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.     

Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca²⁺. It has a K_i of about 30 m. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca²⁺-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.Abbreviation CPD cholesterylphosphoryldimethylethanolamine     

Soil N mineralization and nitrification in relation to nitrogen solution chemistry in a small forested watershed

Spatial variations in soil processes regulating mineral N losses to streams were studied in a small watershed near Toronto, Ontario. Annual net N mineralization in the 0–8 cm soil was measured in adjacent upland and riparian forest stands using in situ soil incubations from April 1985 to 1987. Mean annual rates of soil N mineralization and nitrification were higher in a maple soil (93.8 and 87.0 kg.ha^–1) than in a pine soil (23.3 and 8.2 kg.ha^–1 ). Very low mean rates of mineralization (3.3 kg.ha^–1) and nitrification (3.4 kg.ha^–1) were found in a riparian hemlock stand. Average NO₃-N concentrations in soil solutions were 0.3–1.0 mg.L^–1 in the maple stand and >0.06mg.L^–1 in the pine stand. Concentrations of NO₃–N in shallow ground water and stream water were 3–4× greater in a maple subwatershed than in a pine subwatershed. Rapid N uptake by vegetation was an important mechanism reducing solution losses of NO₃–N in the maple stand. Low rates of nitrification were mainly responsible for negligible NO₃–N solution losses in the pine stand.     

Oligomerization of deoxynucleoside-bisphosphate dimers: Template and linkage specificity

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3–5 pyrophosphate-linked dimer pdAppdAp, in comparison with the 3–3 and 5–5 linked dimers. In the absence of poly(U), the 5–5linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.Nucleic Acid-Like Structures V. For the previous paper in this series see Visscher and Schwartz (1988).     

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.     

Low temperatures stabilize interferon α-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli

Summary The accumulation of interferon (IFN) -2 in transformed strains of Escherichia coli and Methylophilus methylotrophus was greater at 25° C than at 37° C. Interferon -2 catabolism was followed by measuring the change in IFN titre (measured immunoreactively) with time at temperatures between 25° C and 37° C in chloramphenicol-treated cells. The IFN -2 titre remained constant at 29° C and below, while at higher temperatures the titres declined. The t _1/2 values for IFN -2 decreased with increasing incubation temperature. Pulse-chase studies using [³⁵S]methionine, sodium dodecyl sulphate-gel electrophoresis and autoradiography demonstrated that IFN -2 was subjected to degradation at 37° C while at 25° C it was stable. It is proposed that the susceptibility of IFN -2 to degradation in both E. coli and M. methylotrophus is affected by incubation temperature and 30° C may be a transition temperature above which the conformation of the molecule is recognised by the bacterial proteases.     

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.