Copper(II) complexes of the neurotoxic peptide fragments of human and chicken prion proteins were studied by potentiometric, UV-vis, CD, and EPR spectroscopic and ESI-MS methods. The peptides included the terminally blocked native and scrambled sequences of HuPrP106-126 (HuPrPAc106-126NH2 and ScrHuPrPAc106-126NH2) and also the nona- and tetrapeptide fragments of both the human and chicken prion proteins (HuPrPAc106-114NH2, ChPrPAc119-127NH2, HuPrPAc109-112NH2, and ChPrPAc122-125NH2). The histidyl imidazole-N donor atoms were found to be the major copper(II) binding sites of all peptides; 3N and 4N complexes containing additional 2 and 3 deprotonated amide-N donors, respectively, are the major species in the physiological pH range. The complex formation processes for nona- and tetrapeptides are very similar, supporting the fact that successive deprotonation and metal ion coordination of amide functions go toward the N-termini in the form of joined six- and five-membered chelates. As a consequence, the peptide sequences investigated here, related to the neurotoxic region of the human PrP106-126 sequence, show a higher metal-binding affinity than the octarepeat fragments. In the case of the HuPrP peptide sequences, a weak pH-dependent binding of the Met109 residue was also detected in the 3N-coordinated complexes.

Background-The role of inflammatory markers is not well defined for either diagnosis or treatment of pericarditis. The aim of this study is to prospectively evaluate the frequency of high-sensitivity C-reactive protein (hs-CRP) elevation in patients with acute pericarditis, its time course of normalization, and the possible importance for diagnosis, therapy, and prognosis. Key Words: C-reactive protein Ⅲ diagnosis Ⅲ pericarditis Ⅲ prognosis Ⅲ therapy T here are no universally accepted criteria for the diagnosis of pericarditis. In clinical practice, some criteria are usually adopted (chest pain, pericardial rubs, PR depression and diffuse ST-segment elevation on ECG, pericardial effusion; at least 2 of 4 should be present for the diagnosis), and the role of inflammatory markers (eg, C-reactive protein [CRP]) is not well defined. [1][2][3][4][5] Clinical Perspective on p 1097Pericarditis is an inflammatory disease, and evidence of elevated markers of inflammation could support the diagnosis. Moreover, the same length of therapy is empirical, and markers of inflammation (ie, CRP) may be a useful guide for treatment because it can be assumed that anti-inflammatory therapy should be continued until the inflammation is extinguished. On this basis, persistent elevation of inflammatory markers, as evidence of disease activity, could be associated with a worse prognosis.The aim of the present study is to prospectively evaluate the frequency of high-sensitivity CRP (hs-CRP) elevation in patients with acute pericarditis, its time course of normalization, and the possible importance for diagnosis, therapy, and prognosis. Methods PatientsAll consecutive cases of acute pericarditis within 24 hours from symptom onset were screened for inclusion in a prospective cohort study from August 2005 to August 2007. A final diagnosis of idiopathic or viral acute pericarditis was reached at the end of the diagnostic assessment, which included chest x-ray, echocardiography, viral serology, and other specific testing according to the initial clinical presentation. Pericardiocentesis was done when a bacterial or neoplastic etiology was suspected or in case of cardiac tamponade or severe pericardial effusion without response to medical therapy after 1 week. Patients with a final diagnosis of viral and idiopathic pericarditis were included in the Received August 27, 2010; accepted January 10, 2011. Figure 1. The diagnosis of acute pericarditis was done according to available published criteria. [1][2][3][4][5][6][7][8] Diagnostic criteria included pericarditic typical chest pain, pericardial friction rubs, widespread ST-segment elevation or PR depressions not reported previously, and new or worsening pericardial effusion. A clinical diagnosis of acute pericarditis was made when at least 2 of these criteria were present. Criteria for the diagnosis of recurrence included recurrent pain and 1 or more of the following signs: fever, pericardial friction rub, ECG changes, echocardiographic evidence of pericardial effusion, and an elevation in th...

No abstract

Complex formation processes between the 39-mer residue peptide fragment of human prion protein, HuPrP(76-114), and copper(II) ions have been studied by potentiometric, UV-vis, circular dichroism (CD), electron paramagnetic resonance, and electrospray ionization mass spectrometry methods. This peptide consists of 39 amino acid residues and contains two histidines (His77 and His85) belonging to the octarepeat domain and two histidines (His96 and His111) outside this domain. It was found that HuPrP(76-114) is able to bind 4 equiv of metal ions and all histidyl residues are independent, except nonequivalent metal binding sites in the oligonuclear species. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites below pH 5.5 in the form of various macrochelates. The macrochelation slightly suppresses, but cannot prevent, the deprotonation and metal ion coordination of amide functions, resulting in the formation of (N(im),N(-)), (N(im),N(-),N(-)), and (N(im),N(-),N(-),N(-))-coordinated copper(II) complexes in the pH range from 5.5 to 9. CD spectroscopy results gave clear evidence for the differences in the metal binding affinity of the histidyl sites according to the following order: His111 > His96 >> His77 approximately His85. Among the oligonuclear complexes, the formation of di- and tetranuclear species seems to be favored over the trinuclear ones, at pH values beyond the physiological ones. This phenomenon was not observed in the complex formation reactions of HuPrP(84-114), a peptide fragment containing only one histidyl residue from the octarepeat. As a consequence, the data support the existence of cooperativity in the metal binding ability of this peptide probably due to the presence of two octarepeat sequences of the dimeric octarepeat domain of HuPrP(76-114) at basic pH values.

A 31-mer polypeptide, which encompasses residues 84-114 of human prion protein HuPrP(84-114) and contains three histidyl residues, namely one from the octarepeat (His85) and two histidyl residues from outside the octarepeat region (His96 and His111), and its mutants with two histidyl residues HuPrP(84-114)His85Ala, HuPrP(84-114) His96Ala, HuPrP(84-114)His111Ala and HuPrP(91-115) have been synthesised and their Cu2+ complexes studied by potentiometric and spectroscopic (UV/Vis, CD, EPR, ESI-MS) techniques. The results revealed a high Cu2+-binding affinity of all peptides, and the spectroscopic studies made it possible to clarify the coordination mode of the peptides in the different complex species. The imidazole nitrogen donor atoms of histidyl residues are the exclusive metal-binding sites below pH 5.5, and they have a preference for macrochelate structure formation. The deprotonation and metal-ion coordination of amide functions take place by increasing the pH; all of the histidines can be considered to be independent metal-binding sites in these species. As a consequence, di- and trinuclear complexes can be present even in equimolar samples of the metal ion and peptides, but the ratios of polynuclear species do not exceed the statistically expected ones; this excludes the possibility of cooperative Cu2+ binding. The species with a (N(im),N,N)-binding mode are favoured around pH 7, and their stability is enhanced by the macrochelation from another histidyl residue in the mononuclear complexes. The independence of the histidyl sites results in the existence of coordination isomers and the preference for metal binding follows the order of: His111>His96>His85. Deprotonation and metal-ion coordination of the third amide functions were detected in slightly alkaline solutions at each of the metal-binding sites; all had a (N(im),N,N,N)-coordination mode. Spectroscopic measurements also made it clear that the four lysyl amino groups of the peptides are not metal-binding sites in any cases.

No abstract

The flexible N-terminal domain of the prion protein (PrP(c)) is believed to play a pivotal role in both trafficking of the protein through the cell membrane and its pathogenic conversion into the β sheet-rich scrapie isoform (PrP(sc)). Unlike mammalian PrP(c), avian prion proteins are not known to undergo any pathogenic conformational conversions. Consequently, some critical advances in our understanding of the molecular mechanisms underlying prion pathogenesis are expected from comparative studies of the biophysical properties of the N-terminal domains of the two proteins. The present study addresses the role played by different environmental factors, i.e., copper(II), pH, and membrane-mimicking environments, in assisting the conformational preferences of huPrP60-91 and chPrP53-76, two soluble peptides encompassing the N-terminal copper(II) binding domains of the human and chicken prion proteins, respectively. Moreover, the membrane interactions of huPrP60-91, chPrP53-76, and their copper(II) complexes were evaluated by Trp fluorescence in conjunction with measurements of the variation in thermotropic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. Circular dichroism experiments revealed that huPrP60-91 adopts a predominant polyproline II conformation in aqueous solution that is destabilized at basic pH or in the presence of trifluoroethanol (TFE). Unlike anionic sodium dodecyl sulfate (SDS), which seems to stabilize the polyproline II conformation further, zwitterionic dodecylphosphocholine (DPC) micelles do not affect the peptide structure. On the contrary, copper(II) promptly promotes an increase in β-turn-rich structures. Differential scanning calorimetry (DSC) and Trp fluorescence assays carried out on DPPC model membranes after incubation with huPrP60-91 showed a marked tendency of the peptide to slowly penetrate the lipid bilayer with a concomitant conformational transition toward an extended β-sheet-like structure. Such an event, which was ascribed to the hydrophobic Trp side chain residues, was shown to also depend on the level of copper(II) occupancy along the peptide. Conversely, the CD spectra of chPrP53-76 aqueous solutions indicated the presence of a mixture of random-coil/β-turn-like structures whose resulting equilibrium was influenced by SDS and copper(II) addition. Furthermore, chPrP53-76 did not exhibit any tendency to interact with model membranes in either the presence or absence of copper(II). The results reported here provide evidence of the different roles played by environmental factors in affecting the conformation and membrane activity of human and avian prion N-terminal domains.

Copper(II) binding to prion peptides does not prevent Cu redox cycling and formation of reactive oxygen species (ROS) in the presence of reducing agents. The toxic effects of these species are exacerbated in the presence of catecholamines, indicating that dysfunction of catecholamine vesicular sequestration or recovery after synaptic release is a dangerous amplifier of Cu induced oxidative stress. Cu bound to prion peptides including the high affinity site involving histidines adjacent to the octarepeats exhibits marked catalytic activity toward dopamine and 4-methylcatechol. The resulting quinone oxidation products undergo parallel oligomerization and endogenous peptide modification yielding catechol adducts at the histidine binding ligands. These modifications add to the more common oxidation of Met and His residues produced by ROS. Derivatization of Cu-prion peptides is much faster than that undergone by Cu-β-amyloid and Cu-α-synuclein complexes in the same conditions.