EPA,not DHA,prevents fibrosis in pressure overload-induced heart failure: potential role of free fatty acid receptor 4

Heart failure with preserved ejection fraction (HFpEF) is half of all HF, but standard HF therapies are ineffective. Diastolic dysfunction, often secondary to interstitial fibrosis, is common in HFpEF. Previously, we found that supra-physiologic levels of ω3-PUFAs produced by 12 weeks of ω3-dietary supplementation prevented fibrosis and contractile dysfunction following pressure overload [transverse aortic constriction (TAC)], a model that resembles aspects of remodeling in HFpEF. This raised several questions regarding ω3-concentration-dependent cardioprotection, the specific role of EPA and DHA, and the relationship between prevention of fibrosis and contractile dysfunction. To achieve more clinically relevant ω3-levels and test individual ω3-PUFAs, we shortened the ω3-diet regimen and used EPA- and DHA-specific diets to examine remodeling following TAC. The shorter diet regimen produced ω3-PUFA levels closer to Western clinics. Further, EPA, but not DHA, prevented fibrosis following TAC. However, neither ω3-PUFA prevented contractile dysfunction, perhaps due to reduced uptake of ω3-PUFA. Interestingly, EPA did not accumulate in cardiac fibroblasts. However, FFA receptor 4, a G protein-coupled receptor for ω3-PUFAs, was sufficient and required to block transforming growth factor β1-fibrotic signaling in cultured cardiac fibroblasts, suggesting a novel mechanism for EPA. In summary, EPA-mediated prevention of fibrosis could represent a novel therapy for HFpEF.     

Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment

The oxygenase domain of the inducible nitric oxide synthase, Δ65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H₄B) and is the site for NO synthesis. The role of H₄B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple mechanistic account of interaction of the cofactor tetrahydrobiopterin (H₄B) with the bacterially expressed Δ65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Δ65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Δ65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H₄B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these findings confirm that the H₄B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain. (Mol Cell Boichem xxx: 1–10, 2005) Supported by Calcutta University Research Grants.     

Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

'In dopaminergic neurons, α-synuclein (αS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of αS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free state and the lipid-bound αS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s^− 1. Partitioning of the binding modes is dependent on lipid/αS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of αS adopts an α-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this “dark state.” Titration of αS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by αS.     

表达barstar基因及bar基因的转基因油菜的研究

从细菌Ｂａｃｉｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ｂａｒｎａｓｅ抑制剂ｂａｒｓｔａｒ的基因,构建了带有ＴＡ－２９基因５′调控区（－１３００－＋３）与ｂａｒｓｔａｒ基因编码区、ＣａＭＶ３５Ｓ启动子与除草剂抗性基因ｂａｒ两个表达框架的植物表达质粒ｐＢＢＳ。以“双低”油菜“５－４”的子叶柄为受体,通过农杆菌介导的遗传转化,获得了在含有１０ｍｇ／Ｌ卡那霉素和２０ｍｇ／ＬＰＰＴ的筛选培养基上再生的转基因植株。ＰＣＲ分析结果表明,ｂａｒｓｔａｒ基因已整合到油菜染色体上;Ｎｏｒｔｈｅｒｎｂｌｏｔ检测表明,ｂａｒｓｔａｒ基因及ｂａｒ基因在转基因植物中得到了正确的调控与表达。以转基因油菜“５－４”为父本授粉给表达ｂａｒｎａｓｅ基因的雄性不育植株,不育株能正常结实。     

Drin pesticides removal from aqueous solutions using acid-treated date stones

This work describes the potential applicability of chemically and thermally treated date stones for removing drin pesticides (aldrin, dieldrin and endrin) from aqueous solutions. The effect of several parameters, such as sorbent particle size, adsorbent dose, shaking speed, shaking time, concentration of pesticide solution and temperature, was evaluated by batch experiments. Pesticide determination was carried out using stir bar sorptive extraction and gas chromatography coupled with mass spectroscopy. Maximum removal efficiency (93%) was reached using 0.1 g of acid-treated date stones (ATDS) (63–100 μm) and 100 mL of aldrin (0.5 mg L⁻¹). The removal efficiency of drin pesticides decreased in the order of aldrin, dieldrin and endrin, and decreased as the temperature rose. Adsorption data were processed according to various kinetic models. Lagergren and Morris-Weber equations were applied to fit the kinetic results. The second order model was the most suitable on the whole, and intra-particle diffusion was found to be the rate-controlling the adsorption process. According to adsorption kinetic data, 3.5 h were considered as the equilibrium time for determining adsorption isotherms. Adsorption data were analyzed by the Langmuir, Freundlich and Dubinin–Radushkevich adsorption approaches. Experimental results showed that the Freundlich isotherm model best described the adsorption process. In addition, thermodynamic parameters such as ΔH, ΔS and ΔG were calculated. Negative values of ΔH and ΔG indicate the exothermic and spontaneous nature of pesticide adsorption on ATDS.     

A Molecular Mechanism of Bacterial Flagellar Motor Switching

The high-resolution structures of nearly all the proteins that comprise the bacterial flagellar motor switch complex have been solved; yet a clear picture of the switching mechanism has not emerged. Here, we used NMR to characterize the interaction modes and solution properties of a number of these proteins, including several soluble fragments of the flagellar motor proteins FliM and FliG, and the response-regulator CheY. We find that activated CheY, the switch signal, binds to a previously unidentified region of FliM, adjacent to the FliM-FliM interface. We also find that activated CheY and FliG bind with mutual exclusivity to this site on FliM, because their respective binding surfaces partially overlap. These data support a model of CheY-driven motor switching wherein the binding of activated CheY to FliM displaces the carboxy-terminal domain of FliG (FliG_C) from FliM, modulating the FliG_C-MotA interaction, and causing the motor to switch rotational sense as required for chemotaxis.     

Fas Apoptosis Inhibitory Molecule Contains a Novel β-Sandwich in Contact with a Partially Ordered Domain

Fas apoptosis inhibitory molecule (FAIM) is a soluble cytosolic protein inhibitor of programmed cell death and is found in organisms throughout the animal kingdom. A short isoform of FAIM is expressed in all tissue types, while an alternatively spliced long isoform is specifically expressed in the brain. Here, the short isoform is shown to consist of two independently folding domains in contact with each other. The NMR solution structure of the C-terminal domain of murine FAIM is solved in isolation and revealed to be a novel protein fold, a noninterleaved seven-stranded β-sandwich. The structure and sequence reveal several residues that are likely to be involved in functionally significant interactions with the N-terminal domain or other binding partners. Chemical shift perturbation is used to elucidate contacts made between the N-terminal domain and the C-terminal domain.     

Monoamine oxidases (MAO) in the pathogenesis of heart failure and ischemia/reperfusion injury

Recent evidence highlights monoamine oxidases (MAO) as another prominent source of oxidative stress. MAO are a class of enzymes located in the outer mitochondrial membrane, deputed to the oxidative breakdown of key neurotransmitters such as norepinephrine, epinephrine and dopamine, and in the process generate H₂O₂. All these monoamines are endowed with potent modulatory effects on myocardial function. Thus, when the heart is subjected to chronic neuro-hormonal and/or peripheral hemodynamic stress, the abundance of circulating/tissue monoamines can make MAO-derived H₂O₂ production particularly prominent. This is the case of acute cardiac damage due to ischemia/reperfusion injury or, on a more chronic stand, of the transition from compensated hypertrophy to overt ventricular dilation/pump failure. Here, we will first briefly discuss mitochondrial status and contribution to acute and chronic cardiac disorders. We will illustrate possible mechanisms by which MAO activity affects cardiac biology and function, along with a discussion as to their role as a prominent source of reactive oxygen species. Finally, we will speculate on why MAO inhibition might have a therapeutic value for treating cardiac affections of ischemic and non-ischemic origin. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus

The majority of proteins exist in vivo within macromolecular assemblies whose functions are dependent on dynamical processes spanning a wide range of time scales. One such assembly is formed by the molecular chaperone αB-crystallin that exists in a variety of exchanging oligomeric states, centred on a mass of approximately 560 kDa. For many macromolecular assemblies, including αB-crystallin, the inherent dynamics, heterogeneity and high mass contribute to difficulties in quantitative studies. Here, we demonstrate a strategy based on correlating solution-state nuclear magnetic resonance spectroscopy and mass spectrometry data to characterize simultaneously the organization and dynamics of the polydisperse αB-crystallin ensemble. We show that protomeric dimers assemble into oligomers via the binding of extended C-termini, with each monomer donating and receiving one terminus. Moreover, we establish that the C-termini undergo millisecond fluctuations that regulate the interconversion of oligomeric forms. The combined biophysical approach allows construction of an energy profile for a single monomer that completely describes the equilibrium dynamics of the ensemble. It also facilitates an analysis of dynamics spanning the millisecond to hour time scales and secondary to quaternary structural levels, and provides an approach for, obtaining simultaneously detailed structural, thermodynamic and kinetic information on a heterogeneous protein assembly.     

Galectin-1 attenuates cardiomyocyte hypertrophy through splice-variant specific modulation of CaV1.2 calcium channel

Pressure overload-induced cardiac hypertrophy occurs in response to chronic blood pressure increase, and dysfunction of Ca_V1.2 calcium channel involves in cardiac hypertrophic processes by perturbing intracellular calcium concentration ([Ca²⁺]_i) and calcium-dependent signaling. As a carbohydrate-binding protein, galectin-1 (Gal-1) is found to bind with Ca_V1.2 channel, which regulates vascular Ca_V1.2 channel functions and blood pressure. However, the potential roles of Gal-1 in cardiac Ca_V1.2 channel (Ca_V1.2_CM) and cardiomyocyte hypertrophy remain elusive. By whole-cell patch clamp, we find Gal-1 decreases the I_Ca,L with or without isoproterenol (ISO) application by reducing the channel membrane expression in neonatal rat ventricular myocytes (NRVMs). Moreover, Gal-1 could inhibit the current densities of Ca_V1.2_CM by an alternative exon 9*-dependent manner in heterologously expressed HEK293 cells. Of significance, overexpression of Gal-1 diminishes ISO or KCl-induced [Ca²⁺]_i elevation and attenuates ISO-induced hypertrophy in NRVMs. Mechanistically, Gal-1 decreases the ISO or Bay K8644-induced phosphorylation of intracellular calcium-dependent signaling proteins δCaMKII and HDAC4, and inhibits ISO-triggered translocation of HDAC4 in NRVMs. Pathologically, we observe that the expressions of Gal-1 and Ca_V1.2_E9* channels are synchronously increased in rat hypertrophic cardiomyocytes and hearts. Taken together, our study indicates that Gal-1 reduces the channel membrane expression to inhibit the currents of Ca_V1.2_CM in a splice-variant specific manner, which diminishes [Ca²⁺]_i elevation, and attenuates cardiomyocyte hypertrophy by inhibiting the phosphorylation of δCaMKII and HDAC4. Furthermore, our work suggests that dysregulated Gal-1 and Ca_V1.2 alternative exon 9* might be attributed to the pathological processes of cardiac hypertrophy, and provides a potential anti-hypertrophic target in the heart.