Does litomosoides sigmodontis synthesize dimethylethanolamine from choline?

Juvenile female Litomosoides sigmodontis secrete a protein (Juv-p120) highly modified with dimethylethanolamine (DMAE). In an attempt to establish the source of this decoration worms were pulsed with [3H]-choline and [3H]-ethanolamine and the radio-isotope labelled products analysed. Both isotope labels were successfully taken up by the worms, as demonstrated by labelling of phospholipids with [3H]-choline, being predominantly incorporated into phosphatidylcholine and [3H]-ethanolamine into phosphatidylethanolamine. Isotope labelling of phosphatidylethanolamine was particularly striking with the worms taking up ~30 times as much labelled ethanolamine as choline. It was possible to detect faint labelling of Juv-p120 with [3H]-ethanolamine after prolonged exposure periods but, unlike the situation with the phospholipids, it was much more readily labelled with [3H]-choline. When pulsing with [3H]-ethanolamine it was also possible to detect isotope-labelled phosphatidylcholine, which may ultimately account for the low levels of labelling of Juv-p120. Overall our results raise the previously unconsidered but intriguing possibility that in L. sigmodontis, choline may be the precursor of DMAE

Differential interaction of glimepiride and glibenclamide with the β-cell sulfonylurea receptor II

Glimepiride is a novel sulfonylurea for the treatment of type II-diabetic patients exhibiting different receptor binding kinetics to β-cell membranes with 8–9-fold higher koff rate and 2.5–3-fold higher kon rate compared to glibenclamide (see accompanying paper (Müller, G. et al. (1994) Biochim. Biophys. Acta 1191, 267–277)). To elucidate the molecular basis for this differential behaviour of glimepiride and glibenclamide, direct photoaffinity labeling studies using β-cell tumor membranes were performed. [3H]Glimepiride was specifically incorporated into a membrane polypeptide of Mr = 65000 under conditions, which led to predominant labeling of a 140 kDa protein by [3H]glibenclamide (Kramer, W. et al. (1988) FEBS Lett. 229, 355–359). Labeling of the 140 kDa protein by [3H]glibenclamide was inhibited by unlabeled glimepiride and, vice versa, glibenclamide inhibited labeling of the 65 kDa protein by [3H]glimepiride. The 65 kDa protein was also specifically photolabeled by the sulfonylurea [125I]35623, whereas an 4-azidobenzoyl derivative of glibenclamide, N3-[3H]33055, exclusively labeled a 33 kDa protein. Competitive Scatchard analysis of [3H]glimepiride-binding and [3H]glibenclamide-binding to RINm5F cell membranes using glibenclamide and glimepiride, respectively, as heterologous displacing compounds yielded non-linear plots. These findings may be explained by cooperative interactions between the 140 and 65 kDa sulfonylurea-binding proteins. The possibility that sulfonylureas of different structure have different access to the 140 and 65 kDa receptor proteins due to the β-cell membrane barrier was investigated by photoaffinity labeling of solubilized β-cell membrane proteins. Interestingly, solubilization of β-cell tumor membranes led to a shift of specific [3H]glibenclamide binding from the 140 kDa to the 65 kDa binding protein, exclusively, and to an increased labeling of the 65 kDa protein by [3H]glimepiride. The labeling of a unique protein is in agreement with similar Kd values measured for both sulfonylurcas upon solubilization of β-cell tumor and RINm5F cell membranes (see accompanying paper). Furthermore, competitive Scatchard plots of [3H]glimepiride binding to solubilized RINm5F cell membrane proteins in the presence of glibenclamide and vice versa approximate linearity suggesting loss of cooperativity between the 140 kDa glibenclamide-binding and 65 kDa glimepiride-binding proteins upon solubilization. The physiological significance of the differential interaction of glimepiride and glibenclamide with different binding proteins was also substantiated by photoaffinity labeling of RINm5F cells leading to labeling of a 140 kDa protein by [3H]glibenclamide and of a 65 kDa protein by [3H]glimepiride. In conclusion, this report presents the first evidence that different sulfonylurea drugs bind to different components of the sulfonylurea receptor complex which are characterized by different accessibility for the drugs

Elastic Scattering of Pions From the Three-nucleon System

We examine the scattering of charged pions from the trinucleon system at a pion energy of 180 MeV. The motivation for this study is the structure seen in the experimental angular distribution of back-angle scattering for pi+ 3He and pi- 3H but for neither pi- 3He nor pi+ 3H. We consider the addition of a double spin flip term to an optical model treatment and find that, though the contribution of this term is non-negligible at large angles for pi+ 3He and pi- 3H, it does not reproduce the structure seen in the experiment.Comment: 15 pages + 5 figure

Structure functions for the three nucleon system

The spectral functions and light-cone momentum distributions of protons and neutrons in 3He and 3H are given in terms of the three-nucleon wave function for realistic nucleon-nucleon interactions. To reduce computational complexity, separable expansions are employed for the nucleon-nucleon potentials. The results for the light-cone momentum distributions suggest that they are not very sensitive to the details of the two-body interaction, as long as it has reasonable short-range repulsion. The unpolarised and polarised structure functions are examined for both 3He and 3H in order to test the usefulness of 3He as a neutron target. It is found that the measurement of the spin structure function of polarised 3H would provide a very clear test of the predicted change in the polarised parton distributions of a bound proton.Comment: 30 pages, REVTeX, 11 figure

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC 12) permeabilized with staphylococcal α-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC 12 cells. Permeabilization with α-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC 12 cells

Neutrino Breakup of A=3 Nuclei in Supernovae

We extend the virial equation of state to include 3H and 3He nuclei, and predict significant mass-three fractions near the neutrinosphere in supernovae. While alpha particles are often more abundant, we demonstrate that energy transfer cross-sections for muon and tau neutrinos at low densities are dominated by breakup of the loosely-bound 3H and 3He nuclei. The virial coefficients involving A=3 nuclei are calculated directly from the corresponding nucleon-3H and nucleon-3He scattering phase shifts. For the neutral-current inelastic cross-sections and the energy transfer cross sections, we perform ab-initio calculations based on microscopic two- and three-nucleon interactions and meson-exchange currents.Comment: 6 pages, 2 figures, minor additions, to appear in Phys. Rev.

Electromagnetic Structure of the Trinucleons

The electromagnetic form factors of the trinucleons 3H and 3He are calculated with wave functions obtained with the Argonne AV18 two-nucleon and Urbana IX three-nucleon interactions. Full account is taken of the two-body currents required by current conservation with the AV18 interaction as well as those associated with N-Delta transition currents and the currents of Delta resonance components in the wave functions. Explicit three-nucleon current operators associated with the two-pion exchange three-nucleon interaction arising from irreducible S-wave pion-nucleon scattering is constructed and shown to have very little effect on the calculated magnetic form factors. The calculated magnetic form factor of 3H, and charge form factors of both 3H and 3He are in satisfactory agreement with the experimental data. However, the position of the zero in the magnetic form factor of 3He is slightly underpredicted.Comment: 27 pages RevTex file, 19 PostScript figures, submitted to Phys. Rev.

Automated quantitative analysis of single and double label autoradiographs

A method for the analysis of silver grain content in both single and double label autoradiographs is presented. The total grain area is calculated by counting the number of pixels at which the recorded light intensity in transmission dark field illumination exceeds a selected threshold. The calibration tests included autoradiographs with low (3H- thymidin) and high (3H-desoxyuridin) silver grain density. The results are proportional to the customary visual grain count. For the range of visibly countable grain densities in single labeled specimens, the correlation coefficient between the computed values and the visual grain counts is better than 0.96. In the first emulsion of the two emulsion layer autoradiographs of double labeled specimens (3H-14C- thymidin) the correlation coefficient is 0.919 and 0.906. The method provides a statistical correction for the background grains not due to the isotope. The possibility to record 14C tracks by shifting the focus through the second emulsion of the double labeled specimens is also demonstrated. The reported technique is essentially independent of size, shape and density of the grains