Laura Guidolin

Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri.

A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography. This agglutinin does not require metal cations for its activity and is specific for derivatives of D-galactose. On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions. This behavior is due to the establishment of disulfide bridge between the thiols of cysteine, well represented in the molecule as revealed by amino acid analysis. The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition. The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells. The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

Cadmium-thionein in Tetrahymena thermophila and Tetrahymena pyriformis.

The treatment of Tetrahymena thermophila with cadmium causes a reduction in growth rate according to dose; almost all the metal is accumulated in the cytosol where the Zn content is also increased threefold. Bio-Gel and Water 160 (HPLC) column chromatography show that Cd and Zn are bound to a protein with an ultraviolet (UV) spectrum that appears to be similar to that of Cd-metallothioneins isolated by higher organisms, but its molecular weight is greater: about 28 000 D, comparable to that of metallothionein isolated from Tetrahymena pyriformis. Further purification of these proteins by ion exchange chromatography revealed the presence of two peaks, considered as two isoforms of the metallothioneins present in both T. thermophila and T. pyriformis (MT 1 and MT 2). Their amino acid analyses confirmed that they are different isometallothioneins, MT 1 and MT 2, with about 30% cysteine, and aspartic acid, glycine and lysine as major amino acids. From our analyses we may conclude that Tetrahymena pyriformis MTs are similar to those present in invertebrates and vertebrates, while Tetrahymena thermophila MTs are peculiar in that they have cyclic amino acid histidine in both MT 1 and MT 2; furthermore, aromatic amino acid phenylalanine is also present in MT 2.

Effects of a new photoactivatable cationic porphyrin on ciliated protozoa and branchiopod crustaceans, potential components of freshwater ecosystems polluted by pathogenic agents and their vectors

The increasing use of photosensitized processes for disinfection of microbiologically polluted waters requires a precise definition of the factors controlling the degree of photosensitivity in target and non-target organisms. In this regard, tests with protozoa and invertebrates which have a natural habitat in such waters may be used as first screening methods for the assessment of possible hazards for the ecosystem. A new cationic porphyrin, namely meso-tri(N-methyl-pyridyl)mono(N-dodecyl-pyridyl)porphine (C12), is tested in this work on the protozoan Ciliophora Colpoda inflata and Tetrahymena thermophila and the Crustacea Branchiopoda Artemia franciscana and Daphnia magna. The protocol involved 1 h incubation with porphyrin doses in the 0.1-10.0 μM range and subsequent irradiation with visible light at a fluence rate of 10 mW cm(-2). The results indicate that C12 porphyrin has a significant affinity for C. inflata and T. thermophila; this is also shown by fluorescence microscopic analyses. C. inflata cysts were resistant to the phototreatment up to a porphyrin dose of 0.6 μM. The effects of C12 on cysts have been evaluated at 3 and 24 h after the end of the phototreatment; a delay in the excystment process was observed. T. thermophila was fairly resistant to the phototreatment with C12 porphyrin. The data obtained with the two crustaceans indicated that the effects of dark- and photo-treatment with C12 need to be closely examined for every organism. A. franciscana is more resistant, probably owing to its ability to adapt to extreme conditions, while the high level of photosensitivity displayed by Daphnia magna represents a potential drawback, as this organism is often selected as a reference standard for assessing the environmental safety. Thus, while C12 photosensitisation can represent a useful tool for inducing a microbicidal or larvicidal action on polluted waters, the irradiation protocols must be carefully tailored to the nature of the specific water basin, and in particular to its biotic characteristics.

Immunolocation of phenoloxidase in vacuoles of the compound ascidian Botryllus schlosseri morula cells

Abstract Using a polyclonal antibody raised against purified phenoloxidase from the colonial ascidian Botryllus schlossen, we studied its distribution among haemocytes and its intracellular location. The enzyme is present inside granular amoebocytes and morula cells, thus confirming the close relationship between the two cell types, as suggested by previous histochemical and histoenzymatic analysis. Immunocytochemistry in both light and electron microscopy shows that phenoloxidase is located inside the vacuoles of moru‐la cells, known to be the effectors of the rejection reaction.

Chelatins in Euglena gracilis and Ochromonas danica

Amino acid analysis was performed on low molecular weight copper binding proteins purified from two species of Protozoa after exposure to a high level of this metal. The compound from Ochromonas is similar to Cu-chelatins. The two peptides from Euglena have a different molecular weight and a very dissimilar amino acid composition. Peptide No. 1 has a peculiar composition with a high content of aspartic acid and arginine. Some speculations are made about its detoxification role in comparison with other compounds found in blue-green algae.

Chapter 1:Antimicrobial Photodynamic Therapy: Basic Principles

PDT of infectious diseases of microbial origin is based on the property of selected photosensitising agents, especially those characterized by the presence of positively charged functional groups, to readily bind with the outer wall of bacterial, fungal and protozoan cells; once activated by irradiation with visible light, the photosensitisers generate reactive oxygen species which induce the irreversible modification of specific constituents of the wall. The consequent enhanced permeability of the wall allows sufficiently large doses of the photosensitiser to penetrate to the cytoplasmic membrane. The photoprocess then involves a number of endocellular activities (e.g., enzymic catalysis, metabolic pathways) whose integrity is critical for cell survival. The genetic material is generally involved in the late stage of the photoprocess, which minimizes the probability of inducing mutagenic processes. A suitable choice of the irradiation protocol usually leads to an extensive killing of microbial pathogens with no or minimal damage to host tissues. PDT differs from other usually adopted therapeutic modalities for the broad spectrum of action, the efficacy against antibiotic resistant cells, and the lack of selection of photoresistant strains.

Mechanisms involved in the photosensitized inactivation of Acanthamoeba palestinensis trophozoites

Aims:  To advance our understanding of the mechanisms involved in the RLP068 phthalocyanine‐photosensitized inactivation of Acanthamoeba palestinensis trophozoites through a precise identification of the targets of the photoprocess in both the cytosolic and mitochondrial compartments.

Photodynamic Therapy: A Novel Promising Approach for the Treatment of Spontaneous Microbial Infections in Pet Animals

Photodynamic therapy with full spectrum visible light and porphyrin-type photosensitisers appears to efficiently induce the inactivation of a broad spectrum of microbial pathogens, including antibiotic-resistant strains, without in turn promoting the selection of PDT-resistant species. Application of this therapeutic modality to the treatment of spontaneously developed infections in dogs resulted in a very efficient healing of wounds with an extensive drop in the population of pathogens. The treatment appeared to be applicable for both mycotic and bacterial infections and was devoid of detectable undesired side effects. No significant differences in efficacy was observed whether a porphyrin, chlorin, or phthalocyanine photosensitising agent was used in spite of strong differences in the light absorption spectrum among the three tetrapyrrole derivatives.

Chapter 17:Inactivation of Microbial Pathogens by Photosensitized Processes: Environmental Applications

The photodynamic inactivation of microbial pathogens represents a very flexible technology which has a broad scope and potential for tackling a range of problems connected with the microbiological contamination of waters of different origin and physical or chemical characteristics. In this way it is possible to achieve a thorough control of the population of a variety of harmful bacteria, fungi and parasites by using a single protocol, which can be properly modulated, in fish-farming tanks, natural ponds, industrial wastewater, potable water basins. Moreover, the technique can be useful as a tool for the preservation of biodiversity through the protection of endangered species. The process can be considered as based on a natural chemical agent, the porphyrin, and a natural physical agent (sunlight or a solar simulator). Therefore, the impact of the technology on the environment is likely to be very low, especially since the dosages of the porphyrin are generally small and no significant accumulation in the application sites or their surroundings is expected.

Larval and pupal descriptions of Anomalini (Coleoptera: Scarabaeidae: Rutelinae) species from Ecuador

The third instars are described and illustrated for five Anomalini (Coleoptera: Scarabaeidae: Rutelinae) species from Ecuador: Anomala balzapambae Ohaus, 1897, A. popayana Ohaus, 1897, A. valida Burmeister, 1844, Callistethus buchwaldianus (Ohaus, 1908), and C. levii (Blanchard, 1851). The pupae of three Ecuadorian species are also described and illustrated: A. discoidalis Bates, 1888, A. popayana, and C. levii. Diagnostic characters of the species are provided. A key to the known larvae of Anomalini from the New World is provided, which now includes five genera and 31 species.