One step purification of peanut phospholipase D by precipitation with alginate

A simple titrimetric assay with soybean lecithin has been used for screening phospholipase D activity from some plant sources, viz. peanut, wheat germ, cabbage and carrot. The enzyme from peanut has been purified by binding to alginate which is a water soluble polymer. The purification consisted of co-precipitation of enzyme with alginate upon addition of 0.06 M Ca⁺⁺. The enzyme was eluted from the polymer using 0.2 M sodium chloride. The activity recovery was 61% with 34 fold purification.     

Influence of Antioxidants on the Peroxidative Swelling of Mitochondria in vitro

To clarify the role of prooxidative processes during in vitro swelling of freshly isolated rat liver mitochondria, the influence of different antioxidants and free-radical scavengers was tested. Ascorbate below 10 mmol/L without externally added Fe²⁺ acted as a prooxidant and enhanced swelling. Higher concentrations in the presence of Fe²⁺ showed antioxidant properties and a decrease in swelling and lipid peroxidation. Swelling was abolished by -tocopherol and reduced to 50% by butylated hydroxytoluene. Glutathione supplementation decreased both swelling and lipid peroxidation. Oxidized glutathione caused swelling without any effect on peroxidation. Hydrogen peroxide, cumene hydroperoxide and t-butyl hydroperoxide caused progressive decreases in glutathione and reduced niacinamide coenzyme levels, suggesting prooxidative changes. Dithiothreitol was found to abolish this effect. Thus, antioxidants reverse superoxide-induced mito chondrial swelling and lipid peroxidation in vitro.     

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.     

Antiviral Activity of the Human Cathelicidin,LL-37, and Derived Peptides on Seasonal and Pandemic Influenza A Viruses

Human LL-37, a cationic antimicrobial peptide, was recently shown to have antiviral activity against influenza A virus (IAV) strains in vitro and in vivo. In this study we compared the anti-influenza activity of LL-37 with that of several fragments derived from LL-37. We first tested the peptides against a seasonal H3N2 strain and the mouse adapted H1N1 strain, PR-8. The N-terminal fragment, LL-23, had slight neutralizing activity against these strains. In LL-23V9 serine 9 is substituted by valine creating a continuous hydrophobic surface. LL-23V9 has been shown to have increased anti-bacterial activity compared to LL-23 and we now show slightly increased antiviral activity compared to LL-23 as well. The short central fragments, FK-13 and KR-12, which have anti-bacterial activity did not inhibit IAV. In contrast, a longer 20 amino acid central fragment of LL-37 (GI-20) had neutralizing activity similar to LL-37. None of the peptides inhibited viral hemagglutination or neuraminidase activity. We next tested activity of the peptides against a strain of pandemic H1N1 of 2009 (A/California/04/09/H1N1 or “Cal09”). Unexpectedly, LL-37 had markedly reduced activity against Cal09 using several cell types and assays of antiviral activity. A mutant viral strain containing just the hemagglutinin (HA) of 2009 pandemic H1N1 was inhibited by LL-37, suggested that genes other than the HA are involved in the resistance of pH1N1. In contrast, GI-20 did inhibit Cal09. In conclusion, the central helix of LL-37 incorporated in GI-20 appears to be required for optimal antiviral activity. The finding that GI-20 inhibits Cal09 suggests that it may be possible to engineer derivatives of LL-37 with improved antiviral properties.     

Guanidine hydrochloride and urea-induced unfolding of Brugia malayi hexokinase

Guanidine hydrochloride and urea-induced unfolding of B. malayi hexokinase (BmHk), a tetrameric protein, was examined in detail by using various optical spectroscopic techniques, enzymatic activity measurements, and size-exclusion chromatography. The equilibrium unfolding of BmHk by guanidine hydrochloride (GdmCl) and urea proceeded through stabilization of several unique oligomeric intermediates. In the presence of low concentrations of GdmCl, stabilization of an enzymatically active folded dimer of BmHk was observed. However an enzymatically inactive dimer of BmHk was observed for urea-treated BmHk. This is the first report of an enzymatically active dimer of hexokinase from any human filarial parasite. Furthermore, although complete recovery of the native enzyme was observed on refolding of BmHk samples denatured by use of low concentrations of GdmCl or urea, no recovery of the native enzyme was observed for BmHk samples denatured by use of high concentrations of GdmCl or urea.     

The dynamic nature of contact networks in infectious disease epidemiology

Although contact network models have yielded important insights into infectious disease transmission and control throughout the last decade, researchers have just begun to explore the dynamic nature of contact patterns and their epidemiological significance. Most network models have assumed that contacts are static through time. Developing more realistic models of the social interactions that underlie the spread of infectious diseases thus remains an important challenge for both data gatherers and modelers. In this article, we review some recent data-driven and process-driven approaches that capture the dynamics of human contact, and discuss future challenges for the field.