Lipid structure and Ca2+-ATPase function

Effects of lipid structure on the function of the Ca²⁺-ATPase of skeletal muscle of sarcoplasmic reticulum are reviewed. Binding of phospholipids to the ATPase shows little specificity. Phosphatidylcholines with short (C14) or long (C24) fatty acyl chains have marked effects on the activity of the ATPase, including a change in the stoichiometry of Ca binding. Low ATPase activity in gel phase lipid follows from low rate of phosphorylation. Phosphatidylinositol 4-phosphate increases ATPase activity by increasing the rate of dephosphorylation of the phosphorylated ATPase. Stimulation is not seen with other anionic phospholipids; phosphatidic acid decreases ATPase activity in a Mg²⁺-dependent manner.Abbreviations di(C141)PC dimyristoleoylphosphatidycholine - di(C160)PC dipalmitoylphosphatidylcholine - di(C181)PC dioleoylphosphatidylcholine - di(Br₂C180)PC dibromostearoylphosphatidylcholine - di(C241)PC dinervonylphosphatidylcholine - di(C181)PA dioleoylphosphatidic acid - di(C181)PE dioleoylphosphatidylethanolamine - Ptdlns phosphatidylinositol - PtdIns-4P phos-phatidylinositol 4-phosphate     

Human medulloblastoma gangliosides

To establish a model system for the study of ganglioside metabolismof the human brain tumor, medulloblastoma, we have chemicallycharacterized the gangliosides of the Daoy cell line. Thesecells contain a high concentration of gangliosides (143 ±13 nmol LBSA/10⁸ cells). The major species have been structurallyconfirmed to be G_M2 (65.9%), G_M3 (13.0%), and G_Dla (10.3%).Isolation of individual gangliosides homogeneous in both carbohydrateand ceramide moieties by reversed-phase HPLC and analysis bynegative-ion fast atom bombardment collisionally activated dissociationtandem mass spectrometry have allowed us to unequivocally characterizeceramide structures. In the case of G_M2, 10 major ceramide subspecieswere identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0,d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18: 1-C23:1, d18:1-C24:1,and d18:1-C24:0. Taken together with previous studies, thesefindings in human medullo-blastoma cells support the view thathigh expression and marked heterogeneity of ceramide structureare general characteristics of tumor gangliosides, moleculeswhich are shed by the tumor cells and which are biologicallyactive in vivo. medulloblastoma gangliosides ceramide structure HPLC mass spectrometry     

Number and sex of offspring of ΔF508 carriers outside cystic fibrosis families

Number and sex of offspring were determined in a group of 7,841 randomly selected blood donors who were screened for the F508 mutation. We did not find any evidence for differences in number or sex ratio of offspring between F508 carriers and non-carriers.     

Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization.

We describe the combination of hot banding with fluorescence in situ hybridization as a rapid and efficient method to identify integration sites of transfected DNA sequences in chromosomes. As a test system we used SW480 EJ2, a clonal cell line obtained after transfection of SW480 with pSV2neoEJ, a plasmid containing a point-mutated, c-Ha-RAS oncogene. Nick-translated probes were compared with random primed-labeled probes to evaluate their relative efficiency in fluorescence in situ hybridization. The fluorescence signals were quantified in interphase nuclei by confocal scanning laser microscopy. Nick-translated probes were found to yield better results. Hot banding followed by fluorescence in situ hybridization localized the integration site of pSV2neoEJ in SW480 EJ2 at the site of a translocation on a marker chromosome Xp+. The combination of fluorescence in situ hybridization and hot banding can be used to (a) rapidly and efficiently analyze integration sites in large numbers of transfectants, (b) assess the clonality of transfected cell lines, and (c) localize the site of integration of transfected genes in the recipient genome.     

L-pilin variants of Neisseria gonorrhoeae MS11

Phase- and antigenic variation of pilin expression in Neisseria gonorrhoeae is based on the genetic exchange between silent pilin genes (pilS) and the pilin expression locus (pilE). Similarly, the non-piliated L-variants of strain MS11, which show an increased resistance to certain antibiotics, are the result of recombination with the pilE locus. However, this recombination is atypical in that pilE(L) carries a tandem arrangement of a complete pilin gene and additional partial pilin genes under the control of the same pilE promoter. Since the two pilin gene copies are tandemly arranged and are often in the same translational frame, oversized pilin molecules are produced, which do not assemble into pili. The tandem gene copies introduced in a pilE(L) locus originate from silent loci where they are already joint. Upon reversion to the P+ phenotype the L-variants lose one pilin gene copy from the pilE(L) in a process reminiscent of the deletion events that otherwise lead to the formation of the non-revertible and non-piliated Pn mutants of MS11 gonococci. Thus deletion of pilin genes from pilE can be regarded as a third mechanism of pilin variation in gonococci.     

The intracellular distribution of adenylate kinase in the leaves of spinach,wheat and barley

Of the total adenylate-kinase activity in 10-d-old barley and wheat leaves, 40–50% is localised in the chloroplasts, while in mature spinach leaves 50–70% of the enzyme is chloroplastic. The extra-chloroplastic adenylate-kinase activity is associated with the mitochondria, very little, if any, is freely soluble in the cytoplasm. The adenylate pool of the cytoplasm could have access to adenylate-kinase activity in the intermitochondrial space because of the free permeation of adenylates across the outer mitochondrial membrane. Thus the adenylate pool of the cytoplasm could be subject to adenylate-kinase equilibrium. The mitochondrial adenylate kinase appeared to the localised exclusively in the intermembrane space.     

Application of step-wise discriminant analysis and Bayesian classification procedure in determining prognosis of acute myocardial infarction.

A retrospective study was carried out to assess the feasibility of computer-assisted prognostication by discriminant analysis and the Bayesian classification procedure based on clinical information collected on patients with acute myocardial infarction. The overall accuracy was 94.2% in predicting hospital death but the prediction of late death after discharge was less accurate. It was found that not all of the 44 variables used for analysis were necessary to reach the same level of predictive accuracy--16 to 20 variables would result in almost the identical prediction. The Bayesian classification procedure was applied to estimate probabilities of individual patients belonging to the different prognostic categories.