Local atrial natriuretic peptide signaling prevents hypertensive cardiac hypertrophy in endothelial nitric-oxide synthase-deficient mice

The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.     

Expansion of substrate specificity of cytochrome P450 2A6 by random and site-directed mutagenesis

The natural product indole is a substrate for cytochrome P450 2A6. Mutagenesis of P450 2A6 was done to expand its capability in the oxidization of bulky substituted indole compounds, which are not substrates for the wild-type enzyme or the double mutant L240C/N297Q, as determined in our previous work (Wu, Z.-L., Aryal, P., Lozach, O., Meijer, L., and Guengerich, F. P. (2005) Chem. Biodivers. 2, 51-65). Error-prone PCR and site-directed mutagenesis led to the identification of two critical amino acid residue changes (N297Q and I300V) that achieve the purpose. The new mutant (N297Q/I300V) was able to oxidize both 4- and 5-benzyloxy(OBzl)indoles to form colored products. Both changes were required for oxidation of these bulky substrates. The colored product derived from 5-OBzl-indole was mainly 5,5'-di-OBzl-indirubin, whereas the dominant blue dye isolated upon incubations with 4-OBzl-indole was neither an indigo nor an indirubin. Two-dimensional NMR experiments led to assignment of the structure as 4-OBzl-2-(4'-OBzl-1',7'-dihydro-7'-oxo-6'H-indol-6'-ylidene)indolin-3-one, in which a pyrrole ring and a benzene ring are connected with a double bond instead of the pyrrole-pyrrole connection of other indigoids. Monomeric oxidation products were also isolated and characterized; three phenols (4-OBzl-1H-indol-5-ol, 4-OBzl-1H-indol-6-ol, and 4-OBzl-1H-indol-7-ol) and one quinone (4-OBzl-1H-indole-6,7-dione, the postulated immediate precursor of the final blue dye) were identified. The results are interpreted in the context of a crystal structure of a P450 2A6-coumarin complex. The I300V change opens an additional pocket to accommodate the OBzl bulk. The N2297Q change is postulated to generate a hydrogen bond between Gln and the substrate oxygen. Thus, the substrate specificity of P450 2A6 was expanded, and new products were obtained in this study.     

Overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells: role of ERK and p38 mitogen-activated protein kinase

Acetaldehyde, the major ethanol metabolite that is far more toxic and reactive than ethanol, has been postulated to be responsible for alcohol-induced tissue and cell injury. This study was to examine whether facilitated acetaldehyde metabolism affects acetaldehyde-induced oxidative stress and apoptosis. Transgene-encoding human aldehyde dehydrogenase-2 (ALDH2), which converts acetaldehyde into acetate, was constructed under chicken beta-actin promoter and transfected into human umbilical vein endothelial cells (HUVECs). Efficacy of ALDH2 transfection was verified using green fluorescent protein and ALDH2 enzymatic assay. Generation of reactive oxygen species (ROS) was measured using chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride fluorescence microscopy, quantitative DNA fragmentation, and caspase-3 assay. Acetaldehyde (0-200 microm) elicited ROS generation and apoptosis in HUVECs in a time- and concentration-dependent manner, associated with activation of the stress signal molecules ERK1/2 and p38 mitogen-activated protein (MAP) kinase. A close liner correlation was observed between the acetaldehyde-induced ROS generation and apoptosis. Interestingly, the acetaldehyde-induced ROS generation, apoptosis, activation of ERK1/2, and p38 MAP kinase were prevented by the ALDH2 transgene or antioxidant alpha-tocopherol. The involvement of ERK1/2 and p38 MAP kinase in acetaldehyde-induced apoptosis was confirmed by selective kinase inhibitors U0126, SB203580, and SB202190. Collectively, our data revealed that facilitation of acetaldehyde metabolism by ALDH2 transgene overexpression may prevent acetaldehyde-induced cell injury and activation of stress signals. These results indicated therapeutic potential of ALDH2 enzyme in the prevention and detoxification of acetaldehyde or alcohol-induced cell injury.     

Role of protein-water interactions and electrostatics in alpha-synuclein fibril formation

Deposition of misfolded alpha-synuclein is a critical factor in several neurodegenerative disorders. Filamentous alpha-synuclein is the major component of Lewy bodies and Lewy neurites, the intracellular inclusions in the dopaminergic neurons of the substantia nigra, which are considered the pathological hallmark of Parkinson's disease. We show here that anions induce partial folding of alpha-synuclein at neutral pH, forming a critical amyloidogenic intermediate, which leads to significant acceleration of the rate of fibrillation. The magnitude of the accelerating effect generally followed the position of the anions in the Hofmeister series, indicating a major role of protein-water-anion interactions in the process at salt concentrations above 10 mM. Below this concentration, electrostatic effects dominated in the mechanism of anion-induced fibrillation. The acceleration of fibrillation by anions was also dependent on the cation. Moderate concentrations of anions affected both the rates of nucleation and the elongation of alpha-synuclein fibrillation, primarily via their effect on the interaction of the protein with water.     

Casein kinase 1alpha interacts with retinoid X receptor and interferes with agonist-induced apoptosis

Agonists of retinoid X receptors (RXRs), which include the natural 9-cis-retinoic acid and synthetic analogs, are potent inducers of growth arrest and apoptosis in some cancer cells. As such, they are being used in clinical trials for the treatment and prevention of solid tumors and are used to treat cutaneous T cell lymphoma. However, the molecular mechanisms that underlie the anti-cancer effects of RXR agonists remain unclear. Here, we show that a novel pro-apoptotic pathway that is induced by RXR agonist is negatively regulated by casein kinase 1alpha (CK1alpha). CK1alpha associates with RXR in an agonist-dependent manner and phosphorylates RXR. The ability of an RXR agonist to recruit CK1alpha to a complex with RXR in cells correlates inversely with its ability to inhibit growth. Remarkably, depletion of CK1alpha in resistant cells renders them susceptible to RXR agonist-induced growth inhibition and apoptosis. Our study shows that CK1alpha can promote cell survival by interfering with RXR agonist-induced apoptosis. Inhibition of CK1alpha may enhance the anti-cancer effects of RXR agonists.     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.     

Analysis of the Molecular Evolutionary History of the Ascorbate Peroxidase Gene Family: Inferences from the Rice Genome

Ascorbate peroxidase (APx) is a class I peroxidase that catalyzes the conversion of H₂O₂ to H₂O and O₂ using ascorbate as the specific electron donor. This enzyme has a key function in scavenging reactive oxygen species (ROS) and the protection against toxic effects of ROS in higher plants, algae, and Euglena. Here we report the identification of an APx multigene family in rice and propose a molecular evolutionary relationship between the diverse APx isoforms. In rice, the APx gene family has eight members, which encode two cytosolic, two putative peroxisomal, and four chloroplastic isoforms, respectively. Phylogenetic analyses were conducted using all APx protein sequences available in the NCBI databases. The results indicate that the different APx isoforms arose by a complex evolutionary process involving several gene duplications. The structural organization of APx genes also reflects this process and provides evidence for a close relationship among proteins located in the same subcellular compartment. A molecular evolutionary pathway, in which cytosolic and peroxisomal isoforms diverged early from chloroplastic ones, is proposed.Reviewing Editor: Dr. Niles Lehman     

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.     

The emergence of obesity among indigenous Siberians

Once considered a disease of affluence and confined to industrialized nations, obesity is currently emerging as a major health concern in nearly every country in the world. Available data suggest that the prevalence rate of obesity has reached unprecedented levels in most developing countries, and is increasing at a rate that far outpaces that of developed nations. This increase in obesity has also been documented among North American circumpolar populations and is associated with lifestyle changes related to economic development. While obesity has not been well studied among indigenous Siberians, recent anthropological studies indicate that obesity and its associated comorbidities are important health problems.The present study examines recent adult body composition data from four indigenous Siberian populations (Evenki, Ket, Buriat, and Yakut) with two main objectives: 1) to determine the prevalence of overweight and obesity among these groups, and 2) to assess the influence of lifestyle and socioeconomic factors on the development of excess body fat. The results of this study indicate that obesity has emerged as an important health issue among indigenous Siberians, and especially for women, whose obesity rates are considerably higher than those of men (12% vs. 7%). The present study investigated the association between lifestyle and body composition among the Yakut, and documented substantial sex differences in lifestyle correlates of obesity. Yakut men with higher incomes and who owned more luxury consumer goods were more likely to have excess body fat while, among Yakut women, affluence was not strongly associated with overweight and obesity.