Bacterial extract cantastim activates macrophages via TLR-2

CANTASTIM is a second generation bacterial immunomodulator. The aim of this study was to examine the mechanism by which bacterial immunomodulator CANTASTIM induces production of inflammatory cytokines in monocytes/macrophages. Proinflammatory cytokines were induced in PMA-differentiated THP-1 cells by stimulation with TLR agonists and CANTASTIM in the presence or absence of anti-TLR blocking antibodies or isotype matched control antibodies. Also, RNA interference was used to knockdown TLR2 or TLR4 expression in PMA-differentiated THP-1 cells before stimulation. As expected, induction of TNF-alpha and IL-6 by TLR4 agonist LPS was inhibited in a significant manner by anti-TLR4 but not by anti-TLR2 antibody. Unexpectedly, treatment with anti-LR2 blocking antibody inhibited only IL-6 production induced by Pam3CSK4 while the level of TNF-alpha was unchanged. When cells were stimulated by TLR2 agonist heat-killed Listeria monocytogenes the release of TNF-alpha was significantly attenuated by anti-TLR2 antibodies. Silencing of TLR2 led to a statistically significant inhibition of TNF-alpha secretion induced by TLR2 agonist while siRNA silencing of TLR4 did not affect the response to TLR2 agonist. Cells exposed to CANTASTIM produced significant levels of pro-inflammatory cytokines but the levels were lower than LPS-stimulated cells. Production of both cytokines was inhibited by treatment with anti-TLR2 blocking antibody and not by anti-TLR4 antibody. Silencing of TLR2 led to a statistically significant inhibition of TNF-a secretion induced by CANTASTIM while silencing of TLR4 had no effect on the response to CANTASTIM. These results support the hypothesis that CANTASTIM may exert its immunomodulatory and adjuvant activities through interaction of its bacterial components with TLR2.     

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH?HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).     

Rapid ascorbate response to bacterial elicitor treatment in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> cells

An early event of the incompatible plant–pathogen interactions is an oxidative burst. On one hand, the ROS generated during oxidative burst is advantageous. ROS can serve as secondary messengers mediating defence gene activation and establishment of additional defences. On the other hand, the concentration of ROS must be carefully regulated to avoid undesired cellular cytotoxicity. The major water soluble, low molecular weight antioxidant, ascorbic acid plays a crucial role in ROS balancing (scavenging). The regulation of ascorbate level, therefore, can be an important point of the fine-tuning of ROS level during the early phase of plant–pathogen interaction. To evaluate how this interaction affects the biosynthesis, the recycling, and the level of ascorbate, we challenged Arabidopsis thaliana cells with two different harpin proteins (HrpZ_pto and HrpW_pto). HrpZ_pto and HrpW_pto treatments caused a well-defined ROS peak. The expression of the alternative oxidase (AOX1a) and vtc5, one of the paralog genes that encode the rate limiting enzyme of ascorbate biosynthesis, followed the elevation of ROS. Similarly, the activity of ascorbate peroxidase and galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) (GLDH), the enzyme catalysing the ultimate, mitochondria coupled step of ascorbate biosynthesis and the level of ascorbate and glutathione also followed the elevation of ROS due to harpin treatment. The enhanced expression of AOX1a, the elevated activity of GLDH, and the increased level of ascorbate and glutathione all can contribute to the mitigation or absence of programmed cell death. Finally, a new function, the fine-tuning of redox balance during plant–pathogen interaction, can be proposed to vtc5.     

Effects of Chromium Picolinate on Micronucleus Frequency and Morphology of Lymphocytes in Calves

We report the effects of chromium picolinate (CrPic) on micronucleus frequency, morphology of lymphocytes, and lipid peroxidation in calves. Twenty-four Holstein calves were selected for the study. They were kept in a farm and were fed a commercially available calf diet and alfalfa, ad libitum. The animals were divided into three groups of eight subjects each and were treated as follows: The first group was supplemented with a daily dose of 200 μg Cr as chromium picolinate; a second group received 400 μg Cr per day and a third group that served as control received no supplemental chromium. After 12-week supplementation, blood samples were collected to determine the micronucleus frequency, the apoptotic cell percentage, and the malondialdehyde (MDA) and blood chromium levels. In both supplemented groups, the cells had irregularly shaped and segmented nuclei. Supplementation also increased the percentage of apoptotic cells (p < 0.001) and serum MDA (p < 0.01) and slightly increased the chromium levels. The animals supplemented with 400 μg showed a significant increase of micronucleus frequency (p < 0.01). The results of this study suggest that supplementation with 200 and 400 μg chromium as chromium picolinate may lead to cytotoxicity. The higher level of supplementation may also have genotoxic effects. However, further studies investigating the mechanism of the action of CrPic are required.     

Über die Bildung von Bakterienhemmstoffen durch Cosmarium impressulum

1. An already published test method for detecting bactericidal substances in paper chromatograms was further improved. 2. In cultures of Cosmarium impressulum free from bacteria, two bactericidal substances were found in the ether extracts from the algae and two others in the extracts of the culture medium. The are active against some or all bacteria testes (Serratia marcescens, Pseudomonas fluorescens; Aerobacter aerogenes or Bacillus pumilus). 3. If the culture medium of Cosmarium or another desmidiale was inoculated with the test bacteria, a clear bactericidal effect was never observed. 4. Because the activity of the bactericidal substances of Cosmarium is only small and the formation is not constant, it is concluded that the water-soluble bactericidal substances of the alga are not the cause that epiphytic bacteria do not grow normally on Cosmarium.     

Informationstheoretisches lernmodell

Summary The formation of the interconnection stimulus-response in a learning system is analysed. The system, a technical or a biological one establishes this correspondence by processing the information fed back from the medium during the learning process. This information has two aspects: a quantitative one related to the probability of the events, and a qualitative one related to the utility of the events in view of a goal. Both aspects are taken into consideration; by successive experiences the system eliminates the double uncertainty concerning the probabilistic dependence: stimulu — sresponse — outcome and its utility. Learning implies then a system for evaluating the utilities of different outcomes in view of a goal, a memory to record them and a decision system for selecting the corresponding responses upon a given criterion.

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Der Beitrag stellt einen Teil der Untersuchungen dar, die zur Ausarbeitung der Doktor-Dissertation am Polytechnischen Institut Gheorghe Gheorghiu-Dej in Bukarest unternommen wurden.