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

Revision on the effects of an entomopathogen bacteria Bacillus thuringiensis on the immune response of Red Palm Weevil (Rhynchophorus ferrugineus), is it a question of gender?

1 Università degli Studi di Palermo, Italia; 2 Università degli Studi dell’Insubria, Italia Il punteruolo rosso, Rhynchophorus ferrugineus (Olivier 1790) (Coleoptera: Curculionidae) fitofago infestante delle palme e Galleria mellonella L. (Lepidoptera: Pyralidae) parassita delle arnie, sono stati utilizzati come modelli per approfondire le conoscenze della relazione ospite-nematode entomopatogeno. Gli effetti del nematode entomopatogeno Steinernema carpocapsae (Nematoda: Steinernematidae) sull'immuno attività delle larve di R. ferrugineus e di G. mellonella, sono stati messi a confronto. E’ stata anche valutata la capacità di completare il ciclo vitale di S. carpocapsae nei due insetti. Sebbene S. carpocapsae sia efficace nel controllo biologico del punteruolo rosso, non è tuttavia in grado di completare il suo ciclo all’interno dell’ospite, mentre lo stesso nematode si riproduce con successo in G. mellonella. Sulla base di precedenti dati di laboratorio e sulla revisione sistematica della letteratura, gli effetti indotti dal nematode sulla risposta immunitaria del Coleottero e del Lepidottero, sono stati comparati. In particolare sono stati analizzati: la popolazione emocitaria immunocompetente (numero e tipi cellulari), l’attività del sistema profenolossidasi-fenolossidasi (proPO) e infine è stata valutata l’inibizione e proliferazione del batterio simbionte Gram negativo Xenorhabdus nematophila (Enterobacteraceae).Since their discovery, bioinsecticides based on Bacillus thuringiensis (Bt) have demonstrated targeted insect control with minimal environmental impact. Understanding the biology of insect pests, as well as interac- tions with pathogens and immune systems, can help to enhance their effect within these interactions. On the basis of previous laboratory data and a systematic literature review we analyzed the effects induced by the entomopathogen bacteria Bt, on red palm weevil (RPW), an highly invasive pest of palms that had a significant economic, environmental and social impact when introduced into Italy. The bacteria-induced effects on physiology, immune response and stress answers in the RPW. Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) were considered. Previously a Bt strain, commercialized against another family of Coleopters, was found to be a potential pathogen of RPW. Data showed that Bt interacts negatively with the growth and the vitality of RPW moreover with hemocytes of R. ferrugineus whose numbers decreased drastically in the hemolymph both in total number and in type. In particular, we focused on the Bt stress- induced infections considering the genders and the instars. The interaction between Bt and RPW hemocytes was analyzed by evaluating the expression heat shock proteins (HSPs) particularly HSP70 in the supernatant of the hemocyte lysate obtained from larvae and adults. HSPs are rapidly synthesized in the cells after stress exposition including infection by pathogens. The western blot analysis, showed that the HSP70 expression was modulated in the time (3h, 6h, 12h, 19, 24h) in the response to Bt treatment, highlighting that Bt is a stress factor for the larvae but also for adults. The protein expression was increased approximately seven times after 3 hours from treatment and after 6 hours it returning to control value. Data show difference in how males and females invest in Bt infection answer. Our data on immunity support the theory that males choose investment in reproduction over investment in immunity. In fact, B. thuringiensis-induced mortality data show the effect that females mount a stronger immune response and they also suffer lower mortality

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