Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol

Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis.     

First synthesis of phosphonobile acids and preliminary studies on their aggregation properties

The synthesis of three novel phosphonobile acids from natural bile acids is reported. The CMC of phosphonodeoxycholic acid (PDCA) at pH 8.2 was found to be lower than that of the parent deoxycholic acid (DCA). PDCA micelles were also found to have higher microviscosity compared to DCA micelles, suggesting higher hydrophobicity and tighter packing in the interior of PDCA micelles. PDCA aggregated further to form an aqueous gel at pH 4.     

Species Composition of Bacterial Communities Influences Attraction of Mosquitoes to Experimental Plant Infusions

In the container habitats of immature mosquitoes, catabolism of plant matter and other organic detritus by microbial organisms produces metabolites that mediate the oviposition behavior of Aedes aegypti and Aedes albopictus. Public health agencies commonly use oviposition traps containing plant infusions for monitoring populations of these mosquito species, which are global vectors of dengue viruses. In laboratory experiments, gravid females exhibited significantly diminished responses to experimental infusions made with sterilized white oak leaves, showing that attractive odorants were produced through microbial metabolic activity. We evaluated effects of infusion concentration and fermentation time on attraction of gravid females to infusions made from senescent bamboo or white oak leaves. We used plate counts of heterotrophic bacteria, total counts of 4′,6-diamidino-2-phenylindole-stained bacterial cells, and 16S ribosomal DNA (rDNA) polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) to show that changes in the relative abundance of bacteria and the species composition of bacterial communities influenced attraction of gravid A. aegypti and A. albopictus mosquitoes to infusions. DGGE profiles showed that bacterial species composition in infusions changed over time. Principal components analysis indicated that oviposition responses to plant infusions were in general most affected by bacterial diversity and abundance. Analysis of bacterial 16S rDNA sequences derived from DGGE bands revealed that Proteobacteria (Alpha-, Beta-, Delta-, and Gamma-) were the predominant bacteria detected in both types of plant infusions. Gravid A. aegypti were significantly attracted to a mix of 14 bacterial species cultured from bamboo leaf infusion. The oviposition response of gravid mosquitoes to plant infusions is strongly influenced by abundance and diversity of bacterial species, which in turn is affected by plant species, leaf biomass, and fermentation time.     

Theoretical investigation on the glycan‐binding specificity of Agrocybe cylindracea galectin using molecular modeling and molecular dynamics simulation studies

Galectins are β‐galactoside binding proteins which have the ability to serve as potent antitumor, cancer biomarker, and induce tumor cell apoptosis. Agrocybe cylindracea galectin (ACG) is a fungal galectin which specifically recognizes α(2,3)‐linked sialyllactose at the cell surface that plays extensive roles in the biological recognition processes. To investigate the change in glycan‐binding specificity upon mutations, single point and double point site‐directed in silico mutations are performed at the binding pocket of ACG. Molecular dynamics (MD) simulation studies are carried out for the wild‐type (ACG) and single point (ACG1) and double point (ACG2) mutated ACGs to investigate the dynamics of substituted mutants and their interactions with the receptor sialyllactose. Plausible binding modes are proposed for galectin–sialylglycan complexes based on the analysis of hydrogen bonding interactions, total pair‐wise interaction energy between the interacting binding site residues and sialyllactose and binding free energy of the complexes using molecular mechanics–Poisson–Boltzmann surface area. Our result shows that high contribution to the binding in different modes is due to the direct and water‐mediated hydrogen bonds. The binding specificity of double point mutant Y59R/N140Q of ACG2 is found to be high, and it has 26 direct and water‐mediated hydrogen bonds with a relatively low‐binding free energy of −47.52 ± 5.2 kcal/mol. We also observe that the substituted mutant Arg59 is crucial for glycan‐binding and for the preference of α(2,3)‐linked sialyllactose at the binding pocket of ACG2 galectin. When compared with the wild‐type and single point mutant, the double point mutant exhibits enhanced affinity towards α(2,3)‐linked sialyllactose, which can be effectively used as a model for biological cell marker in cancer therapeutics. Copyright © 2015 John Wiley & Sons, Ltd.     

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.     

Bacteria stimulate hatching of yellow fever mosquito eggs

Background

Aedes aegypti Linnaeus is a peridomestic mosquito that lays desiccation-resistant eggs in water-filled human-made containers. Previous investigations connected egg hatching with declining dissolved oxygen (DO) that is associated with bacterial growth. However, past studies failed to uncouple DO from other potential stimulatory factors and they contained little quantitative information about the microbial community; consequently, a direct role for bacteria or compounds associated with bacteria in stimulating egg hatching cannot be dismissed.

Methodology/Principal Findings

Environmental factors stimulating hatch of Ae. aegypti eggs were investigated using non-sterile and sterile white oak leaf (WOL) infusions and a bacterial culture composed of a mix of 14 species originally isolated from bamboo leaf infusion. In WOL infusion with active microbes, 92.4% of eggs hatched in 2-h at an average DO concentration of 2.4 ppm. A 24-h old bacterial culture with a DO concentration of 0.73 ppm also stimulated 95.2% of eggs hatch within 1-h. In contrast, only 4.0% of eggs hatched in sterile infusion, whose DO averaged 7.4 ppm. Effects of bacteria were uncoupled from DO by exposing eggs to bacterial cells suspended in NaCl solution. Over a 4-h exposure period, 93.8% of eggs hatched while DO concentration changed minimally from 7.62 to 7.50 ppm. Removal of bacteria by ultra-filtration and cell-free filtrate resulted in only 52.0% of eggs hatching after 4-h at an average DO concentration of 5.5 ppm.

Conclusions/Significance

Collectively, the results provide compelling evidence that bacteria or water-soluble compounds secreted by bacteria, not just low DO concentration, stimulate hatching of Ae. aegypti eggs. However, the specific cues involved remain to be identified. These research findings contribute new insight into an important aspect of the oviposition biology of Ae. aegypti, a virus vector of global importance, providing the basis for a new paradigm of environmental factors involved in egg hatching.     

Purification of lipase from Cunninghamella verticillata by stepwise precipitation and optimized conditions for crystallization

Lipase from the oil-mill waste isolate Cunninghamella verticillata was purified by stepwise precipitation using acetone, as a sequel to our earlier conventional column chromatographic method [Gopinath et al. (2002)World Journal of Microbiology and Biotechnology 18, 449–458]. The yield of purified lipase was approx. 4-fold higher than by the previous method and the purified lipase was obtained with 70–80% acetone saturations. The enzyme was resolved as a single band with homogeneity both by native and by SDS–PAGE. The optimum condition for the lipase to crystallize was 5 g of enzyme in 0.05 M sodium phosphate buffer (pH 6.5) with 5 mM FeCl₂ and 10% 2-methyl 2,4-pentanediol (MPD).These authors equally contributed to this work     

ExSer: A standalone tool to mine protein data bank (PDB) for secondary structural elements

Detailed structural analysis of protein necessitates investigation at primary, secondary and tertiary levels, respectively. Insight into protein secondary structures pave way for understanding the type of secondary structural elements involved (α-helices, β-strands etc.), the amino acid sequence that encode the secondary structural elements, number of residues, length and, percentage composition of the respective elements in the protein. Here we present a standalone tool entitled "ExSer" which facilitate an automated extraction of the amino acid sequence that encode for the secondary structural regions of a protein from the protein data bank (PDB) file. AVAILABILITY: ExSer is freely downloadable from http://code.google.com/p/tool-exser/