A panel of monoclonal antibodies recognizing GPI-anchored ADP-ribosyltransferase ART4, the carrier of the Dombrock blood group antigens

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.     

Sodium nitroprusside potentiates hydrogen-sulfide-induced contractions in body wall muscle from a marine worm

Hydrogen sulfide (H2S) at concentrations of about 0.05 to 1 mmol.l(-1) appears to function as a gasotransmitter in vertebrates, analogous to nitric oxide (NO) and carbon monoxide, but the actions of H2S in invertebrate tissue have not been well studied. In this study, we investigated the role of H2S in modulating body wall muscle tone in the marine echiuran worm Urechis caupo (Echiuridae). We first determined that U. caupo body wall homogenates produce H2S upon addition of L-cysteine and pyridoxal-5'-phosphate (PLP), and that the rate is increased by addition of 2-mercaptoethanol, suggesting the presence of an activated L-serine sulfhydrase pathway. We then measured the contractile response of U. caupo body wall circular muscle strips to sodium hydrosulfide (NaHS)--which produces H2S in solution--and the NO donor sodium nitroprusside (SNP), both with and without subsequent application of acetylcholine (ACh). We found that NaHS alone stimulated contraction in muscle strips equivalent to about one-third the force of ACh alone, whereas SNP alone had no effect on muscle tone. However, simultaneous addition of NaHS with SNP elicited a much stronger contraction, reaching more than twice that of ACh alone, which could be increased further by subsequent application of ACh.     

Inhibition survey with phenolic compounds against the δ- and η-class carbonic anhydrases from the marine diatom thalassiosira weissflogii and protozoan Plasmodium falciparum

Abstract

The inhibition of δ- and η-class carbonic anhydrases (CAs; EC 4.2.1.1) was poorly investigated so far. Only one δ-CA, TweCA from the diatom Thalassiosira weissflogii, and one η-CA, PfCA, from Plasmodium falciparum, have been cloned and characterised to date. To enrich δ- and η-CAs inhibition profiles, a panel of 22 phenols was investigated for TweCA and PfCA inhibition. Some derivatives showed effective, sub-micromolar inhibition of TweCA (K_Is 0.81–65.4?µM) and PfCA (K_Is 0.62–78.7?µM). A subset of compounds demonstrated a significant selectivity for the target CAs over the human physiologically relevant ones. This study promotes the identification of new potent and selective inhibitors of TweCA and PfCA, which could be considered as leads for finding molecular probes in the study of carbon fixation processes (in which TweCA and orthologue enzymes are involved) or drug candidates in the treatment of malaria.     

Implications of comparative ventral body wall histology on selection of abdominal surgical approach and closure in 12 species of fish

A ventral midline surgical approach for fish celiotomy is commonly performed in veterinary clinical medicine and research, although the relevant ventral body wall anatomy of many fish species is not well documented. Histological evaluation of tissue samples from the ventral body wall of 12 fish species was performed to provide a reference for surgical approach and closure decisions. The width between muscle bundles running parallel to the long axis and total thickness of tissue layers varied among species. An appreciable space between longitudinal muscles of the ventral body wall and a lack of muscle, vessels and nerves on midline in all species examined supports recommendations of ventral midline incisions to spare important structures. Dense connective tissue consistent with an aponeurosis between musculature along the ventral body wall was not observed in any species evaluated. Connective tissue was concentrated within the dermis of all species evaluated, with an additional layer of collagen along the coelomic membrane in Russian sturgeon Acipenser gueldenstaedtii, koi Cyprinus carpio, goldfish Carassius auratus, black drum Pogonias cromis, black seabass Centropristis striata, tomtate Haemulon aurolineatum and scup Stenotomus caprinus. A sufficiently wide space on ventral midline for practical targeting during the surgical approach is present in A. gueldenstaedtii, C. carpio, striped bass Morone saxatilis, H. aurolineatum, P. cromis, rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta. Sand perch Diplectrum formosum, C. auratus, S. caprinus, grey triggerfish Balistes capriscus and black sea bass Centropristis striata have a negligible space between longitudinal muscles on midline. The variation in ventral body wall structure observed in this study helps inform surgical decision making for celiotomy incision and closure in these species.     

Fungi associated with galleries of the emerald ash borer

The emerald ash borer (EAB) is an exotic forest pest that has killed millions of ash trees in the United States and Canada, resulting in an ecological disaster and billions of dollars in economic losses of urban landscape and forest trees. The beetle was first detected in Michigan in 2002 and has spread through much of the Eastern and Midwestern U.S., reaching Minnesota in 2009. Since then, it has spread across the state and poses a great risk to the more than 1 billion ash trees in Minnesota. The larval stage of EAB creates wounds on trees as they feed on the inner bark, causing disruption of water and sap flow that results in tree death. The fungal community associated with EAB larval galleries is poorly understood and the role these fungi may play in tree death is not known. This study describes fungi isolated from EAB larval galleries sampled throughout the main geographic areas of Minnesota where ash is affected by EAB. Fungal cultures were identified by extracting genomic DNA and sequencing the ITS region of the rDNA. Results from 1126 isolates reveal a diverse assemblage of fungi and three functional guilds comprised of canker pathogens, wood decay, and entomopathogenic fungi. The most common canker-associated genera were Cytospora followed by Phaeoacremonium, Paraconiothyrium, Coniothyrium, Nectria, Diplodia, and Botryosphaeria. Fungi in the Basidiomycota were nearly all wood decay causing fungi and many were species of pioneer colonizing genera including Sistotrema, Irpex, Peniophora, Phlebia and Ganoderma. Some of these fungi seriously affect urban trees, having the potential to cause rapid wood decay resulting in hazardous tree situations. Several entomopathogenic genera with the potential for biological control of EAB were also isolated from galleries. Purpureocillium was the most commonly isolated genus, followed by Beauveria, Clonostachys, Lecanicillium, Akanthomyces, Cordyceps, Microcera, Tolypocladium, and Pochonia. The results identify important fungal functional guilds that are occupying a new niche in ash trees resulting from EAB and include fungi that may accelerate decline in tree health, increase hazard tree situations, or may provide options for biological control of this destructive invasive insect.     

Loss of DNA mismatch repair imparts a selective advantage in planarian adult stem cells

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis.