Cross-linking modifications of HDL apoproteins by oxidized phospholipids: structural characterization, in vivo detection, and functional implications.

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2- system. We found that five histidine residues in helices 5-8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3-4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR-/- mice, including cross-link adducts of apoA-I His-165-apoA-I Lys-93, apoA-I His-154-apoA-I Lys-105, apoA-I His-154-apoA-IV Lys-149, and apoA-II Lys-30-apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.

Gut microbe-generated phenylacetylglutamine is an endogenous allosteric modulator of ß2-adrenergic receptors.

Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing ß1- versus ß2-adrenergic receptors (ß1AR and ß2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of ß2AR, but not ß1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on ß2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical ß2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR.

Regulatory role of miRNAs in Wnt signaling pathway linked with cardiovascular diseases.

MicroRNAs (miRNAs) are discovered in science about 23 years ago. These are short, a series of non-coding, single-stranded and evolutionary conserved RNA molecules found in eukaryotic cells. It involved post-transcriptional fine-tune protein expression and repressing the target of mRNA in different biological processes. These miRNAs binds with the 3'-UTR region of specific mRNAs to phosphorylate the mRNA degradation and inhibit the translation process in various tissues. Therefore, aberrant expression in miRNAs induces numerous cardiovascular diseases and developmental defects. Subsequently, the miRNAs and Wnt singling pathway are regulating a cellular process in cardiac development and regeneration, maintain the homeostasis and associated heart diseases. In Wnt signaling pathway majority of the signaling components are expressed and regulated by miRNAs, whereas the inhibition or dysfunction of the Wnt signaling pathway induces cardiovascular diseases. Moreover, inadequate studies about the important role of miRNAs in heart development and diseases through Wnt signaling pathway has been exist still now. For this reason in present review we summarize and update the involvement of miRNAs and the role of Wnt signaling in cardiovascular diseases. We have discussed the mechanism of miRNA functions which regulates the Wnt components in cellular signaling pathway. The fundamental understanding of Wnt signaling regulation and mechanisms of miRNAs is quite essential for study of heart development and related diseases. This approach definitely enlighten the future research to provide a new strategy for formulation of novel therapeutic approaches against cardiovascular diseases.

Structural basis for the recognition of oxidized phospholipids in oxidized low density lipoproteins by class B scavenger receptors CD36 and SR-BI.

Specific oxidized phospholipids (oxPC(CD36)) accumulate in vivo at sites of oxidative stress and serve as high affinity ligands for scavenger receptors class B (CD36 and SR-BI). Recognition of oxPC(CD36) by scavenger receptors plays a role in several pathophysiological processes. The structural basis for the recognition of oxPC(CD36) by CD36 and SR-BI is poorly understood. A characteristic feature of oxPC(CD36) is an sn-2 acyl group that incorporates a terminal gamma-hydroxy (or oxo)-alpha,beta-unsaturated carbonyl. In the present study, a series of model oxidized phospholipids were designed, synthesized, and tested for their ability to serve as ligands for CD36 and SR-BI. We demonstrated that intact the sn-1 hydrophobic chain, the sn-3 hydrophilic phosphocholine or phosphatidic acid group, and the polar sn-2 tail are absolutely essential for high affinity binding. We further found that a terminal negatively charged carboxylate at the sn-2 position suffices to generate high binding affinity to class B scavenger receptors. In addition, factors such as polarity, rigidity, optimal chain length of sn-2, and sn-3 positions and negative charge at the sn-3 position of phospholipids further modulate the binding affinity. We conclude that all three positions of oxidized phospholipids are essential for the effective recognition by scavenger receptors class B. Furthermore, the structure of residues in these positions controls the affinity of the binding. The present studies suggest that, in addition to oxPC(CD36), other oxidized phospholipids observed in vivo may represent novel ligands for scavenger receptors class B.

A new species of Sitana (Squamata: Agamidae) from the Deccan Peninsula Biogeographic Zone of India.

We describe a new species of fan-throated lizard of the genus Sitana from the Deccan peninsula of India. The new species is from the Sitana sivalensis clade and can be readily diagnosed morphologically from S. sivalensis, S. fusca and S. schleichi by having the dewlap extending beyond forearm insertion. The new species differs from all other congeners in the combination of morphological characters such as a feebly serrated dewlap with a dark blue line on the throat in adult males (versus  a well serrated dewlap with a bright blue patch and orange spots in S. ponticeriana complex), small body size (versus a large body size in S. gokakensis and S. thondalu) and a relatively smaller dewlap size (relatively larger in S. laticeps, S. spinaecephalus, S. dharwarensis, S. gokakensis, S. thondalu, S. marudhamneydhal, S. ponticeriana and S. visiri). The new species was found to be commonly distributed in arid and open habitats as well as in farmlands and plantations in northern Andhra Pradesh, eastern Madhya Pradesh and most parts of Chhattisgarh and Odisha states.

Pulsed-electromagnetic-field induced osteoblast differentiation requires activation of genes downstream of adenosine receptors A2A and A3.

Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation.

Ahaetulla nasuta anomala (Annandale, 1906) (Squamata: Colubridae), resurrected as a valid species with marked sexual dichromatism.

In this paper we resolve the taxonomic confusion related to Ahaetulla nasuta anomala (Annandale, 1906). On the basis of molecular and morphological data, we remove it from the synonymy of Ahaetulla nasuta (Lacépède, 1789) and reinstate it as a valid species-Ahaetulla anomala. This species is sexually dichromatic, males are green and females are brown in colour. Though the brown morph morphologically resembles Ahaetulla pulverulenta (Duméril, Bibron & Duméril, 1854) there are significant morphological and genetic differences between these two species. Additional information on taxonomy, natural history and distribution of the species is provided.

Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-kappa B and proinflammatory gene program activation in intestinal epithelial cells.

BACKGROUND: Infection of intestinal epithelial cells by pathogenic Salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. The transcription factor NF-kappa B is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-kappa B during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-kappa B activation in all the cells; invasion of cells by the bacteria is not required to activate NF-kappa B. RESULTS: Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and I kappa B kinase (IKK) signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by Salmonella. Flagellin expression was required for Salmonella invasion of host cells and it activated NF-kappa B via toll-like receptor 5 (TLR5). Surprisingly, a number of cell lines found to be unresponsive to flagellin express TLR5 and expression of exogenous TLR5 in these cells induces NF-kappa B activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-kappa B activation by flagellin. These observations are consistent with the possibility of either a very stable TLR5 signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both. CONCLUSION: These collective results provide the evidence that flagellin acts as the main determinant of Salmonella mediated NF-kappa B and proinflammatory signaling and gene activation by this flagellated pathogen. In addition, expression of the fli C gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express fli C are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in trans cannot restore the ability of the fli C mutant bacteria to invade intestinal epithelial cells. Lastly, TLR5 expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition.

Lyn facilitates glioblastoma cell survival under conditions of nutrient deprivation by promoting autophagy.

Members of the Src family kinases (SFK) can modulate diverse cellular processes, including division, death and survival, but their role in autophagy has been minimally explored. Here, we investigated the roles of Lyn, a SFK, in promoting the survival of human glioblastoma tumor (GBM) cells in vitro and in vivo using lentiviral vector-mediated expression of constitutively-active Lyn (CA-Lyn) or dominant-negative Lyn (DN-Lyn). Expression of either CA-Lyn or DN-Lyn had no effect on the survival of U87 GBM cells grown under nutrient-rich conditions. In contrast, under nutrient-deprived conditions (absence of supplementation with L-glutamine, which is essential for growth of GBM cells, and FBS) CA-Lyn expression enhanced survival and promoted autophagy as well as inhibiting cell death and promoting proliferation. Expression of DN-Lyn promoted cell death. In the nutrient-deprived GBM cells, CA-Lyn expression enhanced AMPK activity and reduced the levels of pS6 kinase whereas DN-Lyn enhanced the levels of pS6 kinase. Similar results were obtained in vitro using another cultured GBM cell line and primary glioma stem cells. On propagation of the transduced GBM cells in the brains of nude mice, the CA-Lyn xenografts formed larger tumors than control cells and autophagosomes were detectable in the tumor cells. The DN-Lyn xenografts formed smaller tumors and contained more apoptotic cells. Our findings suggest that on nutrient deprivation in vitro Lyn acts to enhance the survival of GBM cells by promoting autophagy and proliferation as well as inhibiting cell death, and Lyn promotes the same effects in vivo in xenograft tumors. As the levels of Lyn protein or its activity are elevated in several cancers these findings may be of broad relevance to cancer biology.

Oxidized phospholipids: biomarker for cardiovascular diseases.

Biologically active oxidized phospholipids can initiate and modulate many of the cellular events attributed to inflammation leading to atherosclerosis. Produced by enzymatic or non-enzymatic processes, these molecules interact with various cells via specific receptors and in general give rise to inflammatory signals. There is considerable evidence that oxidized phospholipids accumulate in vivo and play significant roles in atherosclerosis and thrombosis, suggesting that oxidized phospholipids could be biomarkers that reflect the global extent of these diseases in vivo. Thus, understanding the biosynthetic pathways, receptor specificity and signaling processes of oxidized phospholipids is important in understanding atherosclerosis, thrombosis and related inflammatory diseases.

Mapping and characterization of the binding site for specific oxidized phospholipids and oxidized low density lipoprotein of scavenger receptor CD36.

Recent studies have identified a novel family of oxidized phosphatidylcholines (oxPC(CD36)) that serve as highly specific ligands for scavenger receptor CD36. oxPC(CD36) accumulate in vivo and mediate macrophage foam cell formation as well as promote platelet hyper-reactivity in hyperlipidemia via CD36. The structural basis of oxPC(CD36) binding to CD36 has not been elucidated. We used liquid-phase binding to glutathione S-transferase fusion proteins containing various regions of CD36 to initially identify the region spanning CD36 amino acids 157-171 to contain a major binding site for oxPC(CD36). A bell-shaped pH profile and salt concentration dependence suggest an electrostatic mechanism of the binding. Two conserved, positively charged amino acids in the region 157-171 (lysines at positions 164 and 166) were identified as critical for oxPC(CD36) and oxidized low density lipoprotein (oxLDL) binding to CD36. Lysine neutralization with chemical modifier or site-directed mutagenesis of lysine 164/166 to alanine or glutamate, but not to arginine, abolished binding. Cells expressing full-length CD36 with mutated lysines (164 and 166) failed to recognize oxPC(CD36) and oxLDL. Synthetic peptides mimicking the CD36 binding site, but not mutated or scrambled peptides, effectively prevented: (i) oxLDL binding to CD36, (ii) macrophage foam cell formation induced by oxLDL, and (iii) platelet activation by oxPC(CD36). These data indicate that CD36 (160-168) represents the core of the oxPC(CD36) binding site with lysines 164/166 being indispensable for the binding.

Oxidized high-density lipoprotein inhibits platelet activation and aggregation via scavenger receptor BI.

Numerous studies have reported the presence of oxidatively modified high-density lipoprotein (OxHDL) within the intima of atheromatous plaques as well as in plasma; however, its role in the pathogenesis of thrombotic disease is not established. We now report that OxHDL, but not native HDL, is a potent inhibitor of platelet activation and aggregation induced by physiologic agonists. This antithrombotic effect was concentration and time dependent and positively correlated with the degree of lipoprotein oxidation. Oxidized lipoproteins are known ligands for scavenger receptors type B, CD36 and scavenger receptor B type I (SR-BI), both of which are expressed on platelets. Studies using murine CD36(-/-) or SR-BI(-/-) platelets demonstrated that the antithrombotic activity of OxHDL depends on platelet SR-BI but not CD36. Binding to SR-BI was required since preincubation of human and murine platelets with anti-SR-BI blocking antibody abrogated the inhibitory effect of OxHDL. Agonist-induced aggregation of platelets from endothelial nitric oxide synthase (eNOS)(-/-), Akt-1(-/-), and Akt-2(-/-) mice was inhibited by OxHDL to the same degree as platelets from wild-type (WT) mice, indicating that the OxHDL effect is mediated by a pathway different from the eNOS/Akt pathway. These novel findings suggest that contrary to the prothrombotic activity of oxidized low-density lipoprotein (OxLDL), HDL upon oxidation acquires antithrombotic activity that depends on platelet SR-BI.

Specific oxidized phospholipids inhibit scavenger receptor bi-mediated selective uptake of cholesteryl esters.

We have recently demonstrated that specific oxidized phospholipids (oxPC(CD36)) accumulate at sites of oxidative stress in vivo such as within atherosclerotic lesions, hyperlipidemic plasma, and plasma with low high-density lipoprotein levels. oxPC(CD36) serve as high affinity ligands for the scavenger receptor CD36, mediate uptake of oxidized low density lipoprotein by macrophages, and promote a pro-thrombotic state via platelet scavenger receptor CD36. We now report that oxPC(CD36) represent ligands for another member of the scavenger receptor class B, type I (SR-BI). oxPC(CD36) prevent binding to SR-BI of its physiological ligand, high density lipoprotein, because of the close proximity of the binding sites for these two ligands on SR-BI. Furthermore, oxPC(CD36) interfere with SR-BI-mediated selective uptake of cholesteryl esters in hepatocytes. Thus, oxidative stress and accumulation of specific oxidized phospholipids in plasma may have an inhibitory effect on reverse cholesterol transport.

Protein kinase R-dependent regulation of interleukin-10 in response to double-stranded RNA.

The double-stranded RNA-activated protein kinase R (PKR) is an important component of antiviral defense. PKR participates in different signaling pathways in response to various stimuli to regulate translation via phosphorylation of the eukaryotic initiation factor 2alpha, and transcription via activating NF-kappaB and IRF-1, to induce pro-inflammatory cytokines. Here we show PKR regulates interleukin-10 induction in response to double-stranded RNA, bacterial lipopolysaccaride, and Sendai virus infection. Using chemical inhibitors, dominant negative constructs, and genetic knockouts, we demonstrate that the PKR-mediated interleukin-10 induction engages JNK and NF-kappaB. Together, our data demonstrate the role of PKR in regulating an anti-inflammatory cytokine. The findings have significance in antiviral as well as broader innate immune responses.

Temporal activation of NF-kappaB regulates an interferon-independent innate antiviral response against cytoplasmic RNA viruses.

NF-kappaB is known to exert its antiviral innate immune response via the IFN-beta-induced Janus kinase/signal transducers and activators of transcription pathway. However, our current studies have demonstrated that activated NF-kappaB is capable of directly establishing an antiviral state independent of IFN or secreted soluble factor(s) against two highly pathogenic respiratory RNA viruses. Human parainfluenza virus type 3, a mildly cytopathic virus that induced NF-kappaB very early during infection was converted to a virulent virus after NF-kappaB inhibition. In contrast, a highly cytopathic virus, human respiratory syncytial virus that induced NF-kappaB late during infection, was converted to a mildly cytopathic virus after NF-kappaB induction before virus replication. This interconversion of cytopathic phenotypes of viruses after NF-kappaB modulation was further shown to be independent of IFN and soluble secreted factors(s). Moreover, tumor necrosis factor alpha (TNF-alpha) and IL-1beta elicited an antiviral response, which was NF-kappaB-dependent. Thus, NF-kappaB induction directly confers an essential innate antiviral response against human parainfluenza virus type 3 and respiratory syncytial virus, which is independent of IFN-inducible factor(s).