Protein isolation by solution-controlled gel sorption.

Illustrated are the principles for isolating proteins from solution by sorption into a polymer gel phase, driven by the addition of a water-soluble polymer to the protein solution. The separation is shown to be analogous to conventional two-phase aqueous extraction. However, the use of a gel phase rather than a solution for absorbing the protein makes separation of the protein from the polymer and the recycling of the gel phase much simpler. The model system used was linear poly(ethylene glycol) (PEG) and dextran gel. Increasing the molecular weight and concentration of the PEG favored sorption by the gel of ovalbumin, bovine serum albumin, cytochrome c, and hemoglobin. The proteins could be quantitatively recovered by immersing the gel in PEG-free solution.     

Probucol promotes endogenous antioxidant reserve and confers protection against reperfusion injury

The present study examines whether a subchronic probucol treatment of rats offers protection against ischemia-reperfusion (IR) injury in isolated perfused hearts. Sprague-Dawley rats were treated every second day per week with probucol (cumulative dose 120 mg/kg body mass, i.p.) for 4 weeks. In the probucol group, baseline myocardial antioxidant enzyme, glutathione peroxidase (GSHPx), activity was increased (p<0.05), whereas superoxide dismutase (SOD) and catalase (CAT) activities were not changed. Baseline oxidative stress, as indicated by the myocardial lipid peroxidation, was less (p<0.05) in the probucol group. Isolated hearts were subjected to 60 min global I and 20 min R. Recovery of the contractile function in globally ischemic hearts upon reperfusion was 36% in untreated group and 74% in the probucol group. After IR, GSHPx and CAT activities were significantly (p<0.05) higher in the probucol group compared with the control group, whereas SOD did not change. Lipid peroxidation owing to IR was significantly less in the probocol group. These data suggest that probucol treatment improves endogenous antioxidant reserve and protects against increased oxidative stress following IR injury.     

Pattern of nucleotide substitution and divergence of prophenoloxidase in decapods

Despite the unprecedented development in identification and characterization of prophenoloxidase (proPO) in commercially important decapods, little is known about the evolutionary relationship, rate of amino acid replacement and differential selection pressures operating on proPO of different species of decapods. Here we report the evolutionary relationship among these nine decapod species based on proPO gene and types of selective pressures operating on proPO codon sites. Our analyses revealed that all the nine decapod species shared a common ancestor. The mean percentage sequence divergence at proPO gene was 34.4+/-0.6%. Pairwise estimates of nonsynonymous to synonymous ratio (omega) for Homarus americanus-H. gammarus is greater than one, therefore indicating adaptive evolution (functional diversification) of proPO in these two species. In contrast, strong purifying selection (omega<1) was observed in all other species pairs. However, phylogenetically closely related decapods revealed relatively higher omega value (omega=0.15+/-0.3) than the distantly related species pairs (omega=0.0075+/-0.005). These discrepancies could be due to higher fixation probability of beneficial mutation in closely related species. Maximum likelihood-based codon substitution analyses revealed a strong purifying selection operating on most of the codon sites, therefore suggesting proPO is functionally constrained (purifying selection). Codon substitution analyses have also revealed the evidence of strong purifying selection in haemocyanin subunits of decapods.     

Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.     

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

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