In der vorliegenden Arbeit sind Untersuchungen vorgestellt, die sich mit Wirkungen und Interaktionen von zwei ubiquitär im menschlichen Körper vorkommenden Gasen befassen, i.e. Sauerstoff und Stickstoffmonoxid. Im Falle beider Substanzen ermöglicht die geringe Größe der Moleküle eine freie Diffusion über Membranen hinweg, eine Eigenschaft, die für die Funktion der Signaltransduktion geradezu prädestiniert. Aus den vorgelegten Untersuchungen lassen sich die folgenden Folgerungen ableiten: * Stickstoffmonoxid wirkt in-vitro selektiv bakteriostatisch auf Bakterien, die üblicherweise Früh- und Neugeborene besiedeln. Dabei hängt die Selektivität von den jeweiligen bakteriellen Verteidigungsmechanismen ab, die bakteriostatische Wirkung liegt in einem Konzentrationsbereich, der außerhalb desjenigen liegt, der derzeit klinisch angewendet wird. * Hyperoxie führt im Ganztiermodell der unreifen Ratte zu einer zerebralen Hochregulation von iNOS und damit zur Synthese von NO. Soweit dies anhand der Synthese von Peroxynitrit als definitivem Schädigungsmechanismus beurteilbar ist, wird trotz entsprechender iNOS-Expression wenig bis gar kein Peroxynitrit gebildet. Da das Zusammentreffen von NO und Sauerstoff sonst regelhaft zur Entstehung von Peroxynitrit führt, müssen im Gehirn der unreifen Ratte ausreichende antioxidative Schutzmechanismen präsent sein, die diese Reaktion verhindern. * Im in-vitro-Modell der Gasäquilibrierung von Nabelschnur-PMN zeigte sich unter Hyperoxie das ausgeprägteste Aktivierungsmuster aller verglichenen Sauerstoffkonzentrationen. Dies stand im Gegensatz zur Exposition adulter Zellen, hier fand sich eine größere Hyperoxietoleranz bei gleichzeitig stärkster Aktivierung unter Hypoxiebedingungen. Welche Bedeutung diesen Ergebnissen im klinischen Umgang mit Neugeborenen zukommt muß derzeit noch offen bleiben. Allerdings häufen sich Hinweise aus experimentellen Studien, die darauf hindeuten, daß ein restriktiver Umgang mit hohen Sauerstoffkonzentrationen auch im klinischen Umfeld gerechtfertigt sein könnte. / The present investigations deal with the effects and interactions of gases, which are ubiquitous in the human body i.e. oxygen and nitric oxide. Both substances are small enough to freely diffuse across biological membranes. This ability predestines both molecules for the function of signal transduction. The results of our investigations lead to conclusions as follows: * Nitric oxide has selective bacteriostatic effects in-vitro on some bacterial strains typically isolated from preterm and term newborn infants. Selectivity depends on the presence of bacterial defense mechanisms. The bacteriostatic effect takes place at concentrations above those currently used in clinical practice. * Hyperoxia leads to upregulation of iNOS and subsequent NO production in an animal model of the immature rat. Despite this upregulation of iNOS synthesis there is no increased production of peroxynitrite which is known to cause cellular and DNA damage. Since the combination of NO and high concentrations of oxygen lead to peroxynitrite formation on a regular basis, effective antioxidant mechanisms appear to prevent peroxynitrite formation in the brain of the immature rat. * The most pronounced activation of cord blood polymorphonuclear cells (PMN) during conditions of hyperoxia, normoxia, and hypoxia was found for exposure towards high oxygen concentrations in an in-vitro model of gas equilibration. As opposed to that, hypoxia was the most potent trigger for adult PMN. It remains to be determined which clinical implications must be derived from these results. However, increasing experimental evidence indicates that exposure towards high oxygen concentrations should be restricted also in clinical practice and not only in preterm infants, but also in term newborns.

Exogenously supplied organoselenium compounds, capable of propagating a selenium redox cycle, were shown to supplement natural cellular defenses against oxidants generated during biological activity. Phenylaminoalkyl selenides were developed in our laboratory as novel substrate analogs for the enzyme dopamine beta-monooxygenase. Recently, phenylaminoalkyl selenides were found to protect plasmid DNA and Molecular beacons from oxoperoxynitrate – mediated damage by scavenging this oxidant and forming the corresponding selenoxides as the sole selenium – containing products. Rate constants were determined for the reactions of the phenylaminoalkyl selenoxides with GSH at physiological pH and 25 degrees C. The kinetic data obtained in current and previous research was subsequently used in a MatLab simulation, which showed the feasibility of selenium redox cycling by GSH in the presence of a cellular oxidant, oxoperoxynitrate. Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests covering 11.7 million hectares. Somatic embryogenesis (SE) is an effective technique to implement production of high value genotypes of LP. SE is a multi-step process, which includes initiation of somatic embryo (SME) growth from tree tissue, maintenance and multiplication of early stage SMEs and the maturation / germination phase. In this work, we isolated a substance from stage 2 or 3 LP female gametophyte (FG) tissue that stimulates early stage SME growth, and characterized this substance as citric acid on the basis of 1H NMR and mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at p = 0.05 for 3 of the 5 genotypes tested. Phenylketonuria (PKU) is an autosomal recessive disorder caused by an impaired conversion of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr). A novel assay based on enzymatic - colorimetric methodology (ECA) was developed in order to detect elevated concentrations of L-Phe in undeproteinized plasma of PKU patients via continuous spectrophotometric detection. We report here that L-Phe concentrations in undeproteinized plasma measured using our ECA were comparable to those determined on an amino acid analyzer based on Pearson correlation coefficients and a Bland and Altman comparison.

We now report an in depth analysis of the successful in vitro enzymatic synthesis of PHB utilizing the three-enzyme system from the bacteria Cupriavidus necator. Using HPLC methodology developed in this laboratory, and by adding each enzyme in a step-wise manner, we follow each individual stage in the three-enzyme route for PHB synthesis and delineate all stoichiometric relationships. We report the construction of the first metabolic model developed specifically for analyzing in vitro enzymatic PHB synthesis. We developed a hands-on student laboratory for culturing, producing, isolating, and purifying the bacterial biopolyesters PHB. We now report the first structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and beta-hydroxybutyryl-iso-CoA using MS, MS/MS, and homo- and hetero-nuclear NMR analyses.We describe HPLC methodology to separate the isomers of several iso-CoA-containing compounds and report the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl-transfer reactions. We describe a simple regioselective synthesis of iso-CoA from CoA. We also demonstrate a plausible mechanism, which accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. Herein we report that phenylaminoethyl selenide compounds protect DNA from peroxynitrite-mediated single-strand breaks. The mechanism of protection against peroxynitrite mediated DNA damage was investigated by HPLC. The chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. The unique chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. We report the development of novel CDB derivatives, which are selective COX-II inhibitors. A series of compounds were assayed with an in vitro colorimetric inhibitor screening and with a whole blood ELISA screening and the results indicate that MST is a selective inhibitor of COX-II.