Bayesian search of functionally divergent protein subgroups and their function specific residues

MOTIVATION: The rapid increase in the amount of protein sequence data has created a need for an automated identification of evolutionarily related subgroups from large datasets. The existing methods typically require a priori specification of the number of putative groups, which defines the resolution of the classification solution. RESULTS: We introduce a Bayesian model-based approach to simultaneous identification of evolutionary groups and conserved parts of the protein sequences. The model-based approach provides an intuitive and efficient way of determining the number of groups from the sequence data, in contrast to the ad hoc methods often exploited for similar purposes. Our model recognizes the areas in the sequences that are relevant for the clustering and regards other areas as noise. We have implemented the method using a fast stochastic optimization algorithm which yields a clustering associated with the estimated maximum posterior probability. The method has been shown to have high specificity and sensitivity in simulated and real clustering tasks. With real datasets the method also highlights the residues close to the active site. AVAILABILITY: Software 'kPax' is available at http://www.rni.helsinki.fi/jic/softa.html     

Sequence analysis of the cbb3 oxidases and an atomic model for the Rhodobacter sphaeroides enzyme

The cbb3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. To understand the minimal common properties of the superfamily, and to learn about cbb3-type oxidases specifically, we have analyzed a wide set of heme-copper oxidase sequences and built a homology model of the catalytic subunit of the cbb3 oxidase from Rhodobacter sphaeroides. We conclude that with regard to the active site surroundings, the cbb3 oxidases greatly resemble the structurally known oxidases, while major differences are found in three segments: the additional N-terminal stretch of ca. 60 amino acids, the segment following helix 3 to the end of helix 5, and the C-terminus from helix 11 onward. The conserved core contains the active site tyrosine and also an analogue of the K-channel of proton transfer, but centered on a well-conserved histidine in the lower part of helix 7. Modeling the variant parts of the enzyme suggests that two periplasmic loops (between helices 3 and 4 and between helices 11 and 12) could interact with each other as a part of the active site structure and might have an important role in proton pumping. An analogue of the D-channel is not found, but an alternative channel might form around helix 9. A preliminary packing model of the trimeric enzyme is also presented.     

Novel α2β1 Integrin Inhibitors Reveal That Integrin Binding to Collagen under Shear Stress Conditions Does Not Require Receptor Preactivation

The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm²). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm²) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2β1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2β1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation.     

The tricky path to recombining X and Y chromosomes in meiosis

Sex chromosomes are the Achilles' heel of male meiosis in mammals. Mis-segregation of the X and Y chromosomes leads to sex chromosome aneuploidies, with clinical outcomes such as infertility and Klinefelter syndrome. Successful meiotic divisions require that all chromosomes find their homologous partner and achieve recombination and pairing. Sex chromosomes in males of many species have only a small region of homology (the pseudoautosomal region, PAR) that enables pairing. Until recently, little was known about the dynamics of recombination and pairing within mammalian X and Y PARs. Here, we review our recent findings on PAR behavior in mouse meiosis. We uncovered unexpected differences between autosomal chromosomes and the X-Y chromosome pair, namely that PAR recombination and pairing occurs later, and is under different genetic control. These findings imply that spermatocytes have evolved distinct strategies that ensure successful X-Y recombination and chromosome segregation.     

Modified hyaluronan hydrogels support the maintenance of mouse embryonic stem cells and human induced pluripotent stem cells

These studies provide evidence for the ability of a commercially available, defined, hyaluronan-gelatin hydrogel, HyStem-C?, to maintain both mouse embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs) in culture while retaining their growth and pluripotent characteristics. Growth curve and doubling time analysis show that mESCs and hiPSCs grow at similar rates on HyStem-C? hydrogels and mouse embryonic fibroblasts and Matrigel?, respectively. Immunocytochemistry, flow cytometry, gene expression and karyotyping reveal that both human and murine pluripotent cells retain a high level of pluripotency on the hydrogels after multiple passages. The addition of fibronectin to HyStem-C? enabled the attachment of hiPSCs in a xeno-free, fully defined medium.     

Body Size at Birth Is Associated with Food and Nutrient Intake in Adulthood

Background

Small body size at birth is associated with an increased risk of cardiovascular disease and type 2 diabetes. Dietary habits are tightly linked with these disorders, but the association between body size at birth and adult diet has been little studied. We examined the association between body size at birth and intake of foods and macronutrients in adulthood.

Methodology/Principal Findings

We studied 1797 participants, aged 56 to 70, of the Helsinki Birth Cohort Study, whose birth weight and length were recorded. Preterm births were excluded. During a clinical study, diet was assessed with a validated food-frequency questionnaire. A linear regression model adjusted for potential confounders was used to assess the associations. Intake of fruits and berries was 13.26 g (95% confidence interval [CI]: 0.56, 25.96) higher per 1 kg/m³ increase in ponderal index (PI) at birth, and 83.16 g (95% CI: 17.76, 148.56) higher per 1 kg higher birth weight. One unit higher PI at birth was associated with 0.14% of energy (E%) lower intake of fat (95% CI: -0.26, -0.03) and 0.18 E% higher intake of carbohydrates (95% CI: 0.04, 0.32) as well as 0.08 E% higher sucrose (95% CI: 0.00, 0.15), 0.05 E% higher fructose (95% CI: 0.01, 0.09), and 0.18 g higher fiber (95% CI: 0.02, 0.34) intake in adulthood. Similar associations were observed between birth weight and macronutrient intake.

Conclusions

Prenatal growth may modify later life food and macronutrient intake. Altered dietary habits could potentially explain an increased risk of chronic disease in individuals born with small body size.     

Multiple glycines in TCR alpha-chains determine clonally diverse nature of human T cell memory to influenza A virus

Detailed assessment of how the structural properties of T cell receptors affect clonal repertoires of Ag-specific cells is a prerequisite for a better understanding of human antiviral immunity. Herein we examine the alpha TCR repertoires of CD8 T cells reactive against the influenza A viral epitope M1(58-66), restricted by HLA-A2.1. Using molecular cloning, we systematically studied the impact of alpha-chain usage in the formation of T cell memory and revealed that M1(58-66)-specific, clonally diverse VB19 T cells express alpha-chains encoded by multiple AV genes with different CDR3 sizes. A unique feature of these alpha TCRs was the presence of CDR3 fitting to an AGA(G(n))GG-like amino acid motif. This pattern was consistent over time and among different individuals. Further molecular assessment of human CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes led to the conclusion that the poly-Gly/Ala runs in CDR3alpha were a property of immune, but not naive, repertoires and could be attributed to influenza exposure. Repertoires of T cell memory are discussed in the context of clonal diversity, where poly-Gly/Ala runs in the CDR3 of alpha- and beta-chains might provide high levels of TCR flexibility during Ag recognition while gene-encoded CDR1 and CDR2 contribute to the fine specificity of the TCR-peptide MHC interaction.     

Single bioreactor gastrointestinal tract simulator for study of survival of probiotic bacteria

The aim of the present study was to design an in vitro model system to evaluate the probiotic potential of food. A single bioreactor system-gastrointestinal tract simulator (GITS) was chosen for process simulation on account of its considerable simplicity compared to multi-vessel systems used in previous studies. The bioreactor was evaluated by studying the viability of four known probiotic bacteria (Lactobacillus acidophilus La-5, Lactobacillus johnsonii NCC 533, Lactobacillus casei strain Shirota, and Lactobacillus rhamnosus GG) as a function of their physiological state. L. acidophilus and L. johnsonii survived in GITS better when introduced at an early stationary or exponential phase compared to being previously stored for 2 weeks at 4 degrees C. These two species were more resistant to bile salts and survived better than L. casei and L. rhamnosus GG. The latter two species gave large losses (up to 6 log) in plate counts independent of growth state due to the bile. However, experiments with some commercial probiotic products containing Lb. GG bacteria showed much better survival compared with model food (modified deMan-Rogosa-Sharpe growth medium), thus demonstrating the influence of the food matrix on the viability of bacteria. The study demonstrated that GITS can be successfully used for evaluation of viability of probiotic bacteria and functionality of probiotic food.     

Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins.

Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM-4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations. Interestingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different. However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2. Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.