Homology modeling, simulation and molecular docking studies of catechol-2, 3-Dioxygenase from Burkholderia cepacia: Involved in degradation of Petroleum hydrocarbons

Catechol 2, 3-dioxygenase is present in several types of bacteria and undergoes degradation of environmental pollutants through an important key biochemical pathways. Specifically, this enzyme cleaves aromatic rings of several environmental pollutants such as toluene, xylene, naphthalene and biphenyl derivatives. Hence, the importance of Catechol 2, 3-dioxygenase and its role in the degradation of environmental pollutants made us to predict the three-dimensional structure of Catechol 2, 3-dioxygenase from Burkholderia cepacia. The 10ns molecular dynamics simulation was carried out to check the stability of the modeled Catechol 2, 3- dioxygenase. The results show that the model was energetically stable, and it attains their equilibrium within 2000 ps of production MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3-dioxygenase reveals that the benzene, O-xylene, Toluene, Fluorene, Naphthalene, Carbazol, Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl makes strong hydrogen bond and Van der waals interaction with the active site residues of H150, L152, W198, H206, H220, H252, I254, T255, Y261, E271, L276 and F309. Free energy of binding and estimated inhibition constant of these compounds demonstrates that they are energetically stable in their binding cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except Pyrene all the substrates made close contact with Fe atom by the distance ranges from 1.67 to 2.43 Å. In addition to that, the above mentioned substrate except pyrene all other made π-π stacking interaction with H252 by the distance ranges from 3.40 to 3.90 Å. All these docking results reveal that, except Chrysene all other substrate has good free energy of binding to hold enough in the active site and makes strong VdW interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of catalyzing the above-mentioned substrate.     

Structure of the RACK1 dimer from Saccharomyces cerevisiae

Receptor for activated C-kinase 1 (RACK1) serves as a scaffolding protein in numerous signaling pathways involving kinases and membrane-bound receptors from different cellular compartments. It exists simultaneously as a cytosolic free form and as a ribosome-bound protein. As part of the 40S ribosomal subunit, it triggers translational regulation by establishing a direct link between protein kinase C and the protein synthesis machinery. It has been suggested that RACK1 could recruit other signaling molecules onto the ribosome, providing a signal-specific modulation of the translational process. RACK1 is able to dimerize both in vitro and in vivo. This homodimer formation has been observed in several processes including the regulation of the N-methyl-d-aspartate receptor by the Fyn kinase in the brain and the oxygen-independent degradation of hypoxia-inducible factor 1. The functional relevance of this dimerization is, however, still unclear and the question of a possible dimerization of the ribosome-bound protein is still pending. Here, we report the first structure of a RACK1 homodimer, as determined from two independent crystal forms of the Saccharomyces cerevisiae RACK1 protein (also known as Asc1p) at 2.9 and 3.9 Å resolution. The structure reveals an atypical mode of dimerization where monomers intertwine on blade 4, thus exposing a novel surface of the protein to potential interacting partners. We discuss the significance of the dimer structure for RACK1 function.     

Comparison of membranes and glass slides for 16S rDNA array analyses of microbial communities by sequence-specific labeling of DNA probes

Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for a DNA array based analyzes of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions, and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Arrays prepared on positively charged nylon membranes and coated glass slides were compared. The advantage of using membranes is that chromogenic signal amplification can be used for the detection. Furthermore, the chromogenic detection does not require any sophisticated equipment. The advantage of the glass slides is that multiple fluorescence colors can be detected simultaneously, and that internal controls can be used for normalization. This approach is also suited for high throughput screenings.     

Reducing Campylobacter jejuni Colonization of Poultry via Vaccination

Campylobacter jejuni is a leading bacterial cause of human gastrointestinal disease worldwide. While C. jejuni is a commensal organism in chickens, case-studies have demonstrated a link between infection with C. jejuni and the consumption of foods that have been cross-contaminated with raw or undercooked poultry. We hypothesized that vaccination of chickens with C. jejuni surface-exposed colonization proteins (SECPs) would reduce the ability of C. jejuni to colonize chickens, thereby reducing the contamination of poultry products at the retail level and potentially providing a safer food product for consumers. To test our hypothesis, we injected chickens with recombinant C. jejuni peptides from CadF, FlaA, FlpA, CmeC, and a CadF-FlaA-FlpA fusion protein. Seven days following challenge, chickens were necropsied and cecal contents were serially diluted and plated to determine the number of C. jejuni per gram of material. The sera from the chickens were also analyzed to determine the concentration and specificity of antibodies reactive against the C. jejuni SECPs. Vaccination of chickens with the CadF, FlaA, and FlpA peptides resulted in a reduction in the number of C. jejuni in the ceca compared to the non-vaccinated C. jejuni-challenged group. The greatest reduction in C. jejuni colonization was observed in chickens injected with the FlaA, FlpA, or CadF-FlaA-FlpA fusion proteins. Vaccination of chickens with different SECPs resulted in the production of C. jejuni-specific IgY antibodies. In summary, we show that the vaccination of poultry with individual C. jejuni SECPs or a combination of SECPs provides protection of chickens from C. jejuni colonization.     

Using wintergreen oil for mounting mosquito larvae: a safer alternative to xylene

Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.     

Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.