversion of the single hydrophobic residue to a pair was not ([symbol: see text], z = 0.8). Distributions of certain non-LIVFM amino acids around [symbol: see text] positions in strongly favored patterns were also favored or disfavored (Asp, Glu, Lys, Arg, Asn, Cys, Tyr, and Pro; for each magnitude of z > 2.0). While the strongly favored pattern [symbol: see text] was found in both alpha-helical and beta-strand sequences, it associated significantly with alpha-helices (z = 3.6 for the second-position alpha-helical phi and psi angles) but not with beta-strands (z = -1.1). Certain motifs of LIVFM and non-LIVFM residues might be selected if they lead efficiently to the local nucleations hypothesized to characterize molten globule intermediates in the folding of proteins.

Prediction of alpha-helices in proteins with the hydrophobic strip-of-helix template and distributions of other amino acids around the hydrophobic strip.

Upon addition of requirements for highly characteristic distributions of helix-stabilizing residues to an alpha-helix predictor based upon identification of a longitudinal, hydrophobic strip-of-helix pattern, maximal sensitivity and efficiency scores approached 50% levels at a high degree of stringency. The hydrophobic strip-of-helix template ([symbol: see text], joined in a circle) was applied to 247 helices to maximize the strip-of-helix hydrophobicity index (SOHHI; the mean hydrophobicity of residues in [symbol: see text] positions). Statistically significant increases or decreases in the frequencies of certain residues are observed in some [symbol: see text] and [symbol: see text] positions: [symbol: see text] in the helix (including N- and C-terminal [symbol: see text] in the helix): Leu, Ile, Val, Phe, and Met; first [symbol: see text] positions in extensions of the template to surrounding segments: not increased Leu, Ile, Val, Phe, or Met; N-terminal [symbol: see text]: Asp, Glu; C-terminal [symbol: see text] and the first [symbol: see text] after the helix: His, Lys, Arg; N-terminal [symbol: see text]: Asn; C-terminal [symbol: see text]: Gln; smallest residue in longitudinal strip-of-helix: Ala or Val at crossing points between helices. An algorithm was then derived to indicate alpha-helices by merging predictions with templates [symbol: see text] and [symbol: see text], where the SOHHI > or = 3.0 in the Kyte-Doolittle scale. Relative to the baseline prediction using only the template, more elaborate rules using combinations of other structural patterns produced more efficient (49 versus 42%, respectively) but less sensitive (32 versus 42%, respectively) predictions when the prediction was required to overlap substantially with the known helix. These maximal sensitivities and efficiencies imply that some helices may be formed in folding intermediates but are lost upon coalescence of the final form. Better predictions of protein structure from the primary sequence may require modeling of competing interactions of local structures in folding intermediates.

Adsorption and helical coiling of amphipathic peptides on lipid vesicles leads to negligible protection from cathepsin B or cathepsin D.

The processing of antigenic peptides for presentation by MHC molecules to T cells, may depend upon the function of a second, consensus sequence in or near the T cell-presented epitope. One such processing-regulating sequence appears to be composed of amino acids Leu, Ile, Val, Phe, and Met recurring in a fashion to form a longitudinal, hydrophobic strip when the excised peptide is coiled as an alpha-helix. Such a hydrophobic strip-of-helix may: (a) scavenge peptides from lumens onto lipid membranes of digestion vesicles, (b) stabilize peptides there as protease-resistant helices, (c) specify recognition by the antigenic peptide-binding sites of chaperonin proteins, transmembranal transporters, or MHC molecules. By circular dichroism and electron paramagnetic resonance, we demonstrated that peptides with recurrent hydrophobic residues potentially forming longitudinal strips adsorbed to, and partially coiled as helices on, di-O-hexadecyl, D-L-alpha-phosphatidylcholine (DHPC) vesicles. Cathepsin B or cathepsin D cleavages of three such peptides were identified. With either enzyme, it made no significant difference whether a peptide substrate was in solution or bound to vesicles in terms of efficiency and specificity of peptide bond cleavages. We conclude that protease resistance, per se, of membrane-adsorbed, helically coiled peptides is not a major factor in the selection for T cell presentation of epitopes in peptides which have a motif with a longitudinal hydrophobic strip.