Sugar specific cellular lectins of Phallusia mamillata hemocytes: Purification, characterization and evidence for cell surface localization

Cellular lectins (CLs) of Phallusia mamillata were demonstrated in protein preparations obtained by salt fractionation from hemocytes sonicated in a suitable medium. Since the lectins from the precipitated fraction bind sugars containing D-galactosyl groups, they were purified by affinity chromatography on Sepharose. SDS-PAGE under reducing conditions showed that CLs are formed of two components of apparent MWs approximately 36,900 and 35,090 and thus differ from serum lectins (SLs) (MW about 62,200). The "shrinkage" observed when SLs were examined under nonreducing conditions suggest the presence of intrachain disulphide bonds which can affect the molecular structure of the SLs. CL-SL differences were also revealed by the nonidentity reaction of the immuno-precipitate in immunodiffusion using an anti-SL immune serum. The capacity of hemocytes to form rosettes or clumps with erythrocytes demonstrated that they possess α-lactose specific CLs on their surfaces. © 1989

D-Galactose binding lectins from the tunicate Ascidia malaca: Subunit characterization and hemocyte surface distribution

D-galactose specific lectins purified from Ascidia malaca serum contain a major protein component with an apparent molecular weight of about 58,000 daltons, which moves more rapidly under non-reducing conditions. Intramolecular disulfide linkages can explain this behaviour, suggesting a compact protein structure. Membrane lectins have been demonstrated on the surface of about 34% hemocytes by immunofluorescent methods using a rabbit antiserum against the isolated serum lectins. Small, medium and large hemocytes can be positive, as also shown by binding on Sepharose spherules or by rosette formation with sheep and rabbit erythrocytes. Binding is inhibited by the same sugars specific for the serum lectins. Finally, antibodies to the serum lectins specifically agglutinate the hemocytes. This evidence supports the hypothesis that a lectin with the same specificity and certain structural similarities can be found free in the serum and present on hemocyte surfaces. © 1988

Echinoderm Antimicrobial Peptides to Contrast Human Pathogens

Increased attention has been focused in marine invertebrates as a source of bioactive molecules for biomedical applications. Many bioactive molecules are part of the innate immune system. Some more recently isolated compounds, mainly from sea urchin and sea cucumber are antimicrobial peptides (AMP) active against Gram positive, Gram negative and fungi. In this review we described the most recent studies on AMP isolated from echinoderms. The AMP are little peptides <10 kDa with cationic charge and amphipathic structure. Recently, it was demonstrated that in the coelomocyte lysates of Paracentrotus. lividus and Holothuria tubulosa AMP are present with activity against staphylococcal and Pseudomonas aeruginosa antibiofilm. The data show the great potential of application of AMPs in biotechnology for developing novel therapeutic agents and as complements to conventional antibiotic therapy to combat the multi - resistant bacterial strains

In Vitro Release of Lectins From Phallusia mamillata Hemocytes After Their Fractionation on a Density Gradient

Hemocytes were fractionated by centrifugation on a discontinuous Percoll density gradient from the hemolymph of Phallusia mamillata. Results obtained from microcultures of the fractionated hemocytes, sugar-inhibition experiments, SDS-PAGE, and immunoblotting indicate that "compartment cells" release cellular-type (CL) lectins that are specific for a-lactose and lactulose. The released lectins have the same properties as the CL lectins that were previously isolated from sonicated unfractionated hemocytes, but they differ in terms of some molecular and immunological properties from the lectins (SL) purified from the serum. SLs were never found in the supernatants from microcultures ofthe fractionated hemocytes

In vitro release of lectins by Phallusia mamillata hemocytes

α-Lactose specific lectins are released from Phallusia mamillata hemocytes during short-term cultures. The molecular weight of the subunits, the immunological cross-reaction and the sugar specificity suggest that the released lectins are similar to those isolated from the sonicated hemocytes. Because lectin release appears to take place independently of active protein synthesis, the possibility exists that lectins are pre-formed, stored in hemocytes and released when in vitro conditions stimulate the cells. © 1991

Cytotoxic activity of Ciona intestinalis (Tunicata) hemocytes: Properties of the in vitro reaction against erythrocyte targets

Hemocytes (effectors) of Ciona intestinalis showed a natural cytotoxic capacity (HCA) when assayed in vitro against erythrocytes (targets). Cytotoxic cells lysed, to a variable extent, rabbit (RE), human (A, B, O), guinea pig, and sheep (SE) erythrocytes. Hemocyte cytotoxic activity (HCA) assayed against SE is a calcium-dependent reaction, occurs rapidly (15-30 min), at 25-37°C over a wide range of pH (5.4-8.0). Assays were carried out using: 1) the medium in which hemocytes were maintained, 2) the soluble portion of hemocyte lysates, and 3) debris prepared from hemocyte lysates. Results suggest that HCA is a cell-mediated process that requires effector-target cell contacts. Anti-SE (calcium-dependent) and anti-RE (calcium-independent) agglutinins were also found in the reaction medium, probably released by hemocytes as a consequence of the in vitro experiments. The occurrence of HCA was independent of any allogeneic reaction between mixed hemocytes. Various levels of cytotoxic activity reveal hemocyte specificity. © 1993

Antimicrobial peptides from echinoderms as antibiofilm agents: a natural strategy to combat bacterial infections

Increased attention has been focused on marine invertebrates as a source of bioactive molecules for biomedical applications. Many bioactive molecules are part of the innate immune system. Some more recently isolated compounds, mainly from the sea urchin and the sea cucumber, are antimicrobial peptides (AMPs) active against Gram-positive and Gram-negative bacteria, and fungi. In this review we described the most recent studies on AMPs isolated from echinoderms. AMPs are small peptides (< 10 kDa) with cationic charge and amphipathic structure. Recently, it was demonstrated that in the coelomocyte lysates of Paracentrotus lividus and Holothuria tubulosa, AMPs possess activity against staphylococcal and Pseudomonas aeruginosa biofilms. The data shows a great potential for application of AMPs in biotechnology for developing novel therapeutic agents that are either alternative or complementary to conventional antibiotic therapy to combat multiresistant pathogens

Ciona robusta macrophage migration inhibitory factor (Mif1 and Mif2) genes are differentially regulated in the lipopolysaccharide-challenged pharynx

The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (haemapoetic tissue) of Ciona robusta were investigated using quantitative reverse-transcription PCR (qRT-PCR) and in situ hybridisation (ISH). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in the expression of proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were shown to be upregulated 1 h post LPS challenge. The change in the expression of the two Mif paralogs in the pharynx was assessed before and after stimulation, and qRTPCR and ISH results showed that, although Mif2 and Mif2 were expressed in clusters of haemocytes in pharynx vessels, only Mif1 expression increased after LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that need further analysis

Detection of vitellogenin in a subpopulation of sea urchin coelomocytes

Sea urchin vitellogenin is a high molecular weight glycoprotein, which is the precursor of the major yolk protein present in the unfertilized egg. Vitellogenin processing into the major yolk protein and its further enzymatic cleavage during sea urchin embryonic development, has been extensively described, and the adhesive properties of the processed molecule have been studied. The function of vitellogenin in the adult, where it has been found in the coelomic fluid of both male and female individuals, is still unknown, although its role on promoting the adhesion of embryonic cells has been shown. In this report we describe the detection of vitellogenin in lysates of whole circulating coelomocytes of both male and female sea urchins of the species Paracentrotus lividus. By metrizoic acid gradients we purified total coelomocytes into six subpopulations that were tested for the occurrence of the molecule using vitellogenin-specific polyclonal antibodies. We detected vitellogenin only in the coelomocyte subpopulation called colorless spherule cells, packed in kidney-shaped granules located around the nucleus. We also showed that coelomocytes respond to stress conditions by discharging vitellogenin into the medium. This result together with previous observations on the adhesive properties of the molecule suggest a role for vitellogenin in the clotting phenomenon occurring after host invasion

Controlled uptake of PFOA in adult specimens of Paracentrotus lividus and evaluation of gene expression in their gonads and embryos

Perfluorooctanoic acid (PFOA) has been largely used in the manufacturing industry but a few years ago it turned out to be a dangerous pollutant which is now of concern for terrestrial and aquatic environments. Here, we investigated the bioaccumulation of PFOA in the sea urchin Paracentrotus lividus after exposure to different concentrations of the pollutant for 28 days. We observed rapid uptake of PFOA in the coelomic fluid collected weekly during the exposure period and high bioaccumulation in gonads at the end of the experiment. Interestingly, animals were also able to fast depurate when relocated to a clean environment. In addition, to assess the effect of PFOA on sea urchins' physiological pathways, we analysed the expression profile of some marker genes both in the gonads and in the embryos obtained from parents exposed to PFOA. Our results suggest that PFOA is a persistent, bioaccumulative compound that adversely affects the health of the exposed organisms and their offspring by causing significant changes in the expression of some key target genes and the occurrence of developmental anomalies in the embryos