Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis.

We previously reported the discovery of a novel lipid deacetylase in platelets, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), and that its inhibition impairs agonist-induced platelet aggregation, Rap1 GTP loading, protein kinase C (PKC) activation, and ex vivo thrombus growth. However, precise mechanisms by which AADACL1 impacts platelet signaling and function in vivo are currently unknown. Here, we demonstrate that AADACL1 regulates the accumulation of ether lipids that impact PKC signaling networks crucial for platelet activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5'-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12-/- and CalDAG GEFI-/- mice revealed that the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed weak agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 distinct PKC isoforms and blocked PKC kinase activity in vitro. Finally, AADACL1 inhibition in rats reduced platelet aggregation, protected against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage.

Triplication in permanent teeth: A rare case report.

Fusion is an anomaly manifested in both deciduous and permanent dentitions. Triple tooth refers to the union of three separate tooth entities. It can involve the normal dentition or supernumerary teeth. Triplication is rarely encountered in deciduous and permanent dentition with an incidence of 0.02%. The case presented herein describes a rare case of triplication in permanent maxillary incisors and supernumerary teeth in a 15-year-old female.

ß-Arrestin 1-dependent regulation of Rap2 is required for fMLP-stimulated chemotaxis in neutrophil-like HL-60 cells.

ß-Arrestins have emerged as key regulators of cytoskeletal rearrangement that are required for directed cell migration. Whereas it is known that ß-arrestins are required for formyl-Met-Leu-Phe receptor (FPR) recycling, less is known about their role in regulating FPR-mediated neutrophil chemotaxis. Here, we show that ß-arrestin 1 (ArrB1) coaccumulated with F-actin within the leading edge of neutrophil-like HL-60 cells during chemotaxis, and its knockdown resulted in markedly reduced migration within fMLP gradients. The small GTPase Ras-related protein 2 (Rap2) was found to bind ArrB1 under resting conditions but dissociated upon fMLP stimulation. The FPR-dependent activation of Rap2 required ArrB1 but was independent of Gαi activity. Significantly, depletion of either ArrB1 or Rap2 resulted in reduced chemotaxis and defects in cellular repolarization within fMLP gradients. These data strongly suggest a model in which FPR is able to direct ArrB1 and other bound proteins that are required for lamellipodial extension to the leading edge in migrating neutrophils, thereby orientating and directing cell migration.

Tumor treating fields perturb the localization of septins and cause aberrant mitotic exit.

The anti-tumor effects of chemotherapy and radiation are thought to be mediated by triggering G1/S or G2/M cell cycle checkpoints, while spindle poisons, such as paclitaxel, block metaphase exit by initiating the spindle assembly checkpoint. In contrast, we have found that 150 kilohertz (kHz) alternating electric fields, also known as Tumor Treating Fields (TTFields), perturbed cells at the transition from metaphase to anaphase. Cells exposed to the TTFields during mitosis showed normal progression to this point, but exhibited uncontrolled membrane blebbing that coincided with metaphase exit. The ability of such alternating electric fields to affect cellular physiology is likely to be dependent on their interactions with proteins possessing high dipole moments. The mitotic Septin complex consisting of Septin 2, 6 and 7, possesses a high calculated dipole moment of 2711 Debyes (D) and plays a central role in positioning the cytokinetic cleavage furrow, and governing its contraction during ingression. We showed that during anaphase, TTFields inhibited Septin localization to the anaphase spindle midline and cytokinetic furrow, as well as its association with microtubules during cell attachment and spreading on fibronectin. After aberrant metaphase exit as a consequence of TTFields exposure, cells exhibited aberrant nuclear architecture and signs of cellular stress including an overall decrease in cellular proliferation, followed by apoptosis that was strongly influenced by the p53 mutational status. Thus, TTFields are able to diminish cell proliferation by specifically perturbing key proteins involved in cell division, leading to mitotic catastrophe and subsequent cell death.

GPCR-mediated PLCßγ/PKCß/PKD signaling pathway regulates the cofilin phosphatase slingshot 2 in neutrophil chemotaxis.

Chemotaxis requires precisely coordinated polymerization and depolymerization of the actin cytoskeleton at leading fronts of migrating cells. However, GPCR activation-controlled F-actin depolymerization remains largely elusive. Here, we reveal a novel signaling pathway, including Gαi, PLC, PKCß, protein kinase D (PKD), and SSH2, in control of cofilin phosphorylation and actin cytoskeletal reorganization, which is essential for neutrophil chemotaxis. We show that PKD is essential for neutrophil chemotaxis and that GPCR-mediated PKD activation depends on PLC/PKC signaling. More importantly, we discover that GPCR activation recruits/activates PLCγ2 in a PI3K-dependent manner. We further verify that PKCß specifically interacts with PKD1 and is required for chemotaxis. Finally, we identify slingshot 2 (SSH2), a phosphatase of cofilin (actin depolymerization factor), as a target of PKD1 that regulates cofilin phosphorylation and remodeling of the actin cytoskeleton during neutrophil chemotaxis.

Plasmodium falciparum infection induces expression of a mosquito salivary protein (Agaphelin) that targets neutrophil function and inhibits thrombosis without impairing hemostasis.

BACKGROUND: Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear. PRINCIPAL FINDINGS: Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (K(D) ∼ 10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl3-induced arterial thrombosis, without impairing hemostasis. CONCLUSIONS: Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions.

Tempol, an intracellular antioxidant, inhibits tissue factor expression, attenuates dendritic cell function, and is partially protective in a murine model of cerebral malaria.

BACKGROUND: The role of intracellular radical oxygen species (ROS) in pathogenesis of cerebral malaria (CM) remains incompletely understood. METHODS AND FINDINGS: We undertook testing Tempol--a superoxide dismutase (SOD) mimetic and pleiotropic intracellular antioxidant--in cells relevant to malaria pathogenesis in the context of coagulation and inflammation. Tempol was also tested in a murine model of CM induced by Plasmodium berghei Anka infection. Tempol was found to prevent transcription and functional expression of procoagulant tissue factor in endothelial cells (ECs) stimulated by lipopolysaccharide (LPS). This effect was accompanied by inhibition of IL-6, IL-8, and monocyte chemoattractant protein (MCP-1) production. Tempol also attenuated platelet aggregation and human promyelocytic leukemia HL60 cells oxidative burst. In dendritic cells, Tempol inhibited LPS-induced production of TNF-α, IL-6, and IL-12p70, downregulated expression of co-stimulatory molecules, and prevented antigen-dependent lymphocyte proliferation. Notably, Tempol (20 mg/kg) partially increased the survival of mice with CM. Mechanistically, treated mice had lowered plasma levels of MCP-1, suggesting that Tempol downmodulates EC function and vascular inflammation. Tempol also diminished blood brain barrier permeability associated with CM when started at day 4 post infection but not at day 1, suggesting that ROS production is tightly regulated. Other antioxidants-such as α-phenyl N-tertiary-butyl nitrone (PBN; a spin trap), MnTe-2-PyP and MnTBAP (Mn-phorphyrin), Mitoquinone (MitoQ) and Mitotempo (mitochondrial antioxidants), M30 (an iron chelator), and epigallocatechin gallate (EGCG; polyphenol from green tea) did not improve survival. By contrast, these compounds (except PBN) inhibited Plasmodium falciparum growth in culture with different IC50s. Knockout mice for SOD1 or phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (gp91(phox-/-)) or mice treated with inhibitors of SOD (diethyldithiocarbamate) or NADPH oxidase (diphenyleneiodonium) did not show protection or exacerbation for CM. CONCLUSION: Results with Tempol suggest that intracellular ROS contribute, in part, to CM pathogenesis. Therapeutic targeting of intracellular ROS in CM is discussed.

Plasmodium falciparum merozoite surface protein 1 blocks the proinflammatory protein S100P.

The malaria parasite, Plasmodium falciparum, and the human immune system have coevolved to ensure that the parasite is not eliminated and reinfection is not resisted. This relationship is likely mediated through a myriad of host-parasite interactions, although surprisingly few such interactions have been identified. Here we show that the 33-kDa fragment of P. falciparum merozoite surface protein 1 (MSP1(33)), an abundant protein that is shed during red blood cell invasion, binds to the proinflammatory protein, S100P. MSP1(33) blocks S100P-induced NFκB activation in monocytes and chemotaxis in neutrophils. Remarkably, S100P binds to both dimorphic alleles of MSP1, estimated to have diverged >27 Mya, suggesting an ancient, conserved relationship between these parasite and host proteins that may serve to attenuate potentially damaging inflammatory responses.

Subacute effects of carbofuran on enzyme functions in rat small intestine.

The effect of carbofuran administration to rats has been studied on enzymes functions in rat intestine. Carbofuran was administrated 4.0 mg/kg body weight for 7 days or 2.8 mg/kg body weight for 30 days daily by Ryle's tube. Animals given carbofuran for 30 days exhibited retarded growth compared to control group. The activities of sucrase (56%), alkaline phosphatase (62%), leucine aminopeptidase (56%), and gamma-glutamyl trans peptidase (84%) were enhanced in animals given carbofuran for 7 days. Enhancement in the activities of alkaline phosphatase and leucine amino peptidase (92-96%) was also observed in animals exposed to carbofuran for 30 days, but the activities of sucrase (28%) and gamma-glutamyl transpeptidase (49%) were reduced under these conditions. There was no change in activities of maltase, lactase, and trehalase in pesticide-treated animals for 7 or 30 days. The activity of lactate dehydrogenase was enhanced (p < 0.001) in 7 days and 30 days induced carbofuran toxicity. The activities of glucose-6-phosphatase and glutamate pyruvate transaminase were also enhanced (p < 0.001) in pesticide-treated animals for 7 days, but were reduced by 46% and 26%, respectively, after 30 days of carbofuran exposure. The activity of glutamate oxaloacetate transaminase was unaltered in carbofuran toxicity. Kinetic analysis of brush border enzymes revealed a change in V(max) with no change in apparent Km. Western blot analysis of brush border sucrase, alkaline phosphatase, and leucine aminopeptidase corroborated the enzyme activity data. Intestinal histological revealed distruption of the villi, and comet assay showed disintegration of DNA in enterocytes of animals exposed to carbofuran for 30 days. These findings suggest that carbofuran toxicity may modulate digestive functions in rat intestine.