Antagonistic effect of selenite on tumor promoter induced cell proliferation in cultures of rat tongue epithelium

In cultures of rat tongue epithelial cells, cell proliferation following incubation with different doses of the potent tumor promoter TPA has been studied by using a stathmokinetic method counting colchicine arrested metaphases. It was demonstrated that 24 h incubation with concentrations higher than 5 ng TPA/mL medium caused inhibition, whereas below 5 ng TPA/mL medium caused stimulation of the mitotic activity reaching a maximum around 30 h from the start of the incubation period. Based on the evidence of the anticarcinogenic effect of selenium in several animal models, experiments have been performed elucidating the influence of an atoxic dose (1/1.000.000M) of selenite on the observed TPA-induced cell proliferation. Our results indicate that addition to the culture medium of an atoxic dose of selenite, not affecting the mitotic activity of control cultures, inhibits the TPA-induced stimulation of cell proliferation.     

Hepatic 7 alpha-dehydroxylation of bile acid intermediates, and its significance for the pathogenesis of cerebrotendinous xanthomatosis

The bile acid precursor 7 alpha-hydroxy-4-cholesten-3-one was found to be enzymatically dehydroxylated at a slow rate by liver tissues from the rat, human, and guinea pig. The rat liver enzyme is localized in the microsomal fraction, has a pH optimum of about 8.5, an apparent Km of 0.03-0.04 mM, and a Vmax of 10-15 nmoles.mg protein-1.hr-1. The product from 7 alpha-hydroxy-4-cholesten-3-one was identified as cholesta-4,6-dien-3-one by its chromatographic properties and by mass spectrometry. The reaction proceeded both in air and N2, and pyridine nucleotides were not required as cofactors. In addition to the enzymatic reaction, there was a significant nonenzymatic dehydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, in particular at high pH and with high concentrations of protein. No 7 alpha-dehydroxylation occurred with various 7 alpha-hydroxylated 3 beta-hydroxy-delta 5-steroids. We have previously shown that at least part of the accumulation of cholestanol in cerebrotendinous xanthomatosis (CTX) is due to accelerated 7 alpha-dehydroxylation of bile acid intermediate(s), which are further converted into cholestanol. The capacity to dehydroxylate 7 alpha-hydroxy-4-cholesten-3-one was found to be about the same in homogenates of liver biopsies from two patients with CTX as in preparations from control subjects. It is suggested that increased levels of substrate (7 alpha-hydroxy-4-cholesten-3-one) in the liver, rather than increased amounts of 7 alpha-dehydroxylase is the explanation for the accelerated 7 alpha-dehydroxylation in CTX that leads to increased biosynthesis of cholestanol.     

Studies on the link between HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in rat liver

Under most experimental conditions, there is a covariation between the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, and the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7 alpha-hydroxylase. The most simple explanation for the coupling between the two enzymes is that newly synthesized cholesterol is a substrate for an unsaturated cholesterol 7 alpha-hydroxylase and that substrate availability is of major regulatory importance for this enzyme. The following results seem, however, to rule out that such a simple regulatory mechanism is of major importance and that HMG-CoA reductase activity per se is of importance in the regulation of cholesterol 7 alpha-hydroxylase. 1) The apparent degree of saturation of cholesterol 7 alpha-hydroxylase, as measured in vitro in rat liver microsomes, was found to be relatively high (70-90%) under most experimental conditions, including starvation, cholestyramine treatment, and cholesterol treatment. A significant decrease in the degree of saturation was obtained first after a drastic reduction of total concentration of cholesterol in the microsomes by treatment with high doses of triparanol, an inhibitor of cholesterol biosynthesis. 2) The stimulatory effect of cholesterol feeding on cholesterol 7 alpha-hydroxylase activity in rats seems to be an effect on the enzyme activity (enzyme induction?) rather than an effect on substrate availability. Thus, the stimulatory effect of cholesterol feeding was retained also after almost complete removal of the endogenous cholesterol by extraction with acetone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein L. A novel bacterial cell wall protein with affinity for Ig L chains

A novel Ig-binding protein has been isolated from the surface of bacteria belonging to the anaerobic species Peptococcus magnus. To solubilize the protein, peptococci were treated with different proteolytic enzymes (papain, pepsin, and trypsin) or with mutanolysin, a bacteriolytic agent known to digest the cell walls of streptococci. Papain, trypsin, and mutanolysin all solubilized peptides showing affinity for radiolabeled human IgG in Western blot analysis. Compared with papain and trypsin, mutanolysin liberated a more homogeneous material, which also had a higher m.w. This mutanolysin-solubilized protein (Mr 95 kDa) was obtained highly purified by a single isolation step on IgG-Sepharose, and the molecule was found to exhibit unique Ig-binding properties. Thus, in dot blots and in Western blots, human IgG, F(ab')2 and Fab fragments of IgG, and human kappa and lambda L chains all showed affinity for the protein. Moreover, the molecule also bound human IgM and IgA, whereas no binding was recorded for IgG-Fc fragments or IgG H chains. Finally, the protein bound to human polyclonal Ig L chains immobilized on polyacrylamide beads. These different data demonstrate that the isolated peptococcal protein binds Ig through L chain interaction. The name protein L is therefore suggested for this novel Ig-binding bacterial cell wall protein.     

Cholinesterase Activities in Uterus of Normal and Fenchlorphos Treated Blue Foxes (Alopex Lagopus) during Various Reproductive States

The uterine acetylcholinesterase and total cholinesterase (acetylcholinesterase plus butyrylcholinesterase) activities in normal and fenchlorphos treated blue fox vixens were determined during various reproductive states. AChE and Total-ChE of non-medicated vixens in oestrus were about one half of those in anoestrus. In pregnant uteri (luteal phase) the activities were 25 % and 30% compared to anoestrus. In vixens given 100 mg/kg fenchlorphos for 3 weeks during anoestrus, the remaining activity of AChE in uterus were in average 37%. Pregnant and non-pregnant vixens in the luteal phase medicated prior to mating and during time of implantation, displayed AChE activities which were only moderabely reduced (remaining activities 83% and 72% compared to medicated animals in anoestrus: remaining activity 37%). Plasma ChE-activity increased during pregnancy in the controls while enzyme activity was strongly reduced in animals given 100 mg/kg fenchlorphos daily through the whole pregnancy. It was concluded that the previous reported embryotoxic effect of fenchlorphos in the blue fox did not seem to be directed towards the moderate inhibition of the uterine cholinesterases.     

Detection of proopiomelanocortin mRNA by in situ hybridization,using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

Summary A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (MSH/ACTH[4-11]), ws synthesized and labelled in the 3-end by use of terminal transferase. Probes tailed with either [³H]- or biotin-labelled nucleotides could be used for in situ hydridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidinalkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-brome-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridazation (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC, levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

QALYs, age and fairness

... We can therefore conclude that either we should go for equality; and in that case QALYs are unfair because they haven't got enough of an ageist bias. Or we should accept consequentialism; and in that case QALYs have just the right sort of ageist bias. No plausible case can, however, be made for the claim that QALYs have an unfair bias against old people. Other things being equal we ought when distributing resources essential for survival favour the young. This ethical claim can be supported both by reference to equality (the life-time-view) and by reference to consequentialism (and the premises that resources generally will be more useful when given to young people).     

Mutation analysis and prenatal diagnosis in a Lesch-Nyhan family showing non-random X-inactivation interfering with carrier detection tests

Summary A nonsense mutation at the CpG-site in the codon for Arg(169) in the gene for hypoxanthine phosphoribosyltransferase (hprt) was identified by genomic polymerase chain reaction (PCR) and DNA sequencing in cultured fibroblasts from two brothers with Lesch Nyhan's syndrome. The recurrence of mutation at this CpG-site in several unrelated Lesch-Nyhan families suggests that deamination of 5-methylcytosine is a possible mechanism for mutagenesis. The level of hprt-mRNA in the fibroblasts of the patients was similar to that in healthy controls, whereas hprt-enzyme activity was not detectable. The mutation in this family was also identified in five female relatives and prenatally in a male fetus. Unexpectedly, results from hair follicle analyses and fibroblast selection studies in 8-azaguanine and 6-thioguanine medium showed a non-carrier phenotype in three of the female heterozygotes, whereas X-inactivation mosaicism was demonstrated in one heterozygote. A possible explanation for the apparent non-random X-inactivation in this family is the co-existence of the hprt mutation with an undefined X-linked lethal mutation. This observation is of practical relevance for carrier detection in other Lesch-Nyhan families.     

Drug Stimulated DNA Cleavage Mediated by Cauliflower Topoisomerase II

We have investigated cauliflower (Brassica oleracea) topoisomerase II with respect to its interaction with DNA and demonstrate that the enzyme shares the characteristics of topoisomerase II purified from a variety of phylogenetically remote organisms. In the presence of the 2-nitroimidazole Ro 15-0216, cauliflower topoisomerase II-mediated DNA cleavage is extensively stimulated (approximately 20-fold) only at a site recognized as a major cleavage site for the enzyme in the absence of drug. The conservation of the enzyme's DNA specificity in the presence of Ro 15-0216 is in contrast to the effect exerted by traditional topoisomerase II inhibitors, which cause enzyme-mediated cleavage to take place at a multiple number of DNA sites. Ro 15-0216 may therefore prove useful as a tool in the elucidation of the enzyme's DNA interaction sites and its involvement in nucleic acid metabolism in plant cells.