Lysophosphatidylcholine as an intermediate in phosphatidylcholine metabolism and glycerophosphocholine synthesis in cultured cells: an evaluation of the roles of 1-acyl- and 2-acyl-lysophosphatidylcholine

Previous studies in our laboratory have shown that the principal pathway of phosphatidylcholine (PtdCho) degradation in cultured mouse N1E-115 neuroblastoma, C6 rat glioma, primary rat brain glia and human fibroblasts is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----glycerophosphate plus choline (Morash, S.C. et al. (1988) Biochim. Biophys. Acta 961, 194-202). GroPCho is the first quantitatively major degradation product in this pathway, and could be formed by phospholipases A1 or A2, followed by lysophospholipase, or by a co-ordinated attack releasing both fatty acids by phospholipase B. The quality and quantities of lysoPtdCho present in cells reflect the nature of the initial hydrolysis step (A1 or A2), specificities of the lysophospholipases, and activities of acyltransferases that form PtdCho from lysoPtdCho. The present study was undertaken to elucidate the relative importance of these pathways by examining the fate of exogenous 1-acyl and 2-acyl-lysoPtdCho incubated with N1E-115 and C6 cells in culture. By fatty acid composition, endogenous lysoPtdCho was found to be mainly 1-acyl in both cell types based on a predominance of saturated acyl species; this suggested either preferential further deacylation or reacylation of 2-acyl-lysoPtdCho, or that 2-acyl-lysoPtdCho was not formed. Exogenous 1- and 2-acyl-lysoPtdCho specifically radiolabelled with choline and/or fatty acid were incubated either singly or as equimolar mixtures with cells. Cell association was rapid and not reversible by washing and both species were taken up at similar rates. The 2-acyl species was acylated to PtdCho faster than the 1-acyl species in both cell lines. Acylation of both lyso species was higher in C6 compared to N1E-115 cells. Hydrolysis of lysoPtdCho to GroPCho was higher in N1E-115 cells and with 1-acyl-lysoPtdCho. Transacylation between two molecules of lysoPtdCho was a minor pathway. These results document the variety and relative importance of reactions of lysoPtdCho metabolism; under similar conditions, 1- and 2-acyl-lysoPtdCho are handled differently. Both species turn over actively, but only the 1-acyl species accumulates while 2-acyl-lysoPtdCho is likely to be reacylated to form PtdCho.     

Mass measurement of inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 mouse fibroblasts.

Two specific and selective assays were used to measure changes in the mass of Ins(1,4,5)P3 and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 cells. The results demonstrate that the increase in Ins(1,4,5)P3 was extremely rapid, but transient, returning to basal levels by 30 s. In contrast, the increase in sn-1,2-diacylglycerol was biphasic: the first phase mirrored the transient Ins(1,4,5)P3 response, whereas the second phase was sustained and occurred in the absence of elevated Ins(1,4,5)P3. The possible source of the second phase of diacylglycerol is discussed.     

Initial steps in the degradation of benzene sulfonic acid, 4-toluene sulfonic acids,and orthanilic acid in Alcaligenes sp. strain O-1

Alcaligenes sp. strain O-1 grew with benzene sulfonate (BS) as sole carbon source for growth with either NH₄ ⁺ or NH₄ ⁺ plus orthanilate (2-aminobenzene sulfonate, OS) as the source(s) of nitrogen. The intracellular desulfonative enzyme did not degrade 3- or 4-aminobenzene sulfonates in the medium, although the enzyme in cell extracts degraded these compounds. We deduce the presence of a selective permeability barrier to sulfonates and conclude that the first step in sulfonate metabolism is transport into the cell. Cell-free desulfonation of BS in standard reaction mixtures required 2 mol of O₂ per mol. One mol of O₂ was required for a catechol 2,3-dioxygenase. When meta ring cleavage was inhibited with 3-chlorocatechol in desalted extracts, about 1 mol each of O₂ and of NAD(P)H per mol of BS were required for the reaction, and SO₃ ^2- and catechol were recovered in high yield. Catechol was shown to be formed by dioxygenation in an experiment involving ¹⁸O₂. 4-Toluene sulfonate was subject to NAD(P)H-dependent dioxygenation to yield SO₃ ^2- and 4-methylcatechol, which was subject to meta cleavage. OS also required 2 mol of O₂ per mol and NAD(P)H for degradation, and SO₃ ^2- and NH₄ ⁺ were recovered quantitatively. Inhibition of ring cleavage with 3-chrorocatechol reduced the oxygen requirement to 1 mol per mol of OS SO₃ ^2- (1 mol) and an unidentified organic intermediate, but no NH₄ ⁺, were observed.     

Medical problems of adults who were sexually abused in childhood.

The aim of the study was to see whether adults who had been sexually abused in childhood were vulnerable to physical symptoms and therefore investigation and intervention. The case histories of seven patients who were aged 22-39, were under the care of three consultant psychiatrists, had experienced childhood sexual abuse, and had a history of medical or surgical intervention were surveyed. The patients had had a mean of 18 contacts with non-psychiatric consultant teams and a mean of eight operations, with a high rate (66-70%) of normal findings. They had experienced many somatic symptoms, which led to investigations and interventions in the specialties of gynaecology, obstetrics, gastroenterology, urology, rheumatology, haematology, orthopaedics, neurology, and neuropsychiatry. The history of childhood sexual abuse was recognised only in the later stages of this medical and surgical intervention. The possibility of childhood sexual abuse should be considered earlier in such cases to prevent further unnecessary intervention.     

Synthesis and hybridization of a series of biotinylated oligonucleotides.

A series of oligonucleotides containing biotin-11-dUMP at various positions were synthesized and compared in quantitative, colorimetric hybridization-detection studies. A deoxyuridine phosphoramidite containing a protected allylamino sidearm was synthesized and used in standard, automated synthesis cycles to prepare oligonucleotides with allylamino residues at various positions within a standard 17-base sequence. Biotin substituents were subsequently attached to the allylamino sidearms by reaction with N-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester. These oligomers were hybridized to target DNA immobilized on microtiter wells (ELISA plates), and were detected with a streptavidin-biotinylated horseradish peroxidase complex using hydrogen peroxide as substrate and o-phenylenediamine as chromogen. We found that the sensitivity of detection of target DNA by biotin-labeled oligonucleotide probes was strongly dependent upon the position of the biotin label. Oligonucleotides containing biotin labels near or off the ends of the hybridizing sequence were more effective probes than oligonucleotides containing internal biotin labels. An additive effect of increasing numbers of biotin-dUMP residues was found for some labeling configurations.     

Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.