Phosphatidylcholine breakdown in rat liver plasma membranes. Roles of guanine nucleotides and P2-purinergic agonists

Release of P-choline and choline from purified rat plasma membrane preparations was increased by GTP and its less hydrolyzable analogues, whereas other nucleotide triphosphates had little or no effect. Stimulation by guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S) was dependent upon magnesium, inhibited by guanosine 5'-(2-O-thiol)diphosphate, and independent of calcium. ATP and ADP (1-100 microM) markedly enhanced the GTP gamma S stimulation of P-choline plus choline release but had no effect alone. ADP was as effective as ATP and nonhydrolyzable ATP analogues produced a similar or greater stimulation, whereas AMP and adenosine were much less effective. Vasopressin (0.1 microM) also produced a small stimulation. Under conditions in which protein kinase C was activated, PMA also stimulated the response to GTP gamma S but was ineffective in its absence. P-choline was the initial product which was hydrolyzed to choline. Guanine nucleotide and purinergic effects were also apparent on phosphatidylcholine degradation. EGTA, at 0.5 mM, completely removed purinergic stimulation but did not affect P-choline plus choline released in response to GTP gamma S alone. Prior treatment of plasma membranes with cholera toxin or prior injection of animals with islet-activating protein did not affect the stimulation of P-choline plus choline release either by GTP gamma S alone or by GTP gamma S plus ATP. These results indicate that a phosphatidylcholine phospholipase C is coupled to purinergic receptors in rat liver plasma membranes by a GTP-binding protein. Hydrolysis of phosphatidylcholine could contribute to hepatic diacylglycerol levels and thus influence protein kinase C activity.     

Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4

o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of ¹⁸O- and ¹⁶O-labeled 3-methylcatechol from toluene in an atmosphere of ¹⁸O₂ and ¹⁶O₂ established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.     

Biochemical similarity among serologically distinct flagellins of Campylobacter jejuni

Abstract We describe a simplified method for obtaining highly purified flagellin, suitable for biochemical analysis using HPLC-gel permeation. Amino acid composition and N-terminal sequence analyses were performed on flagellins from serologically distinct isolates. The amino acid composition of flagellin from 10 strains was very similar. The N-terminal amino acid sequence is highly conserved. Significant sequence homology was found with flagellin of Bacillus subtilis .     

Current status of pyrethroid resistance in anophelines

Similarities between DDT and pyrethroid insecticides have led to widespread concern that cross-resistance between them might limit the usefulness of the latter. Both types of insecticide have similarities in chemical structure, both have a negative temperature coefficient (ie. they are more active at lower temperatures), both act as neurotoxins on sodium channels, and both produce the twin effects of knockdown and kill. As discussed by Tom Miller (see pages S8-S12) there is firm evidence for Pyrethroid resistance in some species of medical and veterinary importance - especially in the horn fly, Haemotobia irritans. But in the case of anopheline mosquitoes, the evidence for pyrethroid resistance is much less strong. As Colin Malcolm explains here, a critical analysis of available data indicates that true physiological resistance of anophelines to pyrethroids is much less widespread than previous commentaries suggest. Moreover, the risk of cross-resistance between pyrethroids and DDT may have been over-emphasized, since different resistance mechanisms appear to be involved.     

The effect of food consumption on sodium and water balance in the predaceous diving beetle,Dytiscus verticalis

Summary Sodium and water balance ofDytiscus verticalis in fresh water were investigated under three feeding regimes: unfed, and fed a diet either low or high in sodium chloride. Unfed sodium influx was 0.13 and sodium efflux was 0.74 moles/100 gwm·h. These values are low in comparison with most freshwater animals. The electrical potential difference across the integument in artificial soft water (ASW) was about 150 mV smaller than the potential necessary to maintain sodium balance in the absence of active transport. However, sodium influx did not show saturation kinetics over an external concentration range of 91 to 1725 M. Unfed beetles failed to arrest net sodium loss to baths that were initially distilled water or ASW, even when bath sodium concentrations reached 75–298 M. The long-term rate of net sodium loss ranged from 0.61 to 4.4 moles/100 gwm·h for four sets of animals. Beetles decreased sodium efflux during a period of fasting. During subsequent feeding, beetles fed a high sodium diet (HSD) increased sodium efflux while beetles fed a low sodium diet (LSD) maintained low rates of sodium efflux. HSD fed beetles increased body sodium and hemolymph sodium concentration, and expanded extracellular fluid, relative to LSD fed beetles. Thus beetles cannot achieve sodium balance in fresh water without dietary sodium input, although they are able to regulate sodium loss.Abbreviations gwm grams wet mass - ASW artificial soft water - DW distilled water - HSD high sodium diet - LSD low sodium diet - ECF extracellular fluid volume     

Maternal origin of deletion 15q11–13 in 25/25 cases of Angelman syndrome

Summary About half of the cases of Angelman syndrome arise from deletions of chromosome band 15q12. In 25 cases we have been able to determine the parental origin of the deletion and, in line with other reported cases, we have found the deletion to be of maternal origin. There were no exceptions. The parental origin was determined using cytogenetic markers in 13 of the cases, in nine by using the pattern of inheritance of restriction fragment length polymorphisms, and in three using both techniques.     

In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.     

Expression in yeast of a cDNA copy of the K2 killer toxin gene

Summary A cDNA copy of the M2 dsRNA encoding the K2 killer toxin ofSaccharomyces cerevisiae was expressed in yeast using the yeastADH1 promoter. This construct produced K2-specific killing and immunity functions. Efficient K2-specific killing was dependent on the action of the KEX2 endopeptidase and the KEX1 carboxypeptidase, while K2-specific immunity was independent of these proteases. Comparison of the K2 toxin sequence with that of the K1 toxin sequence shows that although they share a common processing pathway and are both encoded by cytoplasmic dsRNAs of similar basic structure, the two toxins are very different at the primary sequence level. Site-specific mutagenesis of the cDNA gene establishes that one of the two potential KEX2 cleavage sites is critical for toxin action but not for immunity. Immunity was reduced by an insertion of two amino acids in the hydrophobic amino-terminal region which left toxin activity intact, indicating an independence of toxin action and immunity.     

Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum

DNA sequencing of the region downstream of the cellulose synthase catalytic subunit gene of Acetobacter xylinum led to the identification of an open reading frame coding for a polypeptide of 86 kDa. The deduced amino acid sequence of this polypeptide matches from position 27 to 40 with the N-terminal amino acid sequence determined for a 93 kDa polypeptide that copurifies with the cellulose synthase catalytic subunit during purification of cellulose synthase. The cellulose synthase catalytic subunit gene and the gene encoding the 93 kDa polypeptide, along with other genes probably, are organized as an operon for cellulose biosynthesis in which the first gene is the catalytic subunit gene and the second gene codes for the 93 kDa polypeptide. The function of the 93 kDa polypeptide is not clear at present, however it appears to be tightly associated with the cellulose synthase catalytic subunit. Sequence analysis of the polypeptide shows that it is a membrane protein with a signal sequence at the N-terminal end and a transmembrane helix in the C-terminal region for anchoring it into the membrane.