ABSTRACT
A polysaccharide (LVP) was purified from fruiting body of Lentinus velutinus by ethanol precipitation fractionation and DEAE and Sephadex G-100 column chromatography. The yield of purified polysaccharide was 0.025%. Molecular characteristics of LVP were determined by gel permeation chromatography, FT-IR spectroscopy, and thin-layer chromatography. Our results revealed that LVP is a polysaccharide composed of only glucose units, and has a molecular weight of 336 kDa. Biological activity assays indicated that LVP exhibits both cytotoxic and antioxidant activity. LVP showed specific cytotoxicity against cancer cells (HeLa and HepG2 cells), and alterations in cancer cell morphology were found after LVP treatment.
ABSTRACT
Five Aphanomyces strains were isolated during suspected outbreaks of crayfish disease in Spain and Italy. Genetic and physiological evidence show that the strains isolated from the freshwater crayfish Procambarus clarkii and Pacifastacus leniusculus, do not fit into any previously identified group of Aphanomyces astaci and are not capable of killing crayfish following standardised experimental infection. RAPD-PCR and ITS sequencing analysis show a high degree of similarity between the new isolates, while they are clearly different from the A. astaci reference strains. They do, however, possess some properties, which are commonly associated with parasitic species such as repeated zoospore emergence and the lack of sexual reproduction. The five isolates share some physiological properties i.e. a high growth rate, and germination in response to nutrients and, in contrast to A. astaci, they do not express chitinase constitutively during growth or sporulation. Until their taxonomic status is fully elucidated we suggest that the new isolates be given the tentative species name Aphanomyces repetans.
Subject(s)
Aphanomyces/physiology , Astacoidea/microbiology , Disease Outbreaks/veterinary , Infections/veterinary , Animals , Aphanomyces/enzymology , Aphanomyces/genetics , Aphanomyces/isolation & purification , Base Sequence , Chitin/metabolism , Chitinases/metabolism , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Intergenic/chemistry , DNA, Intergenic/genetics , Infections/microbiology , Italy/epidemiology , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction/veterinary , Spain/epidemiology , Spores, Fungal/growth & development , Water MicrobiologyABSTRACT
Regulation of hematopoiesis in invertebrates is largely unknown, although the hemocytes are essential in immunity, performing functions such as phagocytosis, encapsulation and lysis of foreign cells. We have developed a method to isolate hematopoietic stem cells from the freshwater crayfish, Pacifastacus leniusculus, and therefore, this animal provides a powerful tool for studies on invertebrate hematopoiesis. The hematopoietic tissue of crayfish was found to be actively proliferating. Injection of a beta1,3-glucan caused a severe loss of hemocytes, followed by a rapid recovery, due to release from the hematopoietic organ. Transcripts for peroxinectin, a hemocyte cell adhesion protein, were present in the hematopoietic cells, whereas mRNA for proPO was not detected. A gene coding for a Runt-domain protein known to be involved in hematopoiesis in Drosophila and mammals, was upregulated prior to hemocyte release.We conclude that hemocytes are synthesised and partly differentiated in the hematopoietic tissue, but the final differentiation into functional hemocytes expressing proPO is not completed until the hemocytes are released into the circulation.
Subject(s)
Astacoidea/physiology , Gene Expression , Hematopoiesis/physiology , Hemocytes/physiology , Neoplasm Proteins , Amino Acid Sequence , Animals , Anticoagulants/pharmacology , Blood Proteins/drug effects , Blood Proteins/genetics , Blood Proteins/metabolism , Blotting, Northern , Catechol Oxidase/genetics , Catechol Oxidase/metabolism , Cell Adhesion Molecules/drug effects , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Lineage , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Flow Cytometry , Glucans , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/physiology , Hemocytes/cytology , Hemocytes/drug effects , Humans , In Situ Hybridization , Molecular Sequence Data , Polysaccharides/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Transcription Factors/drug effects , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Up-RegulationABSTRACT
The crayfish plague (Aphanomyces astaci) susceptible freshwater crayfish Astacus astacus and the resistant species Pacifastacus leniusculus were compared with respect to differential haemocyte count and expression of prophenoloxidase and peroxinectin. A major difference found was that resistant crayfish continuously produced high levels of prophenoloxidase (proPO) transcripts and that these levels could not be further increased, whereas in susceptible crayfish proPO transcript levels and resistance were augmented by immunostimulants. In As. astacus this could be registered as higher proPO transcript levels in the semigranular population of haemocytes and to an increased survival time after experimental infections with A. astaci.
Subject(s)
Astacoidea/enzymology , Astacoidea/microbiology , Catechol Oxidase/genetics , Enzyme Precursors/genetics , Oomycetes/pathogenicity , Animals , Astacoidea/genetics , Catechol Oxidase/biosynthesis , Enzyme Precursors/biosynthesis , Fresh Water , Gene Expression , Glucans , Hemocytes/drug effects , Hemocytes/enzymology , In Situ Hybridization, Fluorescence , Polysaccharides/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Thirty-one isolates of Saprolegnia sp., most originating from infected salmon or trout, were characterised genetically and physiologically. The majority (6 of 31) of the isolates from several widely separated geographical locations was found to be genetically almost identical as assessed by RAPD-PCR. The remaining isolates belonged to 3 different groups with 1 to 3 representatives each. It is suggested that the first group of isolates represents a virulent form of the organism that has been widely spread by clonal propagation. The ability to repeated zoospore emergence, as an alternative to direct germination, seems to characterise specific Saprolegnia genotypes that may have adapted to certain hosts.
