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991.
Haupt C Morgado I Kumar ST Parthier C Bereza M Hortschansky P Stubbs MT Horn U Fändrich M 《Journal of molecular biology》2011,405(2):341-348
Amyloid fibrils are naturally occurring polypeptide scaffolds with considerable importance for human health and disease. These supermolecular assemblies are β-sheet rich and characterized by a high structural order. Clinical diagnosis and emerging therapeutic strategies of amyloid-dependent diseases, such as Alzheimer's, rely on the specific recognition of amyloid structures by other molecules. Recently, we generated the B10 antibody fragment, which selectively binds to Alzheimer's Aβ(1-40) amyloid fibrils but does not explicitly recognize other protein conformers, such as oligomers and disaggregated Aβ peptide. B10 presents poly-amyloid specific binding and interacts with fibrillar structures consisting of different polypeptide chains. To determine the molecular basis behind its specificity, we have analyzed the molecular properties of B10 with a battery of biochemical and biophysical techniques, ranging from X-ray crystallography to chemical modification studies. We find that fibril recognition depends on positively charged residues within the B10 antigen binding site. Mutation of these basic residues into alanine potently impairs fibril binding, and reduced B10-fibril interactions are also observed when the fibril carboxyl groups are covalently masked by a chemical modification approach. These data imply that the B10 conformational specificity for amyloid fibrils depends upon specific electrostatic interactions with an acidic moiety, which is common to different amyloid fibrils. 相似文献
992.
Sexual reproduction was examined in the aflatoxin-producing fungus Aspergillus nomius. Crosses between sexually compatible strains resulted in the formation of multiple nonostiolate ascocarps within stromata, which places the teleomorph in genus Petromyces. Ascocarp and ascospore morphology in Petromyces nomius were similar to that in P. flavus and P. parasiticus, and differences between teleomorphs were insufficient for species separation. Formation of mature ascocarps was infrequent, with only 24% of the 83 crosses producing viable ascospores. The majority of P. nomius strains contained a single mating-type gene (MAT1-1 or MAT1-2), but several strains contained both genes. MAT1-1/MAT1-2 strains were self-sterile and capable of mating with both MAT1-1 and MAT1-2 strains; hence P. nomius appears to be functionally heterothallic. 相似文献
993.
Fonseca DM Paula MO Wowk PF Campos LW Gembre AF Turato WM Ramos SG Dias-Baruffi M Barboza R Gomes E Horn C Marchal G Arruda LK Russo M Bonato VL 《Immunology and cell biology》2011,89(7):777-785
Epidemiological and experimental evidence supports the notion that microbial infections that are known to induce Th1-type immune responses can suppress Th2 immune responses, which are characteristics of allergic disorders. However, live microbial immunization might not be feasible for human immunotherapy. Here, we evaluated whether induction of Th1 immunity by the immunostimulatory sequences of CpG-oligodeoxynucleotides (CpG-ODN), with or without culture filtrate proteins (CFP), from Mycobacterium tuberculosis would suppress ongoing allergic lung disease. Presensitized and ovalbumin (OVA)-challenged mice were treated subcutaneously with CpG, or CpG in combination with CFP (CpG/CFP). After 15 days of treatment, airway inflammation and specific T- and B-cell responses were determined. Cell transfer experiments were also performed. CpG treatment attenuated airway allergic disease; however, the combination CpG/CFP treatment was significantly more effective in decreasing airway hyperresponsiveness, eosinophilia and Th2 response. When an additional intranasal dose of CFP was given, allergy was even more attenuated. The CpG/CFP therapy also reduced allergen-specific IgG1 and IgE antibodies and increased IgG2a. Transfer of spleen cells from mice immunized with CpG/CFP also reduced allergic lung inflammation. CpG/CFP treatment induced CFP-specific production of IFN-γ and IL-10 by spleen cells and increased production of IFN-γ in response to OVA. The essential role of IFN-γ for the therapeutic effect of CpG/CFP was evidenced in IFN-γ knockout mice. These results show that CpG/CFP treatment reverses established Th2 allergic responses by an IFN-γ-dependent mechanism that seems to act both locally in the lung and systemically to decrease allergen-specific Th2 responses. 相似文献
994.
995.
A Combinatorial Histidine Scanning Library Approach to Engineer Highly pH-Dependent Protein Switches
Megan L Murtaugh Sean W Fanning Tressa M Sharma Alexandra M Terry James R Horn 《Protein science : a publication of the Protein Society》2011,20(9):1619-1631
There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein–protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton‐linkage dictate that the presence of multiple ionizable groups, which undergo a pKa change on protein binding, are necessary to result in highly pH‐dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild‐type residue is sampled throughout the interface of a model anti‐RNase A single domain VHH antibody. Antibodies were coselected for high‐affinity binding and pH‐sensitivity using an in vitro, dual‐function selection strategy. The resulting antibodies retained near wild‐type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine “hot‐spots,” which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH‐sensitive protein affinity reagents for a number of different applications. 相似文献
996.
Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B1 when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B1. Reductions in aflatoxin B1 often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B1 when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains. 相似文献
997.
Background
The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained.Methodology/Principal Findings
This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias.Conclusions
Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization. 相似文献998.
Most human exposures to ionising radiation are partial body exposures. However, to date only limited tools are available for rapid and accurate estimation of the dose distribution and the extent of the body spared from the exposure. These parameters are of great importance for emergency triage and clinical management of exposed individuals. Here, measurements of γ-H2AX immunofluorescence by microscopy and flow cytometry were compared as rapid biodosimetric tools for whole and partial body exposures. Ex vivo uniformly X-irradiated blood lymphocytes from one donor were used to generate a universal biexponential calibration function for γ-H2AX foci/intensity yields per unit dose for time points up to 96 hours post exposure. Foci--but not intensity--levels remained significantly above background for 96 hours for doses of 0.5 Gy or more. Foci-based dose estimates for ex vivo X-irradiated blood samples from 13 volunteers were in excellent agreement with the actual dose delivered to the targeted samples. Flow cytometric dose estimates for X-irradiated blood samples from 8 volunteers were in excellent agreement with the actual dose delivered at 1 hour post exposure but less so at 24 hours post exposure. In partial body exposures, simulated by mixing ex vivo irradiated and unirradiated lymphocytes, foci/intensity distributions were significantly over-dispersed compared to uniformly irradiated lymphocytes. For both methods and in all cases the estimated fraction of irradiated lymphocytes and dose to that fraction, calculated using the zero contaminated Poisson test and γ-H2AX calibration function, were in good agreement with the actual mixing ratios and doses delivered to the samples. In conclusion, γ-H2AX analysis of irradiated lymphocytes enables rapid and accurate assessment of whole body doses while dispersion analysis of foci or intensity distributions helps determine partial body doses and the irradiated fraction size in cases of partial body exposures. 相似文献
999.
Christa Kasang Samuel Kalluvya Charles Majinge August Stich Jochen Bodem Gilbert Kongola Graeme B. Jacobs Mathias Mlewa Miriam Mildner Irina Hensel Anne Horn Wolfgang Preiser Gert van Zyl Hartwig Klinker Eleni Koutsilieri Axel Rethwilm Carsten Scheller Benedikt Weissbrich 《PloS one》2011,6(8)
Background
The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients <25 years is representative for HIVDR in the rest of the therapy-naïve population.Methods and Findings
HIVDR was determined in 88 sequentially enrolled ART-naïve patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged <25 years and 68 patients were aged 25–63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072–0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients >25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095–0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma.Conclusions
ART-naïve patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naïve HIV-infected population. 相似文献1000.
Michael V. Miles Philip E. Putnam Lili Miles Peter H. Tang Antonius J. DeGrauw Brenda L. Wong Paul S. Horn Heather L. Foote Marc E. Rothenberg 《Mitochondrion》2011,11(1):127-135
The current study evaluated 23 children (ages 2–16 years) with recurrent food intolerance and allergies for CoQ10 deficiency and mitochondrial abnormalities. Muscle biopsies were tested for CoQ10 levels, pathology, and mitochondrial respiratory chain (MRC) activities. Group 2 (age > 10 years; n = 9) subjects had significantly decreased muscle CoQ10 than Group 1 (age < 10 y; n = 14) subjects (p = 0.001) and 16 controls (p < 0.05). MRC activities were significantly lower in Group 2 than in Group 1 (p < 0.05). Muscle CoQ10 levels in study subjects were significantly correlated with duration of illness (adjusted r2 = 0.69; p = 0.012; n = 23). Children with recurrent food intolerance and allergies may acquire CoQ10 deficiency with disease progression. 相似文献