The effect of chain length on protein solubilization in polymer-based vesicles (polymersomes)

Using a mean-field analysis we derive a consistent model for the perturbation of a symmetric polymeric bilayer due to the incorporation of transmembrane proteins, as a function of the polymer molecular weight and the protein dimensions. We find that the mechanism for the inhibition of protein incorporation in polymeric bilayers differs from that of their inclusion in polymer-carrying lipid vesicles; in polymersomes, the equilibrium concentration of transmembrane proteins decreases as a function of the thickness mismatch between the protein and the bilayer core, whereas in liposomes the presence of polymer chains affects the protein adsorption kinetics. Despite the increased stiffness of polymer bilayers (when compared to lipid ones), their perturbation decay length and range of protein-protein interaction is found to be relatively long. The energetic penalty due to protein adsorption increases relatively slowly as a function of the polymer chain length due to the self-assembled nature of the polymer bilayer. As a result, we predict that transmembrane proteins may be incorporated in significant numbers even in bilayers where the thickness mismatch is large.     

Stimulation of perivascular nitric oxide synthesis by oxygen

We hypothesized that elevated partial pressures of O(2) would increase perivascular nitric oxide (*NO) synthesis. Rodents with O(2)- and.NO-specific microelectrodes implanted adjacent to the abdominal aorta were exposed to O(2) at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA). Exposures to 2.0 and 2.8 ATA O(2) stimulated neuronal (type I) NO synthase (nNOS) and significantly increased steady-state.NO concentration, but the mechanism for enzyme activation differed at each partial pressure. At both pressures, elevations in.NO concentration were inhibited by the nNOS inhibitor 7-nitroindazole and the calcium channel blocker nimodipine. Enzyme activation at 2.0 ATA O(2) appeared to be due to an altered cellular redox state. Exposure to 2.8 ATA O(2), but not 2.0 ATA O(2), increased nNOS activity by enhancing nNOS association with calmodulin, and an inhibitory effect of geldanamycin indicated that the association was facilitated by heat shock protein 90. Infusion of superoxide dismutase inhibited.NO elevation at 2.8 but not 2.0 ATA O(2). Hyperoxia increased the concentration of.NO associated with hemoglobin. These findings highlight the complexity of oxidative stress responses and may help explain some of the dose responses associated with therapeutic applications of hyperbaric oxygen.     

IgE binding synthetic peptides of Alt a 1, a major allergen of Alternaria alternata

Alternaria alternata protein, Alt a 1 is a major allergen associated with allergy in atopic patients. Although the molecule binds strongly to IgE antibody from patients, the epitopes involved have not been identified or defined. In the present study, we synthesized overlapping peptides spanning the whole sequence and evaluated their IgE binding with sera from patients with Alternaria-induced allergy. The results identified four IgE binding linear regions. Two of these regions K41-P50 and Y54-K63 showed consistent reactivity with all four patients studied. The specific epitopes involved in the immune response may be of value in the immunodiagnosis and probably also in specific immunotherapy.     

Immunolocalization of detergent-susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody

The A-type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA-2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA-2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T(1/2) whole cell lysates and nuclear lysates. The epitope for LA-2F9 was narrowed down to amino acid residues 268-278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell-cycle-dependent, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) phase of the cell-cycle. The intensity of staining increased 3.5-fold within 6 h of refeeding, when the maximum number of cells were labeled with LA-2F9. We also checked whether the LA-2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC-35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA-2F9 has a novel and specific reactivity towards detergent-susceptible lower order lamin structures that are likely to be assembly intermediates.     

Purification,characterization, and in vitro mineralization studies of a novel goose eggshell matrix protein,ansocalcin

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 microg/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentration-dependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.     

Effect of d-limonene on three stored-product beetles

d-Limonene was investigated for contact and fumigant toxicity, ovicidal effects, oviposition-deterrent, development inhibition, and feeding-deterrent activities against three stored-product beetles (Coleoptera): lesser grain borer, Rhyzopertha dominica (F.); rice weevil, Sitophilus oryzae (L.); and red flour beetle, Tribolium castaneum (Herbst). Contact and fumigant toxicity decreased as larvae aged. Contact toxicity was similar for adults of the three species tested, but R. dominica was most susceptible to fumigant activity. T. castaneum oviposition decreased as concentration of d-limonene increased and d-limonene reduced oviposition up to 92.3% at the concentration of 2.14 mg/cm2. Hatching of d-limonene-treated eggs of T. castaneum was reduced by 94.5% with no subsequent larval and adult survival at 2.14 mg/cm2 concentration. A flour disc bioassay indicated 87.7 to 96.8% feeding-deterrency by d-limonene toward all three insect species tested at the highest concentration of 60.0 mg/g food. These results suggest that d-limonene can be effectively used to suppress populations of stored-product beetles.     

HLA-DRB1*0402 (DW10) transgene protects collagen-induced arthritis-susceptible H2Aq and DRB1*0401 (DW4) transgenic mice from arthritis

To investigate the role of HLA-DR4 in predisposition to arthritis, we generated transgenic mice carrying DRB1*0401 and DRB1*0402 genes. We have previously shown that DRB1*0401 molecule renders B10.RQB3 (H2A(q)) mice susceptible to porcine and human type II collagen-induced arthritis. We report that the introduction of DRB1*0402 transgene does not lead to development of arthritis in mice when they are immunized with porcine and human type II collagen. In addition, DRB1*0402 protects B10.RQB3 mice against developing arthritis with bovine type II collagen. These data show that DRB1 can modulate the disease mediated by A(q). In vivo depletion of DRB1*0402 did not lead to induction of collagen-induced arthritis in transgenic mice. In vitro cytokine analysis shows that mice protected from collagen-induced arthritis produce lower amounts of Th1 and higher levels of Th2 type cytokines upon immunization with type II collagen. Protection of mice was also related to higher apoptosis in DW10 mice as indicated by higher amounts of BclII in response to type II collagen. On the basis of our observations in HLA transgenic mice, we hypothesize that DRB1 polymorphism can modulate disease by shaping the T cell repertoire in thymus and select autoreactive T cells.     

Crk associates with a multimolecular Paxillin/GIT2/beta-PIX complex and promotes Rac-dependent relocalization of Paxillin to focal contacts

We have previously demonstrated that the CrkII and CrkL adapter proteins are required for the spreading of epithelial colonies and the breakdown of adherens junctions in response to hepatocyte growth factor. When overexpressed, CrkII and CrkL promote lamellipodia formation, cell spreading, and the loss of epithelial adherens junctions in the absence of hepatocyte growth factor. The exact mechanism by which Crk proteins elicit these changes is unclear. We show that the overexpression of CrkII or CrkL, but not Src homology 2 or amino-terminal Src homology 3 domain mutant Crk proteins, promotes the relocalization of Paxillin to focal contacts throughout the cell and within lamellipodia in a Rac-dependent manner. In stable cell lines overexpressing CrkII, enhanced lamellipodia formation and cell spreading correlate with an increased association of CrkII with Paxillin, GIT2 (an ARF-GAP) and beta-PIX (a Rac1 exchange factor). Mutants of Paxillin that fail to associate with Crk or GIT2, or do not target to focal adhesions inhibit Crk-dependent cell spreading and lamellipodia formation. We conclude from these studies that the association of Crk with Paxillin is important for the spreading of epithelial colonies, by influencing the recruitment of Paxillin to focal complexes and promoting the enhanced assembly of Paxillin/GIT2/beta-PIX complexes.     

Lamin A/C speckles mediate spatial organization of splicing factor compartments and RNA polymerase II transcription

The cylindrical shape of the fission yeast cell is generated by linear polarized growth from its cell ends. Using immunofluorescence and live imaging microscopy, we have investigated the roles of the cell end marker tea1p in generating linear polarized growth. We found that tea1p is primarily transported on plus ends of microtubules from the vicinity of the nucleus to the cell ends, and that its movement near the nucleus is independent of the kinesin tea2p. Deletion analysis identified a coiled-coil domain in tea1p essential for its retention at cell ends, and demonstrated that tea1p exerts different functions dependent on its location. On the tips of microtubules, tea1p prevents the curling of microtubules around the cell ends, whereas it is required for maintaining linear cell growth and for retention of polarity factors such as the Dyrk kinase pom1p, the CLIP170-like tip1p, and tea2p at the cell ends. We propose that tea1p has roles in organizing the microtubule cytoskeleton on the tips of microtubules, and in the retention of factors at the cell ends necessary for the cell to grow in a straight line.