Purification of the Endospores and Sporangia of Rhinosporidium Seeberi on Percoll Columns

Human rhinosporidial tissue was used as the source of the various developmental stages of Rhinosporidium seeberi - endospores with electron dense bodies, juvenile, and immature sporangia. After homogenisation in phosphate buffered saline (PBS) and removal of tissue fragments by centrifugation, the rhinosporidial bodies were isolated on centrifuged Percoll columns with gradients of densities or on triple-layered columns of varying density. The separated bands, after repeated washing in PBS gave bodies free from human tissue as shown on Leishman and PAS staining and indirect immunofluorescence with rabbit and human patients' anti-rhinosporidial sera. Sonicates of these bodies were tested on agarose gel for precipitation with antisera, and on SDS-PAG electrophoresis and Coomassie Blue staining. Percoll columns were shown to be capable of isolating these stages of R. seeberi, free from human tissue and contaminating bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Anti-Rhinosporidial Antibody Levels in Patients with Rhinosporidiosis and in Asymptomatic Persons,in Sri Lanka

The only report hitherto, from India in 1982, on anti-rhinosporidial antibody levels in patients with rhinosporidiosis recorded that antibody was not detected in Indian patients. The present report describes the use of the dot-ELISA assay of serum anti-rhinosporidial IgG, IgM and IgA and salivary sIgA in patients with diverse clinical presentations, in rural asymptomatic persons who had bathed in ground waters that probably harboured the causative pathogen, Rhinosporidium seeberi, and in laboratory persons who were exposed to R. seeberi. Ultrasonic extracts of purified endospores and sporangia of R. seeberi were used as antigen. The geometric mean (reciprocal) titres of serum antibody detected in patients were IgM 142.1, IgG 178.5, IgA 84.6, with ranges of 0-640, 30-960 and 0-160 respectively, salivary sIgA titres ranged from 0 to 18 with a mean of 4.6. The levels of antibody had no correlation with the site, the number of sporangia, duration and recurrence of the disease. Asymptomatic persons from the same endemic area as patients showed mean titres of IgM 89.6, IgG 69.1, IgA 95.5, with salivary sIgA titres of 3.1. Asymptomatic personnel who had been working in a laboratory where rhiniosporidial work was being done, showed mean titres of 169.6 IgM, 62.8 IgG, and 6.5 salivary sIgA. These results indicate that an anti-rhinosporidial antibody response occurs in rhinosporidial patients, as well as in asymptomatic persons who were exposed to R. seeberi in the environment. Anti-R. seeberi antibody does not appear to be protective in rhinosporidiosis since appreciable titres were present in patients with recurrent, single, multiple or disseminated lesions of long duration.     

Cell-mediated immune responses (CMIR) to Rhinosporidium seeberi in mice

There is no published data on Cell Mediated Immune Responses in experimental animals to Rhinosporidium seeberi the causative agent of human and animal rhinosporidiosis. The quantitative mouse foot-pad model was used to assay the Delayed-Type Hypersensitivity (DTH) cell-mediated immune response to extracts of purified endospores and sporangia of R. seeberi. Histological examination was used to confirm that the foot-pad reactions were compatible with DTH reactions in the mouse. We report that sonically disintegrated rhinosporidial endospores/sporangia induced DTH responses in the foot-pads of sensitized mice which were comparable in intensity and histological profile to that induced by sheep red blood cells in SRBC sensitized mice. Anti-rhinosporidial antibody was also induced. Filtrates of the soluble antigens in sonicated suspensions failed to evoke a DTH-foot-pad (DTH-FP) response in sensitized mice although an anti-rhinosporidial antibody response to this preparation was detected. Prolonged pre-treatment with sonicated suspensions of endospores and sporangia resulted in a decrease of DTH reactivity as compared with reactions following pre-treatment of a shorter duration. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organisation and expression of a cluster of yolk protein genes in the Australian sheep blowfly, Lucilia cuprina

The Australian sheep blowfly Lucilia cuprina is a major pest for the Australian and New Zealand sheep industries. With the long-term aim of making a strain of L. cuprina suitable for a genetic control program, we previously developed a tetracycline-repressible female lethal genetic system in Drosophila. A key part of this system is a female-specific promoter from a yolk protein (yp) gene controlling expression of the tetracycline-dependent transactivator (tTA). Here we report the sequence of a 14.2 kb genomic clone from L. cuprina that contains a cluster of three complete yp genes and one partial yp gene. The Lcyp genes are specifically expressed in females that have received a protein meal. A bioinformatic analysis of the promoter of one of the yp genes (LcypA) identified several putative binding sites for DSX, a known regulator of yp gene expression in other Diptera. A transgenic strain of L. cuprina was made that contained the LcypA promoter driving the expression of the Escherichia coli lacZ reporter gene. Transgenic females express high levels of β-galactosidase after a protein meal. Thus the LcypA promoter could be used to obtain female-specific expression of tTA in transgenic L. cuprina.     

Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.     

Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph-Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.     

Multiple species delimitation approaches with COI barcodes poorly fit each other and morphospecies – An integrative taxonomy case of Sri Lankan Sericini chafers (Coleoptera: Scarabaeidae)

DNA taxonomy including barcoding and metabarcoding is widely used to explore the diversity in biodiversity hotspots. In most of these hotspot areas, chafers are represented by a multitude of species, which are well defined by the complex shape of male genitalia. Here, we explore how well COI barcode data reflect morphological species entities and thus their usability for accelerated species inventorization. We conducted dedicated field surveys in Sri Lanka to collect the species‐rich and highly endemic Sericini chafers (Coleoptera: Scarabaeidae). Congruence among results of a series of protocols for de novo species delimitation and with morphology‐based species identifications was investigated. Different delimitation methods, such as the Poisson tree processes (PTP) model, Statistical Parsimony Analysis (TCS), Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), and Barcode Index Number (BIN) assignments, resulted in different numbers of molecular operational taxonomic units (MOTUs). All methods showed both over‐splitting and lumping of morphologically identified species. Only 18 of the observed 45 morphospecies perfectly matched MOTUs from all methods. The congruence of delimitation between MOTUs and morphospecies expressed by the match ratio was low, ranging from 0.57 to 0.67. TCS and multirate PTP (mPTP) showed the highest match ratio, while (BIN) assignment resulted in the lowest match ratio and most splitting events. mPTP lumped more species than any other method. Principal coordinate analysis (PCoA) on a match ratio‐based distance matrix revealed incongruent outcomes of multiple DNA delimitation methods, although applied to the same data. Our results confirm that COI barcode data alone are unlikely to correctly delimit all species, in particular, when using only a single delimitation approach. We encourage the integration of various approaches and data, particularly morphology, to validate species boundaries.     

Fungi associated with dried fish in Sri Lanka

The mycoflora of dried-salted fish from markets in Kandy, Sri Lanka was studied with emphasis on visibly spoiled dried fish. A total of 61 fungal isolates from 25 dried-fish were isolated and identified. The most prevalent fungus wasAspergillus niger. Species ofAspergillus flavus, A. fumigatus, A. glaucus, A. restrictus, Aureobasidium spp.Basipetospora halophila (a genuinely halophilic fungus)Cladosporium herbarum, Gliomastix, spp.,Penicillium chalybeum andPenicillium expansum were present. The isolated fungi did not grow in synthetic media containing more than 30% sodium chloride.Aureobasidium spp. andGliomastix spp. did not grow on dried-fish under laboratory conditions. The protective exoskeleton appeared to prevent fungal growth on dried shrimp. TheA. flavus strains isolated were not aflatoxigenic.     

Mass spectrometry based proteomics analyses in kallikrein-related peptidase research: implications for cancer research and therapy

Introduction: Kallikrein-related peptidases (KLKs) are a family of serine peptidases that are deregulated in numerous pathological conditions, with a multitude of KLK-mediated functional roles implicated in the progression of cancer. Advances in multidimensional mass spectrometry (MS)-based proteomics have facilitated the quantitative measurement of deregulated KLK expression in cancer, identifying certain KLKs, as well as their substrates, as potential cancer biomarkers.

Areas covered: In this review, we discuss how these approaches have been utilized for KLK biomarker discovery and unbiased substrate determination in complex protein pools that mimic the in vivo extracellular microenvironment.

Expert commentary: Although a limited number of studies have been performed, the quantity of information generated has greatly improved our understanding of the functional roles of KLKs in cancer progression. In addition, these data suggest additional means through which deregulated KLK expression may be targeted in cancer treatment, highlighting the potential therapeutic value of these state-of-the-art MS-based studies.     

N‐terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells

Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase‐9‐dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N‐terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N‐terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagicEscherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N‐terminal region in addressing certain RTX toxins to mitochondria.