Haemolytic activity and characterization of nematocyst venom fromPelagia noctiluca(Cnidaria: Scyphozoa)

We investigated the haemolytic capacity of the crude venom extracted from isolated nematocysts of Pelagia noctiluca (Cnidaria: Scyphozoa), and evidenced the proteic fractions responsible for this activity. The nematocyst venom was used at various concentrations to evaluate the haemolytic activity and the lysosomal membrane stability of red blood cells of two teleostean species treated with the extract. The nematocyst extract was assayed against erythrocytes of the two teleostean species living in different environments, Carassius auratus as a common freshwater species, and Liza aurata as a representative of seawater species. Experiments on the haemolytic activity of P. noctiluca in the pres…

Inflammatory-like reaction following bacterial injection and antimicrobial peptide isolation from Anemonia sulcata (Cnidaria)

Antimicrobial response in Anemonia sulcata (Cnidaria)

Risposta infiammatoria all’inoculo di batteri ed isolamento di peptidi antimicrobici in Anemonia sulcata (Cnidaria)

Titolo: Novel proteases from marine organisms with potential interest in restoration procedure

Isolamento, caratterizzazione e distribuzione cellulare di una neurotossina ad attività litica di Actinia equina (Anthozoa, Cnidaria

Components of hemocyte extracts from marine invertebrates exert antimicrobial activity

Identification of antimicrobial peptides in the gonad of European sea bass males and females

In vertebrates, the gonad is considered an immunologically privileged site as it triggers lower immune responses aiming to avoid germ cell damage. In fish,several studies reported that leucocytes show conditioned immune response and modulate some reproductive functions, allowing the pathogen to establish chronic and latent infections into reproductive organs. In mammals, antimicrobial peptides (AMPs) have recently been recognised as important effectors in male reproductive tract immunity. In fish, AMPs are increasingly recognized as a critical first line of defence against many pathogens as bacteria, fungi, virus, protozoa and even tumour cells. We have recently determined that the European…

Attività biologica e caratterizzazione molecolare di un peptide neurotossico ad attività litica di Actinia equina (Anthozoa, Cnidaria)

Upregulated transcription of phenoloxidase genes in the pharynx and endostyle of Ciona intestinalis in response to LPS

We investigated the role of phenoloxidases (POs) in ascidians inflammatory reaction, a components of a copper-containing protein family involved in invertebrate immune system. In Ciona intestinalis two phenoloxidases (CinPO-1, CinPO-2) have been sequenced. In the present study, real time PCR analysis showed that both CinPO-1 and CinPO-2 genes were modulated by LPS inoculation suggesting that they are inducible and highly expressed in the inflamed pharynx. In situ hybridization disclosed CinPO-1 and CinPO-2 transcripts in pharynx hemocytes (granulocytes) and, mainly, in unilocular refractile granulocytes (URG) which mainly populated the inflamed tunic matrix. Interestingly, the genes are als…

Further insight on Ciona intestinalis prophenoloxidase system activated during the LPS induced inflammatory response

From Cnidarian immunobiology to cultural heritage applications

The study of cnidarians immunity, as model systems of metazoans, lead additional informations on the first steps of the immunity evolution. The functions of the genes and cellular pathways in higher vertebrates are conserved during the evolution of metazoans, as shown by the discovery of homologues in cnidarians. These basal metazoans in fact, are far from "simples" in the range of methods at their disposal to deal with potential prey but also invading microbes and pathogens. They can give informations about the invertebrates innate immune repertoire. We investigated the immunobiology starting from the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of sub…

Cold-active molecules for a sustainable preservation and restoration of historic-artistic manufacts

In the last decades biotechnology research provides sustainable alternatives to traditional procedures for preventive preservation of cultural assets. Recently, bioactive molecules (BMs) isolated from marine invertebrate organisms have been isolated and tested for bioremoval of protein layers (BMP) or to controlling microbial colonization (BMA), acting at temperature lower than 30°C. The Protease or Antimicrobial activity was tested on ad hoc assembled specimens and on different historic-artistic manufacts. In bio-removing protocol BMP molecules were applied as gelled solutions, in order to guarantees a selective action, respectful of constitutive materials and manufact integrity. Peculiari…

Il sistema della profenolossidasi in Ciona intestinalis. Caratterizzazione e interazione tra fenolossidasi, perossinectina e superossido dismutasi Cu-Zn dipendente nella risposta infiammatoria indotta da LPS

Blue Biotechnology and Cultural Heritage: case studies

In order to promote innovative methodologies for conservation and restoration of historic-artistic manufacts, the efforts are focalized on the development of “sustainable” applications as alternatives to traditional restoration procedures, which can sometimes be detrimental for the artworks, humans and environment [1]. The role of Biotechnology in this field showed very useful applications for diagnosis of bio-deterioration of cultural assets, by an integrated methodology based on molecular and microbiological skills, and in bio-cleaning / bio-removing of organic/inorganic layers from artwork surfaces by enzymes or viable bacteria cells [2, 3]. In this work, bioactive molecules isolated fro…

First evidence of antimicrobial activity of neurotoxin 2 from anemonia sulcata (Cnidaria)

International audience; We investigated the antibacterial activity of Anemonia sulcata (Cnidaria, Anthozoa) tentacle and body acidic extracts. Biochemical purification consisted of first step on solid phase Sep-Pak C8 column followed by several HPLC runs on C18 column using different conditions. Anti-Micrococcus lysodeikticus activity has been detected in 40 % acetonitrile fractions. The resulting purified molecule from tentacles had a molecular mass determined by MALDI-TOF mass spectrum of 4946,299 Da and has been completely sequenced. Its aa sequence revealed identity with the Neurotoxin 2 (ATX-II), a Na + channel blocking toxins. Consequently, ATX-II appeared to display a dual role as to…

Biotecnology and Cultural Heritage: bioactive molecules applied in restoration projects

specific inflammatory response of Anemonia sulcata (cnidaria) after bacterial injection cause tissue rejection and enzymatic activity alteration

The evolution of multicellular organisms was marked by adaptation to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self” have evolved into a long history of cellular and molecular strategies from damage repair to the co-evolution of host-pathogen interaction. The phylum of Cnidaria is one of the first branches in the tree of animal life to provide crucial insights on the evolution of immunity. Sea anemones (Anthozoa, Cnidaria) are benthic sessile species able to maintain the integrity of the tissues and allorecognition in colonial forms and to differentiate between symbionts and pathogenic intruders. We investigated the inflammatory response in sea ane…

Characterization of cellular and molecular responses of Actinia equina (Linnaeus, 1758)

Innovative and Integrated Strategies: Case Studies

In this chapter, case studies related to biodeterioration, bioaerosol, biocide and biocleaning are reported. The aim is highlighting the role of biology and biotechnology tools for the preventive conservation of organic and inorganic artifacts, understanding how traditional as well as innovative methods can help the conservationists to develop integrated strategies considering works of art/environment/ humans as a dynamic system. Particularly, based on the experience acquired during the researches of Laboratory of Biology and Biotechnology for Cultural Heritage (LaBBCH), the authors suggest several approaches to reveal and identify biological systems able to induce biodeterioration of cultu…

Reazione infiammatoria ed isolamento di un peptide antimicrobico da Anemonia sulcata (Cnidaria)

Marine organisms as source of bioactive molecules applied in restoration projects

Abstract In recent decades research in the conservation and restoration field has provided sustainable alternatives to traditional procedures for cleaning or controlling the microbial colonization of works of art. In the present study, for the first time novel bioactive molecules extracted from marine invertebrate organisms (Anthozoa) were tested instead of chemical compounds for removing protein layers or as a biocide for controlling fungal or bacterial colonization. In particular, Bioactive Molecules with Protease activity (BMP), acting in a temperature range of 4- 30°C, were tested for the hydrolysis of protein layers on laboratory specimens. The cleaning protocol provides a selective pr…

Evaluation of waterborne exposure to heavy metals in innate immune defences present on skin mucus of gilthead seabream (Sparus aurata)

Aquatic animals are continuously exposed to chemical pollutants but the effects evoked in skin surfaces, which receive the most direct contact with them, are poorly investigated. Terminal carbohydrate composition and immunological components present in skin mucus of gilthead seabream (Sparus aurata L.) specimens exposed to waterborne sublethal dosages of heavy metals [arsenic (As2O3), cadmium (CdCl2) and mercury (CH3HgCl) at 5, 5 and 0.04 μM, respectively for 2, 10 and 30 days were analysed. Moreover, the presence of a fucose binding lectin (FBL) was evaluated by western blot and the protein profiles were by SDS-PAGE and HPLC. Results showed little effects of heavy metals in the presence of…

Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration

The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ’’self’’ from ’’non-self” have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different ba…

BIOACTIVE MOLECULES FROM MARINE INVERTEBRATE ORGANISMS AS POTENTIAL TOOLS IN RESTORATION PROCEDURES

In the last decades molecular biology has provided innovative approaches in order to set up specific protocols for the conservation and restoration of cultural assets. In this study, which falls within the so called field of Blue-biotechnology, new bio-reactive peptides isolated from marine invertebrate organisrns (Cnidaria and Molluscs) were tested aim to bio-cleaning (proteolytic- peptides) the surfaces or to control (antimicrobial-peptides) the colonization of historic-artistic manufacts by fungi or bacteria. Particularly, the proteolytic-peptides showed hydrolytic activity, specific for animal-glue, in a range of temperatures of 4-37°C; than acting without heating the surface, by a cont…

Saggi sull’attività antimicrobica di molecole bioattive in interventi di foderatura di dipinti

In questo studio è stato valutato l’utilizzo di molecole antimicrobiche estratte da organismi marini invertebrati (Anthozoa), al fine di limitare o inibire la crescita microbica su materiali impiegati per il restauro di manufatti storico-artistici. I ceppi batterici e fungini utilizzati nei saggi antimicrobici, sono stati isolati da porzioni di tele, stratificate con colla, impiegate nella foderatura di dipinti. Mediante un approccio integrato che comprende tecniche di microscopia e tecniche molecolari, sono state identificate colonie batteriche appartenenti ai generi Enterobacter e Micrococcus e fungine appartenenti ai generi Aspergillus e Penicillium. L’attività antimicrobica delle moleco…

Removal of old protein layers from surfaces of works of art by new enzymes

The aim of the present project is to set up bio-cleaning protocols in order to easily remove altered protein layers (e.g. animal glues) by enzymatic proteins (proteases). Protein molecules were isolated from marine invertebrate organisms, characterized by size exclusion highpressure liquid chromatography (Waters: SEC-HPLC, BioSuite 250 to 10㎛ SEC 7.5 x 300 mm) and their gelatinase activity analyzed by zymography on polyacrilamide gel. The remarkably proteolytic activity and the reaction temperature range of these enzymes, 4° to 37℃, made it possible to hypothesize their use in bioremediation projects. Tests were performed on protein coating found on some polychrome wooden artifacts exposed …

Novel proteases from marine organisms with potential interest in restoration procedure

In the last decades, molecular biology allowed the development of innovative protocols in the field of conservation/restoration of cultural assets. In this work new hydrolyses, isolated from marine invertebrate organisms, are applied to remove protein layers form works of art surface. Proteolytic zymography assay evidenced that these enzymes are active in a broad temperature range, between 4 degrees and 37 degrees C. The enzymatic cleaning by these proteases, tested on wooden furniture of the second half of the eighteenth century showed positive results, without needing to heat the enzyme solution or the surface on which they were applied. The present report proposes novel proteases more ap…

Individual variability and gene expression specificity of the mussel antifungal mytimycin (MytM)