No Evidence for Borrelia burgdorferi-Specific DNA in Lesions of Localized Scleroderma

A possible association of Borrelia burgdorferi with localized scleroderma is currently the focus of intense research and discussion. Skin biopsies from 30 patients with localized scleroderma (28 of the plaque type/morphea; two linear scleroderma) were analyzed for the presence of Borrelia burgdoferi using three different polymerase chain reaction systems for amplification of segments of borrelial genes.Formalin-fixed, paraffin-embedded biopsies of 14 patients and fresh-frozen, cryo-conserved biopsies of 16 patients with localized scleroderma were obtained. Lesions of all patients showed clear signs of scleroderma and disease progression at the time of biopsy. Fresh-frozen as well as formalin-fixed biopsies from patients with erythema migrans or acrodermatitis chronica atrophicans were used as positive controls.With all three polymerase chain reaction systems, borrelial DNA was detected in none of the 30 specimens of localized scleroderma. In contrast, with one polymerase chain reaction system, Borrelia burgdorferi-specific DNA was found in 24 of 27 frozen biopsies from patients with erythema migrans and in all 5 analyzed frozen biopsies of patients with acrodermatitis chronica atrophicans. In approximately half of the paraffin-embedded biopsies from patients with erythema migrans (nine of 23) and acrodermatitis chronica atrophicans (13 of 27), Borrelia burgdorferi specific DNA was identified.These results question the association of localized scleroderma with known subtypes of Borrelia burgdorferi

Entwicklung und Charakterisierung von Mikroemulsionen mit aromatischen Heterozyklen für die Elektropolymerisation

For the electropolymerizable aromatic heterocycles thiophene, 3-methoxythiophene and 3,4- ethylenedioxythiophene (EDT) microemulsions were developed . These microemulsions allow to dissolve the aforementioned compounds in high concentrations in water and therefore substitute commonly used solvents such as acetonitrile . Isothermal phase diagrams of the components 0,5 M aqueous lithiumperchlorate, aromatic heterocycle, and nonionic surfactant were developed . Since the used oils are very polar substances, quite hydrophilic surfactants have to be used in order to achieve microemulsions . It was found that the phase diagrams of the différent substances are very similar. However, the substitutes of the oil species have a decisive influence on the formation of liquid crystals. High amounts of surfactant were necessary for the formation of microemulsions . By means of conductivity measurements, viscosity measurements and dynamic light scattering the microemulsions were characterized. It was found that the microemulsion is mainly an oil-in-water system . Obviously the electrolyte ions interact strongly with the surfactant which has complexing properties due to its ethylene oxide groups . Viscosity measurements show that the bicontinuous phase is formed above a >_ 65 % . This result could be proven by means of dynamic light scattering . Moreover, all samples of the system EDT/Lutensol °ON11%lectrolyte which were investigated could be polymerized electrochemically . Current transients show that the deposition on the electrode from the microemulsion with low où content is controlled by diffusion whereas at higher amounts of oil ( > 20 %) it is controlled by the charge-transfer-process through the electrode surface . It was also possible to obtain a film on the electrode from the system 3-methoxythiophene/LutensoloON80/electrolyte . Moreover, the polymerization from a liquid crystal could be achieved. However, the polymerization of thiophene from microemulsion was not possibl

Untersuchungen zur Bedeutung verschiedener Enzyme des Glycin-Stoffwechsels für die Riboflavin-Bildung in Ashbya gossypii

The filamentous fungus Ashbya gossypii is an important riboflavin (vitamin 132) overproducer used in industrial scale. Productivity of the fungus is limited by glycine, a precursor of the de novo purine biosynthesis. Supplementation of the medium with glycine leads to an increase in riboflavin production. The first objective of this thesis was the characterization of the two serine hydroxymethyltransfe rase (SHMT) isoenzymes, which are involved in glycine metabolism, in order to improve the glycine supply for the riboflavin production. For a subcellular localization of both SHMT isoenzymes mutants transformed with HA-fusions were used. By immunodetection SHMT1 was localized in the mitochondria and SHMT2 in the cytosol. Disruption of SHM2 resulted in a significant increase of riboflavin overproduction. The SHMT specific activity decreased about 85 % from 3 mU/mg protein to 0.5 mU/mg protein. After additional inactivation of SHM1 a remaining SHMT activity of 3 % was detected, which was shown to be a side activity of threonine aldolase. The enhanced riboflavin overproduction of SHM2 disruptants was explained by a reduced flux from glycine to serine thus leading to an elevated pool of the riboflavin precursor glycine. Evidence was obtained by $^{13}$C-labeling experiments. When $^{13}$C$_{1}$-threonine was fed, more than 50 % of the label was detected in C, of glycine, resulting from threonine aldolase activity. More than 30 % labeling determined in C$_{1}$ of serine could be explained by a serine synthesis via SHMT. Knockout of SHM1 had no detectable effect on serine labeling but disruption of SHM2 led to a decrease in serine (2 - 5 %) and an increase in glycine (59 - 67 %) labeling in position C$_{1}$. These data indicate the modified carbon flux. Disruption of SHM2 led to a reduced growth rate in minimal medium. Supplementation with 1 mM adenine restored wild-type growth, which showed that growth of Ag$\Delta$SHM2 is one-carbon limited. Since overexpression of the threonine aldolase gene had been shown to replace glycine by threonine limitation of riboflavin synthesis, the second objective of this thesis was a deregulation of threonine biosynthesis in A. gossypii. By heterologous complementation of a Saccharomyces cerevisiae mutant showing threonine auxotrophy the AgHOM3 gene encoding a monofunctional aspartate kinase was rescued. In crude extracts of A. gossypii an aspartate kinase specific activity of 5 mU/mg protein was detected. A mutant disrupted in HOM3 lost this enzyme activity and showed homoserine auxotrophy. The riboflavin production of Ag$\Delta$HOM3 was significantly increased, when the growth was homoserine limited. Supplementation of the medium with homoserine restored wild-type growth but at the same time reduced production of riboflavin below wild-type level. Presumably an enhanced induction of the RIB genes was the reason for the increased riboflavin production of Ag$\Delta$HOM3