Devaraneni, Prasanna K.
Devereaux, Jordan J.
Valiyaveetil, Francis I.
In this contribution, we report in vitro folding of the archaebacterial voltage-gated K(+) channel, K(v)AP. We show that in vitro folding of the K(v)AP channel from the extensively unfolded state requires lipid vesicles and that the refolded channel is biochemically and functionally similar to the native channel. The in vitro folding process is slow at room temperature, and the folding yield depends on the composition of the lipid bilayer. The major factor influencing refolding is temperature, and almost quantitative refolding of the K(v)AP channel is observed at 80 degrees C. To differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition, suggesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the K(v)AP channel provides a good model system for investigating the folding of a multidomain integral membrane protein.
Highlights • The 873 K isothermal section of Co–Ti–V ternary system is determined. • Nine three-phase equilibria are experimentally measured. • All of Co–V and Co–Ti binary phases show large ternary solubility. • The maximum solid solubility of Ti, Co, V in binary phases is determined. Abstract The isothermal section of the Co–Ti–V ternary system at 873 K has been investigated by means of diffusion triple together with electron probe microanalysis technique. Series of tie lines and tie-triangles have been determined and the isothermal section at 873 K has been established, and nine three-phase fields have been figured out. It is found that VCo 3 , VCo, TiCo 3 , TiCo 2 (h), TiCo 2 (c) and TiCo have range of homogeneity at 873 K, and the V 3 Co and Ti 2 Co are line compounds. The solid solubility of Ti in VCo 3 , VCo and V 3 Co is about 5.2 at.% Ti, 8.8 at.% Ti and 6.9 at.% Ti, and that of V in TiCo 3 , TiCo 2 (h), TiCo 2 (c), TiCo and Ti 2 Co is about 6.1 at.% V, 3.7 at.% V, 3.0 at.% V, 9.3 at.% V and 6.6 at.% V, respectively.
Svalo, Julie
Bille, Michala
Theepakaran, Neeraja Parameswaran
Sheykhzade, Majid
Nordling, Jorgen
Bouchelouche, Pierre
K(v)7 channels are involved in smooth muscle relaxation, and accordingly we believe that they constitute potential targets for the treatment of overactive bladder syndrome. We have therefore used myography to examine the function of K(v)7 channels in detrusor, i.e. pig bladder, with a view to determining the effects of the following potassium channel activators: ML213 (K(v)7.2/K(v)7.4 channels) and retigabine (K(v)7.2-7.5 channels). Retigabine produced a concentration dependent relaxation of carbachol- and electric field-induced contractions. The potency was similar in magnitude to that of ML213-induced relaxation, suggesting that K(v)7.2 and/or K(v)7.4 channels constitute the subtypes that are relevant to bladder contractility. The effects of retigabine and ML213 were attenuated by pre-incubation with 10 mu M XE991 (K(v)7.1-7.5 channel blocker) (P<0.05), which in turn confirmed K(v)7 channel selectivity. Subtype-selective effects were further investigated by incubating the detrusor with 10 mu M chromanol 293B (K(v)7.1 channel blocker). Regardless of the experimental protocol, this did not cause a further increase in the evoked contraction. In contrast, the addition of XE991 potentiated the KCl-induced contractions, but not those induced by carbachol or electric field, indicating the presence of a phosphatidyl-inositol-4, 5-biphosphate-dependent mechanism amongst the K(v)7 channels in detrusor. qRT-PCR studies of the mRNA transcript level of K(v)7.3-7.5 channels displayed a higher level of K(v)7.4 transcript in detrusor compared to that present in brain cortex and heart tissues. Thus, we have shown that K(v)7.4 channels are expressed and functionally active in pig detrusor, and that the use of selective K(v)7.4 channel modulators in the treatment or detrusor overactivity seems promising. (C) 2013 Elsevier B.V. All rights reserved.
Vibrio anguillarum has been described as a leading cause of disease in various species of aquatic animals from many parts of the world. Fish vaccination is a useful strategy for several disease problems in aquaculture. Outer membrane proteins of bacteria are considered ideal candidates for vaccine development. In the present study, OmpK gene of V. anguillarum was cloned and sequenced. The recombinant OmpK was overexpressed and purified by affinity chromatography on Ni-NTA Superflow resin. Immunoreactivity of the pure OmpK was studied by western blotting. The fresh water Indian major carp Labeo rohita (Hamilton, 1822) was well protected when challenged with V. anguillarum after vaccination with OmpK compared to non vaccinated fish. Specific antibody against OmpK and bacterial inhibition was detected in the vaccinated fish. Our results suggest OmpK as a candidate vaccine molecule against V. anguillarum.
Vibrio anguillarum has been described as a leading cause of disease in various species of aquatic animals from many parts of the world. Fish vaccination is a useful strategy for several disease problems in aquaculture. Outer membrane proteins of bacteria are considered ideal candidates for vaccine development. In the present study, OmpK gene of V. anguillarum was cloned and sequenced. The recombinant OmpK was overexpressed and purified by affinity chromatography on Ni-NTA Superflow resin. Immunoreactivity of the pure OmpK was studied by western blotting. The fresh water Indian major carp Labeo rohita (Hamilton, 1822) was well protected when challenged with V. anguillarum after vaccination with OmpK compared to non vaccinated fish. Specific antibody against OmpK and bacterial inhibition was detected in the vaccinated fish. Our results suggest OmpK as a candidate vaccine molecule against V. anguillarum. (C) 2012 Elsevier B.V. All rights reserved.