The monomer state of beta-amyloid: where the Alzheimer's disease protein meets physiology.

One hundred years of study have identified beta-Amyloid (A beta) as the most interesting feature of Alzheimer's disease (AD). Since the discovery of A beta as the principal component of amyloid plaques, the central challenge in AD research has been the understanding of A beta involvement in the neurodegenerative process of the disease. The ability of A beta to undergo conformational changes and subsequent aggregation has always been a limiting factor in finding out the activities of the peptide. Extensive research has been carried out to study the molecular mechanisms of amyloid self-assembly. The finding that soluble Abeta concentrations in the brain are correlated with the severity of AD, whereas fibrillar density is not /40,42/, has pointed attention toward the oligomeric forms of Abeta, which are generally considered the most toxic and, therefore, the most important species to be addressed. Despite great efforts in basic AD research, none of the currently available treatments is able to treat the devastating effects of the disease, leading to the consideration that there is more to reason than just A beta production and aggregation. Here we summarize the emerging evidence for the physiological functions of A beta, including our recent demonstration that A beta monomers are endowed with neuroprotective activity, and propose that A beta aggregation might contribute to AD pathology through a "loss-of-function" process. Finally, we discuss the current therapeutics targeting the cerebral load of A beta and possible new ones aimed at preserving the biological functions of A beta.

Focusing on the functional characterization of the anserinase from Oreochromis niloticus.

Carnosine, anserine and homocarnosine are the three most representative compounds of the histidine dipeptides family, widely distributed in mammals in different amounts depending on the species and the tissue considered. Histidine dipeptides are mainly degraded by two different carnosinase homologues: a highly specific metal-ion dependent carnosinase (CN1) located in serum and brain and a non-specific cytosolic form (CN2). The hydrolysis of such dipeptides in prokaryotes and eukaryotes is also catalyzed by the anserinase (ANSN). Such naturally occurring dipeptides represent an interesting topic because they seem to have numerous biological roles such as potential neuroprotective and neurotransmitter functions in the brain and therefore ANSN results to be a very interesting target of study. We here report, for the first time, cloning, expression of ANSN from the fish Oreochromis niloticus both in a mammalian and in a prokaryotic system, in order to perform deep functional studies by enzymatic assays in the presence of different metals and substrates. Furthermore, by means of a mass spectrometry-based proteomic approach, we analysed protein sequence and the potential presence of post-translational modifications in the mammalian recombinant protein. Finally, a preliminary structural characterization was carried out on ANSN produced in Escherichia coli.

Prion proteins leading to neurodegeneration.

Prion diseases are fatal neurodegenerative disorders related to the conformational alteration of the prion protein (PrP C) into a pathogenic and protease-resistant isoform PrP(Sc). PrP(C) is a cell surface glycoprotein expressed mainly in the central nervous system and despite numerous efforts to elucidate its physiological role, the exact biological function remains unknown. Many lines of evidences indicate that prion is a copper binding protein and thus involved in the copper metabolism. Prion protein is not expressed only in mammals but also in other species such as birds, reptiles and fishes. However, it is noteworthy to point out that prion diseases are only observed in mammals while they seem to be spared to other species. The chicken prion protein (chPrP C) shares about 30% of identity in its primary sequence with mammal PrP C. Both types of proteins have an N-terminal domain endowed with tandem amino acid repeats (PHNPGY in the avian protein, PHGGGWQ in mammals), followed by a highly conserved hydrophobic core. Furthermore, NMR studies have highlighted a similar globular domain containing three alpha-helices, one short 3(10)-helix and a short antiparallel beta-sheet. Despite this structural similarity, it should be noted that the normal isoform of mammalian PrP C is totally degraded by proteinase K, while avian PrP C is not, thereby producing N-terminal domain peptide fragments stable to further proteolysis. Notably, the hexarepeat domain is considered essential for protein endocytosis, and it is supposed to be the analogous copper-binding octarepeat region of mammalian prion proteins. The number of copper binding sites, the affinity and the coordination environment of metal ions are still matter of discussion for both mammal and avian proteins. In this review, we summarize the similarities and the differences between mammalian and avian prion proteins, as revealed by studies carried out on the entire protein and related peptide fragments, using a range of experimental and computational approaches. In addition, we report the metal-driven conformational alteration, copper binding modes and the superoxide dismutase-like (SOD-like) activity of the related copper(II) complexes.

Copper binding to naturally occurring, lactam form of angiogenin differs from that to recombinant protein, affecting their activity.

Angiogenin is a member of the ribonuclease family and a normal constituent of human plasma. It is one of the most potent angiogenic factors known and is overexpressed in different types of cancers. Copper is also an essential cofactor in angiogenesis and, during this process, it is mobilized from inside to outside of the cell. To date, contrasting results have been reported about copper(ii) influencing angiogenin activity. However, in these studies, the recombinant form of the protein was used. Unlike recombinant angiogenin, that contains an extra methionine with a free terminal amino group, the naturally occurring protein present in human plasma starts with a glutamine residue that spontaneously cyclizes to pyroglutamate, a lactam derivative. Herein, we report spectroscopic evidence indicating that copper(ii) experiences different coordination environments in the two protein isoforms, and affects their RNase and angiogenic activity differently. These results show how relatively small differences between recombinant and wild type proteins can result in markedly different behaviours.

The insulin degrading enzyme activates ubiquitin and promotes the formation of K48 and K63 diubiquitin.

We report an ATP-dependent ubiquitin conjugation with IDE which, in turn, promotes Ub-Ub linkages in tube tests. We propose a novel function for IDE as a non-canonical ubiquitin activating enzyme.

Targeted photochemical modification of HIV-derived oligoribonucleotides by antisense oligodeoxynucleotides linked to porphyrins.

Antisense oligodeoxynucleotides directed against a 24-mer RNA derived from the long terminal repeat (LTR) region of HIV were linked to proto- and methylpyrroporphyrin and their zinc derivatives. The oligonucleotide-porphyrin conjugates were tested for their ability to induce photodamage on the target RNA. Upon hybridization followed by irradiation at 405 nm, the photochemical reaction led to photocross-linking of the antisense derivative to the RNA substrate. The protoporphyrin exhibited a much higher cross-linking yield than the methylpyrroporphyrin while the Zn-porphyrin derivatives were found to be less efficient than their corresponding nonmetallated congeners. The specificity of the photocross-linking reaction between the porphyrin-oligomer and its target RNA was demonstrated by the following evidence: (1) hybrid formation was required for photocross-linking to occur, (2) the sites of cross-linking on the target RNA were identified at G residues located in close proximity to the porphyrin photoactive center in the hybrid and (3) addition of bulk calf liver RNA did not affect the photocross-linking efficiency.

Cytoprotective effect of copper(II) complexes against ethanol-induced damage to rat gastric mucosa.

The cytoprotective effect of various copper(II) complexes on the gastric mucosa damage induced by acute intragastric administration of ethanol was investigated. For in vitro experiments, the following copper(II) complexes were tested: Cu(II)(L-Trp)(L-Phe), Cu(II)(L-Leu)Cu(II)(L-Leu-Leu)(L-Leu), Cu(II)(L-Phe-L-Leu), Cu(II)(Gly-His-Lys), and Cu(II)(cyHis)2(ClO4)2. Inorganic copper such as CuSO4 was also tested. The free radical generating system, acting for 2 hr on cardial and fundic mucosa scrapings or mucosal microsomes, was Fe++ (20 microM)/ascorbate (0.25 mM). We found a marked inhibition to 75% of lipid peroxidation in the range 10-100 mM, regardless of whether copper was given in complexed or inorganic form. The results suggest that nontoxic copper(II)-amino acid complexes are able to neutralize oxygen-derived free radicals. In addition, copper(II) complexes suppressed membrane lipid peroxidation when mucosa homogenates were exposed to t-butyl hydroperoxide (1-20 microM) plus Fe++ (50 microM). In vivo experiments on rat stomachs, pretreated p.o. by gavage either with Cu(II)(L-Trp)(L-Phe) as paradigmatic agent or with copper sulphate at equivalent doses in the range 3-30 mg/kg body weight showed a significant decrease (30 min after 95% ethanol administration) in the number and severity of mucosal hemorrhagic lesions. In the gastric mucosa scrapings of copper-treated rats after ethanol exposure, we found that malondialdehyde and conjugated diene levels were unchanged compared to those of untreated controls; five enzyme activities released from lysosomes were near control values. In isolated mucosal cells, whether or not pretreated with 200 microM solution of either Cu(II)(L-Trp)(L-Phe) or CuSO4, the release of cathepsin D activity was also unmodified. The results suggest that the cytoprotective effect of Cu(II) complexes against ethanol-induced mucosal lesions was not associated in vivo to lipid peroxidation.

Inhibition of photohemolysis by copper(II) complexes with SOD-like activity.

Red blood cell lysis photosensitized by Ketoprofen, 2-(3-benzoxyphenyl)propionic acid (KPF), was investigated in the presence of some copper(II) complexes with linear and cyclic dipeptides as well as functionalized beta-cyclodextrins with SOD-like activity with the aim of ascertaining their protective activity towards the photoinduced cell damage. The comparison between the decrease of photolytic activity caused by these complexes and their superoxide dismutase activity showed an appreciable correlation. The correct determination of species existing in experimental conditions was obtained through a simulation approach based on the knowledge of the stability constants of the complexes.

Copper(II) complexes encapsulated in human red blood cells.

Copper(II) complexes were encapsulated in human red blood cells in order to test their possible use as antioxidant drugs by virtue of their labile character. ESR spectroscopy was used to verify whether encapsulation in red blood cells leads to the modification of such complexes. With copper(II) complexes bound to dipeptides or tripeptides, an interaction with hemoglobin was found to be present, the hemoglobin having a strong coordinative site formed by four nitrogen donor atoms. Instead, with copper(II) complexes with TAD or PheANN3, which have the greatest stability. ESR spectra always showed the original species. Only the copper(II) complex with GHL gave rise to a complicated behavior, which contained signals from iron(III) species probably coming from oxidative processes. Encapsulation of all copper(II) complexes in erythrocytes caused a slight oxidative stress, compared to the unloaded and to the native cells. However, no significant differences were observed in the major metabolic properties (GSH, glycolytic rate, hexose monophosphate shunt, Ca(2+)-ATPase) of erythrocytes loaded with different copper(II) complexes, with the exception of methemoglobin levels, which were markedly increased in the case of [Cu(GHL)H-1] compared to [Cu(TAD)]. This latter finding suggests that methemoglobin formation can be affected by the type of complex used for encapsulation, depending on the direct interaction of the copper(II) complex with hemoglobin.

Cytotoxic and cytostatic activity of copper(II) complexes. Importance of the speciation for the correct interpretation of the in vitro biological results.

The cytotoxicity of some copper(II) compounds against the mouse cancer cell line B16, murine L929, human KB cells, and fibroblasts was investigated. All the copper(II) systems tested were shown to have pronounced toxicity against transformed cells and a cytostatic effect against untransformed cells, i.e., human fibroblasts. A careful speciation of the actual in vitro conditions reveals that copper(II) is essentially present as mixed complexes formed with the amino acids of the culture medium, [Cu(glutamine)(histidine)] being the main species. It was found that the cytotoxic activity is related to the amount of copper(II) contained in the tested compounds.

Determination of superoxide dismutase-like activity of copper(II) complexes. Relevance of the speciation for the correct interpretation of in vitro O2- scavenger activity.

The superoxide dismutase activity of several copper(II) complexes of linear and cyclic dipeptides has been measured. The results of a classic indirect method (xanthine-xanthine oxidase) have been compared with those obtained by generation of the superoxide radical through 2-(3-benzoylphenyl)propionic acid (ketoprofen) photolysis. A simulation approach, based on the knowledge of the stability constants of the different complex species existing in experimental conditions, has allowed us to obtain the correct speciation and to use these data to calculate the pertinent catalytic constants.

A comparative study of two isoforms of laccase secreted by the "white-rot" fungus Rigidoporus lignosus, exhibiting significant structural and functional differences.

Two isoforms of laccase were obtained as the predominant phenol-oxidases in defined medium liquid cultures of the "white-rot" fungus Rigidoporus lignosus (R. lignosus). A characterization of the two laccases was made in terms of molecular mass, isoelectric point, metal content and N-terminal sequence. Furthermore, in order to gain information on the structural features related to the metal centers, a study of their geometric arrangement and their redox ability was made. It turned out that the two isoenzymes greatly differed with regard to pH stability, catalytic and copper centers features. It is proposed that all such differences are dependent on the amino acid sequences, which cause a distortion of the copper sites, thus accounting for the redox potential values and kinetic properties.

Synthesis and structural characterization of 6I,6II-diamino-6I,6II-dideoxy-cyclomaltoheptaose, a difunctionalized beta-cyclodextrin.

6I,6II-Diamino-6I,6II-dideoxy-cyclomaltoheptaose was prepared using the regioselective procedure described by Tabushi. The difunctionalized beta-cyclodextrin crystallizes as hexadecahydrate in the orthorhombic space group P2(1)2(1)2(1), with a = 11.395(3), b = 32.989(9), c = 17.560(5) A, V = 6601 A3, Z = 4. The structure was solved by molecular replacement techniques using the program PATSEE and was refined to a conventional final R = 0.058 for the 5031 observed reflections with I > or = 3 sigma(I). The beta-CD macrocycle presents only slight differences with respect to uncomplexed hydrated or methylated beta-CD. The macrocycle structure maintains an approximate seven-fold symmetry. The round shape of the cyclodextrin ring is stabilized by intramolecular O-H ... O H-bonds between the secondary hydroxyl groups of neighbouring glucose residues. Along the a axis, the beta-CD molecules are arranged in columns; the macrocycles form a herring-bone pattern, so that the cavity of each beta-CD molecule is closed at each end by neighbouring molecules. The macrocycles are directly linked to each other by H-bonds involving either primary and secondary hydroxyl or amino groups of symmetry-related molecules. The resulting layers are connected to each other by a dense intermolecular hydrogen-bond network, in which solvent molecules participate.

Chemical characterization of sicilian prickly pear (Opuntia ficus indica) and perspectives for the storage of its juice.

In this work, Sicilian cultivars of prickly pear (Opuntia ficus indica) were partially characterized from a chemical point of view, and the possibility of long-term storage of their juice was investigated. The acidity of the prickly pear juice turned out to be very low (0.02%) and the pH very high (6.4-6.5) if compared with values found in other common fruit juices. In the perspective of processing and storage conditions according to Italian law, the acidity has been corrected by adding the proper amount of tartaric and/or phosphoric acid. The sugar content (mainly glucose and fructose) is very high (11-12%), and also L-ascorbic acid is present in considerable amount (31-38 mg/100 g). Among the transition metals, a high content of manganese(II) (1.7-2.9 ppm) and good amounts of iron(III) (0.6-1.2 ppm) and zinc(II) (0.3-0.4 ppm) were found. In particular, such ions appear to be present mainly in the thick skin of the fruit or "trapped" inside the pulp. Pectin methylesterase (PME) seems to be present in very small amount and/or is not highly active. Furthermore, PME activity decreases considerably after the necessary adjustment of the pH and the thermal treatment requested for long-term storage. After approximately 2 months, none of the juices prepared was affected by noticeable sedimentation of the pulp. Finally, different samples of prickly pear juice were sensorially analyzed, employing descriptors such as color, aroma, viscosity, acidity, sweetness, and off-flavors. The results obtained can be considered very satisfactory, and the juice has been widely appreciated when compared with other products commonly available on the market such as pear and peach juices.

A non-linear least-squares approach to the refinement of all parameters involved in acid-base titrations.

A non-linear least-squares computer program has been written for the refinement of the parameters involved in potentiometric acid-base titrations. The program ACBA (ACid-BAse titrations) is applicable under quite general conditions to solutions containing one or more acids or bases. The method of refinement used gives the program several advantages over the other programs described previously.

Protective effect of carnosine during nitrosative stress in astroglial cell cultures.

Formation of nitric oxide by astrocytes has been suggested to contribute, via impairment of mitochondrial function, to the neurodegenerative process. Mitochondria under oxidative stress are thought to play a key role in various neurodegenerative disorders; therefore protection by antioxidants against oxidative stress to mitochondria may prove to be beneficial in delaying the onset or progression of these diseases. Carnosine has been recently proposed to act as antioxidant in vivo. In the present study, we demonstrate its neuroprotective effect in astrocytes exposed to LPS- and INFgamma-induced nitrosative stress. Carnosine protected against nitric oxide-induced impairment of mitochondrial function. This effect was associated with decreased formation of oxidatively modified proteins and with decreased up-regulation oxidative stress-responsive genes, such as Hsp32, Hsp70 and mt-SOD. Our results sustain the possibility that carnosine might have anti-ageing effects to brain cells under pathophysiological conditions leading to degenerative damage, such as aging and neurodegenerative disorders.

Imaging of kinked configurations of DNA molecules undergoing orthogonal field alternating gel electrophoresis by fluorescence microscopy.

The dynamics of individual DNA molecules undergoing orthogonal field alternating gel electrophoresis (OFAGE) have been studied by use of T2 DNA molecules labeled with a dye and visualized with a fluorescence microscope. The mechanism of reorientation used by a molecule to align itself in the direction of the new orthogonal field depends on the degree of extension of the chain immediately before the application of this field. The formation of kinks is promoted when time is allowed between the application of the two orthogonal fields so that the molecule attains a partially relaxed configuration. In this case, the chain appears bunched up in domains moving along the contour of the molecule. These regions are found to be the locations where the kinks are formed upon application of the second field perpendicular to the chain. The formation of kinks provides a significant retardation of the reorientation of the molecules, relative to molecules that do not form kinks, and appears to play an important role in the fractionation attained with OFAGE. A classification of various reorientation mechanisms observed in molecules that form kinks is presented.

Production, purification, and properties of an extracellular laccase from Rigidoporus lignosus.

Laccase from Rigidoporus lignosus, a white-rot basidiomycete, has been isolated from culture filtrates. The enzyme was purified to homogeneity and some of its structural and kinetic parameters have been determined. The effects of pH, temperature, and organic solvents on the activity and stability of the enzyme, under different conditions, were also assayed. The results we have obtained, including the rather broad substrate specificity of enzyme, combined with their relatively easy production and purification, suggest that laccase may be efficiently employed in a variety of biotechnology applications.

Copper(II) binding modes in the prion octapeptide PHGGGWGQ: a spectroscopic and voltammetric study.

The N-terminal octapeptide repeat region of human prion protein (PrPc) is known to bind Cu(II). To investigate the binding modes of copper in PrPc, an octapeptide Ac-PHGGGWGQ-NH2 (1), which corresponds to an octa-repeat sequence, and a tetrapeptide Ac-HGGG-NH2 (2) have been synthesised. The copper(II) complexes formed with 1 and 2 have been studied by circular dichroism (CD) and electron spin resonance (ESR) spectroscopy. Both peptides form 1:1 complexes with Cu(II) at neutral and basic pH. CD, ESR and visible absorption spectra suggest a similar co-ordination sphere of the metal ion in both peptides, which at neutral pH consists of a square pyramidal geometry with three peptidic nitrogens and the imidazole nitrogen as donor atoms. Cyclic voltammetric measurements were used to confirm the geometrical features of these copper(II) complexes: the observation of negative redox potentials are in good agreement with the inferred geometry. All these results taken together suggest that peptide 1 provides a single metal binding site to which copper(II) binds strongly at neutral and basic pH and that the binding of the metal induces the formation of a stiffened structure in the HGGG peptide fragment.

Electrospray mass spectrometric studies of L-carnosine (beta-alanyl-L-histidine) complexes with copper(II) or zinc ions in aqueous solution.

Electrospray ionization mass spectrometry (ESI-MS) was used for the speciation of supramolecular assemblies formed between equimolar amounts of carnosine and copper or zinc ions in dilute aqueous solutions. In the case of pure carnosine and carnosine/copper systems, the effect of pH changes, in the range 2-9, on the complexes surviving in solution was also explored. ESI data, besides supporting previous reported results on the formation of dimeric carnosine/copper and carnosine/zinc complexes, allowed a more complete speciation of the examined systems, bringing to light the existence of bis-complex species and, in the zinc case, the formation of oligomeric species. The data obtained for the systems investigated show that ESI-MS is not only a reliable and fast technique for the analysis of the metal/ligand systems, but also an interesting tool to obtain stoichiometric information on metal complexes formed in very low concentration solutions.