Humoral opsonin from the colonial ascidian Botryllus schlosseri as a member of the galectin family

The synthesis and secretion of the humoral opsonin from the colonial ascidian Botryllus schlosseri was studied by using specific polyclonal antibodies that recognise both of the bands obtained after SDS-PAGE of the purified lectin. The agglutinin was revealed on the surface of agglutinated yeast and red blood cells and in the cytoplasm of the phagocytes, either uniformly distributed or localised inside the cytoplasmic projections. A previous incubation of haemocytes with the agglutinin resulted in intense labelling of the surface that disappeared in the presence of D-galactose, whereas it was not affected by treatment with proteases. These results, together with the already published data, suggest that our molecule is a new member of the galectin family. Received: 16 July 1999 / Accepted: 7 February 2000     

Release of phagocytosis-stimulating factor(s) by morula cells in a colonial ascidian

In vitro yeast phagocytosis by haemocytes of the compound ascidian Botryllus schlosseri was studied, with particular attention to interactions among different immunocyte types. It is demonstrated that the supernatant from haemocyte cultures matched with yeast cells contains factor(s) able to enhance yeast ingestion by Botryllus phagocytes. The increase in phagocytosis is not the consequence of yeast opsonisation, as the phagocytic index does not significantly increase when yeast cells, previously incubated in the culture media, are washed and re-suspended in filtered sea water. When haemocytes were fractionated by density gradient centrifugation and each band was incubated with yeast, the ability to stimulate phagocytosis was found in the supernatants from haemocyte cultures of fractions rich in morula cells (MC). Previous studies have demonstrated that MC express molecules recognised by anti-cytokine antibodies, as a consequence of the recognition of foreign molecules or cells. Our results indicate that molecules immunoreactive with anti-cytokine antibodies are required for modulating phagocyte activity, as the above-reported enhancing effect is completely absent in the presence of anti-IL-1-α and anti-TNF-α, but not of anti-rabbit-IgG antibodies, and they also highlight the presence of ‘cross-talk‘ between MC and phagocytes. A new scenario is therefore sketched, in which MC actively recognise non-self molecular patterns and, upon this recognition, release humoural factor(s) recognised by phagocytes, which modulate phagocytosis.     

Purification and partial characterisation of phenoloxidase from the colonial ascidian Botryllus schlosseri

Phenoloxidase (PO) from the colonial ascidian Botryllus schlosseri was purified using two different chromatographic strategies. A three-step purification was developed in order to maintain enzyme activity, whereas an easier purification procedure was adopted to obtain enough PO for the production of specific polyclonal antibodies. The enzyme showed optimal pH and temperature values of 7.0 to 7.5 and 35 °C, respectively, and a K _m value of 4.62 ± 0.76 mM was estimated using l-DOPA as substrate. A molecular weight of 160 kDa was determined after SDS-PAGE under non-reducing conditions. The addition of the reducing agent β-mercaptoethanol caused the disappearance of the 160 kDa band and the appearance of a new band at 80 kDa, suggesting that active PO is a dimer and the two subunits are linked by disulphide bridges. Received: 14 December 1998 / Accepted: 24 August 1999     

Development of low-cost ammonia gas sensors and data analysis algorithms to implement a monitoring grid of urban environmental pollutants

The present study is focused on the implementation of a novel, low cost, urban grid of nanostructured chemresistor gas sensors for ammonia concentration ([NH(3)]) monitoring, with NH(3) being one of the main precursors of secondary fine particulate. Low-cost chemresistor gas sensors based on carbon nanotubes have been developed, their response to [NH(3)] in the 0.17-5.0 ppm range has been tested, and the devices have been properly calibrated under different relative humidity conditions in the 33-63% range. In order to improve the chemresistor selectivity towards [NH(3)], an Expert System, based on fuzzy logic and genetic algorithms, has been developed to extract the atmospheric [NH(3)] (with a sensitivity of a few ppb) from the output signal of a model chemresistor gas sensor exposed to an NO(2), NO(X) and O(3) gas mixture. The concentration of these pollutants that are known to be the most significant interfering compounds during ammonia detection with carbon nanotube gas sensors has been tracked by the ARPA monitoring network in the city of Milan and the historical dataset collected over one year has been used to train the Expert System.     

Immunotoxicity in ascidians: Antifouling compounds alternative to organotins – II. The case of Diuron and TCMS pyridine

Using short-term hemocyte cultures of the colonial ascidian Botryllus schlosseri exposed to various sublethal concentrations of Diuron (3-(3,4-diclorophenyl)-1,1-dimethylurea) and TCMS pyridine (2,3,5,6-tetrachloro-4-(metylsulphonyl)pyridine), we evaluated their immunotoxic effects through a series of cytochemical assays previously used for organotin compounds. At concentrations higher than 250 μ M and 10 μ M for Diuron and TCMS pyridine, respectively, both biocides exerted immunosuppressant effects on Botryllus hemocytes, causing i) deep changes in the cytoskeleton that irreversibly affect cell morphology and phagocytosis, ii) induction of DNA damage, iii) leakage of oxidative and hydrolytic enzymes due to membrane alteration. Unlike organotin compounds, Diuron and TCMS pyridine do not inhibit cytochrome-c-oxidase, and only TCMS pyridine triggers oxidative stress. When co-present, they exert an antagonistic interaction on cytoskeletal components.     

Irgarol inhibits the synthesis of ATP in mitochondria from rat liver

The interactions of Irgarol with rat liver mithocondrial have been investigated. The results indicate that Irgarol inhibits the ATP synthesis. The analysis of the various steps involved in the ATP synthesis suggests that the inhibition is due to the opening of small-size pores.     

Involvement of quinones and phenoloxidase in the allorejection reaction in a colonial ascidian, Botrylloides simodensis: histochemical and immunohistochemical study

When incompatible colonies of Botrylloides simodensis were brought into contact at their artificially cut surfaces, allorejection occurred and a black line was formed along the contact border. Morula cells (MCs), a type of hemocytes, are the major effector in the allorejection reaction and are known to possess phenoloxidase (PO) that generates quinones. In this rejection reaction, MCs infiltrate the tunic and break down, discharging their vacuolar contents. Ascorbic acid (antioxidant) and benzamidine (protease inhibitor) showed inhibitory effects on MC breakdown, black line formation and new tunic cuticle formation, whereas tropolone (metal chelator) and sodium benzoate (substrate analog) did not. MCs probably store some amount of quinones, as well as PO; oxidants derived from the quinones appear to disintegrate the tissue to form a black line and promote MC breakdown. Histochemical and immunohistochemical studies revealed that MCs contain eosinophilic materials, PO and quinones. Quinones that are stored in MCs and produced by PO probably have a destructive function, forming rejection lesions.     

Immunotoxic effects of organotin compounds in Tapes philippinarum

One of the most harmful groups of coastal pollutants is the organotin compounds (OTCs) which have severe effects on both aquatic organisms and mammals including humans. The immunotoxic effects of OTCs were studied in the cultivated clam Tapes philippinarum by determining the immunosuppressant role on in vitro yeast phagocytosis at low doses (0.01, 0.05, 0.1 μM). The phagocytic index was significantly reduced in an irreversible non-lethal manner depending on concentration and lipophilic affinity. The order of inhibition was TBT≥DBT>MBT for butyltins and TPTC>TPTA≥TPTH for triphenyltins.     

Immunotoxicity in ascidians: antifouling compounds alternative to organotins: III--the case of copper(I) and Irgarol 1051

After the widespread ban of TBT, due to its severe impact on coastal biocoenoses, mainly related to its immunosuppressive effects on both invertebrates and vertebrates, alternative biocides such as Cu(I) salts and the triazine Irgarol 1051, the latter previously used in agriculture as a herbicide, have been massively introduced in combined formulations for antifouling paints against a wide spectrum of fouling organisms. Using short-term (60 min) haemocyte cultures of the colonial ascidian Botryllus schlosseri exposed to various sublethal concentrations of copper(I) chloride (LC₅₀ = 281 μM, i.e., 17.8 mg Cu L⁻¹) and Irgarol 1051 (LC₅₀ > 500 μM, i.e., >127 mg L⁻¹), we evaluated their immunotoxic effects through a series of cytochemical assays previously used for organotin compounds. Both compounds can induce dose-dependent immunosuppression, acting on different cellular targets and altering many activities of immunocytes but, unlike TBT, did not have significant effects on cell morphology. Generally, Cu(I) appeared to be more toxic than Irgarol 1051: it significantly (p < 0.05) inhibited yeast phagocytosis at 0.1 μM (∼10 μg L⁻¹), and affected calcium homeostasis and mitochondrial cytochrome-c oxidase activity at 0.01 μM (∼1 μg L⁻¹). Both substances were able to change membrane permeability, induce apoptosis from concentrations of 0.1 μM (∼10 μg L⁻¹) and 200 μM (∼50 mg L⁻¹) for Cu(I) and Irgarol 1051, respectively, and alter the activity of hydrolases. Both Cu(I) and Irgarol 1051 inhibited the activity of phenoloxidase, but did not show any interactive effect when co-present in the exposure medium, suggesting different mechanisms of action.     

Immunotoxicity in ascidians: antifouling compounds alternative to organotins - II. The case of Diuron and TCMS pyridine

Using short-term hemocyte cultures of the colonial ascidian Botryllus schlosseri exposed to various sublethal concentrations of Diuron (3-(3,4-diclorophenyl)-1,1-dimethylurea) and TCMS pyridine (2,3,5,6-tetrachloro-4-(metylsulphonyl)pyridine), we evaluated their immunotoxic effects through a series of cytochemical assays previously used for organotin compounds. At concentrations higher than 250 micro M and 10 micro M for Diuron and TCMS pyridine, respectively, both biocides exerted immunosuppressant effects on Botryllus hemocytes, causing i) deep changes in the cytoskeleton that irreversibly affect cell morphology and phagocytosis, ii) induction of DNA damage, iii) leakage of oxidative and hydrolytic enzymes due to membrane alteration. Unlike organotin compounds, Diuron and TCMS pyridine do not inhibit cytochrome-c-oxidase, and only TCMS pyridine triggers oxidative stress. When co-present, they exert an antagonistic interaction on cytoskeletal components.