Investigation of the nature of the methionine-pi interaction in beta-hairpin peptide model systems

There are frequent contacts between aromatic rings and sulfur atoms in proteins. However, it is unclear to what degree this putative interaction is stabilizing and what the nature of the interaction is. We have investigated the aryl-sulfur interaction by placing a methionine residue diagonal to an aromatic ring on the same face of a beta-hairpin, which places the methionine side chain in close proximity to the aryl side chain. The methionine (Met)-aryl interaction was compared with an equivalent hydrophobic and cation-pi interaction in the context of the beta-hairpin. The interaction between phenylalanine (Phe), tryptophan (Trp), or cyclohexylalanine (Cha) and Met stabilized the beta-hairpin by -0.3 to -0.5 kcal mole(-1), as determined by double-mutant cycles. The peptides were subjected to thermal denaturations that suggest a hydrophobic driving force for the interactions between Met and Trp or Cha. The observed interaction of Met or norleucine (Nle) with Trp or Cha are quite similar, implying a hydrophobic driving force for the Met-pi interaction. However, the thermodynamic data suggest that there may be some differences between the interaction of Met with Trp and Phe and that there may be a small thermodynamic component to the Met...Phe interaction.     

ATP synthases: insights into their motor functions from sequence and structural analyses

ATP synthases are motor complexes comprised of F₀ and F₁ parts that couple the proton gradient across the membrane to the synthesis of ATP by rotary catalysis. Although a great deal of information has been accumulated regarding the structure and function of ATP synthases, their motor functions are not fully understood. For this reason, we performed the alignments and analyses of the protein sequences comprising the core of the ATP synthase motor complex, and examined carefully the locations of the conserved residues in the subunit structures of ATP synthases. A summary of the findings from this bioinformatic study is as follows. First, we found that four conserved regions in the sequence of subunit are clustered into three patches in its structure. The interactions of these conserved patches with the and subunits are likely to be critical for energy coupling and catalytic activity of the ATP synthase. Second, we located a four-residue cluster at the N-terminal domain of mitochondrial OSCP or bacterial (or chloroplast) subunit which may be critical for the binding of these subunits to F₁. Third, from the localizations of conserved residues in the subunits comprising the rotors of ATP synthases, we suggest that the conserved interaction site at the interface of subunit c and (mitochondria) or (bacteria and chloroplasts) may be important for connecting the rotor of F₁ to the rotor of F₀. Finally, we found the sequence of mitochondrial subunit b to be highly conserved, significantly longer than bacterial subunit b, and to contain a shorter dimerization domain than that of the bacterial protein. It is suggested that the different properties of mitochondrial subunit b may be necessary for interaction with other proteins, e.g., the supernumerary subunits.     

Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels.

An algorithm is presented for the accurate and rapid generation of multiple protein sequence alignments from tertiary structure comparisons. A preliminary multiple sequence alignment is performed using sequence information, which then determines an initial superposition of the structures. A structure comparison algorithm is applied to all pairs of proteins in the superimposed set and a similarity tree calculated. Multiple sequence alignments are then generated by following the tree from the branches to the root. At each branchpoint of the tree, a structure-based sequence alignment and coordinate transformations are output, with the multiple alignment of all structures output at the root. The algorithm encoded in STAMP (STructural Alignment of Multiple Proteins) is shown to give alignments in good agreement with published structural accounts within the dehydrogenase fold domains, globins, and serine proteinases. In order to reduce the need for visual verification, two similarity indices are introduced to determine the quality of each generated structural alignment. Sc quantifies the global structural similarity between pairs or groups of proteins, whereas Pij' provides a normalized measure of the confidence in the alignment of each residue. STAMP alignments have the quality of each alignment characterized by Sc and Pij' values and thus provide a reproducible resource for studies of residue conservation within structural motifs.     

Sequence alignments and pair hidden Markov models using evolutionary history

This work presents a novel pairwise statistical alignment method based on an explicit evolutionary model of insertions and deletions (indels). Indel events of any length are possible according to a geometric distribution. The geometric distribution parameter, the indel rate, and the evolutionary time are all maximum likelihood estimated from the sequences being aligned. Probability calculations are done using a pair hidden Markov model (HMM) with transition probabilities calculated from the indel parameters. Equations for the transition probabilities make the pair HMM closely approximate the specified indel model. The method provides an optimal alignment, its likelihood, the likelihood of all possible alignments, and the reliability of individual alignment regions. Human alpha and beta-hemoglobin sequences are aligned, as an illustration of the potential utility of this pair HMM approach.     

Causes of male sexual trait divergence in introduced populations of guppies

Males from different populations of the same species often differ in their sexually selected traits. Variation in sexually selected traits can be attributed to sexual selection if phenotypic divergence matches the direction of sexual selection gradients among populations. However, phenotypic divergence of sexually selected traits may also be influenced by other factors, such as natural selection and genetic constraints. Here, we document differences in male sexual traits among six introduced Australian populations of guppies and untangle the forces driving divergence in these sexually selected traits. Using an experimental approach, we found that male size, area of orange coloration, number of sperm per ejaculate and linear sexual selection gradients for male traits differed among populations. Within populations, a large mismatch between the direction of selection and male traits suggests that constraints may be important in preventing male traits from evolving in the direction of selection. Among populations, however, variation in sexual selection explained more than half of the differences in trait variation, suggesting that, despite within‐population constraints, sexual selection has contributed to population divergence of male traits. Differences in sexual traits were also associated with predation risk and neutral genetic distance. Our study highlights the importance of sexual selection in trait divergence in introduced populations, despite the presence of constraining factors such as predation risk and evolutionary history.     

A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss)

MicroRNAs (miRNAs) participate in various vitally biological processes via controlling target genes activity and thousands of miRNAs have been identified in many species to date, including 18,698 known animal miRNA in miRBase. However, there are only limited studies reported in rainbow trout (Oncorhynchus mykiss) especially via the computational-based approaches. In present study, we systematically investigated the miRNAs in rainbow trout using a well-developed comparative genome-based homologue search. A total of 196 potential miRNAs, belonging to 124 miRNA families, were identified, most of which were firstly reported in rainbow trout. The length of miRNAs ranged from 17 to 24 nt with an average of 20 nt while the length of their precursors varied from 47 to 152 nt with an average of 85 nt. The identified miRNAs were not evenly distributed in each miRNA family, with only one member per family for a majority, and multiple members were also identified for several families. Nucleotide U was dominant in the pre-miRNAs with a percentage of 30.04%. The rainbow trout pre-miRNAs had relatively high negative minimal folding free energy (MFE) and adjusted MFE (AMFE). Not only the mature miRNAs but their precursor sequences are conserved among the living organisms. About 2466 O. mykiss genes were predicted as potential targets for 189 miRNAs. Gene Ontology (GO) analysis showed that nearly 2093, 2107, and 2081 target genes are involved in cellular component, molecular function, and biological processes respectively. KEGG pathway enrichment analysis illuminated that these miRNAs targets might regulate 105 metabolic pathways, including those of purine metabolism, nitrogen metabolism, and oxidative phosphorylation. This study has provided an update on rainbow trout miRNAs and their targets, which represents a foundation for future studies.     

Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes

Aims: We have developed a PCR‐based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin‐coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin‐coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin‐coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin‐coding sequences present in non‐LAB bacteriocin producers. The results suggest that several potentially new bacteriocin‐coding sequences were found. Significance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.     

葛根抗腹泻药效组分实验研究

目的:验证葛根抗腹泻的药效组分,为葛根的质量评价提供科学数据。方法:在经方葛根芩连汤的基础上分析葛根抗腹泻的药效组分,采用组合药理实验辅助验证药效组分。结果:抗腹泻相关实验中葛根水煎液中、高剂量组、葛根药效组分组、葛根芩连汤组与阴性对照组比较均有显著性差异(P0.01)。结论:葛根素-大豆苷-大豆苷元-3'-羟基葛根素是葛根抗腹泻的药效组分。     

Development of sequential multiple comparison procedure for dose response test

We propose a multiple comparison procedure to identify the minimum effective dose level by sequentially comparing each dose level with the zero dose level in the dose finding test. If we can find the minimum effective dose level at an early stage in the sequential test, it is possible to terminate the procedure in the dose finding test after a few group observations up to the dose level. Thus, the procedure is viable from an economical point of view when high costs are involved in obtaining the observations. In the procedure, we present an integral formula to determine the critical values for satisfying a predefined type I familywise error rate. Furthermore, we show how to determine the required sample size in order to guarantee the power of the test in the procedure. In practice, we compare the power of the test and the required sample size for various configurations of the population means in simulation studies and adopt our sequential procedure to the dose response test in a case study.