Ecological dynamics of the bivalve-killing dinoflagellate Heterocapsa circularisquama and its infectious viruses in different locations of western Japan

We studied the ecological relationships between the bloom-forming dinoflagellate Heterocapsa circularisquama and its infectious viruses in field surveys conducted in western Japan. The occurrence of H. circularisquama blooms in Imari Bay during 2002 and in Ago Bay during 2002 and 2004 was accompanied by specific increase in abundance of viruses lytic to H. circularisquama. Using northern dot-blot analysis, approximately 96% of the clonal virus isolates collected in the field surveys positively reacted with a molecular probe specific for HcRNAV (H. circularisquama RNA virus); hence, viral impacts on H. circularisquama population observed in these field surveys are considered largely due to HcRNAV and/or its closely related viruses. The dynamics of type UA viruses and type CY viruses having complementary host ranges to H. circularisquama clones were different in each survey and considered to reflect fluctuations in abundance of their suitable host cells in situ. The dynamics of H. circularisquama and its viruses in Ago Bay from 2002 to 2004 suggests the concentration of HcRNAV in the sediment prior to the host's blooming season is a significant factor in determining the size and length of the H. circularisquama blooms. These results support the hypothesis that HcRNAV infection is one of the significant factors affecting the population dynamics of H. circularisquama in both quantity (biomass) and quality (clonal composition).     

Staphylococcal enterotoxin induces emesis through increasing serotonin release in intestine and it is downregulated by cannabinoid receptor 1

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable bacterial superantigenic toxins causing food poisoning in humans throughout the world. However, it remains unclear how SEs induce emesis and its emetic signal pathway. We investigated a mechanism of SEA-induced emesis using a small emetic animal model, house musk shrew. SEA-induced emesis in the animals was inhibited by a 5-hydroxytryptamine (5-HT) synthesis inhibitor and a 5-HT(3) receptor antagonist. SEA could increase 5-HT release in the small intestine. Pre-treatment with 5,7-dihydroxytryptamine (5,7-DHT) markedly inhibited SEA-induced emesis. SEA-induced emesis was also abolished by surgical vagotomy. Furthermore, cannabinoid (CB) receptor agonists inhibited SEA-induced emesis, and the action was reversed by a CB1 antagonist. Both 5-HT release and CB1 receptor expression were found in the mucosal and myenteric plexus of the intestine. Moreover, a CB1 receptor agonist significantly decreased the 5-HT release in the intestine. These results demonstrate that SEA induces 5-HT release in intestine, rather than in brain, and that the 5-HT(3) receptors on vagal afferent neurons are essential for SEA-stimulated emesis. In addition, SEA-induced emesis is downregulated by the CB system through decreasing 5-HT release in intestine.     

Protein disulfide isomerase-mediated disulfide bonds regulate the gelatinolytic activity and secretion of matrix metalloproteinase-9

Matrix metalloproteinase-9 (MMP-9) is one of the major MMPs that can degrade extracellular matrix. Besides normal physiological functions, MMP-9 is involved in metastasis and tumor angiogenesis. Although several inhibitors of MMP-9 have been identified, in vivo regulators of MMP-9 activation are unknown. In the present study we intended to investigate novel therapeutic target protein(s) that regulate MMP-9 activation and/or secretion. We have identified protein disulfide isomerase as a novel upstream regulator of MMP-9. Mass spectrometric analysis of post-translational modification in MMP-9 confirmed six disulfide bonds in the catalytic domain and one disulfide bond in the hemopexin domain of MMP-9. Establishment of cells that overexpressed wild-type and mutant forms of MMP-9 revealed that 'cysteine-switch' and disulfide bonds within the catalytic domain are necessary for the secretion and intracellular trafficking of MMP-9. However, the disulfide bond of the hemopexin domain and other cysteines have no significant role in secretion. These insights into the secretion of MMP-9 constitute the basis for the development of potential drugs against metastasis.     

A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity

The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, cysteine endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.     

Tuning the geometries of a de novo blue copper protein by axial interactions

The axial interactions of Cu(2+) in type 1 copper proteins control the physical characteristics of the proteins. We tuned the geometries of a de novo designed blue copper protein with a four-helical bundle structure. The designed protein axially bound various ligands, such as chloride, phosphate, sulfate, acetate, azide, and imidazole, to Cu(2+), exhibiting a blue or green color. The UV-vis spectral bands were observed at approximately 600?nm and approximately 450?nm, with the A (~450)/A (~600) ratios between 0.14 and 1.58. The stronger axial interaction shifted the geometry of the type 1 copper site from trigonal planar geometry (blue copper) toward a tetrahedral-like geometry (green copper). Resonance Raman spectral analyses showed that the phosphate-bound type had the highest-strength Cu-S bond, similar to that of plastocyanin. The chloride-bound type exhibited features similar to those of stellacyanin and nitrite reductase, and the imidazole-bound type exhibited features similar to those of azurin M121E mutant.     

Membrane microdomains in immunity: glycosphingolipid-enriched domain-mediated innate immune responses

Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated.     

Tetrahydro-naphthols as orally available TRPV1 inhibitors

Starting from a naphthol-based lead series with low oral bioavailability, we have identified potent TRPV1 antagonists with oral bioavailability in rats. These compounds emerged from SAR studies aimed at replacing the lead's phenol structure whilst maintaining potency. Compound rac-6a is an orally available TRPV1 antagonist with single-digit nanomolar activity. The enantiomers show a low eudismic ratio at the receptor level.     

Makomotindoline from Makomotake, Zizania latifolia infected with Ustilago esculenta

Makomotindoline (1) was isolated from Makomotake, Zizania latifolia infected with Ustilago esculenta. The structure was determined by the interpretation of spectroscopic data and synthesis. Makomotindoline (1), its l-Glc isomer (2) and its aglycon (3) were synthesized and their effects on rat glioma cells showed adverse effects on the cell growth.