The potential use of DNA probes to identify and type strains within the Mycobacterium tuberculosis complex

DNA was extracted and purified from 11 strains of Mycobacterium bovis isolated from cattle in Ireland. After digestion with restriction endonuclease PvuII and electrophoresis on an agarose gel, the separated DNA fragments were transferred to a nylon membrane and sequentially hybridized with three DNA probes derived from BCG.
None of the three probes detected restriction fragment length polymorphism (RFLP) within the 11 M. bovis strains, indicating a very close genetic relationship. One probe, pBCG12, detected RFLPs between the M. bovis strains and a reference PvuII digest of DNA from M. tuberculosis R37Rv, confirming that M. bovis and M. tuberculosis are closely related though genetically distinct.     

Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.     

The catalytic consequences of experimental evolution. Transition-state structure during catalysis by the evolved beta-galactosidases of Escherichia coli (ebg enzymes) changed by a single mutational event.

1. The first chemical step in the hydrolysis of galactosylpyridinium ions by the evolvant ebg enzyme is less sensitive to leaving-group acidity than in the case of the wild-type ebg enzyme, implying less glycone-aglycone-bond fission at the transition state. 2. The first chemical step in the hydrolysis of aryl galactosides by ebg enzyme is probably less sensitive to leaving-group acidity than in the case of ebg enzyme, possibly as a consequence of resulting in more effective proton donation to the leaving aglycone. 3. alpha-Deuterium kinetic isotope effects of 1.1(0) and beta-deuterium kinetic isotope effects of 1.0(0) were measured for the hydrolysis of galactosyl-enzyme intermediates derived from ebg and ebg enzymes: these effects are not compatible with reaction of the sugar ring through a 4C1-like conformation, or with an ionic glycosyl-enzyme intermediate. 4. The variation with pH of steady-state kinetic parameters for hydrolysis of p-nitrophenyl galactoside by ebg and ebg enzymes and of 3-methylphenyl beta-galactoside, 3,4-dinitrophenyl beta-galactoside and beta-galactosyl-3-bromopyridinium ion by ebg enzyme was measured. The steep, non-classical, fall in activity against p-nitrophenyl galactoside at low pH observed with ebg and ebg enzymes is not observed with ebg enzymes.     

Humoral factors released during trauma ofAplysia body wall

1. Preliminary, general chemical characteristics of substances in artificial sea water (ASW) washed through stimulated body wall (SBW) and in hemolymph taken from noxiously stimulated animals (SHL) were consistent with those of classical neurotransmitters, amino acids, and small- to medium-sized peptides. 2. 5-Hydroxytryptamine (5HT) and acetylcholine (ACh), unlike SBW and SHL, caused relaxation when perfused into isolated body wall. FMRFamide produced a biphasic response--brief contraction followed by prolonged relaxation. 3. Small cardioactive peptide (SCPB) caused body wall contractions similar to those produced by SBW and SHL, except that SCPB contractions displayed more desensitization and were completely blocked by 30 mM CoCl2. SCPB and SBW contractions were synergistic. 4. Dopamine caused persistent body wall contractions similar to those of SBW and SHL. Dopamine contractions were reduced but not blocked by 30 mM CoCl2. Unlike SBW activity, dopamine activity was reduced by alkalinization. 5. Glutamate and taurine produced strong but usually short-lasting body wall contractions. Adenosine, octopamine, arginine vasotocin, and cholecystokinin (CCK-8) caused weak or variable contractions. Met-enkephalin and somatostatin caused no obvious body wall responses. 6. When superfused over the fully sheathed abdominal ganglion, FMRFamide, met-enkephalin, glutamate, aspartate, and taurine reduced the magnitude of the gill-withdrawal reflex elicited by siphon nerve stimulation. 7. Taken together with earlier results, these data suggest a preliminary framework for trauma signal pathways. It is proposed that stress hormones (perhaps including FMRFamide, SCPs, 5HT, and dopamine) are released into hemolymph from neuroendocrine cells. Effective amounts of active intracellular solutes such as amino acids may also be released by extensive cellular rupture. Various humoral signals produce slow effects that contribute to hemostasis, balling up, increased cardiac output, and reflex suppression.     

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

Summary N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2–8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP was consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.     

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Fractional conversion of thromboxane A2 and B2 to urinary 2,3-dinor-thromboxane B2 and 11-dehydrothromboxane B2 in the cynomolgus monkey

Following the intravenous administration of thromboxane (TX) B2, the stable hydration product of TXA2, to human and nonhuman primates the most abundant urinary metabolites are 2,3-dinor-TXB2 and 11-dehydro-TXB2. However, it is not known whether fractional conversion of TXB2 to its enzymatic metabolites is an accurate representation of TXA2 metabolism. Thus, we have compared the metabolic disposition of synthetic TXA2 and TXB2 via the beta-oxidation and 11-OH-dehydrogenase pathways in vivo in the monkey. TXA2 or TXB2 (20 ng/kg) was intravenously administered to four cynomolgus monkeys pretreated with aspirin in order to suppress endogenous TXA2 production. Urinary TXB2, 2,3-dinor-TXB2 and 11-dehydro-TXB2 were measured before, during and up to 24 h after thromboxane administration by means of reversed-phase high-performance liquid chromatography radioimmunoassay. Aspirin treatment suppressed urinary 2,3-dinor-TXB2 and 11-dehydro-TXB2 by approx. 75%. A similar fractional conversion of TXA2 and TXB2 into 2,3-dinor-TXB2 and 11-dehydro-TXB2 was found. These results suggest that TXA2 is hydrolyzed to TXB2 prior to enzymatic degradation and that metabolites of the latter represent reliable indices of TXA2 biosynthesis. Due to the variability in the conversion of thromboxanes into 2,3-dinor-TXB2 and 11-dehydro-TXB2, the measurement of both metabolites seems to represent a more reliable index of acute changes in TXA2 production.     

The importance of worm identity and life history in determining the vertical distribution of tubificids (Oligochaeta) in a riverine mud

The vertical distribution of Tubifex tubifex, Limnodrilus hoffmeisteri and L. udekemianus, in a riverine mud was studied over a year. Distribution was dependent upon species identity, season and locality. The size structure of the population, and hence species life history, was of fundamental importance in determining temporal patterns of vertical distribution. A positive correlation between worm length and depth of penetration was clearly demonstrated for all three species. However, the mid-stream populations of the three species were closer to the mud surface during the summer and autumn than were the near-bank populations, although the size structures of the populations were not significantly different. Thus, another factor, possibly the distribution of food, also influenced the vertical distribution of the worms. Cocoon distribution was similar to that of the adult worms. Cocoons of the larger species, L. udekemianus, occurred deeper in the mud than those of T. tubifex. A comparison between the vertical distributions of tubificids in cores sectioned in situ, on the stream bank, and 30 mins later, showed there to be no significant change in distribution with time.