The association between apolipoprotein E gene polymorphisms and essential hypertension: a meta-analysis of 45 studies including 13,940 cases and 16,364 controls.

The apolipoprotein E single-nucleotide polymorphisms are among the potential candidate genes that may serve as modulators in susceptibility to essential hypertension. In an effort to clarify earlier inconclusive results, we performed a meta-analysis of population-based case-control genetic association studies. Random-effects methods were applied on summary data in order to combine the results of the individual studies. We identified in total 45 studies, including 13 940 hypertensive cases and 16 364 controls. The contrast of E4 carriers versus non-carriers yielded an overall odds ratio (OR) of 1.16 (95% confidence interval (CI): 1.02, 1.31), whereas the contrast of E4 allele versus the others in a subtotal of 6617 cases and 7330 controls, yielded an OR of 1.39 (95% CI: 1.12, 1.72). There was moderate evidence of publication bias in both contrasts, which was eliminated after excluding studies not in Hardy-Weinberg equilibrium. Subgroup analyses revealed that significant estimates arose from studies on Asian populations, as opposed to the Caucasian ones. Furthermore, no evidence of publication bias was demonstrated in the comparisons within this subgroup. Our results are consistent with recent meta-analyses but show that the association is weaker than that has been previously demonstrated. Further studies are needed in order to fully address questions about the etiological mechanism of the particular association, as well as to study the effect in populations of African descent.

Secondary structure of chorion proteins of the Lepidoptera Pericallia ricini and Ariadne merione by ATR FT-IR and micro-Raman spectroscopy.

The gross morphological features of the eggs and eggshells (chorions) of two Lepidoptera species, Pericallia ricini and Ariadne merione were revealed for the first time by scanning and transmission electron microscopy. These two insect pests are extremely serious threats for many crops, mainly in India, but also in several other regions of the world. Micro-Raman and ATR FT-IR spectroscopy were also applied to study in detail the secondary structure of the eggshell (chorion) proteins of these Lepidoptera species. Both techniques indicate that the two species have nearly identical conformations of their chorion proteins with abundant antiparallel ß-pleated sheet. These results are in support of our previous findings that the helicoidal architecture of the proteinaceous chorion of Lepidoptera and fishes is dictated by a common molecular denominator, the antiparallel ß-pleated sheet secondary structure.

Apolipoprotein E polymorphisms and type 2 diabetes: a meta-analysis of 30 studies including 5423 cases and 8197 controls.

Type 2 diabetes mellitus (T2DM) is a highly complicated metabolic disorder for which there is worldwide effort for the identification of susceptibility genes. Polymorphisms of the Apolipoprotein E (ApoE) gene are associated with plasma lipid and lipoprotein levels and influence cardiovascular risk. Since insulin resistance is known to be strongly associated with metabolic dyslipidemia, ApoE polymorphisms have been implicated in predisposition to diabetes but the results of the individual studies were inconclusive. We present here a meta-analysis of population-based case-control genetic-association studies relating ApoE polymorphisms and T2DM. We included in the analysis 30 studies, which reported data of ApoE genotypes in 5423 T2DM patients and 8197 healthy unrelated controls. Multivariate and univariate methods suggest a significant role played by the E2 allele, since carriers of the E2 allele were at elevated risk for T2DM (Odds Ratio=1.18, 95% CI: 1.02, 1.35). There was no evidence for publication bias or other small-study related bias or significant heterogeneity in the analyses. Cumulative meta-analysis revealed no trend of the effect estimates over time and influential analysis excluded the possibility of a single influential study. E2 allele of ApoE seems to be a moderate risk factor for T2DM. Meta-regression analysis provided some weak evidence that the risk conferred by E2 allele is mediated through altering serum lipid levels (Total Cholesterol, LDL and HDL). Further studies are needed in order to elucidate the metabolic mechanism of this association as well as to study its effects on larger populations.

Prediction of signal peptides in archaea.

Computational prediction of signal peptides (SPs) and their cleavage sites is of great importance in computational biology; however, currently there is no available method capable of predicting reliably the SPs of archaea, due to the limited amount of experimentally verified proteins with SPs. We performed an extensive literature search in order to identify archaeal proteins having experimentally verified SP and managed to find 69 such proteins, the largest number ever reported. A detailed analysis of these sequences revealed some unique features of the SPs of archaea, such as the unique amino acid composition of the hydrophobic region with a higher than expected occurrence of isoleucine, and a cleavage site resembling more the sequences of gram-positives with almost equal amounts of alanine and valine at the position-3 before the cleavage site and a dominant alanine at position-1, followed in abundance by serine and glycine. Using these proteins as a training set, we trained a hidden Markov model method that predicts the presence of the SPs and their cleavage sites and also discriminates such proteins from cytoplasmic and transmembrane ones. The method performs satisfactorily, yielding a 35-fold cross-validation procedure, a sensitivity of 100% and specificity 98.41% with the Matthews' correlation coefficient being equal to 0.964. This particular method is currently the only available method for the prediction of secretory SPs in archaea, and performs consistently and significantly better compared with other available predictors that were trained on sequences of eukaryotic or bacterial origin. Searching 48 completely sequenced archaeal genomes we identified 9437 putative SPs. The method, PRED-SIGNAL, and the results are freely available for academic users at http://bioinformatics.biol.uoa.gr/PRED-SIGNAL/ and we anticipate that it will be a valuable tool for the computational analysis of archaeal genomes.

PRED-GPCR: GPCR recognition and family classification server.

The vast cell-surface receptor family of G-protein coupled receptors (GPCRs) is the focus of both academic and pharmaceutical research due to their key role in cell physiology along with their amenability to drug intervention. As the data flow rate from the various genome and proteome projects continues to grow, so does the need for fast, automated and reliable screening for new members of the various GPCR families. PRED-GPCR is a free Internet service for GPCR recognition and classification at the family level. A submitted sequence or set of sequences, is queried against the PRED-GPCR library, housing 265 signature profile HMMs corresponding to 67 well-characterized GPCR families. Users query the server through a web interface and results are presented in HTML output format. The server returns all single-motif matches along with the combined results for the corresponding families. The service is available online since October 2003 at http://bioinformatics.biol.uoa.gr/PRED-GPCR.

Amyloid fibrillogenesis of silkmoth chorion protein peptide-analogues via a liquid-crystalline intermediate phase.

Chorion, the major component of silkmoth eggshell, consists of the A and B classes of low-molecular weight structural proteins. Chorion protects the oocyte and the developing embryo from environmental hazards and this is due to the extraordinary physical and chemical properties of its constituent proteins. We have shown previously [FEBS Lett. 479 (2000) 141; 499 (2001) 268] that peptide-analogues of the A and B classes of chorion proteins form amyloid fibrils under a variety of conditions, which led us to propose that silkmoth chorion is a natural, protective amyloid. In this work, we present data showing conclusively that, the first main step of amyloid-like fibrillogenesis of chorion peptides is the formation of nuclei of liquid crystalline nature, which is reminiscent of spider-silk formation. We show that these liquid-crystalline nuclei (spherulites) 'collapse'/deteriorate to form amyloid fibrils in a spectacular manner, important, it seems, for chorion morphogenesis and amyloid fibrillogenesis in general. The molecular 'switch' causing this spectacular transformation is, most probably, a conformational transition to the structure of chorion peptides, from a left-handed parallel beta-helix to an antiparallel beta-pleated sheet. Apparently, these peptides were suitably designed to play this role, after millions of years of molecular evolution.

A novel method for GPCR recognition and family classification from sequence alone using signatures derived from profile hidden Markov models.

G-protein coupled receptors (GPCRs) constitute a broad class of cell-surface receptors, including several functionally distinct families, that play a key role in cellular signalling and regulation of basic physiological processes. GPCRs are the focus of a significant amount of current pharmaceutical research since they interact with more than 50% of prescription drugs, whereas they still comprise the best potential targets for drug design. Taking into account the excess of data derived by genome sequencing projects, the use of computational tools for automated characterization of novel GPCRs is imperative. Typical computational strategies for identifying and classifying GPCRs involve sequence similarity searches (e.g. BLAST) coupled with pattern database analysis (e.g. PROSITE, BLOCKS). The diagnostic method presented here is based on a probabilistic approach that exploits highly discriminative profile Hidden Markov Models, excised from low entropy regions of multiple sequence alignments, to derive potent family signatures. For a given query, a P-value is obtained, combining individual hits derived from the same family. Hence a best-guess family membership is depicted, allowing GPCRs' classification at a family level, solely using primary structure information. A web-based version of the application is freely available at URL: http:/bioinformatics.biol.uoa.gr/PRED-GPCR.

A Web-based classification system of DNA-binding protein families.

Rational classification of proteins encoded in sequenced genomes is critical for making the genome sequences maximally useful for functional and evolutionary studies. The family of DNA-binding proteins is one of the most populated and studied amongst the various genomes of bacteria, archaea and eukaryotes and the Web-based system presented here is an approach to their classification. The DnaProt resource is an annotated and searchable collection of protein sequences for the families of DNA-binding proteins. The database contains 3238 full-length sequences (retrieved from the SWISS-PROT database, release 38) that include, at least, a DNA-binding domain. Sequence entries are organized into families defined by PROSITE patterns, PRINTS motifs and de novo excised signatures. Combining global similarities and functional motifs into a single classification scheme, DNA-binding proteins are classified into 33 unique classes, which helps to reveal comprehensive family relationships. To maximize family information retrieval, DnaProt contains a collection of multiple alignments for each DNA-binding family while the recognized motifs can be used as diagnostically functional fingerprints. All available structural class representatives have been referenced. The resource was developed as a Web-based management system for online free access of customized data sets. Entries are fully hyperlinked to facilitate easy retrieval of the original records from the source databases while functional and phylogenetic annotation will be applied to newly sequenced genomes. The database is freely available for online search of a library containing specific patterns of the identified DNA-binding protein classes and retrieval of individual entries from our WWW server (http://kronos.biol.uoa.gr/~mariak/dbDNA.html).

A novel tool for the prediction of transmembrane protein topology based on a statistical analysis of the SwissProt database: the OrienTM algorithm.

OrienTM is a computer software that utilizes an initial definition of transmembrane segments to predict the topology of transmembrane proteins from their sequence. It uses position-specific statistical information for amino acid residues which belong to putative non-transmembrane segments derived from statistical analysis of non-transmembrane regions of membrane proteins stored in the SwissProt database. Its accuracy compares well with that of other popular existing methods. A web-based version of OrienTM is publicly available at the address http://biophysics.biol.uoa.gr/OrienTM.

"Soft"-cuticle protein secondary structure as revealed by FT-Raman, ATR FT-IR and CD spectroscopy.

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of a beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). In this study, we examined experimentally the secondary structure of intact and guanidine hydrochloride extracted cuticle and the cuticular protein extract. The studied cuticle came from the larval dorsal abdomen of the lepidopteran Hyalophora cecropia, a classical example of "soft" cuticle. Analysis with FT-Raman, ATR FT-IR and CD spectroscopy indicates that antiparallel beta-pleated sheet is the predominant molecular conformation of "soft-cuticle" proteins both in situ in the cuticle and following extraction. It seems that this conformation dictates the modes of chitin-protein interaction in cuticle, in agreement with earlier proposals (Atkins, J. Biosci. 8 (1985) 375).

PRED-CLASS: cascading neural networks for generalized protein classification and genome-wide applications.

A cascading system of hierarchical, artificial neural networks (named PRED-CLASS) is presented for the generalized classification of proteins into four distinct classes-transmembrane, fibrous, globular, and mixed-from information solely encoded in their amino acid sequences. The architecture of the individual component networks is kept very simple, reducing the number of free parameters (network synaptic weights) for faster training, improved generalization, and the avoidance of data overfitting. Capturing information from as few as 50 protein sequences spread among the four target classes (6 transmembrane, 10 fibrous, 13 globular, and 17 mixed), PRED-CLASS was able to obtain 371 correct predictions out of a set of 387 proteins (success rate approximately 96%) unambiguously assigned into one of the target classes. The application of PRED-CLASS to several test sets and complete proteomes of several organisms demonstrates that such a method could serve as a valuable tool in the annotation of genomic open reading frames with no functional assignment or as a preliminary step in fold recognition and ab initio structure prediction methods. Detailed results obtained for various data sets and completed genomes, along with a web sever running the PRED-CLASS algorithm, can be accessed over the World Wide Web at http://o2.biol.uoa.gr/PRED-CLASS.

Amyloid-like fibrils from an 18-residue peptide analogue of a part of the central domain of the B-family of silkmoth chorion proteins.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties, and protect the oocyte and the developing embryo from the environment. We present data from negative staining, Congo red binding, X-ray diffraction, Fourier transform-Raman, attenuated total reflectance infrared spectroscopy and modelling studies of a synthetic peptide analogue of a part of the central domain of the B family of silkmoth chorion proteins, indicating that this peptide folds and self-assembles, forming amyloid-like fibrils. These results support further our proposal, based on experimental data from a synthetic peptide analogue of the central domain of the A family of chorion proteins, that silkmoth chorion is a natural, protective amyloid [Iconomidou et al., FEBS Lett. 479 (2000) 141-145].

Amyloids protect the silkmoth oocyte and embryo.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of proteins that have remarkable mechanical and chemical properties protecting the oocyte and the developing embryo from a wide range of environmental hazards. We present data from electron microscopy (negative staining and shadowing), X-ray diffraction and modeling studies of synthetic peptide analogues of silkmoth chorion proteins indicating that chorion is a natural amyloid. The folding and self-assembly models of chorion peptides strongly support the beta-sheet helix model of amyloid fibrils proposed recently by Blake and Serpell [Structure 4 (1996) 989-998].

Secondary structure of chorion proteins of the teleostean fish Dentex dentex by ATR FT-IR and FT-Raman spectroscopy.

FT-Raman spectroscopy and ATR-IR spectroscopy were applied to study the secondary structure of the eggshell (chorion) proteins of the teleostean fish Dentex dentex. Raman and IR spectra clearly indicate an abundance of antiparallel beta-pleated sheet conformation in chorion proteins. This finding is further supported by analysis of the vibrational data by regression techniques and deconvolution procedures. Thus, the common morphological characteristics of D. dentex, Salmo gairdneri, and other teleostean fish chorions may be explained on the basis of common secondary structure features of their constituent proteins. A detailed understanding of the interactions that dictate the self-assembly of fish chorion proteins to form the fish eggshell awaits determination of amino acid sequences.

An hierarchical artificial neural network system for the classification of transmembrane proteins.

This work presents a simple artificial neural network which classifies proteins into two classes from their sequences alone: the membrane protein class and the non-membrane protein class. This may be important in the functional assignment and analysis of open reading frames (ORF's) identified in complete genomes and, especially, those ORF's that correspond to proteins with unknown function. The network described here has a simple hierarchical feed-forward topology and a limited number of neurons which makes it very fast. By using only information contained in 11 protein sequences, the method was able to identify, with 100% accuracy, all membrane proteins with reliable topologies collected from several papers in the literature. Applied to a test set of 995 globular, water-soluble proteins, the neural network classified falsely 23 of them in the membrane protein class (97.7% of correct assignment). The method was also applied to the complete SWISS-PROT database with considerable success and on ORF's of several complete genomes. The neural network developed was associated with the PRED-TMR algorithm (Pasquier,C., Promponas,V.J., Palaios,G.A., Hamodrakas,J.S. and Hamodrakas,S.J., 1999) in a new application package called PRED-TMR2. A WWW server running the PRED-TMR2 software is available at http://o2.db.uoa.gr/PRED-TMR2

Molecular, crystal and solution structure of a beta-cyclodextrin complex with the bromide salt of 2-(3-dimethylaminopropyl)tricyclo[3.3.1.1(3,7)]decan-2-ol, a potent antimicrobial drug.

The pharmacological properties of a cyclomaltoheptaose (beta-cyclodextrin) series of adamantane-group-bearing compounds that exhibit potent antibacterial activity have been studied, both isolated and in complex with beta-cyclodextrins (betaCDs). In this work, the structure of the bromide salt of 2-(3-dimethylaminopropyl)-tricyclo[3.3.1.1(3,7)]decan-2-ol(A DM-10) complexed with betaCD and ten water molecules was studied in the solid state by X-ray crystallography and in solution by NMR spectroscopy. X-ray crystallographic studies of the complex were performed both at room and cryogenic temperatures. The long aliphatic chain of ADM-10 adopts a single conformation at low temperature in contrast to what is observed at room temperature, where two side chain conformations are seen. Both NMR and X-ray crystallography studies indicate that the adamantane moiety of ADM-10 is buried in the betaCD cavity. Chemical shifts in NMR experiments can be explained on the basis of the crystal structure of the complex.

A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm.

We present a novel method that predicts transmembrane domains in proteins using solely information contained in the sequence itself. The PRED-TMR algorithm described, refines a standard hydrophobicity analysis with a detection of potential termini ('edges', starts and ends) of transmembrane regions. This allows one both to discard highly hydrophobic regions not delimited by clear start and end configurations and to confirm putative transmembrane segments not distinguishable by their hydrophobic composition. The accuracy obtained on a test set of 101 non-homologous transmembrane proteins with reliable topologies compares well with that of other popular existing methods. Only a slight decrease in prediction accuracy was observed when the algorithm was applied to all transmembrane proteins of the SwissProt database (release 35). A WWW server running the PRED-TMR algorithm is available at http://o2.db.uoa. gr/PRED-TMR/

Is beta-pleated sheet the molecular conformation which dictates formation of helicoidal cuticle?

Over 100 sequences for cuticular proteins are now available, but there have been no formal analyses of how these sequences might contribute to the helicoidal architecture of cuticle or to the interaction of these proteins with chitin. A secondary structure prediction scheme (Hamodrakas, S.J., 1988. A protein secondary structure prediction scheme for the IBM PC and compatibles. CABIOS 4, 473-477) that combines six different algorithms predicting alpha-helix, beta-strands and beta-turn/loops/coil has been used to predict the secondary structure of chorion proteins and experimental confirmation has established its utility (Hamodrakas, S.J., 1992. Molecular architecture of helicoidal proteinaceous eggshells. In: Case, S.T. (Ed.), Results and Problems in Cell Differentiation, Vol. 19, Berlin-Heidelberg, Springer Verlag, pp. 116-186 and references therein). We have used this same scheme with eight cuticular protein sequences associated with hard cuticles and nineteen from soft cuticles. Secondary structure predictions were restricted to a conserved 68 amino acid region that begins with a preponderance of hydrophilic residues and ends with a 33 amino acid consensus region, first identified by Rebers and Riddiford (Rebers, J.F., Riddiford, L.M., 1988. Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. J. Mol. Biol. 203, 411-423). Both classes of sequences showed a preponderance of beta-pleated sheet, with four distinct strands in the proteins from 'hard' cuticles and three from 'soft'. In both cases, tyrosine and phenylalanine were found on one face within a sheet, an optimal location for interaction with chitin. We propose that this beta-sheet dictates formation of helicoidal cuticle.

CoPreTHi: a Web tool which combines transmembrane protein segment prediction methods.

CoPreTHi is a Java based web application, which combines the results of methods that predict the location of transmembrane segments in protein sequences into a joint prediction histogram. Clearly, the joint prediction algorithm, produces superior quality results than individual prediction schemes. The program is available at http://o2.db.uoa.gr/CoPreTHi.

Laser-Raman and FT-IR spectroscopic studies of peptide-analogues of silkmoth chorion protein segments.

Silkmoth chorion, the proteinaceous major component of the eggshell, with extraordinary mechanical and physiological properties, consists of a complex set of proteins, which have a tripartite structure: a central, evolutionarily conserved, domain and two more variable 'arms'. Peptide-analogues of silkmoth chorion protein central domain segments have been synthesized. Laser-Raman and infrared spectroscopic studies suggest the preponderance of antiparallel beta-pleated sheet structure for these peptides, both in solution and in the solid state.