Relationships of ligand binding,redox properties,and protonation in Coprinus cinereus peroxidase

The pH dependence of the redox potentials and kinetics for CO association and dissociation was determined between pH 3.0 and 13.0 at 25 degrees C for the wild-type Coprinus cinereus fungal peroxidase and for a site-directed mutant in which Asp245, which is H-bonded to N delta of the imidazole of the proximal His183, was substituted with Asn. The determination of these functional properties allowed this information to be merged in a self-consistent fashion and to formulate for the first time a complete scheme employing the minimum number of groups required to describe the whole proton-linked behavior of both redox and ligand binding properties. The overall pH dependence can be accounted for by four redox- and ligand-linked groups. The proximal H-bond, which is strictly conserved in all peroxidases, will still be present in the site-specific mutant, but will no longer have an ionic character, and this event will bring about an alteration of redox equilibria and CO binding kinetics, envisaging a relevant role played by this H-bond also in modulating redox properties and ligand binding equilibria.     

Analysis of protein expression in pure cell nuclei populations isolated from human breast cancer tissue by DNA flow cytometric sorting

In this study, cell nuclei from aneuploid breast cancer samples were sorted with respect to DNA content into pure diploid and aneuploid fractions using flow cytometry. The nuclear proteins were then separated by one-dimensional gel electrophoresis (1D-PAGE) and differences in protein expression patterns, between diploid and aneuploid nuclei from the same tumours, were compared. Using a combination of peptide finger printing and peptide identification by MALDI-TOF mass spectrometry, we identified proteins and confirmed that the proteins were of nuclear origins. The results in this study add further information to the knowledge about the breast cancer disease complexity and heterogeneity at molecular level. For some of the tumours studied different nuclei protein patterns were obtained, in the diploid respective aneuploid nuclei populations, whilst other tumours did not show these differences.     

Bacterial catalase-peroxidases are gene duplicated members of the plant peroxidase superfamily

Bacterial catalase-peroxidases are enzymes containing 0.5-1.0 heme per subunit. The identical subunits are generally 80 kDa in size, and the sequenced subunits of E. coli, S. typhimurium and B. stearothermophilus contain 726-731 amino acid residues per subunit. The heme-containing peroxidases of plants, fungi and yeast are monomeric, homologous and 290-350 residues in size. Analyses of the amino acid sequences indicate that the double length of the bacterial peroxidases can be ascribed to gene duplication. Each half is homologous to eukaryotic, monomeric peroxidase and can be modelled into the high-resolution crystal structure of yeast cytochrome c peroxidase. The comparisons and modelling have predicted: (1) the C-terminal half does not bind heme, and bacterial peroxidases have one heme per subunit; (2) the ten dominating helices observed in the yeast enzyme are highly conserved and connected by surface loops which are often longer in the bacterial peroxidases; and (3) yeast cytochrome c peroxidase has evolved more slowly than other known peroxidases. The study has revealed ten invariant residues and a number of highly conserved residues present in peroxidases of the plant peroxidase superfamily and provides a basis for rationally engineered peroxidases.     

Plasminogen activator inhibitor type-1: reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing

Both the urokinase-type and tissue-type plasminogen activator can convert their approximately 54 kDa type-1 inhibitor (PAI-1) to an inactive form with a lower apparent molecular mass. We have determined the amino-terminal amino acid sequences of human native and converted PAI-1, and isolated PAI-1 cDNA and determined the nucleotide sequence in regions corresponding to the amino-terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg-Met bond 33 residues from the carboxy-terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino-terminus, with a Ser-Ala-Val-His-His form and a two-residue shorter form (Val-His-His-) occurring in approximately equal quantities.     

Biobank resources for future patient care: developments, principles and concepts

The aim of the overview is to give a perspective of global biobank development is given in a view of positioning biobanking as a key resource for healthcare to identify new potential markers that can be used in patient diagnosis and complement the targeted personalized drug treatment. The fast progression of biobanks around the world is becoming an important resource for society where the patient benefit is in the focus, with a high degree of personal integrity and ethical standard. Biobanks are providing patient benefits by large scale screening studies, generating large database repositories. It is envisioned by all participating stakeholders that the biobank initiatives will become the future gateway to discover new frontiers within life science and patient care. There is a great importance of biobank establishment globally, as biobanks has been identified as a key area for development in order to speed up the discovery and development of new drugs and protein biomarker diagnostics. One of the major objectives in Europe is to establish concerted actions, where biobank networks are being developed in order to combine and have the opportunity to share and build new science and understanding from complex disease biology. These networks are currently building bridges to facilitate the establishments of best practice and standardizations.     

Interpreting peptide mass spectra by VEMS

Most existing Mass Spectra (MS) analysis programs are automatic and provide limited opportunity for editing during the interpretation. Furthermore, they rely entirely on publicly available databases for interpretation. VEMS (Virtual Expert Mass Spectrometrist) is a program for interactive analysis of peptide MS/MS spectra imported in text file format. Peaks are annotated, the monoisotopic peaks retained, and the b-and y-ion series identified in an interactive manner. The called peptide sequence is searched against a local protein database for sequence identity and peptide mass. The report compares the calculated and the experimental mass spectrum of the called peptide. The program package includes four accessory programs. VEMStrans creates protein databases in FASTA format from EST or cDNA sequence files. VEMSdata creates a virtual peptide database from FASTA files. VEMSdist displays the distribution of masses up to 5000 Da. VEMSmaldi searches singly charged peptide masses against the local database.