Characterization of the arginine deiminase of Streptococcus pyogenes M49 and potential application in tumor therapy

Im Rahmen dieser Arbeit wurde die Auswirkung der Arginin Deiminase (AD) auf den Aminosäure- und Glukosestoffwechsel sowie die Virulenz von Streptococcus pyogenes untersucht. Weiterhin wurde die AD von S. pyogenes M49 heterolog exprimiert und kinetisch charakterisiert. Zudem wurde die Auswirkung dieser rekombinanten AD in Hinblick auf Tumortherapie untersucht. Die AD zeigte antiproliferative Effekte auf verschiedene Glioblastom-Zelllinien sowie auf hepatozelluläre und kolorektale Karzinomzelllinien. In Kombinationsversuchen mit Chemotherapeutika ließen sich diese Effekte signifikant verstärken.In this study we investigated the influence of the Arginine Deiminase on the amino acid and glucose metabolism from S. pyogenes but also on the virulence. Furthermore, the AD from S. pyogenes M49 was heterologously expressed, purified, and kinetically characterized. Futhermore, the impact of the purified AD on cell proliferation of arginine auxotrophic tumors was investigated. The AD showed an antiproliferative effects on glioblastoma cell lines and hepatocellular and colorectal carcinoma cell lines. The effects were significantly increased in combination with chemotherapeutics

Study of nascent NaH in sodium-hydrogen mixture using resonant CARS and FWM processes

The Coherent anti-Stokes Raman Spectroscopy (CARS) technique was applied to measure the O Q, and S branches from the first, second and third overtones of the XICt ground state of NaH. Moreover, P and R doublets were also measured when the anti-Stokes signal was rcsonant with some lines of the electronic transition AIC+- XIC+ of NaH. The high sensitivity resulting from pump laser resonances allowed us to study the Na(nl)+H2 -i NaH+H reaction dynarnics

Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile

During postnatal development, hyperplastic and hypertrophic processes of skeletal muscle growth depend on the activation, proliferation, differentiation, and fusion of satellite cells (SC). Therefore, molecular and functional SC heterogeneity is an important component of muscle plasticity and will greatly affect long-term growth performance and muscle health. However, its regulation by cell intrinsic and extrinsic factors is far from clear. In particular, there is only minor information on the early postnatal period which is critical for muscle maturation and the establishment of adult SC pools. Here, we separated two SC subpopulations (P40/50, P50/70) from muscle of 4-day-old piglets. Our results characterize P40/50 as homogeneous population of committed (high expression of Myf5), fast-proliferating muscle progenitors. P50/70 constituted a slow-proliferating phenotype and contains high numbers of differentiated SC progeny. During culture, P50/70 is transformed to a population with lower differentiation potential that contains 40% Pax7-positive cells. A reversible state of low mitochondrial activity that results from active down-regulation of ATP-synthase is associated with the transition of some of the P50/70 cells to this more primitive fate typical for a reserve cell population. We assume that P40/50 and P50/70 subpopulations contribute unequally in the processes of myofiber growth and maintenance of the SC pool

Study of nascent NaH in sodium-hydrogen mixture using resonant CARS and FWM processes

The Coherent anti-Stokes Raman Spectroscopy (CARS) technique was applied to measure the O Q, and S branches from the first, second and third overtones of the XICt ground state of NaH. Moreover, P and R doublets were also measured when the anti-Stokes signal was rcsonant with some lines of the electronic transition AIC+- XIC+ of NaH. The high sensitivity resulting from pump laser resonances allowed us to study the Na(nl)+H2 -i NaH+H reaction dynarnics

PEGylation increases antitumoral activity of arginine deiminase of Streptococcus pyogenes

Arginine auxotrophy is a metabolic defect that renders tumor cells vulnerable towards arginine-depleting substances, such as arginine deiminase (ADI) from Streptococcus pyogenes (SpyADI). Previously, we confirmed SpyADI susceptibility on patient-derived glioblastoma multiforme (GBM) models in vitro and in vivo. For application in patients, serum half-life of the enzyme has to be increased and immunogenicity needs to be reduced. For this purpose, we conjugated the S. pyogenes-derived SpyADI with 20 kDa polyethylene glycol (PEG20) moieties, achieving a PEGylation of seven to eight of the 26 accessible primary amines of the SpyADI. The PEGylation reduced the overall activity of the enzyme by about 50% without affecting the Michaelis constant for arginine. PEGylation did not increase serum stability of SpyADI in vitro, but led to a longer-lasting reduction of plasma arginine levels in mice. Furthermore, SpyADI-PEG20 showed a higher antitumoral capacity towards GBM cells in vitro than the native enzyme

Regulation of the activity of lactate dehydrogenases from four lactic acid bacteria.

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (P(i)), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (K(act)) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of P(i) are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of P(i) inhibits E. faecalis LDH2, whereas in the absence of FBP, P(i) is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of P(i) to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of P(i), FBP, and pH that results in different regulatory effects on the LDHs of different LABs