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PubMed2004

Gene expression profiling of human lung tissue from smokers with severe emphysema.

The mechanism by which inhaled smoke causes the anatomic lesions and physiologic impairment of chronic obstructive pulmonary disease remains unknown. We used high-density microarrays to measure gene expression in severely emphysematous lung tissue removed from smokers at lung volume reduction surgery (LVRS) and normal or mildly emphysematous lung tissue from smokers undergoing resection of pulmonary nodules. Class prediction algorithms identified 102 genes that accurately distinguished severe emphysema from non-/mildly emphysematous lung tissue. We also defined a number of genes whose expression levels correlated strongly with lung diffusion capacity for carbon monoxide and/or forced expiratory volume at 1 s. Genes related to oxidative stress, extracellular matrix synthesis, and inflammation were increased in severe emphysema, whereas expression of endothelium-related genes was decreased. To identify candidate genes that might be causally involved in the pathogenesis of emphysema, we linked gene expression profiles to chromosomal regions previously associated with chronic obstructive pulmonary disease in genome-wide linkage analyses. Unsupervised hierarchical clustering of the LVRS samples revealed distinct molecular subclasses of severe emphysema, with body mass index as the only clinical variable that differed between the groups. Class prediction models established a set of genes that predicted functional outcome at 6 mo after LVRS. Our findings suggest that the gene expression profiles from human emphysematous lung tissue may provide insight into pathogenesis, uncover novel molecular subclasses of disease, predict response to LVRS, and identify targets for therapeutic intervention.

Aged↗

PubMedDate not supplied

[More efficient investigation in suspected acute coronary syndrome possible. New diagnostic methods can improve the quality of care and save resources].

The immediate evaluation of patients with suspected acute coronary syndrome (ACS) in the emergency department (ED) has remained almost unchanged for decades. At the same time, therapy for established ACS has undergone a remarkable and successful change towards early active intervention. Studies show that 7 out of 10 patients admitted with a suspicion of ACS do not have it, and that 2-5% of the patients with ACS are incorrectly sent home from the ED. With new diagnostic strategies, including e.g. risk prediction algorithms, new blood samples for plaque instability, special investigations like echocardiography, myocardial perfusion imaging and magnetic resonance imaging, as well as the Chest Pain Unit concept, improvements should definitely be possible. With the structured and evidence-based use of such strategies, it is our belief that more patients can be managed as outpatients, that length of stay can be shortened for those admitted, and that some patients with ACS can get an earlier adequate intervention.

Acute Disease↗

PubMed2003

A CTL epitope from human cytomegalovirus IE1 defined by combining prediction of HLA binding and proteasomal processing is the target of dominant immune responses in patients after allogeneic stem cell transplantation.

OBJECTIVE AND METHODS: In an attempt to define HCMV IE1-derived, HLA-A(*)0201-restricted epitopes, an advanced computer-based epitope prediction combining HLA binding and proteasomal cleavages in silico was performed. RESULTS: This prediction algorithm clearly confirmed VLEETSVML to be the most likely CTL epitope. By tetramer staining, HCMV pp65 NLVPMVATV-specific CD8(+) T cells were detectable in 18/24 HCMV seropositive HLA-A(*)0201-expressing individuals (median frequency 0.58%; range 0.1%-4.7%), and IE1 VLEETSVML-specific CD8(+) T cells in 5/24 (median frequency 2.1%; range 0.1%-4.3%), respectively (p<0.01). Also in recipients of an allogeneic SCT, VLEETSVML- and NLVPMVATV-specific CD8(+) T cells were detectable in comparable frequencies, but again the number of patients with detectable pp65-specific CD8(+) T cells was higher (p=0.014). In 4/15 individuals, all demonstrating IE1 VLEETSVML-specific CD8(+) T cells prior to peptide stimulation, VLEETSVML-specific T cell lines (purity of 42.6%-98.6% of all CD3(+)/CD8(+) T cells) were successfully generated after 2-4 weeks of culture using the IFN-gamma secretion assay. CONCLUSION: In conclusion, this novel prediction strategy efficiently predicted an immunodominant viral T-cell epitope.

Adult↗

PubMed2005

Experimental trial for diagnosis of pancreatic ductal carcinoma based on gene expression profiles of pancreatic ductal cells.

Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies, mainly because of the lack of sensitive detection methods. Although gene expression profiling by DNA microarray analysis is a promising tool for the development of such detection systems, a simple comparison of pancreatic tissues may yield misleading data that reflect only differences in cellular composition. To directly compare PDC cells with normal pancreatic ductal cells, we purified MUC1-positive epithelial cells from the pancreatic juices of 25 individuals with a normal pancreas and 24 patients with PDC. The gene expression profiles of these 49 specimens were determined with DNA microarrays containing >44 000 probe sets. Application of both Welch's analysis of variance and effect size-based selection to the expression data resulted in the identification of 21 probe sets corresponding to 20 genes whose expression was highly associated with clinical diagnosis. Furthermore, correspondence analysis and 3-D projection with these probe sets resulted in separation of the transcriptomes of pancreatic ductal cells into distinct but overlapping spaces corresponding to the two clinical classes. To establish an accurate transcriptome-based diagnosis system for PDC, we applied supervised class prediction algorithms to our large data set. With the expression profiles of only five predictor genes, the weighted vote method diagnosed the class of samples with an accuracy of 81.6%. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC.

Carcinoma, Pancreatic Ductal↗

PubMed2025

Unraveling the complex genetic landscape of OTOF-related hearing loss: a deep dive into cryptic variants and haplotype phasing.

BACKGROUND: Pathogenic variants in OTOF are a major cause of auditory synaptopathy. However, challenges remain in interpreting OTOF variants, including difficulties in confirming haplotype phasing using traditional short-read sequencing (SRS) due to the large gene size, the potential incomplete penetrance of certain variants, and difficulties in assessing variants at non-canonical splice sites. This study aims to revisit the genetic landscape of OTOF variants in a Taiwanese non-syndromic auditory neuropathy spectrum disorder (ANSD) cohort using a combination of sequencing technologies, predictive tools, and experimental validations. METHODS: We performed SRS to analyze OTOF variants in 65 unrelated Taiwanese patients diagnosed with non-syndromic ANSD, complemented by long-read sequencing (LRS) for haplotype phasing. A prediction-to-validation pipeline was implemented to assess the pathogenicity of cryptic variants using SpliceAI software and minigene assays. RESULTS: Biallelic pathogenic OTOF variants were identified in 33 patients (50.8%), while monoallelic variants were found in five patients. Three novel variants, c.3864G > A (p.Ala1288 =), c.4501G > A (p.Ala1501Thr), and c.5813 + 2T > C, were detected. The pathogenicity of two non-canonical mis-splicing variants, c.3894 + 5G > C and c.3864G > A (p.Ala1288 =), was confirmed by minigene assays. LRS-based haplotype phasing revealed that the common missense variant c.5098G > C (p.Glu1700Gln) and the novel variant c.5975A > G (p.Lys1992Arg) are in cis and form a founder pathogenic allele in the Taiwanese population. CONCLUSIONS: Our study highlights the genetic heterogeneity of DFNB9 and emphasizes the importance of population-specific variant interpretation. The integration of advanced sequencing technologies, predictive algorithms, and functional validation assays will improve the accuracy of molecular diagnosis and inform personalized treatment strategies for individuals with DFNB9.

Humans↗

PubMed1999

Functional complementation analysis of yeast bc1 mutants. A study of the mitochondrial import of heterologous and hybrid proteins.

Previous complementation studies with yeast bc1 mutants, defective in subunit VII or VIII, using heterologous and hybrid subunits, suggested that the requirement for import into mitochondria might significantly restrict the scope of this test for compatible proteins. Prediction algorithms indicate that the N-terminal domain of subunit VII contains all known characteristics of a mitochondrial targeting signal, whereas in subunit VIII such a signal is absent from the N-terminal domain, but possibly present in an internal region of the protein. Despite the fact that the characteristics of a mitochondrial import signal are found in the N-terminus of all known subunit-VII orthologues, in vitro import experiments show that the protein of human origin is not imported into yeast mitochondria. In vitro import can be restored, however, by replacement of the N-terminal part of the human protein by the N-terminus of the Saccharomyces cerevisiae orthologue, indicating a requirement for species-specific elements. Similar experiments were performed with subunit VIII and orthologues thereof, including a hybrid protein in which the N-terminus of the bovine heart orthologue was replaced by that of S. cerevisiae. The ability of yeast mitochondria to import this hybrid protein, in contrast with the bovine subunit-VIII orthologue itself, indicates that for subunit VIII also the N-terminus, in contradiction of theoretical predictions, contributes to the targeting signal, most likely via species-specific elements. Our findings expose the limitations of the currently available criteria for prediction of the presence and location of a mitochondrial targeting sequence and highlight the necessity of performing separate import studies for interpreting complementation studies as long as the species-specific characteristics of the import signals have not been identified.

Amino Acid Sequence↗

PubMed2005

Structural organization and interactions of transmembrane domains in tetraspanin proteins.

BACKGROUND: Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. RESULTS: Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. CONCLUSION: Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that defines TM1-TM2 interaction in tetraspanins is the specific packing of bulky residues against small residues.

Algorithms↗

PubMed1997

Solution conformation of a peptide corresponding to bovine kappa-casein B residues 130-153 by circular dichroism spectroscopy and 1H-nuclear magnetic resonance spectroscopy.

The peptide Pro130-Thr-Ser-Thr-Pro-Thr-Ile-Glu-Ala-Val-Glu140- Ser-Thr-Val-Ala-Thr-Leu-GLu-Ala-Ser-Pro150-Glu-Val-Ile, which corresponds to residues 130-150 of kappa-casein B, was synthesized and the conformation of the peptide in solution investigated by circular dichroism (CD) spectroscopy, structure prediction algorithms and 1H-nuclear magnetic resonance spectroscopy. In a solution containing the structure-enhancing solvent trifluoroethanol the CD spectrum was typical of a peptide in the alpha-helical conformation and nuclear magnetic resonance showed that the amino acids between Ile136 and Ser149 (kappa-casein numbering) were predominantly in the alpha-helical conformation but that Pro130 to Thr135 and Pro150 to Ile153 were not. In addition, Thr133-Pro134 and Ser-149-Pro150 were primarily in the trans conformation, the residues from Thr131 to Thr135 were in unordered structures and the residues from Glu151 to Ile153 were in an extended conformation. Residues Glu137 to Glu140 and Thr145 to Ala148 also displayed some 3(10)-helix character. When the peptide was dissolved in 10 mM-cetyltrimethylammonium chloride solution at pH 6, the CD spectra indicated that the proportion of helical structure was comparable to that of the peptide in trifluoroethanol solution (400 ml/l), whereas when the peptide was dissolved in buffer alone in 10 mM-SDS solution, the CD spectra were consistent with a low helical content. Acidification of these solutions to pH 2.85 resulted in a slight increase in the helical content of the peptide in buffer and more markedly in buffer containing SDS. When the peptide was in 5 mM-CaCl2 solution at neutral pH, the CD spectrum indicated that some ordered structure was present. Taken together these results indicate that the ionizable residues Glu137, Glu140, Glu147 and Glu151 could be important in determining the stability of the putative helix. The structure predictions found that the sequence from Glu137 to Pro150 would be more likely to be in a helical than any other conformation in the intact bovine protein, but that pig, sheep and goat kappa-caseins did not give a prediction of a strongly helical region in this part of the molecule.

Amino Acid Sequence↗

PubMed1996

Evaluation of a pencil-beam dose calculation technique for charged particle radiotherapy.

PURPOSE: The purpose of this article is to evaluate a pencil-beam dose calculation algorithm for protons and heavier charged particles in complex patient geometries defined by computed tomography (CT) data and to compare isodose distributions calculated with the new technique to those calculated with conventional algorithms in selected patients with skull-base tumors. METHODS AND MATERIALS: Monte Carlo calculations were performed to evaluate the pencil-beam algorithm in patient geometries for a modulated 150-MeV proton beam. A modified version of a Monte Carlo code described in a previous publication (18) was used for these comparisons. Tissue densities were inferred from patient CT data on a voxel-by-voxel basis, and calculations were performed with and without tissue compensators. A dose calculation module using the new algorithm was written, and treatment plans using the new algorithm were compared to plans using standard ray-tracing techniques for 10 patients with clival chordoma and three patients with nasopharyngeal carcinoma who were treated with helium lons at Lawrence Berkeley National Laboratory (LBL). RESULTS: Pencil beam calculations agreed well with Monte Carlo calculations in the patient geometries. The pencil-beam algorithm predicted several multiple-scattering effects that are not modeled by conventional ray-tracing calculations. These include (a) the widening of the penumbra as a function of beam penetration, (b) the degradation in the sharpness of the dose gradient at the end of the particle range in highly heterogeneous regions, and (c) the appearance of hot and cold dose regions in the shadow of complex heterogeneities. In particular, pencil-beam calculations indicated that the dose distribution within the target was not as homogeneous as expected on the basis of ray-tracing calculations. On average, for the 13 patients considered, only about 72% of the conedown target volume received at least 99% of the prescribed dose, whereas, 93% of the conedown volume was contained within the 95% isodose surface. This may be significant because in standard charged particle dose calculations, the dose across the spread-Bragg peak is assumed to be uniform and equal to the maximum or prescribed dose. CONCLUSIONS: Dose distributions computed with the pencil-beam model are more accurate than ray-tracing calculations, providing additional information to clinicians, which may influence the doses they prescribe. In particular, these calculations indicate that for some patients with skull-base tumors, it may be advantageous to prescribe proton doses to a lower isodose level than is commonly done.

Algorithms↗

PubMed2004

Predictive value for the Chinese population of the Framingham CHD risk assessment tool compared with the Chinese Multi-Provincial Cohort Study.

CONTEXT: The Framingham Heart Study helped to establish tools to assess coronary heart disease (CHD) risk, but the homogeneous nature of the Framingham population prevents simple extrapolation to other populations. Recalibration of Framingham functions could permit various regions of the world to adapt Framingham tools to local populations. OBJECTIVE: To evaluate the performance of the Framingham CHD risk functions, directly and after recalibration, in a large Chinese population, compared with the performance of the functions derived from the Chinese Multi-provincial Cohort Study (CMCS). DESIGN, SETTING, AND PARTICIPANTS: The CMCS cohort included 30 121 Chinese adults aged 35 to 64 years at baseline. Participants were recruited from 11 provinces and were followed up for new CHD events from 1992 to 2002. Participants in the Framingham Heart Study were 5251 white US residents of Framingham, Mass, who were 30 to 74 years old at baseline in 1971 to 1974 and followed up for 12 years. MAIN OUTCOME MEASURES: "Hard" CHD (coronary death and myocardial infarction) was used as the end point in comparisons of risk factors (age, blood pressure, smoking, diabetes, total cholesterol, and high-density lipoprotein cholesterol [HDL-C]) as evaluated by the CMCS functions, original Framingham functions, and recalibrated Framingham functions. RESULTS: The CMCS cohort had 191 hard CHD events and 625 total deaths vs 273 CHD events and 293 deaths, respectively, for Framingham. For most risk factor categories, the relative risks for CHD were similar for Chinese and Framingham participants, with a few exceptions (ie, age, total cholesterol of 200-239 mg/dL [5.18-6.19 mmol/L], and HDL-C less than 35 mg/dL [0.91 mmol/L] in men; smoking in women). The discrimination using the Framingham functions in the CMCS cohort was similar to the CMCS functions: the area under the receiver operating characteristic curve was 0.705 for men and 0.742 for women using the Framingham functions vs 0.736 for men and 0.759 for women using the CMCS functions. However, the original Framingham functions systematically overestimated the absolute CHD risk in the CMCS cohort. For example, in the 10th risk decile in men, the predicted rate of CHD death was 20% vs an actual rate of 3%. Recalibration of the Framingham functions using the mean values of risk factors and mean CHD incidence rates of the CMCS cohort substantially improved the performance of the Framingham functions in the CMCS cohort. CONCLUSIONS: The original Framingham functions overestimated the risk of CHD for CMCS participants. Recalibration of the Framingham functions improved the estimates and demonstrated that the Framingham model is useful in the Chinese population. For regions that have no established cohort, recalibration using CHD rates and risk factors may be an effective method to develop CHD risk prediction algorithms suited for local practice.

Adult↗

PubMed1997

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy.

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a 15N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an alpha-helix content of approximately 42%. The alpha-helix comprises an uninterrupted hydrophobic stretch of < or = 12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15-75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled 15N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of 15N solid-state NMR spectroscopy (10(-4) seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed.

Amino Acid Sequence↗

PubMed2000

A model for type II collagen fibrils: distinctive D-band patterns in native and reconstituted fibrils compared with sequence data for helix and telopeptide domains.

The periodical D-band pattern is generally considered a unique ultrastructural feature shared by all fibril-forming collagens, which correlates with the intrafibril, paracrystalline array of tropocollagen monomers. Distinct band patterns have been reported, however, for collagen stained long-spacing (SLS) crystallites of genetic types I, II, and III. Moreover, D-band patterns of negatively stained, native type II collagen fibrils were found to be not identical to those of type I in our previous research. Because of (a) these distinctive features, (b) tropocollagen heterotrimeric conditions (type I) vs homotrimeric conditions (type II), and (c) different lengths and poor homology between extrahelical telopeptides, the molecular array or telopeptide conformation within the extensively studied type I collagen fibrils could be not the same as those in the very much less intensively studied type II collagen fibrils. In this investigation, a distinctive positive-staining D-band pattern was found for type II collagen fibrils obtained from human cartilages. A fibril model was developed by analyzing actual D-band patterns, and matching them against simulated patterns based on the primary structure of extrahelical and helical domains in human type II tropocollagen. In particular, a more prominent b(1) band was apparent in native type II collagen fibrils than in type I. This distinctive feature was also observed for native-type collagen fibrils reconstituted from purified type II collagen, i.e., free from associated minor type XI collagen. On modeling possible monomer arrays, the best fit between microdensitograms and simulation traces was found for 234 amino acid staggering, as is also the case for type I collagen fibrils. On comparing this model with an analogous one for type I collagen fibrils, there was a higher intraband distribution of charged residues for band b(1), consistent with the higher electrondensity observed for this band in type II collagen fibrils. N- and C-telopeptide displacement in the model corresponded to D-locations of a c(2) subband, which we named c(2.0), and band a(3), respectively. In simulation profiles, c(2.0) -like and a(3) -like peaks mimicked the corresponding peaks in microdensitograms when molecular reversals were adopted at positions 10N-12N, 12C-14C, and 17C-19C for N- and C-telopeptides. Hydrophobic interactions and algorithmic predictions of protein secondary structure, according to Chou and Fasman and Rost and Sander criteria, were consistent with these conformational models, and suggest that an additional molecular reversal may occur at positions 3N-5N. These telopeptide "S-fold" conformations, interpreted as axial projections of tridimensional conformation, may represent starting points for further investigation into the still unresolved tridimensional conformation of telopeptides in monomers arrayed within type II collagen fibrils.

Amino Acid Sequence↗

PubMed2000

Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1.

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two-dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane-spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell-cell signalling and pathogen-host interactions in a variety of P. aeruginosa strains.

Bacterial Proteins↗

PubMed2007

Recent progress in engineering alpha/beta hydrolase-fold family members.

The members of the alpha/beta hydrolase-fold family represent a functionally versatile group of enzymes with many important applications in biocatalysis. Given the technical significance of alpha/beta hydrolases in processes ranging from the kinetic resolution of enantiomeric precursors for pharmaceutical compounds to bulk products such as laundry detergent, optimizing and tailoring enzymes for these applications presents an ongoing challenge to chemists, biochemists, and engineers alike. A review of the recent literature on alpha/beta hydrolase engineering suggests that the early successes of "random processes" such as directed evolution are now being slowly replaced by more hypothesis-driven, focused library approaches. These developments reflect a better understanding of the enzymes' structure-function relationship and improved computational resources, which allow for more sophisticated search and prediction algorithms, as well as, in a very practical sense, the realization that bigger is not always better.

Epoxide Hydrolases↗

PubMed1990

Conformational analysis of the immunodominant epitopes of the circumsporozoite protein of Plasmodium falciparum and knowlesi.

All of the coat proteins of the sporozoite and merozoite stages of Plasmodium, determined to date, contain tandem repeats and most of these contain at least one proline residue. These tandemly repeated segments of the circumsporozite (CS) proteins of P. falciparum and P. knowlesi have been shown to constitute an immunodominant epitope. Antibodies to these peptide segments have been shown to be protective and cause the shedding of the CS protein, known as the CSP reaction. In this study, four synthetic peptides were prepared by solid-phase peptide synthesis. The first peptide corresponds to the tetrapeptide tandem repeat in the CS protein of P. falciparum, repeated eight times, (NANP)8. The second peptide is an analogue of the first in which glycine is substituted for proline, (NANG)8. The third peptide corresponds to the tandem repeat of P. knowlesi, PK(1-24), which is repeated twice (QAQGDGANAGQP)2. The fourth peptide is a tetrapeptide repeat, corresponding to the C-terminal tetrapeptide of PK(1-24) and is repeated eight times, (AGQP)8. It is shown by CD measurements that the presence of proline in these repeats induces an increase in beta-sheet (beta-turn) content in the (NANP)8 peptide relative to the repeat of (NANG)8 and PK(1-24) peptide in aqueous media. The (AGQP)8 peptide has the highest beta-sheet (beta-turn) content of all peptides studied. The Chou-Fasman predictive algorithm indicates a high beta-turn content in the synthetic peptides. It is concluded that this increase in defined structure correlates well with and hence may contribute to the increased antigenicity in these repeats.

Amino Acids↗

PubMed2003

Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data.

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.

Algorithms↗

PubMed1996

Specificity of an HLA-DRB1*0401-restricted T cell response to type II collagen.

A panel of HLA-DRB1*0401-restricted CD4+ mouse T cell hybridomas specific for bovine type II collagen were generated from transgenic mice expressing the human HLA-DRA1*0101/-DRB1*0401 and CD4 molecules. The vast majority recognized a single peptide determinant corresponding to residues 261-273 (CII 261-273). This determinant was rapidly defined by the use of a predictive algorithm for peptide binding to DRB1*0401. CII 261-273 is conserved in bovine and human type II collagen and overlaps with an important I-A q - restricted T cell determinant in mice with collagen-induced arthritis. This study demonstrates how HLA-DR and human CD4-transgenic mice can be used to identify a T cell epitope in a potential or candidate autoantigen.

Amino Acid Sequence↗

PubMed2004

Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1.

Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer-dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein.

Alleles↗