Species differences in cytotoxic and genotoxic effects of phenacetin and paracetamol in primary monolayer cultures of hepatocytes

The cytotoxic and genotoxic effects of phenacetin and paracetamol were examined in monolayer cultures of hepatocytes isolated from the mouse, hamster, rat and guinea pig. No marked increase in unscheduled DNA synthesis (UDS) after exposing hepatocytes from any of the species to phenacetin was observed. At cytotoxic concentrations of paracetamol, an increased UDS in mouse hepatocytes in vitro was observed. Pretreatment of the mice by inducers of drug-metabolizing enzymes, such as 3-methylcholanthrene and Aroclor 1254, lowered the concentration threshold for the toxic responses. With rat hepatocytes only a minor increase in UDS was noted, while with hepatocytes from hamsters and guinea pigs in fact a decrease was seen. The narrow range observed between the cytotoxic and genotoxic effects of paracetamol makes it difficult to predict whether the initial DNA damage could lead to a mutation or whether the cells will die before the mutation is expressed. With respect to the cytotoxic effects, hamster hepatocytes were found to be most susceptible to paracetamol, followed by mouse, while rat and guinea pig were less affected. These data were in accordance with in vivo findings (Davis et al., 1974), indicating the potential value of hepatocyte culture when screening for possible liver toxic substances.     

Interaction of tumour promoters with epithelial cells in culture : An immunofluorescence study

12-O-tetradecanoyl phorbol-13-acetate (TPA) has a profound and rapid influence on the cytoskeleton of Madin-Darby Canine Kidney (MDCK) cells. Within 10 min, TPA induces a rapid change in morphology, from a flat, cuboidal state to a rounded or elongated morphology in which the cell membranes become convoluted. Concomitant with this morphological change is a rapid dissolution of stress fibres and a redistribution of F-actin from microfilament bundles to a membrane or sub-membranous location. The rearrangement of actin is paralleled by a rearrangement of alpha-actinin and a reduction in the number of vinculin-containing adhesion plaques. Unusual F-actin configurations are often found emanating from a perinuclear location, usually containing alpha-actinin and terminating in a vinculin-containing adhesion plaque. The cytoskeletal rearrangements occur in the presence of inhibitors of protein synthesis or oxidative phosphorylation, but do not occur if glycolysis is also inhibited. The rearrangements are partly abrogated by the presence of cytochalasin B (CB). Despite these dramatic changes in microfilaments the polymerization state of actin remained unaltered after TPA treatment. Furthermore, although changes in the movement of membrane lipids have been reported, no obvious differences in the ability of glycoproteins to redistribute in the plane of the membrane were found as judged by FITC-concanavalin A (conA) induced patching. The rapidity of the morphological response of MDCK cells to TPA indicates that the cytoskeleton is one of the primary targets of TPA, but that tumour promoters differ from RNA tumour viruses in their effect on the state of actin polymerization.     

Influence of O Side Chains on the Attachment of the Felix O-1 Bacteriophage to Salmonella Bacteria

The adsorption rate constant (ARC) of the Felix O-1 (FO) bacteriophage to sensitive Salmonella strains was used to determine the effect of variations in surface antigens on phage attachment. The N-acetylglucosamine of the common-core polysaccharide of the Salmonella lipopolysaccharide (LPS) was found to be an essential part of the receptor for the FO phage in conformation with earlier reports. It was found that (i) the ARC was low for strains having O side chains containing two or three non core monosaccharides, (ii) the ARC varied when the O side chain contained no, or only one, noncore monosaccharide, (iii) the ARC was high when the O side chain contained only one repeating unit, and (iv) the ARC was high to mutants of chemotype Ra in which the N-acetylglucosamine was the terminal sugar of the LPS. Since a good correlation was found between the ARC of the FO phage and the phage-inactivating capacity of phenol water-extracted LPS, the results suggest that only the structure and composition of the LPS determines the adsorption rate of the FO phage. The phage-inactivating capacity of LPS from the Ra mutants increased in parallel with higher glucosamine contents in the core polysaccharide. In smooth strains having long and numerous O side chains, the access of the FO phage to its receptor is probably blocked by the presence of the side chains, whereas short and numerous side chains or T1 side chains do not interfere with the FO attachment.     

Segregation and manifestations of the mtDNA tRNA(Lys) A-->G(8344) mutation of myoclonus epilepsy and ragged-red fibers (MERRF) syndrome.

We have studied the segregation and manifestations of the tRNA(Lys) A-->G(8344) mutation of mtDNA. Three unrelated patients with myoclonus epilepsy and ragged-red fibers (MERRF) syndrome were investigated, along with 30 of their maternal relatives. Mutated mtDNA was not always found in the offspring of women carrying the tRNA(Lys) mutation. Four women had 10%-33% of mutated mtDNA in lymphocytes, and no mutated mtDNA was found in 7 of their 14 investigated children. The presence of mutated mtDNA was excluded at a level of 3:1,000. Five women had a proportion of 43%-73% mutated mtDNA in lymphocytes, and mutated mtDNA was found in all their 12 investigated children. This suggests that the risk for transmission of mutated mtDNA to the offspring increases if high levels are present in the mother and that, above a threshold level of 35%-40%, it is very likely that transmission will occur to all children. The three patients with MERRF syndrome had, in muscle, both 94%-96% mutated mtDNA and biochemical and histochemical evidence of a respiratory-chain dysfunction. Four relatives had a proportion of 61%-92% mutated mtDNA in muscle, and biochemical measurements showed a normal respiratory-chain function in muscle in all cases. These findings suggest that > 92% of mtDNA with the tRNA(Lys) mutation in muscle is required to cause a respiratory-chain dysfunction that can be detected by biochemical methods. There was a positive correlation between the levels of mtDNA with the tRNA(Lys) mutation in lymphocytes and the levels in muscle, in all nine investigated cases. The levels of mutated mtDNA were higher in muscle than in lymphocytes in all cases. In two of the patients with MERRF syndrome, muscle specimens were obtained at different times. In both cases, biochemical measurements revealed a deteriorating respiratory-chain function, and in one case a progressive increase in the amount of cytochrome c oxidase-deficient muscle fibers was found.     

Lack of transmission of deleted mtDNA from a woman with Kearns-Sayre syndrome to her child.

We have investigated the daughter of a woman with Kearns-Sayre syndrome. The woman had a high percentage of deleted mtDNA in muscle, but no deleted mtDNA was detected in fibroblasts, bone marrow, and peripheral blood cells by Southern blot analysis. With PCR, analytical sensitivity was significantly increased, and deleted mtDNA was detected in all examined tissues from this patient. The patient had healthy parents and nine healthy siblings. No deleted mtDNA was detected in blood from the mother of the patient. The patient had an uneventful pregnancy and delivered at term. Deleted mtDNA could not be detected in placenta by Southern blot analysis. With PCR, deleted mtDNA was detected in the majority of placental specimens. This finding may, however, be due to contamination with maternal DNA. The patient's daughter was healthy at age 5 mo, and morphologic examination of muscle was normal. No transmission of deleted mtDNA to the daughter could be detected by Southern blot and PCR analysis of peripheral blood cells, bone marrow, fibroblasts, and muscle. The presence of deleted mtDNA was excluded at a fractional level of less than 1:100,000 in all examined tissues from the daughter.     

Biological activity of leukotriene sulfones on respiratory tissues

The biological activity of synthetic leukotriene C4, D4 and E4 sulfone has been determined in respiratory smooth muscle in vitro and in vivo. The sulfones of LTC4, LTD4 and LTE4 were potent contractile agonists on indomethacin-treated guinea pig tracheal chains with respective pD2-values of 8.2, 8.0 and 7.9. Contractions were submaximal (75-85% of the cholinergic maximum), slow in onset, prolonged in duration, slowly reversed by washing (compared to acetylcholine or histamine) and were partially reversed by 2 muM FPL-55712. The sulfones of LTC4, LTD4 and LTE4 also contracted indomethacin-treated guinea pig parenchyma (respective pD2's of 7.9 8.2 and 7.8) and rat parenchyma (respective pD2's of 7.1, 7.2 and 7.2) but were inactive on rat trachea (0.01-2.0 muM). When administered intravenously to anaesthetized guinea pigs, the sulfones of LTD4, LTE4 and to a lesser degree LTC4 (respective ED50's - 0.5; 2.0 and 4.6 microgram/kg) elicited dose-dependent increases in inflation pressure which were antagonized by FPL-55712 and indomethacin. Leukotriene C4, D4 and E4 sulfones display a qualitatively similar profile of biological activity to that of their corresponding sulfides.     

A biometric study of some hybridizing Crataegus populations in Denmark

Crataegus curvisepala Lindm., C. laevigata (Poiret) DC. and C. monogyna Jacq. (Rosaceae) form hybrid complexes in Denmark due to introgression. C. palmstruchii Lindm. seems to be variously introgressed individuals of C. laevigata. C. eremitagensis Raunk., C. raavadensis Raunk. and C. schumacheri Raunk. apparently belong to C. curvisepala x laevigata. The delimitation between C. curvisepala x laevigata and C. laevigata x monogyna is discussed.     

Bursty Communication Patterns Facilitate Spreading in a Threshold-Based Epidemic Dynamics

Records of social interactions provide us with new sources of data for understanding how interaction patterns affect collective dynamics. Such human activity patterns are often bursty, i.e., they consist of short periods of intense activity followed by long periods of silence. This burstiness has been shown to affect spreading phenomena; it accelerates epidemic spreading in some cases and slows it down in other cases. We investigate a model of history-dependent contagion. In our model, repeated interactions between susceptible and infected individuals in a short period of time is needed for a susceptible individual to contract infection. We carry out numerical simulations on real temporal network data to find that bursty activity patterns facilitate epidemic spreading in our model.     

Small molecule sensitization to TRAIL is mediated via nuclear localization,phosphorylation and inhibition of chaperone activity of Hsp27

The small chaperone protein Hsp27 confers resistance to apoptosis, and therefore is an attractive anticancer drug target. We report here a novel mechanism underlying the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitizing activity of the small molecule {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, an inactive analog of the phosphoinositide 3-kinase inhibitor inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, in HeLa cells that are refractory to TRAIL-induced apoptosis. On the basis of the fact that {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511 is derived from {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, itself derived from quercetin, and earlier findings indicating that quercetin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 affected Hsp27 expression, we investigated whether {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511 sensitized cancer cells to TRAIL via a conserved inhibitory effect on Hsp27. We provide evidence that upon treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, Hsp27 is progressively sequestered in the nucleus, thus reducing its protective effect in the cytosol during the apoptotic process. {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511-induced nuclear translocation of Hsp27 is linked to its sustained phosphorylation via activation of p38 kinase and MAPKAP kinase 2 and the inhibition of PP2A. Furthermore, Hsp27 phosphorylation leads to the subsequent dissociation of its large oligomers and a decrease in its chaperone activity, thereby further compromising the death inhibitory activity of Hsp27. Furthermore, genetic manipulation of Hsp27 expression significantly affected the TRAIL sensitizing activity of {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, which corroborated the Hsp27 targeting activity of {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511. Taken together, these data indicate a novel mechanism of small molecule sensitization to TRAIL through targeting of Hsp27 functions, rather than its overall expression, leading to decreased cellular protection, which could have therapeutic implications for overcoming chemotherapy resistance in tumor cells.