Hydration pressure and phase transitions of phospholipids: I. Piezotropic approach

Dehydration reduces the main phase transition pressure of phospholipids. An analysis based on the Gibbs-Duhem equation shows how the shift of the transition pressure is correlated to the hydration pressure.By using Fourier transform infrared (FT-IR) spectroscopy we determined the hydration-dependent phase transition pressure. The application of our new approach gives hydration pressure values which agree with the values obtained with the osmotic stress method.     

Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.     

Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients

The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions.     

Timm-staining intensity is correlated with the density of Timm-positive presynaptic structures in the cerebral cortex of lizards

In cortical areas of the lizard, Podarcis hispanica, Timm staining reveals a distinct pattern of lamination. At the electron-microscope level, virtually all of the reaction product is located in the synaptic vesicles of Timm-positive boutons. Using linear-regression analysis, the area density of Timm-positive bouton profiles as well as the numerical and volume density of stained vesicles were found to be closely correlated with the light-microscopic densitometric values obtained for each Timm-positive cortical zone. We discuss the possibility of estimating stereological electron-microscopic data parameters from densitometric measurements at the light-microscope level.     

Fluorometric determination of histamine with OPT: optimum reaction conditions and tests of identity

Histamine reacts with OPT at an alkaline pH giving fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 40 min reaction at 0°C under continuous gassing with nitrogen. After acidification with sulfuric acid to pH 2–5 the fluorescence was stable for hours. Reagent blanks were reduced by the lowering of the reaction temperature and by decreasing the amount of OPT added. These modifications permitted the determination of 2 ng histamine/ml and gave far better reproducibility than the original procedure of Shore, Burkhalter, and Cohn. The fluorometric assay for histamine is nonspecific; the major interfering OPT-reactive tissue component is believed to be spermidine. Specificity was secured by adding formaldehyde before acidification, thus abolishing the fluorescence of histamine but not that of spermidine, or by adding CdCl₂ or SrCl₂ together with OPT, thus preventing the formation of the spermidine fluorophore but not that of the histamine fluorophore.     

Distribution of newly synthesized DT-diaphorase in rat liver

The distribution, synthesis transport, and glycosylation of rat-liver DT-diaphorase has been investigated. The enzyme could be isolated using specific antibodies, mainly from the soluble supernatant but also from microsomal vesicles, Golgi membrane, and mitochondria. 40% of the microsomal enzyme was located in the lumen or on the interior side of the membrane, the rest remaining as an integral non-extractable part of the membrane. Synthesis of DT-diaphorase takes place on both free and bound ribosomes, although it was found to be transported in a sequential manner from the rough to the smooth endoplasmic reticulum and also subsequently to the mitochondria. The rough and smooth microsomal DT-diaphorase contains covalently bound carbohydrate, but no sugar moiety could be detected bound to the cytoplasmic form of the enzyme.     

Dissection of the role of macrophages in triggering T lymphocytes for interleukin 2 production by monoclonal antibody OKT3

Unfractionated human peripheral blood mononuclear cells produce a small amount of interleukin 2 (IL 2) by stimulation with a monoclonal anti-T3 antibody (OKT3) in vitro. The IL 2 production could be greatly augmented by the addition of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In the presence of TPA, the T cell enriched fraction deprived of macrophages did not produce IL 2, but the T cells pulse-incubated with OKT3 and reconstituted with macrophages efficiently produced IL 2 in subsequent culture in the presence of TPA as did T cells reconstituted with OKT3-pulse-incubated macrophages. The stimulating effect of OKT3 in the presence of macrophages was inhibited dose-dependently by the addition of immunoglobulins, particularly by mouse IgG2a which is the same isotype as that of the OKT3 antibody, showing that it inhibits by blocking the binding of OKT3 to Fc receptors on macrophages. The same extent of IL 2 production was induced in T cells when paraformaldehyde-fixed macrophages were substituted for intact macrophages. Remarkable IL 2 production was also induced by OKT3 when latex beads coated with rabbit anti-mouse IgG2a antibody and TPA were added to the culture. It was confirmed that the production induced by these stimulations was due to an increase of IL 2 mRNA. These results show that effective signals for IL 2 production are generated by efficient crosslinking of T3 molecules which results from multi-interaction of T3 molecules on the T cell membrane and anti-T3 antibody molecules on macrophage membrane or on the surface of the latex particle.     

Developmental regulation and properties of the cGMP-specific phosphodiesterase in Dictyostelium discoideum

A simple assay has been developed to measure cGMP-specific phosphodiesterase (cGPD) activity in crude soluble extracts of amoebae of Dictyostelium discoideum. When amoebae of different wild-type strains were starved on buffered agar, all strains exhibited an 8- to 12-fold increase in cGMP-specific hydrolyzing activity during development, with the major increase occurring at aggregation. cGMP-specific activity was found in both prestalk and prespore cells. To determine if the elevated cGMP-specific hydrolyzing activity observed during late development was associated with the same enzyme present in vegetative cells, cGMP-specific activities were partially purified from cells at different developmental stages and characterized. Activity in vegetative cells was fractionated by gel filtration into three components with molecular weights of approximately 172,000, 115,000 and 56,000. In contrast, cells starved 4 hr in suspension or 18 hr on agar possessed only the 172,000 or 115,000 Mr forms, respectively. The low-molecular-weight enzyme differed from the two larger forms in kinetic properties and in sensitivity to sulfhydryl reagents. Nevertheless, the three activities probably represent different forms of the same enzyme because mutants defective at the stmF locus lacked appreciable cGMP-specific hydrolyzing activity throughout development. These results indicate that D. discoideum produces a single cGPD which is strongly developmentally regulated. These findings further suggest that intracellular cGMP might be involved in regulating postaggregative as well as preaggregative development.