Adhesion contact kinetics of HepG2 cells during Hepatitis B virus replication: Involvement of SH3-binding motif in HBX

It has been shown that Hepatitis B virus (HBV) replication directly alters the expression of key cytoskeleton-associated proteins which play key roles in mechanochemical signal transduction. Nevertheless, little is known on the correlation between HBV replication and the subsequent adhesion mechanism of HBV-replicating cells. In this study, it is demonstrated that the lag time of adhesion contact evolution of HepG2 cells with HBV replication is significantly increased by two times compared to that of normal HepG2 cell on collagen coated substrate. During the initial 20 min of cell seeding, only diffuse forms of vinculin was detected in HBV replicating cells while vinculin-associated focal complexes were found in normal and control cells. Similar delay in cell adhesion in HBV-replicating cells was observed in cells transfected with HBX, the smallest HBV protein, suggesting its involvement in this cellular process. In addition, a proline rich region found in many SH3 binding proteins was identified in HBX. HBX was found to interact with the focal adhesion protein, vinexin-beta, through the SH3 binding. Furthermore, HepG2 cells with HBV replication showed evidence of cell rounding up, possibly resulting from cytoskeletal reorganizations associated with interaction between HBX and vinexin-beta. Taken together, our results suggest that HBX is involved in the cytoskeletal reorganization in response to HBV replication.     

Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge

Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B‐factors of residue pairs in CalB wild type (CalB‐WT) followed by simple flexibility analysis of residues in CalB‐WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162‐K308, N169‐F304, Q156_‐L163, S50‐A273, and S239C‐D252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C‐K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB‐WT. Remarkably, the temperature at which 50% of its activity remained after 60‐min incubation (T) of CalB A162C_K308C was increased by 8.5°C compared to that of CalB‐WT (55 and 46.5°C, respectively). Additionally, the half‐life at 50°C of CalB A162C‐K308C was 4.5‐fold higher than that of CalB‐WT (220 and 49 min, respectively). The improvement of thermostability of CalB A162C‐K308C was elucidated at the molecular level by molecular dynamics (MD) simulation. Biotechnol. Bioeng. 2012; 109:867–876. © 2011 Wiley Periodicals, Inc.     

Two amino acids in each of D1 and D2 dopamine receptor cytoplasmic regions are involved in D1-D2 heteromer formation

D(1) and D(2) dopamine receptors exist as heteromers in cells and brain tissue and are dynamically regulated and separated by agonist concentrations at the cell surface. We determined that these receptor pairs interact primarily through discrete amino acids in the cytoplasmic regions of each receptor, with no evidence of any D(1)-D(2) receptor transmembrane interaction found. Specifically involved in heteromer formation we identified, in intracellular loop 3 of the D(2) receptor, two adjacent arginine residues. Substitution of one of the arginine pair prevented heteromer formation. Also involved in heteromer formation we identified, in the carboxyl tail of the D(1) receptor, two adjacent glutamic acid residues. Substitution of one of the glutamic acid pair prevented heteromer formation. These amino acid pairs in D(1) and D(2) receptors are oppositely charged, and presumably interact directly by electrostatic interactions.     

Microsatellite markers for Grosmannia alacris (Ophiostomataceae, Ascomycota) and other species in the G. serpens complex

? Premise of the study: Polymorphic microsatellite markers were developed for the pine-infecting fungus, Grosmannia alacris. ? Methods and Results: Sixteen microsatellite markers were developed by using inter-simple sequence repeat (ISSR)-PCRs and 454 sequencing methods. Seven of these markers showed polymorphisms for a South African population of G. alacris, and 13 markers showed polymorphism when European isolates were included. Most of the primer pairs also amplified four closely related species: G. serpens, Leptographium gibbsii, L. castellanum, and L. yamaokae. ? Conclusions: These new markers will be useful for population studies of G. alacris and other species in the G. serpens complex.     

Grosmannia and Leptographium spp. associated with conifer-infesting bark beetles in Finland and Russia, including Leptographium taigense sp. nov.

Species of Grosmannia with Leptographium anamorphs include important forest pathogens and agents of blue stain in timber. They are commonly found in association with forest pests, such as bark beetles. During a survey of ophiostomatoid fungi in eastern parts of Finland and neighboring Russia, species belonging to the genus Grosmannia were isolated from 12 different bark beetle species infesting Picea abies and Pinus sylvestris, the most economically important conifers in the region. Identification of these fungi was based on morphology, DNA sequence comparisons for three gene regions and phylogenetic analyses. A total of ten taxa were identified. These belonged to six different species complexes in Grosmannia. The phylogenetic analyses provided an opportunity to redefine the G. galeiformis-, L. procerum-, L. lundbergii-, G. piceiperda-, G. olivacea- and G. penicillata-complexes, and to consider the species emerging from the survey within the context of these complexes. The species included G. galeiformis, G. olivacea, L. chlamydatum, L. lundbergii, L. truncatum and a novel taxon, described here as L. taigense sp. nov. In addition, species closely related to G. cucullata, G. olivaceapini comb. nov., G. piceiperda and L. procerum were isolated but their identity could not be resolved. The overall results indicate that the diversity of Grosmannia species in the boreal forests remains poorly understood and that further studies are needed to clarify the status of several species or species complexes.     

Disruption of the mu-delta opioid receptor heteromer

The crystal structure of the mu and kappa opioid receptors has revealed dimeric structural arrangements. Mu-delta receptors heteromers also exist and we have identified discrete cytoplasmic regions in each receptor required for oligomer formation. In the carboxyl tail of the delta receptor we identified three glycine residues (-GGG), substitution of any of these residues prevented heteromer formation. In intracellular loop 3 of both mu and delta receptors we identified three residues (-SVR), substitution of any of these residues prevented heteromer formation.