Prevalence and intensity of Thelazia spp. (Nematoda: Thelazioidea) in a Musca autumnalis (Diptera: Muscidae) population from Central Alberta

A face fly (Musca autumnalis) population near Wetaskiwin, central Alberta, Canada, was sampled 9 times from 26 July to 29 September 1988 for the early larval stages of Thelazia spp. Of 426 female flies examined, 159 (37%) contained Thelazia spp. (almost exclusively T. skrjabini), with an average worm burden of 4.2 larvae per infected fly. Prevalence ranged from 17 to 56% over 9 collections. This is the first report of Thelazia skrjabini in flies from western North America and the highest Thelazia prevalence in face flies yet reported in North America. The face fly population was also parasitized by Heterotylenchus autumnalis, with a prevalence of 5.5%.     

Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle

vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.     

Nucleotide sequence of the chromosomal gene coding for the aminoglycoside 6-adenylyltransferase from Bacillus subtilis Marburg 168

Gene aadK of Bacillus subtilis is 855 bp long and codes for aminoglycoside 6-adenylyltransferase.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis

NB176 is a Bacillus thuringiensis mutant derived by λ-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn 1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by γ-irradiation. Integration of additional copies of the cryIIIA gene into the native 90MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.     

Definition of a domain of GLVR1 which is necessary for infection by gibbon ape leukemia virus and which is highly polymorphic between species.

Expression of human GLVR1 in mouse cells confers susceptibility to infection by gibbon ape leukemia virus (GALV), while the normally expressed mouse Glvr-1 does not. Since human and murine GLVR1 proteins differ at 64 positions in their sequences, some of the residues differing between the two proteins are critical for infection. To identify these, a series of hybrids and in vitro-constructed mutants were tested for the ability to confer susceptibility to infection. The results indicated that human GLVR1 residues 550 to 551, located in a cluster of seven of the sites that differ between the human and mouse proteins, are the only residues differing between the two which must be in the human protein form to allow infection. Sequencing of a portion of GLVR1 from the rat (which is infectible) confirmed the importance of this cluster in that it contained the only notable differences between the rat and mouse proteins. This region, which also differs substantially between the rat and the human proteins, therefore exhibits a pronounced tendency for polymorphism.     

Phosphorus-31 nuclear magnetic resonance of phosphoenzymes of sodium- and potassium-activated and of calcium-activated adenosinetriphosphatase

Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.     

Direct evidence for the interaction between papaverine and alpha 2-adrenergic receptors in canine platelets

The effects of papaverine on specific [3H]-yohimbine binding to canine platelet alpha 2-adrenergic receptors and on the platelet aggregation were assessed and compared with those of verapamil. Both compounds concentration-dependently inhibited [3H]-yohimbine binding with KI values for respective compounds of 0.39 +/- 0.05 microM (n = 3) and 15 +/- 0.19 microM (n = 3). In the presence of either compound KD values in Scatchard analysis of the equilibrium ligand binding increased in concentration-dependent manner, whereas Bmax did not change, indicating competitive inhibition of the ligand binding by these compounds. (-)-Epinephrine (3 microM) potentiation of adenosine diphosphate (ADP, 0.1 microM) aggregation was inhibited by papaverine with IC50 of 11 +/- 3.6 microM (n = 4). In the same experiments verapamil inhibited the platelet aggregation with lower IC50 (3.1 +/- 0.87 microM, n = 4) in comparison with that for papaverine. These results suggest that papaverine, like verapamil, inhibits physiological response of canine platelets through alpha-adrenergic receptor stimulation by direct interaction with the receptors.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.