Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro

BACKGROUND: The effect of non-steroidal anti-inflammatory drugs (NSAIDs) for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated. METHODS: Whole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay. RESULTS: There were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol. CONCLUSIONS: Accordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets.     

Temporal progression and extent of the return of sensation in the foot provided by the saphenous nerve after sciatic nerve transection and repair in the rat - implications for nociceptive assessments

Sensory testing, by providing stimuli for nociceptors of the foot, is a popular method of evaluating sensory regeneration after damage to the sciatic nerve in the rat. In the following study, 20 rats were submitted to double transection of the sciatic nerve. The subsequent 14 mm gap was repaired through guidance interponation. In order to evaluate nerve regeneration, sensory testing was performed additionally to other methods, which included motor testing, morphometry, and electron microscopic assessments of nerves. Somatosensory testing revealed that all animals exhibited next to the same amount of sensory reinnervation on their foot regardless of their experimental group. In motor tests, however, two out of the three experimental groups did not improve at all. These groups also failed to show neural regrowth in morphometric and electron microscopic assessments of the associated nerve. Retrograde tracing was able to prove the saphenous nerve as an alternative source of sensory reinnervation in animals with failed sciatic regeneration. This means that results of sensory testing in the rat should be treated with caution, taking into account the areas tested and the likelihood that in these areas saphenous sprouting could have taken place. Furthermore, it is strongly advised that somatosensory testing should be conducted only on toe 5.     

New and fast method to quantify respiration rates of bacterial and plankton communities in freshwater ecosystems by using optical oxygen sensor spots

A new method of respiration rate measurement based on oxygen luminescence quenching in sensor spots was evaluated for the first time for aquatic bacterial communities. The commonly used Winkler and Clark electrode methods to quantify oxygen concentration both require long incubation times, and the latter additionally causes signal drift due to oxygen consumption at the cathode. The sensor spots proved to be advantageous over those methods in terms of precise and quick oxygen measurements in natural bacterial communities, guaranteeing a respiration rate estimate during a time interval short enough to neglect variations in organism composition, abundance, and activity. Furthermore, no signal drift occurs during measurements, and respiration rate measurements are reliable even at low temperatures and low oxygen consumption rates. Both a natural bacterioplankton sample and a bacterial isolate from a eutrophic river were evaluated in order to optimize the new method for aquatic microorganisms. A minimum abundance of 2.2 x 10(6) respiring cells ml(-1) of a bacterial isolate was sufficient to obtain a distinct oxygen depletion signal within 20 min at 20 degrees C with the new oxygen sensor spot method. Thus, a culture of a bacterial isolate from a eutrophic river (OW 144; 20 x 10(6) respiring bacteria ml(-1)) decreased the oxygen saturation about 8% within 20 min. The natural bacterioplankton sample respired 2.8% from initially 94% oxygen-saturated water in 30 min. During the growth season in 2005, the planktonic community of a eutrophic river consumed between 0.7 and 15.6 micromol O(2) liter(-1) h(-1). The contribution of bacterial respiration to the total plankton community oxygen consumption varied seasonally between 11 and 100%.     

Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3. Major differences in substrate specificity between eukaryotic and bacterial homologs

Sequence alignments of human molybdopterin synthase sulfurase, MOCS3, showed that the N-terminal domain is homologous to Escherichia coli MoeB, whereas the C-terminal domain is homologous to rhodanese-like proteins. Previous studies showed that the activity of the separately purified rhodanese-like domain of MOCS3 displayed 1000-fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. When the six amino acid active site loop of MOCS3 rhodanese-like domain was exchanged with the loop found in bovine rhodanese, thiosulfate:cyanide sulfurtransferase activity was increased 165-fold. Site-directed mutagenesis of each individual residue of the active site loop of the MOCS3 rhodanese-like domain showed that the charge of the last amino acid determines thiosulfate sulfurtransferase activity. Replacing Asp417 by threonine resulted in 90-fold increased activity, whereas replacing it by arginine increased the activity 470-fold. Using a fully defined in vitro system containing precursor Z, MOCS2A, E. coli MoaE, E. coli MoeB, Mg-ATP, MOCS3 rhodanese-like domain, and thiosulfate, it was shown that sulfur transfer to MOCS2A was also affected by the alterations, but not as drastically. Our studies revealed that in humans and most eukaryotes thiosulfate is not the physiologic sulfur donor for MOCS3, whereas in bacterial homologs, which have an arginine at the last position of the active site loop, thiosulfate can be used as a sulfur source for molybdenum cofactor biosynthesis. The phylogenetic analysis of MoeB homologs showed that eukaryotic homologs are of bacterial origin. Furthermore, it could be shown that an MoeB homolog named MoeZ, where the dual CXXC zinc-binding motif of the MoeB domain is not present, arose independently several times during evolution.     

Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genes cop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate,a component of the mycolic acid layer of the cell envelope

By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C. glutamicum strain ATCC 17965. The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 ( cmt from corynebacterium mycolyl transferases). cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids. Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains. Proteome analyses of the cell envelope of C. glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4. All six mycolyltransferase genes were used for mutational analysis. cmt4 could not be mutated and is considered to be essential. cop1 was found to play an additional role in cell shape formation. A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium. This mutant was also no longer able to synthesize trehalose di coryno mycolate (TDCM). Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis. The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.     

Contact angle, WAXS, and SAXS analysis of poly(beta-hydroxybutyrate) and poly(ethylene glycol) block copolymers obtained via Azotobacter vinelandii UWD

This study investigated and correlated physical properties and cell interactions of copolymers obtained by a poly(ethylene glycol) (PEG)-modulated fermentation of Azotobacter vinelandii UWD. PEGs with molecular weights of 400 and 3400 Da and di(ethylene glycol) (DEG) were used to modulate the bacterial synthesis of poly(beta-hydroxybutyrate) (PHB). The PHB crystallinity was determined by wide-angle X-ray scattering (WAXS). Small-angle X-ray scattering (SAXS) showed that lamellar distances decreased between the PHB and the PHB modulated with PEG or DEG. Furthermore, the contact angle of water on the PHB/PEG polymer surfaces decreased when compared to that of PHB. The significant decrease of the contact angle and corresponding increase in surface tension, as well as significant decrease in cell adhesion, suggest the presence of hydrophilic PEG and DEG within the hydrophobic surface.