Drosophila Lipophorin Receptors Recruit the Lipoprotein LTP to the Plasma Membrane to Mediate Lipid Uptake

Lipophorin, the main Drosophila lipoprotein, circulates in the hemolymph transporting lipids between organs following routes that must adapt to changing physiological requirements. Lipophorin receptors expressed in developmentally dynamic patterns in tissues such as imaginal discs, oenocytes and ovaries control the timing and tissular distribution of lipid uptake. Using an affinity purification strategy, we identified a novel ligand for the lipophorin receptors, the circulating lipoprotein Lipid Transfer Particle (LTP). We show that specific isoforms of the lipophorin receptors mediate the extracellular accumulation of LTP in imaginal discs and ovaries. The interaction requires the LA-1 module in the lipophorin receptors and is strengthened by a contiguous region of 16 conserved amino acids. Lipophorin receptor variants that do not interact with LTP cannot mediate lipid uptake, revealing an essential role of LTP in the process. In addition, we show that lipophorin associates with the lipophorin receptors and with the extracellular matrix through weak interactions. However, during lipophorin receptor-mediated lipid uptake, LTP is required for a transient stabilization of lipophorin in the basolateral plasma membrane of imaginal disc cells. Together, our data suggests a molecular mechanism by which the lipophorin receptors tether LTP to the plasma membrane in lipid acceptor tissues. LTP would interact with lipophorin particles adsorbed to the extracellular matrix and with the plasma membrane, catalyzing the exchange of lipids between them.     

Macro and micro diversity of Clostridium difficile isolates from diverse sources and geographical locations

Clostridium difficile has emerged rapidly as the leading cause of antibiotic-associated diarrheal disease, with the temporal and geographical appearance of dominant PCR ribotypes such as 017, 027 and 078. Despite this continued threat, we have a poor understanding of how or why particular variants emerge and the sources of strains that dominate different human populations. We have undertaken a breadth genotyping study using multilocus sequence typing (MLST) analysis of 385 C. difficile strains from diverse sources by host (human, animal and food), geographical locations (North America, Europe and Australia) and PCR ribotypes. Results identified 18 novel sequence types (STs) and 3 new allele sequences and confirmed the presence of five distinct clonal lineages generally associated with outbreaks of C. difficile infection in humans. Strains of animal and food origin were found of both ST-1 and ST-11 that are frequently associated with human disease. An in depth MLST analysis of the evolutionary distant ST-11/PCR ribotype 078 clonal lineage revealed that ST-11 can be found in alternative but closely related PCR ribotypes and PCR ribotype 078 alleles contain mutations generating novel STs. PCR ribotype 027 and 017 lineages may consist of two divergent subclades. Furthermore evidence of microdiversity was present within the heterogeneous clade 1. This study helps to define the evolutionary origin of dominant C. difficile lineages and demonstrates that C. difficile is continuing to evolve in concert with human activity.     

Rotavirus Infection of Cells in Culture Induces Activation of RhoA and Changes in the Actin and Tubulin Cytoskeleton

Rotavirus infection induces an increase in [Ca²⁺]_cyto, which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca²⁺ chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca²⁺ homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.     

Natural disulfide bond-disrupted mutants of AVR4 of the tomato pathogen Cladosporium fulvum are sensitive to proteolysis,circumvent Cf-4-mediated resistance,but retain their chitin binding ability

The extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. Here, the four disulfide bonds of AVR4 were identified as Cys-11-41, Cys-21-27, Cys-35-80, and Cys-57-72 by partial reduction with Tris-(2-carboxyethyl)-phosphine hydrochloride, subsequent cyanylation, and base-catalyzed chain cleavage. The resulting peptide fragments were analyzed by mass spectrometry. Sequence homology and the disulfide bond pattern revealed that AVR4 contains an invertebrate (inv) chitin-binding domain (ChBD). Binding of AVR4 to chitin was confirmed experimentally. The three disulfide bonds encompassing the inv ChBD motif are also required for protein stability of AVR4. Independent disruption of each of the three conserved disulfide bonds in AVR4 resulted in a protease-sensitive protein, whereas the fourth disulfide bond appeared not to be required for protein stability. Most strains of C. fulvum virulent on Cf-4 tomato contain Cys to Tyr substitutions in AVR4 involving two (Cys-11-41, Cys-35-80) of the three disulfide bonds present in the inv ChBD motif. These natural Cys to Tyr mutant AVR4 proteins did retain their chitin binding ability and when bound to chitin were less sensitive to proteases. Thus, the widely applied tomato Cf-4 resistance gene is circumvented by C. fulvum by amino acid substitutions affecting two disulfide bonds in AVR4 resulting in the absence of the corresponding AVR4 isoforms in apoplastic fluid. However, these natural isoforms of AVR4 appear to have retained their intrinsic function, i.e. binding to chitin present in the cell wall of C. fulvum, most likely to protect it against the deleterious effects of plant chitinases.     

Screening methods to determine biophysical properties of proteins in structural genomics

We have developed and tested a simple and efficient protein purification method for biophysical screening of proteins and protein fragments by nuclear magnetic resonance (NMR) and optical methods, such as circular dichroism spectroscopy. The method constitutes an extension of previously described protocols for gene expression and protein solubility screening [M. Hammarstr?m et al., (2002), Protein Science 11, 313]. Using the present purification scheme it is possible to take several target proteins, produced as fusion proteins, from cell pellet to NMR spectrum and obtain a judgment on the suitability for further structural or biophysical studies in less than 1 day. The method is independent of individual protein properties as long as the target protein can be produced in soluble form with a fusion partner. Identical procedures for cell culturing, lysis, affinity chromatography, protease cleavage, and NMR sample preparation then initially require only optimization for different fusion partner and protease combinations. The purification method can be automated, scaled up or down, and extended to a traditional purification scheme. We have tested the method on several small human proteins produced in Escherichia coli and find that the method allows for detection of structured proteins and unfolded or molten globule-like proteins.     

Bioimpedance analysis: a useful technique for assessing appendicular lean soft tissue mass and distribution.

The present study was aimed at evaluating the feasibility and reliability of lower limb skeletal muscle (SM) mass estimates obtained by bioimpedance analysis (BIA). BIA estimates were compared with the estimates obtained by dual-energy X-ray absorptiometry (DXA). Ten normal weight and 10 obese women had BIA and DXA evaluations. Lower limb SM mass was then derived from DXA appendicular lean soft tissue estimates. Lower limb SM mass and SM distribution were also estimated from BIA modeling that fits measured resistance values along the leg. SM mass (mean +/- SD) was 5.8 +/- 1.0 kg by BIA vs. 5.8 +/- 1.1 kg by DXA in normal weight subjects and 7.2 +/- 1.4 kg by BIA vs. 7.2 +/- 1.2 kg by DXA in obese subjects. Mean +/- SD of the absolute value of the relative error was 7.0 +/- 3.4 and 5.9 +/- 3.4% in the two groups, respectively. Similar results were obtained by using five resistance values for the analysis. In conclusion, the proposed BIA model provides an adequate means of evaluating appendicular SM mass.     

Dichotomy between CD1a+ and CD83+ dendritic cells in lymph nodes during SIV infection of macaques

The prevalence and differentiation of dendritic cells (DC) in lymphoid tissue of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys was studied during disease progression. Lymph node biopsies were consecutively obtained from clinical rapid and slow progressors until the development of disease consistent with simian acquired immunodeficiency syndrome (sAIDS) occurred. Quantitative evaluation of CD1a+ DC and the expression of DC antigens related to maturation (CD83, DC-LAMP and S100b) were performed at the single cell level by in situ image analysis. Despite a persistent prevalence of CD1a+ DC in lymphoid tissue during disease progression, there was a subsequent drop of mature CD83+, DC-LAMP+ and S100b+ DC, correlating with the decline of CD4+ T cells in blood. Thus, disease progression to sAIDS was associated with impaired maturation of DC, and lack of CD83, DC-LAMP and S100b expression.     

Riverside wren pairs jointly defend their territories against simulated intruders

Duets are a jointly produced signal where two or more individuals coordinate their vocalizations by overlapping or alternating their songs. Duets are used in a wide array of contexts within partnerships, ranging from territory defence to pair bond maintenance. It has been proposed that pairs that coordinate their songs might also better coordinate other activities, including nest building, parental care and defending shared resources. Here, we tested in the riverside wren (Cantorchilus semibadius), a neotropical duetting species that produces highly coordinated duet songs, whether males and females show similar responses to playback. During territorial disputes in songbird species, individuals tend to direct their attention towards same‐sex territorial intruders, but this bias might be less pronounced in duetting species. We performed a dual‐speaker playback experiment to examine how mated individuals respond to speakers broadcasting female‐versus‐male duet contributions. We found that riverside wrens have high levels of converging behaviour by duetting and remaining in close proximity to one another when responding to simulated paired intruders. Males and females spent more than 80% of their time less than 1 m apart while defending their territory. Both individuals in a pair aggressively engaged with both male and female simulated trespassers by approaching equally close and spending equal time near the two speakers. These results suggest that both sexes perceive a paired territorial intrusion as a similar threat and that both partners are highly invested in defending the shared resources. This study is one of the few to demonstrate equal attention and aggression from mated pairs towards simulated same‐sex and opposite‐sex intruders. We suggest that pairs responding together, in close proximity to one another, might be favourable in duetting species when defending the territory because maintaining a close distance between partners facilitates the extreme coordination of their joint territorial signals.