Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66–68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX–Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX–Hvf17, at concentrations up to 1 μM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca²⁺ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX–Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed β-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.     

The growing importance of fat in regenerative medicine

A recent publication by Michael Longaker and colleagues represents a landmark for the use of adipose tissue as a source of cells for tissue regeneration. The authors investigated the ability of adipose tissue-derived cells (ADCs) to regenerate critical size calvarial (superior portion of the skull) defects in mice by using a novel osteoconducive apatite-coated Poly-lactic-co-glycolic acid (PLGA) scaffold for cell delivery. Direct comparison of this osteogenic ability was performed with bone marrow stromal cells and juvenile calvarial-derived osteoblasts.     

Distinct bacterial dissemination and disease outcome in mice subcutaneously infected with Borrelia burgdorferi in the midline of the back and the footpad

Subcutaneous inoculation of mice with Borrelia burgdorferi, the causative agent of Lyme disease, results in established infection and the development of acute arthritis and carditis, hallmarks of human disease. Because conflicting results may originate from the site of subcutaneous inoculation, we addressed the dissemination capacity of spirochetes injected in the shoulder region versus the footpad. Spirochetes inoculated in the footpad disseminated to a lesser extent to distant organs, such as the ear and the heart. This resulted in distinct degrees of joint and cardiac inflammation at the peak of the disease. The differences eventually leveled out. These results suggest that caution must be exercised in the interpretation of results obtained with routes of inoculation that do not closely represent the natural site of infection.     

Expression of cardiomyocytic markers on adipose tissue-derived cells in a murine model of acute myocardial injury

Animal and early clinical studies have provided evidence suggesting that intracoronary administration of autologous bone marrow-derived cells results in improved outcome following myocardial infarction. Animal studies with cultured marrow stromal cells (MSC) have provided similar data. Cells with properties that are similar to MSC have been identified in adipose tissue. Other groups have demonstrated in vivo differentiation of adipose tissue-derived cells (ADC) into cells exhibiting biochemical and functional markers of cardiac myocytes, including spontaneous beating.Based on these observations, the objective of the present study was to determine whether ADC might undergo similar differentiation in vivo in the context of myocardial injury.ADC were isolated from subcutaneous adipose tissue of Rosa26 mice (which express the beta-galactosidase transgene in almost every tissue) and injected into the intraventricular chamber of B6129S recipient mice immediately following induction of myocardial cryoinjury. Groups of recipients were euthanized at 24 hours, 7 and 14 days post surgery and examined for the presence of donor-derived cells within the heart.Beta-gal positive cells were identified in the infarcts of ADC-treated animals. No staining was observed in uninjured myocardium or in infarcts of control animals. Immunohistochemical analysis revealed co-expression of beta-gal with Myosin Heavy Chain, Nkx2.5 and with Troponin I. Co-expression of beta-galactosidase with Connexin 43, CD31, von Willebrand factor, MyoD or CD45 was not detected.Thus, these data indicate that adipose tissue contains a population of cells that has the ability to engraft injured myocardium and that this engraftment is associated with expression of cardiomyocytic markers by donor-derived cells.     

Profiling the morphological distribution of O-linked oligosaccharides

The morphological distribution of oligosaccharides is determined in the egg jelly surrounding Xenopus laevis eggs. This biological system is used to illustrate a method for readily identifying and quantifying oligosaccharides in specific tissues. The extracellular matrix surrounding X. laevis eggs consists of a vitelline envelope and a jelly coat. The jelly coat contains three morphologically distinct layers designated J1, J2, and J3 from the innermost to the outermost and is composed of 9-11 distinct glycoproteins. Each jelly layer is known to have specific functions in the fertilization of the egg. We developed a rapid method to separate and identify the oligosaccharides from X. laevis egg jelly layers. Identification was based on the retention times in high-performance liquid chromatography (porous graphitized carbon column), exact masses, and tandem mass spectrometry. Over 40 neutral and 30 sulfated oligosaccharides were observed in the three jelly layers. Neutral oligosaccharide structures from different jelly layers were both unique and overlapping, while sulfated oligosaccharides were detected only in layers J1 and J2. Neutral oligosaccharides unique to jelly layer J3 and the combined layers J1+J2 had similar core structures and similar residues. However, differences between these two sets of unique oligosaccharides were also observed and were primarily due to the branching carbohydrate moieties rather than the core structures.     

Targeted deletion of protein kinase C lambda reveals a distribution of functions between the two atypical protein kinase C isoforms

Protein kinase C lambda (PKClambda) is an atypical member of the PKC family of serine/threonine kinases with high similarity to the other atypical family member, PKCzeta. This similarity has made it difficult to determine specific roles for the individual atypical isoforms. Both PKClambda and PKCzeta have been implicated in the signal transduction, initiated by mediators of innate immunity, that culminates in the activation of MAPKs and NF-kappaB. In addition, work from invertebrates shows that atypical PKC molecules play a role in embryo development and cell polarity. To determine the unique functions of PKClambda, mice deficient for PKClambda were generated by gene targeting. The ablation of PKClambda results in abnormalities early in gestation with lethality occurring by embryonic day 9. The role of PKClambda in cytokine-mediated cellular activation was studied by making mouse chimeras from PKClambda-deficient embryonic stem cells and C57BL/6 or Rag2-deficient blastocysts. Cell lines derived from these chimeric animals were then used to dissect the role of PKClambda in cytokine responses. Although the mutant cells exhibited alterations in actin stress fibers and focal adhesions, no other phenotypic differences were noted. Contrary to experiments using dominant interfering forms of PKClambda, mutant cells responded normally to TNF, serum, epidermal growth factor, IL-1, and LPS. In addition, no abnormalities were found in T cell development or T cell activation. These data establish that, in vertebrates, the two disparate functions of atypical PKC molecules have been segregated such that PKCzeta mediates signal transduction of the innate immune system and PKClambda is essential for early embryogenesis.     

Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase

Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A(2) (cPLA(2)). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor alpha (PPARalpha) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARalpha null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA(2) and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases.