Loredana Stabili

Antibacterial activity in the coelomocytes of the sea urchin Paracentrotus lividus

Naturally present antibacterial activity directed against Vibrio alginolyticus was demonstrated in coelomocytes lysate (CL) and cell-free coelomic fluid (CF) of the marine echinoderm Paracentrotus lividus. Kinetic analysis revealed that 5 min of contact was enough to induce significant bactericidal effect. Maximum activity required 30 min of contact. Nonsensitive to the effect of trypsin, the activity was almost completely suppressed by incubation with subtilisin. Purified from CL by three successive steps of chromatography (gel filtration, anion exchange, reverse phase), active antibacterial protein appeared as a single polypeptide chain of approximate molecular weight of 60 kDa.

Characterization of Proteolytic Activity in Coelomic Fluid of Lumbricus terrestris L. (Annelida, Lumbricidae)

Abstract Dose-response curves, time course, pH dependence and temperature sensitivity of proteolytic activity are presented for the coelomic fluid of untreated (CF) and stimulated (CFst) earthworms ( L. terrestris ). Proteolytic enzymes were observed in four fractions after HPLC gelfiltration and DEAE-ion exchange chromatography in CFst. Eight proteases of different electrophoretic mobility were identified after native PAGE and gelatin-agar overlay. The ability of proteolytic fractions to react with specific protease substrates Suc-Ala-Ala-Pro-Phe-thiobenzyl-ester (SPT) and N-benzylcarbonyl-ester (BLT) shows their chymotrypsin- and trypsin-like character. The occurrence of an increased number of proteases after stimulating the earthworms coelomic cavity where leukocytes reside is discussed.

Purification of three serine proteases from the coelomic cells of earthworms (Eisenia fetida)

Abstract 1. 1. Proteinase activity is evidenced in coelomic cell lysate of Eisenia fetida andrei. 2. 2. Tested on gelatin plates, the proteolysis develops rapidly during the first hr of incubation at 37°C. 3. 3. The proteolysis is pH-dependent with two optima for pH 7.0 and 10.0. 4. 4. The proteolysis is dose-dependent. 5. 5. The proteolysis is not influenced by ionic strength. 6. 6. The proteolysis is thermo-sensitive but not totally suppressed by 15 min at 100°C. 7. 7. Three serine proteases, A2 (Mr 42 kDa), B2 (Mr 46 kDa) and C2 (Mr 48 kDa), were purified by affinity chromatography on benzamidine-Sepharose. 8. 8. According to various inhibition and activity tests, A2 is of trypsin-like type, Ca2+-independent but Mg2+- and Mn2+-dependent and C2 is of chymotrypsin-like type.

Comparative aspects of echinoidea cytolysins: The cytolytic activity of Spherechinus granularis (echinoides) coelomic fluid

Abstract 1. 1. Cytolytic activity has been detected in coelomic fluid of the echinoid Spherechinus granularis. It was directed against both erythrocytes and malignant cell lines, but not against autologous and heterologous echinoid dechorionated eggs. 2. 2. The activity depends on molecules of proteic nature that are strongly thermolabile and calcium-dependent. Fungal (zymosan) and algal (laminarin) components appear to reduce, depending on dose, the hemolytic potency of the samples. Moreover, treatment with lipid vesicles, constituted by phosphatidyl-ethanolamine, completely abrogate the lytic activity. 3. 3. The addition of critical Zn2+ ions concentrations produces a loss of the lytic activity in CF2+ samples.

Zinc effect on the sea urchin Paracentrotus lividus immunological competence

Pollution by heavy metals has become one of the most important problems in marine coastal areas as a consequence of anthropogenic inputs. Among metal contaminants, zinc, being considered not very toxic, is sometimes released into the sea in appreciable quantities and its concentration is loosely regulated. In this work we analyzed the effects of a high zinc concentration on the sea urchin Paracentrotus lividus immune system. In particular, after 24 h of zinc treatment, we evaluated coelomocytes morphology and composition as well as the zinc influence on some humoral parameters such as hemolysis, lysozyme-like activity and antibacterial activity on Vibrio alginolyticus. Our results evidenced that the presence of zinc affected both cellular and acellular components of the sea urchin immune system. The P. lividus coelomocytes changed in morphology and number; moreover, the amebocytes changed from a petaloid to a filipodial-like shape and the red spherula cells increased in number. Among the considered humoral effectors lysozyme-like activity and antibacterial activity on V. alginolyticus decreased in short-term to zinc treatment. The modifications in the sea urchin immunological competence might give an early indication of disease susceptibility thus suggesting to consider the examined defence mechanisms as potential biological indicators of metal pollution.

Potential effects of an invasive seaweed ( Caulerpa cylindracea , Sonder) on sedimentary organic matter and microbial metabolic activities

Caulerpa cylindracea (Sonder), among the most successful marine bio-invaders on a global scale, poses severe threats to biodiversity. However, the effects of this seaweed on the quantity and the biochemical composition of sedimentary organic matter are still poorly known. Since the whole set of sedimentary features affects the availability of substrates for benthic microbial communities, we: i) investigated the biochemical composition of sediments colonized and not-colonized by C. cylindracea, and ii) compared the metabolic patterns of the microbial communities associated with C. cylindracea and in the sediments colonized and not-colonized by the seaweed. Our results show that C. cylindracea can influence the quantity and biochemical composition of sedimentary organic matter (OM), and that microbial populations associated with colonized sediments do have specific metabolic patterns and degradation capacities. Caulerpa cylindracea can also influence the metabolic patterns of the microbial community specifically adapted to degrade compounds released by the seaweed itself, with possible consequences on C cycling.

The sea urchin Paracentrotus lividus immunological response to chemical pollution exposure: The case of lindane

In the marine environment organochlorine insecticides can be broadly detected in water, sediments, and biota. These pollutants may have major ecological consequences since they may affect marine organisms and endanger organismal growth, reproduction or survival. In this study we investigated the modification of some sea urchin immunological parameters in response to subchronic lindane (γ-HCH) exposure. Adult specimens of the sea urchin Paracentrotus lividus were exposed to two different concentrations (0.1 and 0.5 mg L(-1)) of lindane. After 24 and 48h of treatment, we examined the lindane influence on coelomocytes vitality and enumeration as well on some humoral parameters. Our results showed that the presence of the pesticide affected both cellular and humoral components of the immune system. In particular, P. lividus coelomocytes vitality did not change but a decrease of the total cell number and an increase of the red cells was recorded. Haemolytic and lysozyme-like activities as well as antibacterial activity on Vibrio alginolyticus of treated animals decreased. Sea urchin immunological competence modifications might represent a tool for monitoring disease susceptibility thus providing biological criteria for the implementation of water quality standards to protect marine organisms.

PROTEASE INHIBITOR IN HOLOTHURIA POLII COELOMIC FLUID

Abstract 1. 1. A coelomic fluid protease inhibitor (CFI) for trypsin and subtilisin, but not for α-chymotrypsin, was found in Holothuria polii . 2. 2. The factor cannot be dialysed and is relatively heat-stable. 3. 3. Purified to apparent homogeneity by gel filtration and hydrophobic interaction chromatography, the inhibitor appears as a monomeric protein with a mol. wt of about 26,000.

Echinodermata: The Complex Immune System in Echinoderms

The Echinodermata are an ancient phylum of benthic marine invertebrates with a dispersal-stage planktonic larva. These animals have innate immune systems characterized initially by clearance of foreign particles, including microbes, from the body cavity of both larvae and adults, and allograft tissue rejection in adults. Immune responsiveness is mediated by a variety of adult coelomocytes and larval mesenchyme cells. Echinoderm diseases from a range of pathogens can lead to mass die-offs and impact aquaculture, but some individuals can recover. Genome sequences of several echinoderms have identified genes with immune function, including expanded families of Toll-like receptors, NOD-like receptors, and scavenger receptors with cysteine-rich domains, plus signaling pathways and cytokines. The set of transcription factors that regulate proliferation and differentiation of the cellular immune system are conserved and indicate the ancestral origins of hematopoiesis. Both larval and adult echinoderms are in constant contact with potential pathogens in seawater, and they respond to infection by phagocytosis and encapsulation, and employ proteins that function in immune detection and response. Antipathogen responses include activation of the SpTransformer genes, a complement system, and the production of many types of antimicrobial peptides. Echinoderms have homologues of the recombinase activating genes plus all associated genes that function in vertebrates for immunoglobulin gene family rearrangement, although their gene targets are unknown. The echinoderm immune system has been characterized as unexpectedly complex, robust, and flexible. Many echinoderms have very long life-spans that correlate with an excellent capacity for cell damage repair. In many marine ecosystems, echinoderms are keystone predators and herbivores, and therefore are species that can serve as optimal sentinels of environmental health. Coelomocytes can be employed in sensor systems to test for the presence of marine pollutants. When Elie Metchnikoff inserted a rose prickle into a larval sea star and observed chemotaxis, phagocytosis, and encapsulation by the mesenchyme cells, he initiated not only the field of immunology but also that of comparative immunology, of which the echinoderms have been an important part.