Comparison of effects of oxytocin and prostaglandin F2-alpha on circular and longitudinal myometrium from the pregnant rat

Previous studies showed that circular (CM) and longitudinal myometrium (LM) have different physiological and pharmacological characteristics. To determine if such differences also exist with respect to the actions of oxytocin and prostaglandin F2-alpha (PGF2 alpha), we compared the effects of these agents on the spontaneous contractions of CM and LM from rats on Days 15, 17, and 21 (term) of pregnancy. Both agents stimulated CM and LM on all gestation days, but the responses differed as follows: 1) the CM response to oxytocin was all-or-nothing on Days 15 and 17, to PGF2 alpha on Day 15, and was graded to both agents only at term; 2) the LM response to both agents was always graded; 3) the maximum response to oxytocin was always less in CM than in LM, and to PGF2 alpha was less in CM except at term, when it was greater than in LM; 4) the EC50 (effective concentration that produced 50% of the maximum response) for PGF2 alpha in CM was greater than in LM, indicating a lesser sensitivity of CM for this agent. Therefore, the heterogeneity between CM and LM extends to the effects of oxytocin and PGF2 alpha, further emphasizing the importance of distinguishing between the two muscle layers in studies of uterine activity.     

Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues

Acetylcholinesterase extracted with Triton X-100 from bovine brain caudate nuclei was purified by affinity chromatography to apparent homogeneity. The purified enzyme was labeled with [3H]diisopropyl fluorophosphate at the active sites and with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a compound which has been shown to be selective for the hydrophobic membrane-binding domains of several other proteins. The subunit structure was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide reduction. After reduction, a single 3H-labeled band at 70 kDa was stained by silver, but most of the 125I label corresponded to a 20-kDa species. Prior to reduction, five 3H-labeled and silver-stained bands were apparent at 70, 140, 160, 260, and greater than 360 kDa. These species were presumed to represent monomer and disulfide-linked oligomers of 70-kDa catalytic subunits. 125I label was selectively associated with the 160-, 260-, greater than 360-, and a 90-kDa species. Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalytic subunit monomers and the second of which contains two disulfide linkages through an intervening 125I-labeled 20-kDa noncatalytic subunit. This 20-kDa subunit is proposed to contain the membrane attachment site. The brain enzyme did not contain components characteristic of the glycolipid anchors of erythrocyte acetylcholinesterases. However, part of the 125I label was associated with fatty acids, indicating that at least a portion of the brain enzyme membrane anchor is composed of nonamino acid components.     

Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

The presence of a glycoinositol phospholipid anchor in Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [125I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence [Hall, L.M.C., & Spierer, P. (1986) EMBO J. 5, 2949-2954] confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine.     

Identification of Campylobacter coli isolates from animals and humans by bacterial restriction endonuclease DNA analysis.

Ninety-nine Campylobacter coli isolates were examined by bacterial restriction endonuclease DNA analysis (BRENDA) with HindIII. Isolates from poultry from the same environment had identical patterns, patterns of isolates carried by suckling piglets were generally the same as those of isolates recovered from their dams, and one human patient yielded the same BRENDA type when sampled 6 weeks later. The 14 human isolates examined produced 11 distinct BRENDA types. Forty-three C. coli isolates from pigs were represented by 20 BRENDA types. Ten C. coli isolates from the feces of gulls yielded five different BRENDA types. Thirty-two C. coli isolates from live chickens and processed chicken yielded five different BRENDA types. Three human isolates had identical DNA patterns; two were from brothers living in the same house, and the third was from a human with no apparent relationship to the brothers. Another human isolate was identical to a poultry isolate. None of the pig strains had DNA patterns resembling those of human strains, nor were the DNA patterns like those of any strains recovered from poultry or gulls. Four C. coli isolates were subcultured onto agar 23 times over a period of 45 days, and their BRENDA patterns were preserved. BRENDA shows great promise for use in epidemiological studies of C. coli.     

Purification and properties of glycerol:NADP+ 2-oxidoreductase from Schizosaccharomyces pombe

Glycerol:NADP+ 2-oxidoreductase (EC 1.1.1.156) was isolated from Schizosaccharomyces pombe, purified and characterized. It had an Mr of 57,000, and SDS-PAGE revealed two polypeptides, of Mr 25,000 and 30,000. Its coenzyme requirement was satisfied exclusively by NADP. The pH optimum for glycerol oxidation was 9.5, for dihydroxyacetone reduction 6.0. Rates of oxidation with some structurally related diols were three- to six-fold lower than for glycerol, while glyceraldehyde and other carbonyl compounds showed negligible rates of reduction. Neither monovalent nor divalent cations activated the enzyme. Apparent Km and Vmax values were determined. The enzyme is similar to glycerol dehydrogenases isolated from Mucor javanicus and from Dunaliella parva but differs considerably from the glycerol:NAD+ 2-oxidoreductase of S. pombe.     

Three different activated ras genes in mouse tumours; evidence for oncogene activation during progression of a mouse lymphoma.

Five unrelated mouse tumours have been shown to carry activated transforming genes using the NIH/3T3 transfection assay. Three of these tumours, a T-cell lymphoma, a fibrosarcoma and a macrophage tumour, were found to carry an activated c-Ki-ras gene. A c-Ha-ras gene was shown to be activated in a myeloid leukaemia and a recently identified member of the 'ras' gene family, N-ras, was found to be activated in a lung carcinoma. The T-cell lymphoma, L5178Y-ES, is a more aggressively growing metastatic variant which arose spontaneously from the parental tumour, L5178Y-E. Although DNA from both parental and variant tumours was shown to transfer a genetic marker to recipient cells equally well, only the metastatic variant carried an activated c-Ki-ras gene detectable by transfection. The altered growth behaviour of the L5178Y-ES cells may therefore be the result of the spontaneous activation of the c-Ki-ras gene after the lymphoma cells had already become tumorigenic.