LC-MS characterization and cell-binding properties of chelate modified somatropin

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is recently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. In some of these cancers, over-expression of the human growth hormone receptor (hGHR) is described. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. As site-specific labelling is necessary to avoid active region interactions (1-16, 41-68, 103-119 and 167-175), characterization of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and a combination of both enzymes. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact amino acid modifications. The use of a mixture of trypsin and chymotrypsin gave an enhanced information efficiency. Moreover, the intact protein, without enzymatic degradation, was analysed on a protein HPLC column using UV detection for quantification and ESI-MS/MS for characterization. Based upon the HPLC-MSn results of the digested somatropin, the chelating molecule is mainly bound to a specific lysine amino acid that is located away from the receptor binding site. Therefore, the cell-binding functionality of the characterized NOTA-somatropin is measured, using a HepG2 cell line

HPLC-MS characterisation of chelate modified somatropin

Somatropin is a recombinant human growth hormone, consisting of 191 amino acids. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is currently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. The modification of the protein with the chelating agent NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein for diagnostic (e.g. 68Ga) or therapeutic (e.g. 90Y) purposes. The NOTA unit is selectively introduced on a lysine side chain. This yields 9 possible labelling sites for somatropin: FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF We have applied an enzymatic digestion procedure for the characterisation of the modified somatropin, using trypsin, chymotrypsin and Staphylococcus aureus V-8 proteases. The resulting peptides were then monitored using HPLC-MS2, allowing the characterisation of the modified protein

The retinal relaxing factor : update on an enigmatic regulator of the retinal circulation

The retinal circulation is regulated by different local factors and might include the retinal relaxing factor (RRF). This factor is found to be continuously released by the retina and relaxes smooth muscle cells. This review describes the current knowledge about the RRF. Despite many research efforts, the cellular source, identity, mechanism, and physiological role of the RRF remain largely unknown. Thus far, it seems that the RRF is a hydrophilic, thermostable, diffusible chemical messenger, which characteristics do not correspond with most well-known endogenous vasorelaxants. The RRF-induced relaxation seems to rely on activation of the inward rectifier K+ channels and the Rho kinase Ca2+ sensitization mechanism. Voltage-dependent K+ channels and plasma membrane Ca2+-ATPase might also be involved, whereas the involvement of cyclooxygenase is still a point of discussion. Furthermore, it appears that the RRF is involved in other relaxation pathways, namely those of hypoxia, adenosine, and adenosine triphosphate, hydrogen sulfide, c-aminobutyric acid, and dorzolamide

Vasorelaxing effect of resveratrol on bovine retinal arteries

PURPOSE: Resveratrol is a red wine polyphenol that causes vasorelaxation, which could be of interest in the treatment or prevention of eye diseases with an impaired blood flow. In this study, the vasorelaxant capacity of resveratrol (cis and trans) on bovine retinal arteries, its vasorelaxing mechanism, and its influence on the relaxation induced by the retinal relaxing factor (RRF) were examined. METHODS: Isolated bovine retinal arteries were mounted into wire myographs for isometric tension measurements. Concentration-response curves of cis- and trans-resveratrol and concentration-response curves of resveratrol in the absence or presence of the endothelium or different inhibitors were constructed. Relaxations elicited by the RRF with and without resveratrol incubation were also compared. RESULTS: Both resveratrol isomers caused a similar strong concentration-dependent relaxation. Removal of the endothelium or blocking endothelium-dependent pathways did not change the relaxation. Also, K+ channel blockers did not reduce the relaxation, except the 120 mM K+ Krebs Ringer bicarbonate solution. Phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate blocked the relaxation partially and so did the inhibition of heme oxygenase-1. Blocking adenylyl cyclase, AMP-activated protein kinase, estrogen receptors, sirtuin 1, or sarco/endoplasmic reticulum Ca2+ ATPase did not have an effect. The relaxation caused by the RRF was not altered by resveratrol incubation. CONCLUSIONS: Cis- and trans-resveratrol relax bovine retinal arteries similarly and concentration dependently. The main relaxation mechanism remains unclear, but K+ channels, carbon monoxide, and the myosin phosphatase pathway may be involved. Resveratrol does not have an influence on the RRF

Specific Migratory Dendritic Cells Rapidly Transport Antigen from the Airways to the Thoracic Lymph Nodes

Antigen transport from the airway mucosa to the thoracic lymph nodes (TLNs) was studied in vivo by intratracheal instillation of fluorescein isothiocyanate (FITC)-conjugated macromolecules. After instillation, FITC+ cells with stellate morphology were found deep in the TLN T cell area. Using flow cytometry, an FITC signal was exclusively detected in CD11cmed-hi/major histocompatibility complex class II (MHCII)hi cells, representing migratory airway-derived lymph node dendritic cells (AW-LNDCs). No FITC signal accumulated in lymphocytes and in a CD11chiMHCIImed DC group containing a CD8αhi subset (non–airway-derived [NAW]-LNDCs). Sorted AW-LNDCs showed long MHCIIbright cytoplasmic processes and intracytoplasmatic FITC+ granules. The fraction of FITC+ AW-LNDCs peaked after 24 h and had reached baseline by day 7. AW-LNDCs were depleted by 7 d of ganciclovir treatment in thymidine kinase transgenic mice, resulting in a strong reduction of FITC-macromolecule transport into the TLNs. Compared with intrapulmonary DCs, AW-LNDCs had a mature phenotype and upregulated levels of MHCII, B7-2, CD40, and intracellular adhesion molecule (ICAM)-1. In addition, sorted AW-LNDCs from FITC-ovalbumin (OVA)–instilled animals strongly presented OVA to OVA-TCR transgenic T cells. These results validate the unique sentinel role of airway DCs, picking up antigen in the airways and delivering it in an immunogenic form to the T cells in the TLNs

Classification of quorum-sensing peptides

Quorum-sensing (QS) enables bacterial cells to establish cell-cell communication and to regulate the expression of specific genes in response to local changes in cell density. It provides a means to coordinate the activities of cells in order to function as a multicellular unit. Quorum-sensing chemicals up till now identified as key components in bacterial cell-cell communication can broadly be divided in three groups, which contain peptidic-derived structural characteristics: N-acyl homoserine lactone derivatives (auto-inducer-1), larger quorum-sensing peptides and boron-furanone derivatives (auto-inducer-2). A quorum-sensing peptide database (Quorumpeps®) was constructed encompassing the structures as well as functionality responses. After three-dimensional optimisation of these structures, chem-informatic molecular descriptors were calculated. The resulting data-matrix was subjected to diverse multivariate analyses to cluster these peptides as well as to extract fundamental relationships between biological activity responses and structural properties (quantitative structure-property relations, QSPR)