Phloretin-Induced Reduction in Dipole Potential of Sterol-Containing Bilayers

The phloretin-induced reduction in the dipole potential of planar lipid bilayers containing cholesterol, ergosterol, stigmasterol, 7-dehydrocholesterol and 5α-androstan-3β-ol was investigated. It is shown that effects depend on the type and concentration of membrane sterol. It is supposed that the effectiveness of phloretin in reducing the dipole potential of the bilayers that contain cholesterol, ergosterol and 7-dehydrocholesterol correlates with the ordering and condensing effects. The role of the concentration-dependent ability of different sterols to promote lateral heterogeneity in membranes is also discussed.     

NHERF2 Protein Mobility Rate Is Determined by a Unique C-terminal Domain That Is Also Necessary for Its Regulation of NHE3 Protein in OK Cells

Na⁺/H⁺ exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.     

Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO₄ or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.     

Role of Bacterial Communities in the Natural Suppression of Rhizoctonia solani Bare Patch Disease of Wheat (Triticum aestivum L.)

Rhizoctonia bare patch and root rot disease of wheat, caused by Rhizoctonia solani AG-8, develops as distinct patches of stunted plants and limits the yield of direct-seeded (no-till) wheat in the Pacific Northwest of the United States. At the site of a long-term cropping systems study near Ritzville, WA, a decline in Rhizoctonia patch disease was observed over an 11-year period. Bacterial communities from bulk and rhizosphere soil of plants from inside the patches, outside the patches, and recovered patches were analyzed by using pyrosequencing with primers designed for 16S rRNA. Taxa in the class Acidobacteria and the genus Gemmatimonas were found at higher frequencies in the rhizosphere of healthy plants outside the patches than in that of diseased plants from inside the patches. Dyella and Acidobacteria subgroup Gp7 were found at higher frequencies in recovered patches. Chitinophaga, Pedobacter, Oxalobacteriaceae (Duganella and Massilia), and Chyseobacterium were found at higher frequencies in the rhizosphere of diseased plants from inside the patches. For selected taxa, trends were validated by quantitative PCR (qPCR), and observed shifts of frequencies in the rhizosphere over time were duplicated in cycling experiments in the greenhouse that involved successive plantings of wheat in Rhizoctonia-inoculated soil. Chryseobacterium soldanellicola was isolated from the rhizosphere inside the patches and exhibited significant antagonism against R. solani AG-8 in vitro and in greenhouse tests. In conclusion, we identified novel bacterial taxa that respond to conditions affecting bare patch disease symptoms and that may be involved in suppression of Rhizoctonia root rot and bare batch disease.     

Molinate biodegradation in soils: natural attenuation versus bioaugmentation

The aims of the present study were to assess the potential of natural attenuation or bioaugmentation to reduce soil molinate contamination in paddy field soils and the impact of these bioremediation strategies on the composition of soil indigenous microbiota. A molinate mineralizing culture (mixed culture DC) was used as inoculum in the bioaugmentation assays. Significantly higher removal of molinate was observed in bioaugmentation than in natural attenuation microcosms (63 and 39 %, respectively) after 42 days of incubation at 22 °C. In the bioaugmentation assays, the impact of Gulosibacter molinativorax ON4^T on molinate depletion was observed since the gene encoding the enzyme responsible for the initial molinate breakdown (harboured by that actinobacterium) was only detected in inoculated microcosms. Nevertheless, the exogenous mixed culture DC did not overgrow as the heterotrophic counts of the bioaugmentation microcosms were not significantly different from those of natural attenuation and controls. Moreover, the actinobacterial clone libraries generated from the bioaugmentation microcosms did not include any 16S rRNA gene sequences with significant similarity to that of G. molinativorax ON4^T. The multivariate analysis of the 16S rRNA DGGE patterns of the soil microcosm suggested that the activity of mixed culture DC did not affect the soil bacterial community structure since the DGGE patterns of the bioaugmentation microcosms clustered with those of natural attenuation and controls. Although both bioremediation approaches removed molinate without indigenous microbiota perturbation, the results suggested that bioaugmentation with mixed culture DC was more effective to treat soils contaminated with molinate.     

Cuticular fatty acid profile analysis of three Rhipicephalus tick species (Acari: Ixodidae)

Cuticular fatty acids (CFA) are important constituents of the arthropod exoskeleton, serving as structural and defense components, and participating in intra-species communication. Here we describe for the first time a comparative analysis of the CFA profiles of three tick species of the genus Rhipicephalus: R. annulatus, R. bursa and R. sanguineus. CFA profiles were determined for R. bursa and R. sanguineus grown both on rabbit or calf, and for R. annulatus grown on calf. CFA composition was compared for each species before and after ethanol treatment, for different hosts of each species, and between the different species. Our data suggest that adsorption of the host’s fatty acids changes the apparent CFA composition. Ethanol treatment efficiently removed the unbound fatty acids from the ticks and revealed the actual composition. Comparison between ticks grown on rabbit versus calf showed significant difference in the relative abundance of fatty acids C14 and 9,12-C18:2 for R. bursa, and a difference in the relative abundance of C14 for R. sanguineus. Comparison of the CFA between the three species revealed significant differences in the abundance of fatty acids C16, 9,12-C18:2, 9-C18:1, C18 and C20. Our results show that while the host had a minor effect on CFA composition within each species, significant differences were observed in the CFA profiles of different species. We suggest that CFA profiles may be used to distinguish between related species. CFA analysis can also be used in studies of communication and defense mechanisms in ticks and other arthropods.     

Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in?Vitro and in Silico

Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid’s physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔH_ind = 11.5–12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔS_ind = 5.1–5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses’ biological function.     

Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid’s physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state ΔH_ind = 11.5–12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending; the entropy change TΔS_ind = 5.1–5.8 MJ/mol is due to coherent in-plane rearrangements of protein chains, which mediate the capsid stiffening. Direct coupling of these modes defines the extent of (ir)reversibility of capsid indentation dynamics correlated with its (in)elastic mechanical response to the compressive force. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses’ biological function.