SEARCH · PubMed Health

Results for “Feature selection stability”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 775 records · Page 43Linked to original sources

PubMed2002

Prediction of food protein allergenicity: a bioinformatic learning systems approach.

Food hypersensitivity is constantly increasing in Western societies with a prevalence of about 1-2% in Europe and in the USA. Among children, the incidence is even higher. Because of the introduction of foods derived from genetically modified crops on the marketplace, the scientific community, regulatory bodies and international associations have intensified discussions on risk assessment procedures to identify potential food allergenicity of the newly introduced proteins. In this work, we present a novel biocomputational methodology for the classification of amino acid sequences with regard to food allergenicity and non-allergenicity. This method relies on a computerised learning system trained using selected excerpts of amino acid sequences. One example of such a successful learning system is presented which consists of feature extraction from sequence alignments performed with the FASTA3 algorithm (employing the BLOSUM50 substitution matrix) combined with the k-Nearest-Neighbour (kNN) classification algorithm. Briefly, the two features extracted are the alignment score and the alignment length and the kNN algorithm assigns the pair of extracted features from an unknown sequence to the prevalent class among its k nearest neighbours in the training (prototype) set available. 91 food allergens from several specialised public repositories of food allergy and the SWALL database were identified, pre-processed, and stored, yielding one of the most extensively characterised repositories of allergenic sequences known today. All allergenic sequences were classified using a standard one-leave-out cross validation procedure yielding about 81% correctly classified allergens and the classification of 367 non-allergens in an independent test set resulted in about 98% correct classifications. The biocomputational approach presented should be regarded as a significant extension and refinement of earlier attempts suggested for in silico food safety assessment. Our results show that the framework described here is powerful enough to become useful as part of a multiple-procedure test scheme that also depicts other evaluation approaches such as solid phase immunoassay and tests for stability to digestions.

Algorithms↗

PubMed1972

Improved experimental model for measuring skin degerming activity on the human hand.

A gloved-hand method is presented for evaluating the interaction of antimicrobial agents with the normal resident bacterial flora of human skin. One of the key features of the experimental model is a simplified technique for sampling the skin, which involves the addition of eluting fluid to the gloved hand. As with other skin sampling techniques, the number of bacteria recovered from the hands showed considerable variation from subject to subject. However, no significant differences were observed between the numbers of bacteria recovered from the right and left hands of individual subjects. The mean number of bacteria recovered from the hand before and after washing with nonmedicated soap was consistent and reproducible over a period of at least 5 consecutive days. The number of recoverable bacteria from the hand was greatly reduced by a single treatment with a surgical scrub preparation containing hexachlorophene. The extent of skin degerming achieved was little affected by the use of a surgical brush, and was maximal at approximately 30 min after contact with the hexachlorophene-containing formulation. It was determined that the level of transient bacteria on the hands could be controlled by a simple wash with nonmedicated soap, resulting in a stabilized base-line level from which treatment interactions with the resident microflora could be measured more precisely. The basic elements of the method presented fulfill the requirements of a satisfactory experimental model for the in vivo evaluation of skin-degerming agents on the hand. The selection of appropriate experimental designs allows treatment comparisons to be made with a high degree of statistical confidence.

Anti-Infective Agents, Local↗

PubMed1998

Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change.

Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.

Animals↗

PubMed2003

Organization and dynamics of tryptophan residues in erythroid spectrin: novel structural features of denatured spectrin revealed by the wavelength-selective fluorescence approach.

We have investigated the organization and dynamics of the functionally important tryptophan residues of erythroid spectrin in native and denatured conditions utilizing the wavelength-selective fluorescence approach. We observed a red edge excitation shift (REES) of 4 nm for the tryptophans in the case of spectrin in its native state. This indicates that tryptophans in spectrin are localized in a microenvironment of restricted mobility, and that the regions surrounding the spectrin tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited state tryptophans. Interestingly, spectrin exhibits a REES of 3 nm even when denatured in 8 M urea. This represents the first report of a denatured protein displaying REES. Observation of REES in the denatured state implies that some of the structural and dynamic features of this microenvironment around the spectrin tryptophans are retained even when the protein is denatured. Fluorescence quenching data of denatured spectrin support this conclusion. In addition, we have deduced the organization and dynamics of the hydrophobic binding site of the polarity-sensitive fluorescent probe PRODAN that binds erythroid spectrin with high affinity. When bound to spectrin, PRODAN exhibits a REES of 9 nm. Because PRODAN binds to a hydrophobic site in spectrin, such a result would directly imply that this region of spectrin offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. The results of our study could provide vital insight into the role of tryptophans in the stability and folding of spectrin.

Animals↗

PubMed2000

A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions.

A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv)(2) (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.

Angiostatins↗

PubMed2005

Synthesis and characterization of water-soluble phenylene-vinylene-based singlet oxygen sensitizers for two-photon excitation.

[reaction: see text] The synthesis and characterization of water-soluble singlet oxygen sensitizers with a phenylene-vinylene motif is presented. The principal motivation for this study was to better understand specific features of a water-soluble molecule that influence the photosensitized production of singlet oxygen upon nonlinear, two-photon excitation of that molecule. To achieve water solubility, sensitizers were synthesized with ionic as well as nonionic substituents. In the ionic approach, salts of N-methylated pyridine, benzothiazole, and 1-methyl-piperazine moieties were used, as were aryl-substituted sulfonic acid moieties. In the nonionic approach, aryl-substituted triethylene glycol moieties were used. Selected photophysical properties of the compounds synthesized were determined, including singlet oxygen quantum yields. Of the molecules examined, the most efficient singlet oxygen sensitizers had triethylene glycol units as the functional group that imparted water solubility. Molecules containing the ionic moieties did not make singlet oxygen in appreciable yield nor did they efficiently fluoresce. Rather, for these latter molecules, rapid charge-transfer-mediated non-radiative processes appear to dominate excited state deactivation.

Drug Stability↗

PubMed2002

RNA loop-loop interactions as dynamic functional motifs.

RNA loop-loop interactions are frequently used to trigger initial recognition between two RNA molecules. In this review, we present selected well-documented cases that illustrate the diversity of biological processes using RNA loop-loop recognition properties. The first one is related to natural antisense RNAs that play a variety of regulatory functions in bacteria and their extra-chromosomal elements. The second one concerns the dimerization of HIV-1 genomic RNA, which is responsible for the encapsidation of a diploid RNA genome. The third one concerns RNA interactions involving double-loop interactions. These are used by the bicoid mRNA to form dimers, a property that appears to be important for mRNA localization in drosophila embryo, and by bacteriophage phi29 pRNA which forms hexamers that participate in the translocation of the DNA genome through the portal vertex of the capsid. Despite the high diversity of systems and mechanisms, some common features can be highlighted. (1) Efficient recognition requires rapid bi-molecular binding rates, regardless of the RNA pairing scheme. (2) The initial recognition is favored by particular conformations of the loops enabling a proper presentation of nucleotides (generally a restricted number) that initiate the recognition process. (3) The fate of the initial reversible loop-loop complex is dictated by both functional and structural constraints. RNA structures have evolved either to "freeze" the initial complex, or to convert it into a more stable one, which involves propagation of intermolecular interactions along topologically feasible pathways. Stabilization of the initial complex may also be assisted by proteins and/or formation of additional contacts.

Animals↗

PubMed1983

Designing prostacyclin analogues.

A series of prostacyclin analogues were synthesized and investigated for influence on blood pressure in rats, in vivo inhibition of platelet aggregation in rats, and in vitro inhibition of platelet aggregation in human platelet-rich plasma. The common feature of the analogues described is a replacement of C1-C4 of prostacyclin by a carboxyphenylene residue. The following structure-activity relationships were obtained. Only the meta-carboxyphenylene derivatives yield substantial prostacyclin activity. The 2,3,4-trinor-1,5-inter-m-phenylene prostacyclin analogues in contrast to the natural prototype are reasonably stable against hydrolysis of the enolether bond. The corresponding 2,3,4-trinor-1,5-inter-m-phenylene analogues of carbaprostacyclin have a somewhat lower specific activity but are superior in stability at acid pH values. With regard to the stereoisomerism at the delta 5 double bond, the Z-isomers of the oxa-cyclic prostacyclin series and the E-isomers of the carba-cyclic prostacyclin series are substantially more active than their counterparts. As with natural prostacyclin, the OH group at C15 has to be present in S-configuration. The "wrong" isomers do not inhibit prostacyclin-dependent effects. Resistance against 15-hydroxyprostaglandin dehydrogenase is achieved by substitutions at or near C15. Optimum specific activity combined with resistance against all known prostaglandin-activating enzymes is observed in prostacyclin and carbaprostacyclin analogues, in which the terminal n-pentyl residue is replaced by cyclohexyl. Duration of action, i.e. lowering of blood pressure in anaesthesized rats and inhibition of platelet aggregation in anaesthesized rats, was investigated with selected analogues in order to check the consequences of chemical and metabolic stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1996

Model assessment and refinement using strategies from biochemical systems theory: application to metabolism in human red blood cells.

Models of biochemical systems are typically formulated with kinetic data obtained from isolated enzymes studied in vitro, and one has always to question whether or not all the relevant metabolites, processes and regulatory interactions have been identified and whether the parameter values obtained in vitro reflect the actual intracellular environment. In this paper we extend and further test strategies for model assessment and refinement that take advantage of the power-law formalism, which provides the systematic structure underlying biochemical systems theory. Our purpose is three fold. First, we introduce an algorithm for systematically scanning a model for putative errors, which, if corrected, would reconcile its behavior with the experimental system. Second, we further test the working hypothesis that systems in nature are selected to be robust and, hence, that the profile of parameter sensitivities can be used to identify poorly defined regions of a model. Third, we illustrate the use of these strategies within the context of a relatively large and realistic biochemical system--the metabolic pathways of the human red blood cell. Our results show that the reference model we have used is neither locally stable nor robust. The algorithm identifies a number of putative regulatory interactions that, when added to the model, are capable of stabilizing the nominal steady state. We include one of these, the feedback inhibition of hexokinase by fructose-6-phosphate, in a first refinement of the model because there is experimental support for it in the literature. Careful re-examination of the most sensitive section in this model, the pathways of nucleotide metabolism, reveals two mechanisms that were omitted from the reference model: membrane transport of adenosine and inosine, and regulation of phosphoribosyl pyrophosphate synthetase by adenosine diphosphate, 2,3 diphosphoglycerate and 5-phosphoribosyl-1-pyrophosphate. It was also found that the concentration of inorganic phosphate had been inappropriately assumed to be a constant. Modifications to correct these deficiencies produced a second refinement of the model whose parameter sensitivities are reduced on average by 10-fold. Although these refinements are modest and there is substantial room for further improvement, this application identified several biochemically relevant features of the model that had been overlooked. It also points to nucleotide metabolism as the area most in need of further experimental study.

Algorithms↗

PubMed2004

Random dynamics of the Morris-Lecar neural model.

Determining the response characteristics of neurons to fluctuating noise-like inputs similar to realistic stimuli is essential for understanding neuronal coding. This study addresses this issue by providing a random dynamical system analysis of the Morris-Lecar neural model driven by a white Gaussian noise current. Depending on parameter selections, the deterministic Morris-Lecar model can be considered as a canonical prototype for widely encountered classes of neuronal membranes, referred to as class I and class II membranes. In both the transitions from excitable to oscillating regimes are associated with different bifurcation scenarios. This work examines how random perturbations affect these two bifurcation scenarios. It is first numerically shown that the Morris-Lecar model driven by white Gaussian noise current tends to have a unique stationary distribution in the phase space. Numerical evaluations also reveal quantitative and qualitative changes in this distribution in the vicinity of the bifurcations of the deterministic system. However, these changes notwithstanding, our numerical simulations show that the Lyapunov exponents of the system remain negative in these parameter regions, indicating that no dynamical stochastic bifurcations take place. Moreover, our numerical simulations confirm that, regardless of the asymptotic dynamics of the deterministic system, the random Morris-Lecar model stabilizes at a unique stationary stochastic process. In terms of random dynamical system theory, our analysis shows that additive noise destroys the above-mentioned bifurcation sequences that characterize class I and class II regimes in the Morris-Lecar model. The interpretation of this result in terms of neuronal coding is that, despite the differences in the deterministic dynamics of class I and class II membranes, their responses to noise-like stimuli present a reliable feature.

Animals↗

PubMed1993

Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a.

The production of a capsular polysaccharide (CPS; K antigen) is characteristic of Klebsiella pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK21a), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-alpha-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumoniae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by approximately 50 nm.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1998

The three-dimensional structure of a helix-less variant of intestinal fatty acid-binding protein.

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic 15.1-kDa protein that appears to function in the intracellular transport and metabolic trafficking of fatty acids. It binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two five-stranded antiparallel beta-sheets and a helix-turn-helix domain. To investigate the role of the helical domain, we engineered a variant of I-FABP by deleting 17 contiguous residues and inserting a Ser-Gly linker (Kim K et al., 1996, Biochemistry 35:7553-7558). This variant, termed delta17-SG, was remarkably stable, exhibited a high beta-sheet content and was able to bind fatty acids with some features characteristic of the wild-type protein. In the present study, we determined the structure of the delta17-SG/palmitate complex at atomic resolution using triple-resonance 3D NMR methods. Sequence-specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 25 degrees C and used to define the consensus 1H/13C chemical shift-derived secondary structure. Subsequently, an iterative protocol was used to identify 2,544 NOE-derived interproton distance restraints and to calculate its tertiary structure using a unique distance geometry/simulated annealing algorithm. In spite of the sizable deletion, the delta17-SG structure exhibits a backbone conformation that is nearly superimposable with the beta-sheet domain of the wild-type protein. The selective deletion of the alpha-helical domain creates a very large opening that connects the interior ligand-binding cavity with exterior solvent. Unlike wild-type I-FABP, fatty acid dissociation from delta17-SG is structurally and kinetically unimpeded, and a protein conformational transition is not required. The delta17-SG variant of I-FABP is the only wild-type or engineered member of the intracellular lipid-binding protein family whose structure lacks alpha-helices. Thus, delta17-SG I-FABP constitutes a unique model system for investigating the role of the helical domain in ligand-protein recognition, protein stability and folding, lipid transfer mechanisms, and cellular function.

Animals↗

PubMed2003

Treatment considerations for anxiety in the elderly.

Anxiety is common in the elderly and can present as a primary anxiety disorder or as a symptom of another disorder. Generalized anxiety disorder, in particular, is a common syndrome in late life. Anxiety symptoms are also common features of late-life depression and dementia. Treatment of anxiety in elderly persons has typically involved the use of benzodiazepines, which are often effective but problematic because they are associated with increased risk of cognitive impairment, falls, and fractures. Based on their safety and efficacy, antidepressants, particularly serotonergic medications, are considered first-line treatment for most anxiety disorders as well as anxiety symptoms of major depressive disorder. Psychotherapy, particularly cognitive-behavioral therapy, may be effective in these disorders as well; research is underway to adapt this therapy to the needs of elderly persons. Anxiety symptoms in dementia are poorly understood but may respond to atypical antipsychotics, serotonergic antidepressants, or mood stabilizers. Overall, the research in late-life anxiety has not caught up to the literature in late-life depression and, for the most part, treatment recommendations must be extrapolated from studies in young adults.

Aged↗

PubMed2006

Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway.

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.

Amino Acid Sequence↗

PubMed1999

Gramicidin D conformation, dynamics and membrane ion transport.

The linear pentadecapeptide antibiotic, gramicidin D, a heterogeneous mixture of six components, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The conformation of gramicidin A in the solid state, in organic solvents, and in planar lipid bilayers and the relationship between the composition and the conformation of gramicidin and its selective transport of ions across membranes has been the subject of intense investigation for over 50 years. The x-ray crystal structure and nmr solution spectroscopy agree fully with one another and reveal that entirely different conformations of gramicidin are present in uncomplexed and ion complexed forms. Precise refinements of the three-dimensional structures of naturally occurring gramicidin D in crystals obtained from methanol, ethanol, and n-propanol demonstrate the unexpected presence of stable left-handed antiparallel double-helical heterodimers that vary with the crystallization solvent. The side chains of Trp residues in the three structures exhibit sequence-specific patterns of conformational preference. Tyr substitution for Trp at position 11 appears to favor beta ribbon formation and stabilization of the antiparallel double helix. This conformation acts as a template for gramicidin folding and nucleation of the different crystal forms. The fact that a minor component in a heterogeneous mixture influences aggregation and crystal nucleation has potential applications to other systems in which anomalous behavior is exhibited by aggregation of apparently homogeneous materials, such as the enigmatic behavior of prion proteins. The crystallographically determined structures of cesium, potassium, rubidium, and hydronium ion complexes of gramicidin A are in excellent agreement with the nmr structure determination of the cesium ion gramicidin complex in a methanol chloroform mixture (50 : 50). The right-handed antiparallel double stranded double helical structures (DSDHR) also exhibit geometric features compatible with the solid-state 15N and 2H nmr data recorded for gramicidin in planar lipid bilayers and attributed to the active form of gramicidin A. The DSDHR crystal structures reveal an ion channel with a single partially solvated cation distributed over three ion binding sites. The channel lumen is relatively smooth and electrostatically negative as required for cation passage, while the exterior is electrostatically neutral, a requirement for membrane insertion. The "coordination" of the Cs+ ion is achieved by interaction with the pi orbitals of the carbonyls which do not point toward the ions. The K+ binding sites, which are similar in position to Cs+ binding sites, are shifted off center slightly toward the wall of the channel.

Amino Acid Sequence↗

PubMed2004

Complete sequence of virulence plasmid pEIB1 from the marine fish pathogen Vibrio anguillarum strain MVM425 and location of its replication region.

AIMS: The aim of this study was to determine the whole DNA sequence of pEIB1, one pJM1-like virulence plasmid from Vibrio anguillarum MVM425 and locate the replication region. METHODS AND RESULTS: DNA sequence of virulence plasmid pEIB1 from V. anguillarum MVM425 was determined using the methods of restriction endonuclease digestion, subcloning, and primer walking. The whole nucleotide sequence of pEIB1 comprises 66,164 bp, encoding 44 open reading frames (>400 bp) containing the genes of DNA replication, biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. With no demonstrated replication origin, the Sau3AI partial digested plasmid DNA fragments of pEIB1 were ligated into the BamHI-fragment containing the kanamycin-resistance gene (Kmr). For there is no effective transformation in V. anguillarum, the ligated DNA was first introduced into E. coli JM83, and the transfomants were selected for resistance to kanamycin. It was demonstrated with southern blotting and DNA sequencing that plasmid pEIB7 containing the Sau3AI DNA fragment of pEIB1 (from 12516 to 13957) has the ability to replicate in E. coli JM83 and V. anguillarum MVM425sh. The segregational stability of plasmid pEIB7 kept in 100 and 4% in E. coli JM83 and V. anguillarum MVM425sh respectively when the cells were cultured in 200th generation. In following experiments, we also found that plasmid pEIB7 replicated at a middle-copy number of 10-40 in JM83, while at a high-copy number of 100-300 in MVM425sh. Moreover, pEIB7 can survive in V. alginolyticus, another fish pathogenic. CONCLUSIONS: With the whole DNA sequence of pEIB1 determining, it was found that pEIB1 showed microheterogeneity in its restriction endonuclease patterns with pJM1 though their DNA sequences had slight difference. According to the complete DNA sequence of pEIB1, its replication region was located from 12516 to 13957. And this replication region is compatible to pUC18 (pMB1), pKA3 (pSC101) and p15A: caiE (p15A). SIGNIFICANCE AND IMPACT OF THE STUDY: The worldwide vibriosis marine pathogen V. anguillarum strains contain common virulence, pJM1-like plasmids, independent on the geographical source. The pEIB1 was the second common virulence plasmid, which sequence was determined. Its sequence is highly homologous to pJM1 as they both encode biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. Some interesting features as in pJM1 were also identified, such as transposon-like structures. So it can be deferred that the whole DNA sequences of virulent plasmid pEIB1 will be great helpful to future revealing these V. anguillarum virulence-related genes derived during evolution from transposition events or horizontal transfer of genes potentially originating in other organisms. Another result, replication region of pEIB1 locating is the first report about replication of pJM1-like plasmid. This work will be useful for researching pJM1-like plasmid replication mechanism in V. anguillarum.

Animals↗

PubMed2001

The phenomenological stability of depersonalization: comparing the old with the new.

The view that depersonalization is a stable syndrome became well established during the first half of the 20th century. Current operational definitions restrict depersonalization to the experience of unreality. This is likely to neglect clinical features of potential neurobiological relevance. By using the year 1946 as the dividing line, 200 cases of depersonalization disorder reported in the medical literature since 1898 were divided into two historical groups (1 and 2). The groups were then compared in terms of 18 phenomenological variables with a sample of 45 prospective cases of DSM-IV depersonalization disorder (group 3 or gold standard). Groups 1 and 2 differed in terms of their symptom profile, but the highest frequency that symptoms achieved in either group did not differ from the rates identified in group 3. A core of (invariable) symptoms, including emotional numbing, visual derealization, and altered body experience, was present throughout. These high rates of spontaneous reporting in all three groups may be explained by the fact that they all are accompanied by specific distress. With the exception of heightened self-observation and altered time experiencing, all other symptoms were significantly lower in group 2. The results suggest that the phenomenology of depersonalization has remained stable over the last 100 years. Our study found differences in frequency for some symptoms, but these are likely to have resulted from reporting biases, themselves governed by changing theoretical views. Clinical descriptions became poorer as the present is approached. This cannot be solely explained on the basis of empirical progress, and it is likely that theoretical biases also play a role. Because the neurobiological relevance of the symptoms of depersonalization remains unknown, it makes sense to continue collecting as many symptoms as possible, thereby avoiding both biased selection or premature closure.

Adult↗

PubMed2000

Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase.

Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)). These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.

Alanine↗