A 28695 B

Objective: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. Conclusions: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

The lactose permease of Escherichia coli transports protons and lactose across the plasma membrane and uses a transmembrane ion gradient as the energy source to drive the uphill accumulation of lactose. In this report, the effect of the electrochemical gradient on the permease has been studied. Bacteriorhodopsin was co-reconstituted with the lactose permease to provide a light-triggered electrochemical gradient. Reaction-induced Fourier transform infrared spectra were acquired, and bacteriorhodopsin contributions were subtracted. In previous work, positive bands in the 1765-1730 cm(-1) region of the reaction-induced FT-IR spectrum were attributed to the perturbation of carboxylic acid residues in the permease [Patzlaff, J. S., Brooker, R. J., and Barry, B. A. (2000) J. Biol. Chem. 275, 28695-28700]. In this study, we have globally labeled the permease with (13)C or (15)N. Isotopic labeling demonstrates that features in the reaction-induced FT-IR spectrum arise from permease carboxylic acid, amide I, and amide II vibrational modes. In addition, isotope labeling leads to a tentative assignment of spectral features to lysine, arginine, histidine, glutamine, and/or asparagine in the permease. These results indicate that the electrochemical gradient causes changes in the environment or protonation state of carboxylic acid residues in the permease and suggest an interaction between these carboxylic acid side chains and nitrogen-containing amino acid side chains. Evidence for a change in secondary structure, corresponding to an interconversion of secondary structural elements, a change in the hydrogen-bonding strength, or coupling of peptide vibrational modes, is also presented. These experiments demonstrate the usefulness of reaction-induced spectroscopy in the study of transmembrane transport.

The contamination of soil with heavy metals is a severe problem due to adverse impact of heavy metals on environmental safety and human health. It is essential to remediate soil contaminated with heavy metals. This study has evaluated the effects of pine biochar, kaolin, and triple super phosphate (TSP) on multiple heavy metals (Ni, Zn, Cu, and Cd) in contaminated soil and accumulation of heavy metals in plants. The amendments can reduce availability of heavy metals in soil by increasing pH, adsorption, complexation, or co-precipitation. Different amendments have variable effects on accumulation of heavy metals in plants and in soil due to its diverse mechanism of stability. The results showed that application of triple super phosphate (TSP) has significant reduced soil Cd exchangeable (EXC) fraction from 58.59 to 21.30%. Bound to carbonates (CAR) fraction decreased from 9.84 to 5.11%, and bound to Fe-Mn oxides (OX) fraction increased from 29.61 to 69.86%. The triple super phosphate (TSP) has the ability to stabilize Cu and especially Cd. However, triple super phosphate (TSP) has enhanced ecological risk of Zn and Ni. Application of pine biochar has significantly enhanced soil pH. The kaolin has significantly reduced EXC fraction of Cd and increased OX fraction of Cu. The amendments and heavy metals have not caused significant effect on SPAD value of Buxus microphylla Siebold & Zucc (B. microphylla). The triple super phosphate (TSP) has significant decreased biomass of B. microphylla and bamboo-williow (Salix sp.) by 24.91 and 57.43%, respectively. Pine biochar and kaolin have increased the accumulation of Zn and Cd in plants. It is concluded that triple super phosphate (TSP) was effective in remediation of Cd and kaolin was effective in remediation of Cd and Cu. Pine biochar was effective in remediation of Cd, Cu, and Zn.