Detection of peroxyl and alkoxyl radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

E.s.r. spin trapping using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect peroxyl, alkoxyl and carbon-centred radicals produced by reaction of t-butyl hydroperoxide (tBuOOH) with rat liver microsomal fraction. The similarity of the hyperfine coupling constants of the peroxyl and alkoxyl radical adducts to those obtained previously with isolated enzymes suggests that these species are the tBuOO. and tBuO. adducts. The effects of metal-ion chelators, heat denaturation, enzyme inhibitors and reducing equivalents demonstrate that these species arise from reaction of tBuOOH with a haem enzyme such as cytochrome P-450 or cytochrome b5. In the absence of NADPH or NADH the previously undetected peroxyl radical adduct is the major species observed. In the presence of these reducing equivalents the alkoxyl and carbon-centred radical adducts predominate, which is in accord with product studies on similar systems. These results demonstrate that both reductive and oxidative decomposition of tBuOOH can occur in rat liver microsomal fraction with the reductive pathway favoured in the presence of NADH or NADPH.     

Joint report of the Third International Bovine Lymphocyte Antigen (BoLA) Workshop, Helsinki, Finland, 27 July 1986

Two hundred and eighty-two alloantisera were submitted by 20 participating laboratories from 13 countries and tested against lymphocytes of 1298 cattle. The cell panel consisted of samples from 38 Bos taurus breeds, 11 Bos taurus crossbreeds, 4 Bos indicus breeds, 6 Bos taurus x Bos indicus, and a variety of other crossbred populations. Using a standardized lymphocytotoxicity test, all 17 previously identified BoLA specificities were confirmed. The workshop produced agreement on 16 new lymphocyte alloantigenic specificities. Three of the new specificities behaved as splits of previously identified BoLA specificities. Four of the new specificities behaved as alleles at the agreed BoLA-A locus. Seven new specificities are tentatively assigned to the BoLA-A locus but require further definition. Two new specificities may represent products of a second closely-linked BoLA locus.     

Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene.

Human neutrophils and HL-60 leukaemic cells possess an NADPH oxidase which catalyses superoxide (O2-) formation and is activated by the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). In dibutyryl cyclic AMP-differentiated HL-60 cells, ATP and UTP in the presence of cytochalasin B activated O2- formation with EC50 values of 5 microM and efficacies amounting to 30% of that of fMet-Leu-Phe. The potency order of purine nucleotides in activating O2- generation was ATP = adenosine 5'-O-(3-thiotriphosphate) greater than ITP greater than dATP = ADP. Pyrimidine nucleotides activated NADPH oxidase in the potency order UTP greater than dUTP greater than CTP = TTP = UDP. Pertussis toxin completely prevented activation of NADPH oxidase by fMet-Leu-Phe and UTP, whereas the effect of ATP was only partially inhibited. ATP and UTP enhanced O2- generation induced by fMet-Leu-Phe by up to 8-fold, and primed the cells to respond to non-stimulatory concentrations of fMet-Leu-Phe. Activation of NADPH oxidase by UTP but not by ATP was inhibited by various activators of adenylate cyclase. In dimethyl sulphoxide-differentiated HL-60 cells and in human neutrophils, ATP and UTP per se did not activate NADPH oxidase, but they potentiated the effect of fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via purino- and novel pyrimidinoceptors respectively, which are coupled to guanine nucleotide-binding proteins leading to the activation of NADPH oxidase. As ATP and UTP are released from cells under physiological and pathological conditions, these nucleotides may play roles as intercellular signal molecules in the activation of O2- formation.     

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.     

The potential role of human kidney cortex cysteine proteinases in glomerular basement membrane degradation

(1) The degradation of glomerular basement membrane and some of its constituent macromolecules by human kidney lysosomal cysteine proteinases has been investigated. Three cysteine proteinases were extracted from human renal cortex and purified to apparent homogeneity. These proteinases were identified as cathepsins B, H and L principally by their specific activities towards Z-Arg-Arg-NHMec, Leu-NNap and Z-Phe-Arg-NHMec, respectively, and their Mr on SDS-polyacrylamide gel electrophoresis under reducing conditions. (2) Cathepsins B and L, at acid pH, readily hydrolysed azocasein and degraded both soluble and basement membrane type IV and V collagen, laminin and proteoglycans. Their action on the collagens was temperature-dependent, suggesting that they are only active towards denatured collagen. Cathepsin L was more active in degrading basement membrane collagens than was cathepsin B but qualitatively the action of both proteinases were similar, i.e., at below 32 degrees C the release of an Mr 400,000 hydroxyproline product which at 37 degrees C was readily hydrolysed to small peptides. (3) In contrast, cathepsin H had no action on soluble or insoluble collagens or laminin but did, however, hydrolyse the protein core of 35S-labelled glomerular heparan sulphate-rich proteoglycan. (4) Thus renal cysteine proteinases form a family of enzymes which together are capable of degrading the major macromolecules of the glomerular extracellular matrix.     

Sensitivity of the cell cycle to TGFβ1 does not correlate with transformation of a rat liver epithelial cell line

The effects of TGF1 on cell cycle events in a rat liver derived epithelial cell line (BL9) and in two in vitro transformants of this line were studied by flow cytometry. Using either ethidium bromide staining or the incorporation of bromodeoxyuridine to evaluate DNA synthesis it was shown that TGF1 prevented the entry of G0/G1 phase BL9 cells into S phase. TGF1 did not exert its inhibitory effect(s) on DNA synthesis by the modulation of early events in the cell cycle. The tumorigenic transformed BL9 cell lines gave contrasting responses to the effects of TGF1. DNA synthesis in a BL9 cell line derived by transfection with an active N-ras oncogene was unaffected by TFG1 and thus appeared refractory to its growth controlling effects. On the other hand cells from a BL9 cell line derived by in vitro transformation with activated aflatoxin B₁ retained their sensitivity to the effects of TGF1. Thus the loss of the inhibitory effect of TGF1 on DNA synthesis is not obligatory for the malignant transformation of rat liver epithelial cells.Abbreviations TGF1 transforming growth factor 1 - BSA bovine serum albumin - FBS foetal bovine serum - BrdUrd bromodeoxyuridine - PI propidium iodide - PBS phosphate buffered saline