Helicity propensity and interaction of synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H: a circular dichroism study

The secondary structure of two synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H was determined by circular dichroism. In particular, the propensity of these peptides to assume an ordered structure was investigated upon by changing the solvent's polarity and the temperature. A reduction of solvent polarity led to a significant increase in the alpha-helix content in the case of HR1, whereas only a slight change in the secondary structure was observed in the case of HR2. In both cases the conformational change followed a two-state transition model. The interaction of the peptides was monitored by the conformational change in the mixture with respect to the single peptides. However, formation of the complex did not significantly enhance thermal stability. A reliable estimation of the secondary structure was obtained by optimising the experimental conditions to collect CD data down to 180 nm, and by comparing the structure data yielded by different software packages.     

Microbial Interactions within a Cheese Microbial Community

The interactions that occur during the ripening of smear cheeses are not well understood. Yeast-yeast interactions and yeast-bacterium interactions were investigated within a microbial community composed of three yeasts and six bacteria found in cheese. The growth dynamics of this community was precisely described during the ripening of a model cheese, and the Lotka-Volterra model was used to evaluate species interactions. Subsequently, the effects on ecosystem functioning of yeast omissions in the microbial community were evaluated. It was found both in the Lotka-Volterra model and in the omission study that negative interactions occurred between yeasts. Yarrowia lipolytica inhibited mycelial expansion of Geotrichum candidum, whereas Y. lipolytica and G. candidum inhibited Debaryomyces hansenii cell viability during the stationary phase. However, the mechanisms involved in these interactions remain unclear. It was also shown that yeast-bacterium interactions played a significant role in the establishment of this multispecies ecosystem on the cheese surface. Yeasts were key species in bacterial development, but their influences on the bacteria differed. It appeared that the growth of Arthrobacter arilaitensis or Hafnia alvei relied less on a specific yeast function because these species dominated the bacterial flora, regardless of which yeasts were present in the ecosystem. For other bacteria, such as Leucobacter sp. or Brevibacterium aurantiacum, growth relied on a specific yeast, i.e., G. candidum. Furthermore, B. aurantiacum, Corynebacterium casei, and Staphylococcus xylosus showed reduced colonization capacities in comparison with the other bacteria in this model cheese. Bacterium-bacterium interactions could not be clearly identified.     

Estimation of hourly average solar radiation on tilted surface via ANNs

An innovative approach is proposed for the estimation of hourly average solar radiation on tilted surface. The proposed approach, which is based on artificial neural networks and problem decomposition, demonstrates the following characteristics: Accuracy. Superior estimation is attained over both conventional approaches and theoretical models. Simplicity and efficiency. A small training set is employed and the training/test patterns involve few easily obtainable parameters. Generalization capability and robustness to noise. Apart from being of interest in meteorology, the accurate and efficient estimation of hourly average solar radiation on tilted surface is especially important in solar energy applications.     

Conventional kinesin holoenzymes are composed of heavy and light chain homodimers

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.     

From pull-down data to protein interaction networks and complexes with biological relevance

Motivation: Recent improvements in high-throughput Mass Spectrometry(MS) technology have expedited genome-wide discovery of protein–proteininteractions by providing a capability of detecting proteincomplexes in a physiological setting. Computational inferenceof protein interaction networks and protein complexes from MSdata are challenging. Advances are required in developing robustand seamlessly integrated procedures for assessment of protein–proteininteraction affinities, mathematical representation of proteininteraction networks, discovery of protein complexes and evaluationof their biological relevance. Results: A multi-step but easy-to-follow framework for identifyingprotein complexes from MS pull-down data is introduced. It assessesinteraction affinity between two proteins based on similarityof their co-purification patterns derived from MS data. It constructsa protein interaction network by adopting a knowledge-guidedthreshold selection method. Based on the network, it identifiesprotein complexes and infers their core components using a graph-theoreticalapproach. It deploys a statistical evaluation procedure to assessbiological relevance of each found complex. On Saccharomycescerevisiae pull-down data, the framework outperformed othermore complicated schemes by at least 10% in F₁-measure and identified610 protein complexes with high-functional homogeneity basedon the enrichment in Gene Ontology (GO) annotation. Manual examinationof the complexes brought forward the hypotheses on cause offalse identifications. Namely, co-purification of differentprotein complexes as mediated by a common non-protein molecule,such as DNA, might be a source of false positives. Protein identificationbias in pull-down technology, such as the hydrophilic bias couldresult in false negatives. Contact: samatovan{at}ornl.gov Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Jonathan Wren Present address: Department of Biomedical Informatics, VanderbiltUniversity, Nashville, TN 37232. The authors wish it to be known that, in their opinion, thefirst two authors should be regarded as joint First Authors.     

Distinct effects of IL-18 on the engraftment and function of human effector CD8 T cells and regulatory T cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8(+) effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Ralpha was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies.     

A new chromosomal race (2n=44) of Nannospalax xanthodon from Turkey (Mammalia: Rodentia)

A new karyotype for blind mole rats was recorded in Tunceli province in Eastern Turkey. The karyotype contained 44 chromosomes, including 13 biarmed pairs, 7 acrocentric pairs, and one heteromorphic pair with a submetacentric and an acrocentric homologue in the autosomal complement (FNa=69). The X chromosome was submetacentric and the Y chromosome medium-sized subtelocentric (FN=73). Distinct dark centromeric C-bands were observed on most of the biarmed and three pairs of the acrocentric autosomes. The NORs were detected on short arms of three subtelocentric pairs and one acrocentric pair of autosomes. The diploid number of chromosomes and the karyotype characteristics observed are obviously unique among hitherto studied populations of blind mole rats and the complement can be evaluated as a new chromosome race of Nannospalax xanthodon. The distribution ranges of individual chromosome races of the species recorded in Eastern Anatolia are revised and possible interracial hybridization is discussed in respect of the finding of a new race.     

Karyotypic conservatism in five species of Prochilodus (Characiformes,Prochilodontidae) disclosed by cytogenetic markers

The family Prochilodontidae is considered a group with well conserved chromosomes characterized by their number, morphology and banding patterns. Thence, our study aimed at accomplishing a cytogenetic analysis with conventional methods (Giemsa staining, silver staining of the nucleolus organizer regions-AgNOR, and C-banding) and fluorescence in situ hybridization (FISH) with 18S and 5S ribosomal DNA probes in five species of the Prochilodus genus (Prochilodus argenteus, Prochilodus brevis, Prochilodus costatus, Prochilodus lineatus and Prochilodus nigricans) collected from different Brazilian hydrographic basins. The results revealed conservatism in chromosome number, morphology, AgNORs 18S and 5S rDNAs location and constitutive heterochromatin distribution patterns. The minor differences observed in this work, such as an Ag-NOR on a P. argenteus chromosome and a distinct C-banding pattern in P. lineatus, are not sufficient to question the conservatism described for this group. Future work using repetitive DNA sequences as probes for FISH will be interesting to further test the cytogenetic conservatism in Prochilodus.     

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.