Laccase from Melanocarpus albomyces binds effectively to cellulose

A laccase from the thermophilic fungus Melanocarpus albomyces was shown to bind to softwood and pure microcrystalline cellulose. The binding isotherm fitted well the Langmuir type one-site binding model. The adsorption parameters indicated that M. albomyces laccase binds with high affinity to cellulose with a relatively low maximum binding capacity, as compared to the values for various cellulases. The binding was shown to be reversible and not influenced by non-specific protein or 0.1-0.5 M Na2SO4. No binding was detected with laccases from Trametes hirsuta or Mauginiella sp., which suggests that binding to cellulose is typical for only some laccases.     

Prenatal alcohol exposure reduces the proportion of newly produced neurons and glia in the dentate gyrus of the hippocampus in female rats

Prenatal alcohol exposure (PAE) alters adult neurogenesis and the neurogenic response to stress in male rats. As the effects of stress on neurogenesis are sexually dimorphic, the present study investigated the effects of PAE on adult hippocampal neurogenesis under both nonstressed and stressed conditions in female rats. Pregnant females were assigned to one of three prenatal treatments: (1) alcohol (PAE)—liquid alcohol (ethanol) diet ad libitum (36% ethanol-derived calories); (2) pair-fed—isocaloric liquid diet, with maltose–dextrin substituted for ethanol, in the amount consumed by a PAE partner (g/kg body wt/day of gestation); and (3) control—lab chow ad libitum. Female offspring were assigned to either nonstressed (undisturbed) or stressed (repeated restraint stress for 9 days) conditions. On day 10, all rats were injected with bromodeoxyuridine (BrdU) and perfused either 24 hours (cell proliferation) or 3 weeks (cell survival) later. We found that PAE did not significantly alter cell proliferation or survival, whereas females from the pair-fed condition exhibited elevated levels of cell survival compared to control females. Importantly, however, the proportion of both new neurons and new glial cells in the hippocampal dentate gyrus was reduced in PAE compared to control females. Exposure to stress did not alter neurogenesis in any of the prenatal treatment groups. In summary, compared to females from the control condition, prenatal dietary restriction enhanced the survival of new neurons, whereas PAE altered the differentiation of newly produced cells in the adult dentate gyrus. Alterations in hippocampal neurogenesis following PAE may contribute to learning and memory deficits seen in individuals with fetal alcohol spectrum disorders.     

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     

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.     

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.     

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.