Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.     

Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence

Background

Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors.

Methods and Results

Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C₁₂HSL and C₄HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C₄HSL. F7 also showed antagonistic activity against 3-oxo-C₁₂HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract.

Conclusions

This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.     

Toxicity of mercury: A comparative study in air-breathing and non air-breathing fish

Annual primary production was estimated from two years data. The results indicate a high primary productivity and a short turn over time for nitrogen.

     

Role of Spm–Cer‐S1P signalling pathway in MMP‐2 mediated U46619‐induced proliferation of pulmonary artery smooth muscle cells: protective role of epigallocatechin‐3‐gallate

During remodelling of pulmonary artery, marked proliferation of pulmonary artery smooth muscle cells (PASMCs) occur s , which contributes to pulmonary hypertension. Thromboxane A₂ (TxA₂) has been shown to produce pulmonary hypertension. The present study investigates the inhibitory effect of epigallocatechin‐3‐gallate (EGCG) on the TxA₂ mimetic, U46619‐induced proliferation of PASMCs. U46619 at a concentration of 10 nM induces maximum proliferation of bovine PASMCs. Both pharmacological and genetic inhibitors of p³⁸MAPK, NF‐κB and MMP‐2 significantly inhibit U46619‐induced cell proliferation. EGCG markedly abrogate U46619‐induced p³⁸MAPK phosphorylation, NF‐κB activation, proMMP‐2 expression and activation, and also the cell proliferation. U46619 causes an increase in the activation of sphingomyelinase (SMase) and sphingosine kinase (SPHK) and also increase sphingosine 1 phosphate (S1P) level. U46619 also induces phosphorylation of ERK1/2, which phosphorylates SPHK leading to an increase in S1P level. Both pharmacological and genetic inhibitors of SMase and SPHK markedly inhibit U46619‐induced cell proliferation. Additionally, pharmacological and genetic inhibitors of MMP‐2 markedly abrogate U46619‐induced SMase activity and S1P level. EGCG markedly inhibit U46619‐induced SMase activity, ERK1/2 and SPHK phosphorylation and S1P level in the cells. Overall, Sphingomyeline–Ceramide–Sphingosine‐1‐phosphate (Spm–Cer–S1P) signalling axis plays an important role in MMP‐2 mediated U46619‐induced proliferation of PASMCs. Importantly, EGCG inhibits U46619 induced increase in MMP‐2 activation by modulating p³⁸MAPK–NFκB pathway and subsequently prevents the cell proliferation. Copyright © 2015 John Wiley & Sons, Ltd.     

Role of PKCα−p38MAPK−Giα axis in peroxynitrite-mediated inhibition of β-adrenergic response in pulmonary artery smooth muscle cells

In the context of cross-talk between transmembrane signaling pathways, we studied the loci within the β-adrenergic receptor/G protein/adenyl cyclase system at which PKC exerts regulatory effects of peroxynitrite (ONOO^?) on isoproterenol stimulated adenyl cyclase activity in pulmonary artery smooth muscle cells. Treatment of the cells with ONOO^? stimulated PKC-α activity and that subsequently increased p³⁸MAPK phosphorylation. Pretreatment with Go6976 (PKC-α inhibitor) and SB203580 (p³⁸MAPK inhibitor) eliminated ONOO^? caused inhibition on isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976, but not SB203580, prevented ONOO^? induced increase in PKC-α activity. Studies using genetic inhibitors of PKC-α (PKC-α siRNA) and p³⁸MAPK (p³⁸MAPK siRNA) also corroborated the findings obtained with their pharmacological inhibitors in eliminating the attenuation of ONOO^? effect on isoproterenol stimulated adenyl cyclase activity. This inhibitory effect of ONOO^? was found to be eliminated upon pretreatment of the cells with pertussis toxin thereby pointing to a G_i dependent mechanism. This hypothesis was reinforced by G_iα phosphorylation as well as by the observation of the loss of the ability of Gpp(NH)p (a measure of G_i mediated response) to stimulate adenyl cyclase activity upon ONOO^? treatment to the cells. We suggest the existence of a pertussis toxin sensitive G protein (G_i)-mediated mechanism in isoproterenol stimulated adenyl cyclase activity, which is regulated by PKCα-p³⁸MAPK axis dependent phosphorylation of its α-subunit (G_iα) in the pulmonary artery smooth muscle cells.     

Magnesian calcite and chamositic ooids forming shoals peripheral to Late Ordovician (Ashgill) muddy siliciclastic shores: southern Ontario

In southern Ontario, ooids are associated with two distinct facies associations in the Queenston Formation, the final stage of Late Ordovician (Ashgill) Taconic basin fill. One facies consists of thin ooid and bioclastic grainstones interbedded with mudrock, and lies near the base of the formation, and, in southwestern Ontario, also forms a local NW-thickening wedge near the middle of the formation. Ooids have radial-fibrous and radial-concentric fabrics (Type A), with chamosite, illite, and Fe-oxide laths at intercrystalline sites. Vertical lithologic and ooid abundance patterns indicate that thresholds to carbonate production were sensitive to changes in terrigenous sediment supply, sea level, circulation, accommodation space, and tectonism.

Ooids in the second facies association are admixed with abraded fragments of open-marine biota, or occur burrow fills, within a <30-cm-thick interval of mudrock near the top of the preserved Queenston succession, a few metres below the Ordovician–Silurian unconformity. Ooids have radial concentric and crosscutting patchy microcrystalline fabrics (Type B). This unit may represent a transgressive or stillstand deposit modified by bioturbation.

The extent of preserved fabric suggests that both ooid types were originally magnesian calcite, but Type A ooids underwent greater burial alteration. This is shown by crystalline mosaics that cross-cut relict primary fabrics; δ¹³C values (−1.82‰ to +0.67‰) and δ¹⁸O values (−4.46‰ to −10.57‰) more negative than marine calcite of similar age; Mn and Fe concentrations more elevated above expected marine values; and a luminescence similar to that of intergranular cements. Burial meteoric diagenesis was likely promoted by excellent permeability of the host sand. We interpret authigenic chamosite and Fe-oxide to reflect diagenesis of iron-bearing and clay detritus trapped during ooid growth. Type B ooids suffered less alteration: δ¹³C (+1.1‰ to +6.64‰) and δ¹⁸O (−3.04‰ to −4.81‰) values overlap the expected marine range, including ¹³C enrichment that occurs within the Hirnantian (latest Ordovician) excursion. Although Mn and Fe values are still higher than those of modern calcitic ooids, negligible luminescence suggests that recrystallization occurred in the presence of marine-derived pore fluids. Further burial alteration was inhibited due to low permeability of the host mud.

Type A ooid facies in the Queenston Formation forms an ancient analogue for lesser known Quaternary ooid shoals peripheral to tropical deltaic systems. The facies of Type B ooids, while more enigmatic, may preserve a geochemical herald of latest Ordovician climate change. The presence of minor chamosite in Type A ooids defines a possible distal facies of the well-known oolitic ironstones of similar age in the mid-continental USA.     

Solubilization, purification, and reconstitution of α2β1 isozyme of Na+/K+-ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C12E8, and Triton X-100

We identified α₂, α₁, and β₁ isoforms of Na⁺/K⁺-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the α₂β₁ isozyme of Na⁺/K⁺-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C₁₂E₈, whereas C₁₂E₈ was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified α₂β₁ isozyme of Na⁺/K⁺-ATPase elicited higher E1Na?E2 K transition compared with that of the C₁₂E₈- and Triton X-100-purified enzyme. The rate of Na⁺ efflux in DHPC–DOPC-reconstituted isozyme was higher compared to the C₁₂E₈–DOPC- and Triton X100–DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified α₂β₁ isozyme of Na⁺/K⁺-ATPase possessed more organized secondary structure compared to the C₁₂E₈- and Triton X-100-purified isozyme.