Flexibility of KorA,a plasmid-encoded,global transcription regulator,in the presence and the absence of its operator

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.     

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.     

Resveratrol induces catalytic bioscavenger paraoxonase 1 expression and protects against chemical warfare nerve agent toxicity in human cell lines

Current advances in enzyme bioscavenger prophylactic therapy against chemical warfare nerve agent (CWNA) exposure are moving towards the identification of catalytic bioscavengers that can degrade large doses of organophosphate (OP) nerve agents without self destruction. This is a preferred method compared to therapy with the purified stoichiometric bioscavenger, butyrylcholinesterase, which binds OPs 1:1 and would thus require larger doses for treatment. Paraoxonase-1 (PON-1) is one such catalytic bioscavenger that has been shown to hydrolyze OP insecticides and contribute to detoxification in animals and humans. Here we investigated the effects of a common red wine ingredient, Resveratrol (RSV), to induce the expression of PON-1 in the human hepatic cell line HC04 and evaluated the protection against CWNA simulants. Dose-response curves showed that a concentration of 20 microM RSV was optimal in inducing PON-1 expression in HC04 cells. RSV at 20 microM increased the extracellular PON-1 activity approximately 150% without significantly affecting the cells. Higher doses of RSV were cytotoxic to the cells. Resveratrol also induced PON-1 in the human lung cell line A549. RSV pre-treatment significantly (P = 0.05) protected the hepatic cells against exposure to 2x LD(50) of soman and sarin simulants. However, lung cells were protected against soman simulant exposure but not against sarin simulant exposure following RSV treatment. In conclusion, these studies indicate that dietary inducers, such as RSV, can up-regulate PON-1, a catalytic bioscavenger, which can then hydrolyze and protect against CWNA-induced toxicity, providing a prospective new method to protect against CWNA exposure.     

Using least median of squares for structural superposition of flexible proteins

Background

The conventional superposition methods use an ordinary least squares (LS) fit for structural comparison of two different conformations of the same protein. The main problem of the LS fit that it is sensitive to outliers, i.e. large displacements of the original structures superimposed.

Results

To overcome this problem, we present a new algorithm to overlap two protein conformations by their atomic coordinates using a robust statistics technique: least median of squares (LMS). In order to effectively approximate the LMS optimization, the forward search technique is utilized. Our algorithm can automatically detect and superimpose the rigid core regions of two conformations with small or large displacements. In contrast, most existing superposition techniques strongly depend on the initial LS estimating for the entire atom sets of proteins. They may fail on structural superposition of two conformations with large displacements. The presented LMS fit can be considered as an alternative and complementary tool for structural superposition.

Conclusion

The proposed algorithm is robust and does not require any prior knowledge of the flexible regions. Furthermore, we show that the LMS fit can be extended to multiple level superposition between two conformations with several rigid domains. Our fit tool has produced successful superpositions when applied to proteins for which two conformations are known. The binary executable program for Windows platform, tested examples, and database are available from https://engineering.purdue.edu/PRECISE/LMSfit.     

Receptor Specificity and Transmission of H2N2 Subtype Viruses Isolated from the Pandemic of 1957

Influenza viruses of the H2N2 subtype have not circulated among humans in over 40 years. The occasional isolation of avian H2 strains from swine and avian species coupled with waning population immunity to H2 hemagglutinin (HA) warrants investigation of this subtype due to its pandemic potential. In this study we examined the transmissibility of representative human H2N2 viruses, A/Albany/6/58 (Alb/58) and A/El Salvador/2/57 (ElSalv/57), isolated during the 1957/58 pandemic, in the ferret model. The receptor binding properties of these H2N2 viruses was analyzed using dose-dependent direct glycan array-binding assays. Alb/58 virus, which contains the 226L/228S amino acid combination in the HA and displayed dual binding to both alpha 2,6 and alpha 2,3 glycan receptors, transmitted efficiently to naïve ferrets by respiratory droplets. Inefficient transmission was observed with ElSalv/57 virus, which contains the 226Q/228G amino acid combination and preferentially binds alpha 2,3 over alpha 2,6 glycan receptors. However, a unique transmission event with the ElSalv/57 virus occurred which produced a 226L/228G H2N2 natural variant virus that displayed an increase in binding specificity to alpha 2,6 glycan receptors and enhanced respiratory droplet transmissibility. Our studies provide a correlation between binding affinity to glycan receptors with terminal alpha 2,6-linked sialic acid and the efficiency of respiratory droplet transmission for pandemic H2N2 influenza viruses.     

Comparative study on removal of enteric pathogens from domestic wastewater using Typha latifolia and Cyperus rotundus along with different substrates

A comparative study was carried out to evaluate the efficiency of different substrate materials along with macrophytes Typha latifolia and Cyperus rotundus in treating domestic wastewater intended for reuse in agriculture. The study was conducted over a period of 6 months with different retention times, and observations were taken twice per month. One-way analysis of variance and Tukey's Honest Significant Difference (HSD) tests were used to determine statistical significant differences between experimental columns. Treatment with T. latifolia planted in sand and mix substrate with 4-day retention time remarkably reduced the concentration of all bacterial pathogens. Log reductions observed were approximately 5.01 and 4.82 for total coliform (TC), 4.46 and 3.93 for Escherichia coli, and 5.52 and 5.48 for Shigella, respectively. Moreover, these treatments were also efficient in completely removing fecal coliform (FC) and Salmonella.Maximum parasites were removed by the treatment having sand alone as a substrate containing C. rotundus, but the difference was not significant from those planted with T. latifolia in the same substrate. The results suggest that T. latifolia aids in bacterial pathogens removal, while C. rotundus aids in parasites removal. Thus, wastewater treatment through constructed wetland having mix plantation of these species along with sand can eliminate some of the major enteric pathogens.     

The metalloprotease of Listeria monocytogenes is regulated by pH

Listeria monocytogenes is an intracytosolic bacterial pathogen. Among the factors contributing to escape from vacuoles are a phosphatidylcholine phospholipase C (PC-PLC) and a metalloprotease (Mpl). Both enzymes are translocated across the bacterial membrane as inactive proproteins, whose propeptides serve in part to maintain them in association with the bacterium. We have shown that PC-PLC maturation is regulated by Mpl and pH and that Mpl maturation occurs by autocatalysis. In this study, we tested the hypothesis that Mpl activity is pH regulated. To synchronize the effect of pH on bacteria, the cytosolic pH of infected cells was manipulated immediately after radiolabeling de novo-synthesized bacterial proteins. Immunoprecipitation of secreted Mpl from host cell lysates revealed the presence of the propeptide and catalytic domain in samples treated at pH 6.5 but not at pH 7.3. The zymogen was present in small amounts under all conditions. Since proteases often remain associated with their respective propeptide following autocatalysis, we aimed at determining whether pH regulates autocatalysis or secretion of the processed enzyme. For this purpose, we used an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain and antibodies that can distinguish N-terminal and non-N-terminal Flag. By fluorescence microscopy, we observed the Mpl zymogen associated with the bacterium at physiological pH but not following acidification. Mature Mpl was not detected in association with the bacterium at either pH. Using purified proteins, we determined that processing of the PC-PLC propeptide by mature Mpl is also pH sensitive. These results indicate that pH regulates the activity of Mpl on itself and on PC-PLC.