Synthesis and Application of Methyl N,O-Hydroxylamine Muramyl Peptides.

The innate immune system's interaction with bacterial cells plays a pivotal role in a variety of human diseases. Carbohydrate units derived from a component of bacterial cell wall, peptidoglycan (PG), are known to stimulate an immune response. Nonetheless, access to modified late-stage peptidoglycan intermediates is limited due to their synthetic complexity. A method to rapidly functionalize PG fragments is needed to better understand the natural host-PG interactions. Here methyl N,O-hydroxylamine linkers are incorporated onto a synthetic PG derivative, muramyl dipeptide (MDP). The modification of MDP maintained the ability to stimulate a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) immune response dependent on the expression of nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Intrigued by this modification's maintenance of biological activity, several applications were explored. Methyl N,O-hydroxylamine MDP was amendable to N-hydroxylsuccinimide (NHS) chemistry for bioconjugation to fluorophores as well as a self-assembled monolayer for Nod2 surface plasmon resonance analysis. Finally, linker incorporation was applicable to larger PG fragments, both enzymatically generated from Escherichia coli or chemically synthesized. This methodology provides rapid access to PG probes in one step and allows for the installation of a variety of chemical handles to advance the molecular understanding of PG and the innate immune system.

Postsynthetic Modification of Bacterial Peptidoglycan Using Bioorthogonal N-Acetylcysteamine Analogs and Peptidoglycan O-Acetyltransferase B.

Bacteria have the natural ability to install protective postsynthetic modifications onto its bacterial peptidoglycan (PG), the coat woven into bacterial cell wall. Peptidoglycan O-acetyltransferase B (PatB) catalyzes the O-acetylation of PG in Gram (-) bacteria, which aids in bacterial survival, as it prevents autolysins such as lysozyme from cleaving the PG. We explored the mechanistic details of PatB's acetylation function and determined that PatB has substrate specificity for bioorthgonal short N-acetyl cysteamine (SNAc) donors. A variety of functionality including azides and alkynes were installed on tri-N-acetylglucosamine (NAG)3, a PG mimic, as well as PG isolated from various Gram (+) and Gram (-) bacterial species. The bioorthogonal modifications protect the isolated PG against lysozyme degradation in vitro. We further demonstrate that this postsynthetic modification of PG can be extended to use click chemistry to fluorescently label the mature PG in whole bacterial cells of Bacillus subtilis. Modifying PG postsynthetically can aid in the development of antibiotics and immune modulators by expanding the understanding of how PG is processed by lytic enzymes.

Membrane Association Dictates Ligand Specificity for the Innate Immune Receptor NOD2.

The human gut must regulate its immune response to resident and pathogenic bacteria, numbering in the trillions. The peptidoglycan component of the bacterial cell wall is a dense and rigid structure that consists of polymeric carbohydrates and highly cross-linked peptides which offers protection from the host and surrounding environment. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a human membrane-associated innate immune receptor found in the gut epithelium and mutated in an estimated 30% of Crohn's disease patients, binds to peptidoglycan fragments and initiates an immune response. Using a combination of chemical synthesis, advanced analytical assays, and protein biochemistry, we tested the binding of a variety of synthetic peptidoglycan fragments to wild-type (WT)-NOD2. Only when the protein was presented in the native membrane did binding measurements correlate with a NOD2-dependent nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) response, supporting the hypothesis that the native-membrane environment confers ligand specificity to the NOD2 receptor for NF-κB signaling. While N-acetyl-muramyl dipeptide (MDP) has been thought to be the minimal peptidoglycan fragment necessary to activate a NOD2-dependent immune response, we found that fragments with and without the dipeptide moiety are capable of binding and activating a NOD2-dependent NF-κB response, suggesting that the carbohydrate moiety of the peptidoglycan fragments is the minimal functional epitope. This work highlights the necessity of studying NOD2-ligand binding in systems that resemble the receptor's natural environment, as the cellular membrane and/or NOD2 interacting partners appear to play a crucial role in ligand binding and in triggering an innate immune response.

Degradation of short-chain alkyl- and alkanolamines by TiO2- and Pt/TiO2-assisted photocatalysis.

The photooxidation of C2H5NH2, (C2H5)2NH, HOC2H4NH2, (HOC2H4)2NH and (HOC2H4)3N using TiO2 and Pt/TiO2 as photocatalysts has been investigated. A laboratory set up was designed and a study on the influence of the concentration of the photocatalyst, the pH-value and the structure of the amine performed. The photocatalytic process was optimized with respect to the concentrations of the model substances during degradation. The decrease of the amine concentrations was found to be maximum at a pH of 10. The time-dependence of the formation of cationic breakdown products, such as NH3/NH4 and short-chain alkyl- and alkanolamines was studied by analyses with single column ion chromatography. The experimental data show that the photodegradation follows a Langmuir-Hinshelwood kinetic. The mineralization of the model substances also was monitored by measurements of the decrease of the TOC and of the formation of NO2 and NO3. The different mineralization efficiencies for the model substances studied are discussed with regard to their structure and adsorption behaviour on the photocatalyst. A possible breakdown mechanism involving the electrophilic attack of the hydroxyl radical is given. The applicability of the TiO2-assisted photocatalytic degradation of C2H5NH2 and (C2H5)2NH was tested at the pilot plant-scale with real solar radiation. The degradation rates and products obtained were similar to those found in the laboratory experiments.

Degradation of nitrogen containing organic compounds by combined photocatalysis and ozonation.

The combination of TiO2-assisted photocatalysis and ozonation in the degradation of nitrogen-containing substrates such as alkylamines, alkanolamines, heterocyclic and aromatic N-compounds has been investigated. A laboratory set-up was designed and the influence of the structure of the N-compound, the TiO2 and ozone concentration on the formation of breakdown products were examined. The experimental results showed that a considerable increase in the degradation efficiency of the N-compounds is obtained by a combination of photocatalysis and ozonation as compared to either ozonation or photocatalysis only. The mineralization of the model substances was monitored by measurements of the TOC and ion-chromatographic determinations of the formed NO2- and NO3-. The temporal changes of concentrations of breakdown products, such as NH4+, short chain alkyl- and alkanolamines were determined by single column ion chromatography (SCIC) and as well as by electrospray mass spectrometry (EI-MS).

[The problem of factor VIII inhibitors in patients with congenital coagulopathies]. / Zum Problem der Faktor VIII-Inhibitoren bei Patienten mit angeborenen Koagulopathien.

It is reported on a 19-year-old patient with haemophilia A, in whom after a tooth extraction haemorrhages developed which could not be commanded. Factor-VIII-inhibitors could be proved qualitatively and quantitatively. As prospective for the therapy proved to be the administration of activated prothrombin complex preparations (FEIBA). The haemorrhages stopped, the partial thromboplastin time shortened. After three weeks the patient could be dismissed from hospital treatment.

[Coagulation factor XII deficiency, a contribution to thrombophilia]. / Zum Problem des Gerinnungsfaktor XII-Mangels, ein Beitrag zur Thrombophilie.

The hereditary deficiency of Hageman's factor rarely occurs. The patients are mostly detected by chance. The leading symptom is the considerably prolonged partial thromboplastin time. A haemorrhagic diathesis is not present. Since in these patients thromboemoblic diseases are possible, in an ascertained diagnosis the prevention of thrombosis should be performed.

[The intravenous fat tolerance test in patients with peripheral arteriosclerotic disorders of the blood supply]. / Untersuchungen zum intravenösen Fettoleranztest bei Patienten mit peripheren arteriosklerotischen Durchblutungsstörungen.

Latent metabolic disturbances become evident only by specific loads. As functional test of the fat metabolism the intravenous fat tolerance test is suited in which after an injection of 0.1 g fat/kg body weight via the decreasing plasma turbidity the degradation rate K2 (%/min) may be calculated. In 29 patients with peripheral arteriosclerotic disturbances of the blood supply we did not find any changed degradation rates in comparison to 29 reference persons, so that a disturbance of the clarification system is not to be supposed in these patients. It is reported on the behaviour of other metabolic parameters. At the same time by the slight changes in the intermediary metabolism after fat injection the good tolerability of the emulsion of soya bean oil lipofundin S which is known from clinical experience is supported.

[Immunocoagulopathies as a possible cause of acquired hemorrhagic diathesis]. / Immunkoagulopathien als mögliche Ursache erworbener hämorrhagischer Diathesen.

Spontaneous factor VIII inhibitors may be responsible for severe life-threatening haemorrhages. It is reported on two patients with inhibitors directed against factor VIII: C. In one female patient a rheumatoid arthritis could be made evident as basic disease. Diagnostics, therapy and course are reported and discussed.

[Hemorheologic studies for evaluating microcirculation in patients with peripheral arterial circulatory disorders and diabetes mellitus]. / Hämorheologische Untersuchungen zur Beurteilung der Mikrozirkulation bei Patienten mit peripheren arteriellen Durchblutungsstörungen und Diabetes mellitus.

Haemorheological investigations in 73 male patients with chronic disturbances of the arterial blood supply of the lower extremities and 54 male non-insulin-requiring (type-II-) diabetics with and without micro- and macroangiopathy resulted in significant changes of the flow properties of the blood in contrast to 20 comparable clinically healthy males. In all groups of patients a clear increase of the aggregation of erythrocytes and the viscosity of plasma as well as a decrease of the deformability of erythrocytes were to be proved. The size of the deviations of all three parameters from the normal correlated with the degree of severity of the arterial obstructive disease and in diabetics with the occurrence of vascular complications (macroangiopathy, retinopathy) as well as with the situation of metabolism. The decreased fluidity of blood may lead to the development of further enlargement of disturbances of microcirculation. Therefore, haemorheological viewpoints should be included into the total diagnostic and therapeutic concept of the arterial obstructive disease and the diabetes mellitus.

[Kasabach-Merritt syndrome]. / Das Kasabach-Merritt-Syndrom.

The Kasabach-Merritt syndrome is a rare, congenital disease, characterized by giant haemangiomatosis and disseminated intravasal coagulation. It is reported on an at present 34-year-old patient with such a disease and the long-term course is described. Complications with haemorrhages can be prevented by means of anticoagulants.