Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.     

Effects of yohimbine and desipramine on cardiac noradrenaline release and ventricular arrhythmias during acute coronary artery occlusion and reperfusion in anesthetized dogs

Effects of yohimbine (YHMB, an alpha 2-antagonist) and desipramine (DMI, a neuronal uptake inhibitor) were compared on cardiac noradrenaline (NA) release either upon left ansa subclavia nerve stimulation during acute occlusion of the left anterior descending coronary artery (LAD) or upon subsequent LAD reperfusion without stimulation in anesthetized dogs. In control dogs, before LAD occlusion, coronary sinus (CS) NA output increased from 5.4 +/- 1.0 to 26.8 +/- 4.0 ng/min (p less than 0.05) upon stimulation (2 Hz, 30 s). The response to stimulation remained unchanged 25 min after LAD occlusion. During reperfusion 60 min after occlusion, the output of CS-NA and lactate increased from 6.1 +/- 0.8 to 51.3 +/- 19.4 ng/min (p less than 0.05) and from 2.7 +/- 0.5 to 6.7 +/- 1.3 mg/min (p less than 0.05), respectively. In dogs treated with YHMB, the stimulation-induced increase in NA output was potentiated at least fourfold (p less than 0.05) either before or during LAD occlusion, but not during reperfusion. In dogs receiving DMI, stimulation-induced CS-NA output was enhanced to a similar extent (approximately twofold, p less than 0.05) either before or during occlusion, while reperfusion-induced NA output was markedly potentiated by approximately ninefold (p less than 0.05). Maximum dP/dt of left ventricular pressure remained unchanged upon reperfusion in all groups. The total arrhythmic ratio in the drug-treated groups did not significantly differ from the ratio in control dogs upon either stimulation or reperfusion. The data suggest that an abrupt increase in NA output upon reperfusion may result from a washout of NA locally accumulated in the ischemic and (or) peri-ischemic region during the preceding occlusion period, and that NA thus released does not have substantial hemodynamic effects. The results indicate that in the presence of YHMB or DMI, the potentiated increase in NA release in response to either nerve stimulation during LAD occlusion or to reperfusion without stimulation did not aggravate ventricular arrhythmia, most probably owing to the antiarrhythmic properties of these substances.     

The Putative Oncogene Pim-1 in the Mouse: Its Linkage and Variation among t Haplotypes

Pim-1, a putative oncogene involved in T-cell lymphomagenesis, was mapped between the pseudo-alpha globin gene Hba-4ps and the alpha-crystallin gene Crya-1 on mouse chromosome 17 and therefore within the t complex. Pim-1 restriction fragment variants were identified among t haplotypes. Analysis of restriction fragment sizes obtained with 12 endonucleases demonstrated that the Pim-1 genes in some t haplotypes were indistinguishable from the sizes for the Pim-1b allele in BALB/c inbred mice. There are now three genes, Pim-1, Crya-1 and H-2 I-E, that vary among independently derived t haplotypes and that have indistinguishable alleles in t haplotypes and inbred strains. These genes are closely linked within the distal inversion of the t complex. Because it is unlikely that these variants arose independently in t haplotypes and their wild-type homologues, we propose that an exchange of chromosomal segments, probably through double crossingover, was responsible for indistinguishable Pim-1 genes shared by certain t haplotypes and their wild-type homologues. There was, however, no apparent association between variant alleles of these three genes among t haplotypes as would be expected if a single exchange introduced these alleles into t haplotypes. If these variant alleles can be shown to be identical to the wild-type allele, then lack of association suggests that multiple exchanges have occurred during the evolution of the t complex.     

The cytotoxicity of chrysotile asbestos fibers to pulmonary alveolar macrophages I. Effects of inhibitors of ADP-ribosyl transferase

Pulmonary alveoler macrophages exposedto very short chrysotile asbestos fibers present a typical cytotoxic response: extracellular releases of lactate dehydrogenase and -galactosidase, and a decrease in cellular ATP content. The objective of this study was to determine if nicotinamide and 3-aminobenzamide, two inhibitors of the ADP-ribosyl transferase, could modify the in vitro toxicity of chrysotilee fibers. After 30 min of pre-exposure with each of the two inihibitors, pulmonary alveolar macrophage monolayers were concominantly exposed for 18 hours to 50g of fibers. It was observed that, in a dose-effect relationship (5 to 30 mM), nicotinamide was very effective in reducing the extracellular liberation of the marker enzymes. At 30 mM, the enzyme releases in the medium had returned to control values; the restoration of cell viability was confirmed by ATP levels. Up to 5 mM 3-aminobenzamide did not provide any protection against chrysotile cytotoxicity. Nicotinic acid, a structural analogue of nicotinamide, but not an inhibitor of the ADP-ribosyl transferase, also showed no protective effect. Nicotinamide and 3-aminobenzamide increased the intracellular NAD⁺ pools, respectively by 350% and 250%. However, with or without additives, the chrysotile fibers caused a constant and significant decrease in NAD⁺ levels (40–55 pmoles). These results suggest that the inhibition of the nuclear ADP-ribosyl transferase is not the major mechanism by which nicotinamide protects pulmonary alveolar macrophages against the chrysotile asbestos fibers.Abbreviations 3-AB 3-aminobenzamide - ADPRT ADP-ribosyl transferase - -GAL -galactosidase - DTT dithiothreitol - FBS fetal bovine serum - FMN flavin mononucleotide - HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid - LDH lactate dehydrogenase - NAD⁺ nicotinamide adenine dinucleotide (oxidized form) - NADH nicotimide adenine dinucleotide (reduced forms) - NADPH nicotimide adenine dinucleotide phosphate (reduced form) - NAM nicotinamide - NIC nicotinic acid - ORS oxygen radical species - PAM pulmonary alveolar macrophages - S.E. standard error of the mean - TAPS tris (hydroxymethyl) methylamino-propane sulfonic acid - TRIS tris (hydroxymethyl) aminomethane - VSF very short chrysotile fibers     

Mouse Chromosome 14

Chair of Committee for Mouse Chromosome 14     

H-2 Polymorphisms Are More Uniformly Distributed than Allozyme Polymorphisms in Natural Populations of House Mice

Patterns of H-2 and allozyme polymorphism in natural populations of house mice from Europe, North Africa and South America were analyzed. The purpose of the analysis was to determine whether H-2 and allozyme polymorphisms were similarly distributed both geographically and temporally in wild mice. Two subspecies of house mice, Mus musculus domesticus and M. m. musculus were sampled and the polymorphisms of two H-2 class I genes, H-2K and H-2D, and 34 allozyme-encoding genes were surveyed. The three kinds of analyses that were conducted included a hierarchical gene diversity analysis, an analysis of the effects of barriers to gene flow, and an analysis of similarity networks. Each of the comparisons demonstrated that H-2 polymorphisms were more uniformly distributed than allozyme polymorphisms and provided additional evidence that H-2 and allozyme polymorphisms are subject to different evolutionary pressures. The analysis of similarity networks also demonstrated that H-2 genes provide little information about the phylogeny of wild mice.     

Absence of increased oxygen consumption in brown adipose tissue of rats exhibiting "cafeteria" diet-induced thermogenesis

Young male Sprague-Dawley rats were induced to overeat (approximately 45%) by provision of a "cafeteria" (CAF) diet of palatable human foods. Normophagic rats fed a commercial chow or a semisynthetic diet served as controls. The CAF rats exhibited (a) the reduced food efficiency and the propranolol-inhibitable elevation in resting metabolic rate (resting VO2) that are indicative of a facultative diet-induced thermogenesis (DIT) by which excess energy gain is resisted, and (b) certain changes in brown adipose tissue (BAT) that are among those taken as evidence for BAT as the effector of DIT, e.g., increased protein content and increased mitochondrial binding of GDP. To assess directly and quantitatively the contribution by BAT to the elevation in VO2 (apparent DIT) of the CAF rats, BAT O2 consumption was determined (Fick principle) from measurements of tissue blood flow (microsphere method) and the arteriovenous difference in blood O2 across interscapular BAT (IBAT). To obtain the measurements, the animals were fitted under halothane anesthesia with vascular cannulas for intraventricular injection of microspheres and sampling of arterial blood and the venous effluent of IBAT. After recovery from anesthesia and rewarming to normal body temperature the animals were placed singly in a temperature-controlled metabolic chamber and the measurements, which also included determination of resting VO2, were made 1.5-2 h later about 11:30 h. As determined from measurements made at 28 degrees C (thermoneutrality) mean values of resting VO2 for the cannulated rats were unchanged from those of intact (unoperated) CAF or control rats.(ABSTRACT TRUNCATED AT 250 WORDS)