Localization of deletion breakpoints in radiation-induced mutants of the hprt gene in hamster cells

DNA was analysed from a large set of hamster hprt gene mutants, some induced by ionising radiations and others occurring naturally, to identify those with large alterations in part of the gene. DNA from these mutants was restricted further with different endonucleases and probed to establish the patterns of restriction fragments remaining. Of 15 mutants characterized, one showed a duplication of part of the 5' end of the gene, and the remainder showed deletions of various sizes. It was possible to approximately locate the breakpoints of the deletions by comparison of fragment patterns to a recently-established map of the hamster gene. The relatively small number of mutants examined precludes rigorous analysis of the distribution of breakpoints in the hprt gene, but taken with other recent studies of deletion mutagenesis it is suggested that non-random induction or selection of this type of mutation may occur.     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.     

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT₁ receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT₁ receptor subtype. Pretreatment with the specific AT₄ receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT₁ receptor subtype.     

The ability of the biological control agent Bacillus subtilis, strain BB, to colonise vegetable brassicas endophytically following seed inoculation

The ability of Bacillus subtilis, strain BB, to colonise cabbage seedlings endophytically was examined following seed inoculation. Strain BB was recovered from different plant parts including leaves (cotyledons), stem (hypocotyl) and roots. While high bacterial populations persisted in the roots and lower stem, they were lower in the upper stem and leaves through time. In addition to cabbage, strain BB colonised endophytically the roots of 5 other vegetable brassicas. Fatty acid methyl ester (FAME) and PCR fingerprinting analysis confirmed the reliability of the detection method. Studies conducted with transmission electron microscope (TEM) showed that BB mainly colonised intercellular spaces of cortical tissues including intercellular spaces close to the conducting elements of roots and stem of cabbage seedlings. Gold labelling was specifically associated with BB and the fibrillar material filling the intercellular spaces where bacterial cells were found.     

Species-dependent features of Dogiel type II neurones in the mammalian enteric nervous system.

Although autonomic gastrointestinal reflex movements, which occur in all mammalian species, have been described almost a century ago, little was known on the mechanisms underlying this behaviour. Recently, however, intrinsic primary afferent neurones, functioning as the first relay in the reflex arches embedded in the intestinal wall, have been identified in the guinea pig ileum. In guinea pig, such neurones display a Dogiel type II morphology and behave electrophysiologically as slow AHP neurones. In other gastrointestinal regions, in both guinea pig and rat, Dogiel type II cells are also encountered, but the strong correlation with slow AHP neuronal features seems less strict. In large mammals, a correlation of the cellular morphology with intracellular el ectrophysiological recordings has only been obtained in the pig small intestine. Surprisingly, in these experiments aberrant electrophysiological behaviour of Dogiel type II neurones is even more striking since the majority of these cells display electrophysiological features considered typical of S neurones. Furthermore, in those rare cases in which a slow afterhyperpolarization (AHP) could be recorded in porcine Dogiel type II cells, its amplitudes were negligible. This has led us to the conclusion that the differences in electrophysiological behaviour of neurones with comparable morphology in different species are most probably due to the modulating influence of the neurotransmitter substances present. This seems to be the most likely hypothesis in view of the considerable differences in neurotransmitter content of neurones with comparable functions throughout the species.     

The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum reveals its substrate and reaction specificity.

Cystathionine gamma-synthase catalyses the committed step of de novo methionine biosynthesis in micro-organisms and plants, making the enzyme an attractive target for the design of new antibiotics and herbicides. The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum has been solved by Patterson search techniques using the structure of Escherichia coli cystathionine gamma-synthase. The model was refined at 2.9 A resolution to a crystallographic R -factor of 20.1 % (Rfree25.0 %). The physiological substrates of the enzyme, L-homoserine phosphate and L-cysteine, were modelled into the unliganded structure. These complexes support the proposed ping-pong mechanism for catalysis and illustrate the dissimilar substrate specificities of bacterial and plant cystathionine gamma-synthases on a molecular level. The main difference arises from the binding modes of the distal substrate groups (O -acetyl/succinyl versusO -phosphate). Central in fixing the distal phosphate of the plant CGS substrate is an exposed lysine residue that is strictly conserved in plant cystathionine gamma-synthases whereas bacterial enzymes carry a glycine residue at this position. General insight regarding the reaction specificity of transsulphuration enzymes is gained by the comparison to cystathionine beta-lyase from E. coli, indicating the mechanistic importance of a second substrate binding site for L-cysteine which leads to different chemical reaction types.