Benedetto Salvato

Evidence for a major role of plasma lipoproteins as hematoporphyrin carriers in vivo.

Hematoporphyrin (5 mg/ml), administered intravenously to tumor-bearing patients, becomes associated with different serum proteins, including lipoproteins (mainly HDL), globulin and albumin. No residual porphyrin is bound to the two latter classes of proteins after 48 h, whereas the complexation with the lipoproteins appears to be particularly stable probably owing to the hydrophobic nature of hematoporphyrin. The late persistence of hematoporphyrin in serum is due to the binding to the VLDL fraction with special regard to its cholesterol moiety. The importance of hematoporphyrin transport by lipoproteins for the photodynamic therapy of tumors is briefly discussed.

HEMATOPORPHYRIN-SENSITIZED PHOTOINACTIVATION OF Streptococcus faecalis

Abstract— The photosensitizing action of hematoporphyrin (HP) on the cells of some bacterial strains belonging to the species Streptococcus faecalis, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, as well as of strains of yeast Candida albicans, was studied in air‐equilibrated aqueous media at pH 7.2. All Gram‐negative strains appeared to be resistant even to prolonged irradiation both in the presence and in the absence of EDTA. On the other hand. Gram‐positive and yeast strains were photosensitive. In the case of Str. faecalis, whose strains displayed a particularly high photosensitivity, the efficiency of the photoprocess was mainly controlled by the HP concentration in the incubation medium (0.1‐1 μg/ml) and by the irradiation time (0–10 min); temperature (18–37oC) exerted a minor effect, whereas the incubation time (6–30 min) had no detectable influence. The photolability of Str. faecalis cells was only slightly different in the logarithmic and stationary phase of growth. The type of photoinduced ultrastructural modifications and the largely preferential binding of HP to the cytoplasmic membrane, as observed by cell fractionation studies, suggest that one important target of the HP‐photosensitized process is represented by membrane components.

THE O-DIPHENOL OXIDASE ACTIVITY OF ARTHROPOD HEMOCYANIN

Arthropod hemocyanin (isolated from the crab Carcinus maenas and the lobster Homarus americanus) is usually referred to as an oxygen transport‐storage protein. The protein, however, also catalyses with low efficiency the oxidation of o‐diphenol to quinone, similarly to tyrosinase (monophenol,o‐diphenol: oxygen oxidoreductase). The enzymatic parameters of hemocyanin are affected by the aggregation state of the protein; namely V max exhibited by a dissociated subunit is one order of magnitude greater than that of aggregated species. The reaction velocity is increased by the presence of perchlorate, an anion of the Hofmeister series. The results are also discussed on the basis of active site accessibility in comparison with tyrosinase.

Effects of copper and cadmium on growth, superoxide dismutase and catalase activities in different yeast strains.

1. Three strains of Saccharomyces cerevisiae have been adapted in vitro upon treatment with copper or cadmium. Growth rate, cellular size, metal uptake, superoxide dismutase and catalase activities were measured. 2. Growth rate and metal uptake are quite different among the yeast strains and also for copper and cadmium treatment. At the employed concentrations, only cadmium chiefly affects the cellular volume. 3. Cu, ZnSOD activity is stimulated in the presence of copper, while it is lightly inhibited in the presence of cadmium. Catalase level remains almost unchanged in the conditions tested. This lack of correlation is then discussed.

Structural properties of Rapana thomasiana grosse hemocyanin: Isolation, characterization and N-terminal amino acid sequence of two different dissociation products

1. The native Rapana thomasiana grosse hemocyanin is dissociated under mild conditions and fractionated into two dissociation products, RHSS1 and RHSS2, with an apparent molecular mass of approximately 250 and approximately 450 kDa, respectively. The two species are present in approximately equivalent amounts. SDS-PAGE analysis reveals that the latter component is a dimer of approximately 250 kDa polypeptide chains. 2. The amino acid compositions, as well as some spectroscopic properties of RHSS1, are very similar to those of RHSS2. After dissociation under mild conditions of the native hemocyanin both species preserve their capability of binding reversibly molecular oxygen. 3. RHSS1 and RHSS2 are sequenced directly from the amino-terminus for 15 and 20 steps, respectively. These parts of the two polypeptide chains are highly homologous but with microheterogeneity associated with some positions. They also exhibit high homology with the N-terminal region of subunits or functional domains of other gastropod Hcs.

A key structural role for active site type 3 copper ions in human ceruloplasmin.

Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.

Characterization of the carbohydrate moieties of the functional unit RvH1-a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion.

The primary structures of two biantennary N -glycans of the glycoprotein Rapana venosa (marine snail) haemocyanin were determined. Two different structural subunits have been found in R. venosa haemocyanin: RvH1 and RvH2. The carbohydrate content of the N-terminal functional unit RvH1-a of RvH1 was studied and compared with the N-terminal functional unit RvH2-a of RvH2. Oligosaccharide fragments were released from the glycoprotein by Smith degradation of a haemocyanin pronase digest and separated on a Superdex 300 column. The glycopeptide fragments, giving a positive reaction for the orcinol/H2SO4 method, were separated by HPLC. In order to determine the linked sugar chains to the hinge glycopeptides isolated from the functional unit RvH1-a, several techniques were applied, including capillary electrophoresis, matrix-assisted laser desorption ionization-MS and electrospray ionization-MS in combination with glycosidase digestion. On the basis of these results and amino acid sequence analysis, we concluded that the functional unit RvH1-a contains 7% oligosaccharides N-glycosidically attached to Asn262 and Asn401, and the following structures were suggested:[structure: see text]

Cannibalistic behaviour in the harpacticoid copepod Tigriopus fulvus

The results reported here demonstrate the existence of cannibalistic behaviour in laboratory-reared Tigriopus fulvus (Fischer, 1860) females towards first-stage non-related nauplii; mothers, sparing their own offspring, do not share this behaviour. The phenomenon is related to the probability of nauplius/female encounters. It is suggested that mother/offspring recognition is mediated by a chemical compound.

Cuprizone neurotoxicity, copper deficiency and neurodegeneration

Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico-chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.

The hemocyanin of Aplysia limacina: Chemical and functional characterization

1. 1. The Hcy of a mediterranean opisthobranch gastropod, Aplysia limacina, has been characterized. Amino acid composition, sedimentation coefficients, the reaction with oxygen and the ultrastructure have been studied. 2. 2. This Hcy is very similar to other gastropod Hey, as are amino acid composition and general structure. 3. 3. Differences are observed both in the ability to form supramolecular aggregates and in the size and shape of the subunits observed by electron microscopy.