Novel chemically modified analogues of neuropeptide Y for tumor targeting

The successful use of peptides as potential radiopharmaceuticals essentially requires the modification of the bioactive peptide hormones to introduce chelators for radiolabeling. In this study, four Y 1/Y 2 receptor-selective NPY analogues with different receptor subtype specificities have been investigated. For in vitro studies, the cold metal surrogate was used. Gallium and indium complexes were introduced by using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid as bifunctional chelator. The peptides were synthesized by solid-phase peptide synthesis (SPPS), the chelator was coupled either at the N-terminus or at the N(epsilon) side chain of Lys(4) of the resin-bound peptide, and the labeling was performed in solution after cleavage. Competitive binding assays showed high binding affinity of the receptor-selective analogues at NPY receptor expressing cells. To test internalization of the novel peptide analogues and the metabolic stability in human blood plasma, the corresponding 5(6)-carboxyfluorescein (CF) analogues were prepared and investigated. One of the most promising analogues, the Y 1-receptor selective [Lys(DOTA)(4), Phe(7), Pro(34)]NPY was labeled with (111)In and injected into nude mice that bear MCF-7 breast cancer xenografts, and biodistribution studies were performed. In vitro and in vivo studies suggest that receptor-selective analogues of NPY have promising characteristics for future applications in nuclear medicine for breast tumor diagnosis and therapy.     

Oxidative stress in skin fibroblasts cultures from patients with Parkinson's disease

Background  

In the substantia nigra of Parkinson's disease (PD) patients, increased lipid peroxidation, decreased activities of the mitochondrial complex I of the respiratory chain, catalase and glutathione-peroxidase, and decreased levels of reduced glutathione have been reported. These observations suggest that oxidative stress and mitochondrial dysfunction play a role in the neurodegeneration in PD. We assessed enzymatic activities of respiratory chain and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with PD.     

Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

Biosynthesis of the lantibiotic Pep5. Isolation and characterization of a prepeptide containing dehydroamino acids

Pep5 is a tricyclic peptide antibiotic which contains the unusual amino acids dehydrobutyrine, lanthionine and 3-methyllanthionine. It is matured from a 60-amino-acid precursor peptide (pre-Pep5) deduced from the sequence of the structural gene pepA. To study the biosynthesis of Pep5 we tried to isolate the primary translation product. We identified a peptide in crude extracts of the Pep5-producing Staphylococcus epidermidis strain using antibodies raised against a synthetic 26-residue peptide representing the leader peptide region of pre-Pep5. The putative precursor was purified by reversed-phase HPLC. The isolated peptide did not react with antibodies directed against a C-terminal fragment of mature Pep5 containing two sulfide bridges. Neither lanthionine nor 3-methyllanthionine was detected in amino acid analysis of the isolated precursor. Its amino acid sequence was identical with the sequence predicted from pepA, but Edman degradation stopped at the first threonine residue of the prolantibiotic region indicating a posttranslational modification at this position. The molecular mass of the isolated peptide was 6575.4 +/- 1.7 Da, determined by ion-spray mass spectrometry. This is in agreement with a molecule being dehydrated at the four threonine and the two serine residues in the propeptide region; such a peptide has a calculated molecular mass of 6576.7 Da. The results strongly suggest that maturation of the lantibiotic Pep5 is initiated by selective dehydration of hydroxyamino acids in the propeptide region of the primary translation product and that thioether ring formation is not closely linked to dehydration.     

Molecular characterization of the human neuropeptide Y Y2-receptor.

Five neuropeptide Y receptors, the Y1-, Y2-, Y4-, Y5- and y6-subtypes, have been cloned, which belong to the rhodopsin-like G-protein-coupled, 7-transmembrane helix-spanning receptors and bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. In this study, the Y2-receptor subtype expressed in a human neuroblastoma cell line (SMS-KAN) and in transfected Chinese hamster ovary cells (CHO-hY2) was characterized on the protein level by using photoaffinity labeling and antireceptor antibodies. Two photoactivatable analogues of NPY were synthesized, in which a Tyr residue was substituted by the photoreactive amino acid 4-(3-trifluoromethyl)-3H-diazirin-3-ylphenylalanine ((Tmd)Phe), [Nalpha-biotinyl-Ahx2,(Tmd)Phe36]NPY (Tmd36), and the Y2-receptor subtype selective [Nalpha-biotinyl-Ahx2,Ahx5-24,(Tmd)Phe27]N PY (Tmd27). Both analogues were labeled with [3H]succinimidyl-propionate at Lys4 and bind to the Y2-receptor with affinity similar to that of the native ligand. A synthetic fragment of the second (E2) extracellular loop was used to generate subtype selective antireceptor antibodies against the Y2-receptor. Photoaffinity labeling of the receptor followed by SDS-PAGE and detection of bound radioactivity and SDS-PAGE of solubilized receptors and subsequent Western blotting revealed the same molecular masses. Two proteins correspondingly have been detected for each cell line with molecular masses of 58 +/- 4 and 50 +/- 4 kDa, respectively.     

Functional role of the extracellular N-terminal domain of neuropeptide Y subfamily receptors in membrane integration and agonist-stimulated internalization

The N terminus is the most variable element in G protein-coupled receptors (GPCRs), ranging from seven residues up to approximately 5900 residues. For family B and C GPCRs it is described that at least part of the ligand binding site is located within the N terminus. Here we investigated the role of the N terminus in the neuropeptide Y receptor family, which belongs to the class A of GPCRs. We cloned differentially truncated Y receptor mutants, in which the N terminus was partially or completely deleted. We found, that eight amino acids are sufficient for full ligand binding and signal transduction activity. Interestingly, we could show that no specific amino acids but rather the extension of the first transmembrane helix by any residues is sufficient for receptor activity but also for membrane integration in case of the hY(1) and the hY(4) receptors. In contrast, the complete deletion of the N terminus in the hY(2) receptors resulted in a mutant that is fully integrated in the membrane but does not bind the ligand very well and internalizes much slower compared to the wild type receptor. Interestingly, also these effects could be reverted by any N-terminal extension. Accordingly, the most important function of the N termini seems to be the stabilization of the first transmembrane helix to ensure the correct receptor structure, which obviously is essential for ligand binding, integration into the cell membrane and receptor internalization.     

Prospects for estimating nucleotide divergence with RAPDs

The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.