Evidence for operation of nicotinic and muscarinic acetylcholine receptor-dependent survival pathways in human coronary artery endothelial cells

Nicotinic acetylcholine receptors (nAChRs) have recently emerged as critical players in modulation of endothelial function. In particular, studies on endothelial cells from different vascular beds have shown anti-apoptotic actions of nicotinic stimulation, but whether there is actually activation of survival signaling downstream nAChR function has not been explored. In the present work we used human coronary artery endothelial cells (HCAECs) and a pharmacological approach to examine the impact of cholinergic stimulation on survival signaling pathways. Our findings show that cholinergic receptors promote activation of three typical survival routes: the phosphatidyl-inositol-3-kinase (PI3K)/AKT axis, activated downstream muscarinic and nAChRs; the JAK2/STAT3 axis, activated downstream nAChR; and ERK1/2 MAP kinases, activated by both muscarinic acetylcholine receptor (mAChR) and nAChR. Based on their sensitivity to α-bungarotoxin, nicotinic regulation of JAK2/STAT3 and ERK1/2 occurs downstream α7-nAChRs. The present findings suggest that in HCAECs the two cholinergic receptors may act concertedly to induce an efficient survival response of coronary cells when exposed to pro-apoptotic stimuli.     

Rapid, large-scale purification and characterization of 'Ada protein' (O6 methylguanine-DNA methyltransferase) of E. coli.

The E. coli Ada protein (O6-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E280 nm (1%)) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from O6-methylguanine in DNA. Its reaction with O6-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 x 10(9) M-1 min-1 at O degree C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low alpha-helical content and the radius of gyration of 23 A indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.     

Physiological Studies on Thiobacilli

Thiosulfate was oxidized to tetrathionate and an unknown oxidized sulfur compound. The activity was found in the particulate fraction (a precipitate obtained by centrifuging (at 90,000 × g for 60 min) the supernatant of centrifugation at 10,000 × g for 30 min). Treatment with deoxycholate caused inactivation of the enzyme. Considering from these points and electronmicrograph, the enzyme would be located on the cell membrane.     

Untersuchungen zur Tiergesundheit,Leistung und zum Rückstandsverhalten beim Schwein bei gleichzeitiger Aufnahme der Mykotoxine Ochratoxin A,Deoxynivalenol, Zearalenon über das Futter im 90-Tage-Test

The effect of the administration of the mycotoxins OTA, ZEA and DON alone resp. in combination on animal health and the residue behavior of pigs from 50 – 60 kg living weight over 90 days was investigated in 4 separate studies. Due to its fast metabolisation the administration of 1000 µg DON resp. 250 µg ZEA per kg feed alone or in combination with other mycotoxins does not lead to detectable residues of these mycotoxins in organs and tissues. Therefore these mycotoxins should not be relevant to the consumer.There is an effect of the simultaneous administration of ZEA resp. DON on the metabolisation resp. secretion of OTA. OTA is of relevance from the point of view of residue toxicology.     

A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System

Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%–0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process.     

Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans.

A sulfur:ferric ion oxidoreductase that utilizes ferric ion (Fe3+) as an electron acceptor of elemental sulfur was purified from iron-grown Thiobacillus ferrooxidans to an electrophoretically homogeneous state. Under anaerobic conditions in the presence of Fe3+, the enzyme reduced 4 mol of Fe3+ with 1 mol of elemental sulfur to give 4 mol of Fe2+ and 1 mol of sulfite, indicating that it corresponds to a ferric ion-reducing system (T. Sugio, C. Domatsu, O. Munakata, T. Tano, and K. Imai, Appl. Environ. Microbiol. 49:1401-1406, 1985). Under aerobic conditions, sulfite, but not Fe2+, was produced during the oxidation of elemental sulfur by this enzyme because the Fe2+ produced was rapidly reoxidized chemically by molecular oxygen. The possibility that Fe3+ serves as an electron acceptor under aerobic conditions was ascertained by adding o-phenanthroline, which chelates Fe2+, to the reaction mixture. Sulfur:ferric ion oxidoreductase had an apparent molecular weight of 46,000, and it is composed of two identical subunits (Mr = 23,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sulfur oxidation by this enzyme was absolutely dependent on the presence of reduced glutathione. The enzyme had an isoelectric point and a pH optimum at pH 4.6 and 6.5, respectively. Almost all the activity of sulfur:ferric ion oxidoreductase was observed in the osmotic shock fluid of the cells, suggesting that it was localized in the periplasmic space of the cells.     

Malaria transmission dynamics in central Côte d'Ivoire: the influence of changing patterns of irrigated rice agriculture

The dynamics of malaria transmission was studied comparatively in the villages of Zatta and Tiemelekro, central Cote d'Ivoire, from February 2002 to August 2003. Prominent agroecosystems in these villages are irrigated rice growing and vegetable farming, respectively. Mosquitoes (Diptera: Culicidae) were collected on human bait at night and by pyrethrum knock-down spray sheet collections at four randomly selected sentinel sites in each village. In 2002, for a total of 96 man-nights per village, 7716 mosquitoes were collected in Zatta and 3308 in Tiemelekro. In 2003, with half the sampling effort, 859 and 2056 mosquitoes were collected in Zatta and Tiemelekro, respectively. Anopheles gambiae Giles s.l. was the predominant mosquito and the key malaria vector throughout, followed by An. funestus Giles. Anthropophily among adult female Anopheles exceeded 95% in both villages. Comparison between years revealed that the biting rate of An. gambiae s.l. in Zatta decreased several-fold from 49.3 bites per person per night (b/p/n) in 2002 to 7.9 b/p/n in 2003 (likelihood ratio test (LRT) = 1072.66; P < 0.001). Although the biting rate remained fairly constant in Tiemelekro, the difference between years was significant (16.1 vs. 18.2 b/p/n; LRT = 148.06; P < 0.001). These observations were paralleled by a marked decrease in the infective rate of An. gambiae s.l. in Zatta (4.6-1.2%), and an increase in Tiemelekro (3.1-7.6%). Meanwhile, the entomological inoculation rate of An. gambiae s.l. decreased 21-fold in Zatta, from 789 to 38 infective bites per person per year (ib/p/y), whereas it remained high in Tiemelekro (233 vs. 342 ib/p/y). The interruption of irrigated rice growing in Zatta in 2003, consequential to a farmers' conflict over land, might be the underlying cause for the significant reduction in malaria transmission, whereas more stable conditions occurred in Tiemelekro.     

Generation and characterization of cells that can be conditionally depleted of mitochondrial SOD2

Manganese-dependent superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide, and decreased SOD2 activity results in a range of pathologies. To investigate the events occurring soon after depletion of SOD2, we generated SOD2 gene knockout chicken DT40 cells complemented with a human SOD2 (hSOD2) cDNA, whose expression can be switched off by doxycycline (Dox). When SOD2 was depleted by the addition of Dox, the cells grew slightly slower and formed fewer colonies than cells expressing hSOD2. In addition, these cells showed a high sensitivity to paraquat, which produces superoxide, and died through apoptosis. In contrast to results obtained with mouse and DrosophilaSod2 mutants, we found no indication of an increase in DNA lesions due to depletion of SOD2.