Exocyclic DNA adducts as oxidative stress markers in colon carcinogenesis: potential role of lipid peroxidation,dietary fat and antioxidants

Molecular pathways to colorectal cancer involve multiple genetic changes, whereby extensive oxyradical damage causes mutations in cancer-related genes and leads to a cycle of cell death and regeneration. Besides direct oxidative DNA-damage, reactive oxygen and nitrogen species can induce etheno (epsilon)-DNA adducts mainly via trans-4-hydroxy-2-nonenal, generated as the major aldehyde by lipid peroxidation (LPO) of omega-6 PUFAs. Patients with familial adenomatous polyposis (FAP) develop multiple colorectal adenomas. In affected tissues increased LPO could be triggered due to increased arachidonic acid metabolism as a result of elevated cyclooxygenases. Our studies demonstrated an increased epsilon-DNA adduct level in affected colon epithelia of FAP patients. Epsilon-DNA adducts are promutagenic and can cause genomic instability that drives colorectal adenoma to malignancy. We have further investigated the potential chemopreventive properties of olive oil and its polyphenolic components. 'Mediterranean diet', of which olive oil is a major fatty acid source, has protective effects against human breast and colorectal cancers. Olive oil extracts and the newly identified lignan fractions showed high antioxidant capacity in vitro. As epsilon-DNA adducts are biomarkers for oxidative stress and LPO induced DNA damage, they can verify the efficacy of newly identified antioxidants, e.g. from olive oil, as chemopreventive agents against colon carcinogenesis.     

Teaching fluid shifts during orthostasis using a classic paper by Foux et al

Hypovolemic and orthostatic challenge can be simulated in humans by the application of lower body negative pressure (LBNP), because this perturbation leads to peripheral blood pooling and, consequently, central hypovolemia. The classic paper by Foux and colleagues clearly shows the effects of orthostasis simulated by LBNP on fluid shifts and homeostatic mechanisms. The carefully carried out experiments reported in this paper show the interplay between different physiological control systems to ensure blood pressure regulation, failure of which could lead to critical decreases in cerebral blood flow and syncope. Here, a teaching seminar for graduate students is described that is designed in the context of this paper and aimed at allowing students to learn how Foux and colleagues have advanced this field by addressing important aspects of blood regulation. This seminar is also designed to put their research into perspective by including important components of LBNP testing and protocols developed in subsequent research in the field. Learning about comprehensive protocols and carefully controlled studies can reduce confounding variables and allow for an optimal analysis and elucidation of the physiological responses that are being investigated. Finally, in collaboration with researchers in mathematical modeling, in the future, we will incorporate the concepts of applicable mathematical models into our curriculum.     

Influence of different spacers on the biological profile of a DOTA-somatostatin analogue

Radiolabeled somatostatin analogues have been successfully used for targeted radiotherapy and for imaging of somatostatin receptor (sst1-5)-positive tumors. Nevertheless, these analogues are subject to improving their tumor-to-nontarget ratio to enhance their diagnostic or therapeutic properties, preventing nephrotoxicity. In order to understand the influence of lipophilicity and charge on the pharmacokinetic profile of [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)]-somatostatin-based radioligands such as [DOTA,1-Nal3]-octreotide (DOTA-NOC), different spacers (X) based on 8-amino-3,6-dioxaoctanoic acid (PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG4), N-acetyl glucosamine (GlcNAc), triglycine, beta-alanine, aspartic acid, and lysine were introduced between the chelator DOTA and the peptide NOC. All DOTA-X-NOC conjugates were synthesized by Fmoc solid-phase synthesis. The partition coefficient (log D) at pH = 7.4 indicated that higher hydrophilicity than [111In-DOTA]-NOC was achieved with the introduction of the mentioned spacers, except with triglycine and beta-alanine. The high affinity of [InIII-DOTA]-NOC for human sst2 (hsst2) was preserved with the structural modifications, while an overall drop for hsst3 affinity was observed, except in the case of [InIII-DOTA]-beta-Ala-NOC. The new conjugates preserved the good affinity for hsst5, except for [InIII-DOTA]-Asn(GlcNAc)-NOC, which showed decreased affinity. A significant 1.2-fold improvement in the specific internalization rate in AR4-2J rat pancreatic tumor cells (sst2 receptor expression) at 4 h was achieved with the introduction of Asp as a spacer in the parent compound. In sst3-expressing HEK cells, the specific internalization rate at 4 h for [111In-DOTA]-NOC (13.1% +/- 0.3%) was maintained with [111In-DOTA]-beta-Ala-NOC (14.0% +/- 1.8%), but the remaining derivatives showed <2% specific internalization. Biodistribution studies were performed with Lewis rats bearing the AR4-2J rat pancreatic tumor. In comparison to [111In-DOTA]-NOC (2.96% +/- 0.48% IA/g), the specific uptake in the tumor at 4 h p.i. was significantly improved for the 111In-labeled sugar analogue (4.17% +/- 0.46% IA/g), which among all the new derivatives presented the best tumor-to-kidney ratio (1.9).     

A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

Effect of novel non-peptidic delta opioid receptor antagonists on human T and B cell activation

The effects of the antagonist naltrindole (NTI) on cells of the immune system have been largely studied although the mechanisms of action are still unclear. The aim of this study is to evaluate, in vitro, the immunomodulatory activity of four new delta-selective opioid compounds structurally related to naltrindole. The effects at different concentrations of these opioid antagonists on proliferative response were studied on normal human peripheral blood mononuclear cells (PBMC) stimulated with different stimuli: mitogens, the antigen PPD, the anti-CD3 monoclonal antibodies (mAb), the superantigen Staphylococcus aureus Cowan strain 1 (SAC) and alloantigens in the mixed lymphocyte cultures (MLR). The immunomodulatory capacity of these compounds was evaluated by determining the interleukin-2 (IL-2) release in mitogen activated PBMC. The present study shows that all the new delta opioid antagonists at 10(-5) M concentration are immunosuppressive. The inhibitory action is also evident at lower concentrations when anti-CD3 mAb and SAC were used as stimulators. In addition, the production of IL-2 was inhibited by the opioid treatment, but this might not be the only mechanism of action.     

Grenzen und Anwendungsmöglichkeiten der colorimetrischen CO2-Bestimmung

Ohne ZusammenfassungMit 6 Textabbildungen     

Ein einfaches Mikrokolorimeter

Ohne Zusammenfassung     

Self-association of nucleotides

The self-association of nucleosides decreases within the series adenosine>guanosine>inosine>cytidine ≈uridine. The same trend is observed for the corresponding nucleotides, though less pronounced, as the charge effect governs series like adenosine ? AMP^2?>ADP^3??ATP^4?. Protonation of adenosine considerably reduces its self-stacking tendency: this is different with ATP^4?, where a maximum is reached for H₂(ATP)^2? caused by additional ionic interactions in the [H₂(ATP)]₂ ^4? dimer. Metal ion coordination may promote self-association, e.g., of ATP^4? via (mainly) charge neutralization (Mg²⁺) and the formation of intermolecular bridges in dimeric stacks (Zn²⁺, Cd²⁺). These results allow definition of conditions with negligible self-association and thus the determination of the stability and structure of monomeric nucleotide complexes in aqueous solution, e.g., quantification of macrochelate formation in M(ATP)^2? complexes. Some biological implications of the results are indicated.