Evidence for the expression of two distinct MHC class II DRβ like molecules in the sheep

This study used monoclonal antibodies to sheep MHC class II molecules as well as an L cell transfectant (T8.1) which expresses DRA and DRB genes to show that two distinct DRβ chains are expressed in the sheep. Two anti-β chain specific monoclonal antibodies VPM37 and VPM43 react with DR antigen but not DQ antigen by ELISA. These two antibodies do not react with the DRβ chain expressed in the T8.1 cell line. Two-dimensional immunoblotting shows that these antibodies recognize a subgroup of the spots recognized by the DR-specific monoclonal antibody VPM57 which does react with the T8.1 β chain. Amino-terminal sequence analysis of the α chain associated with VPM37β chain shows that this α chain is homologous to the human DRα chain strongly indicating that the β chain is DR-like. VPM37 and VPM43 are shown to be directed against different epitopes on sheep MHC class II molecules so it is highly unlikely that the data can be explained by the presence of posttranslational modifications or the existence of a very common allele. These data provide clear evidence for the expression of two distinct DRP chains in the sheep.     

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.     

Calorimetric and Fourier transform infrared spectroscopic studies on the interaction of glycophorin with phosphatidylserine/dipalmitoylphosphatidylcholine-d62 mixtures

Glycophorin has been isolated in pure form from human erythrocyte membranes and reconstituted into lipid vesicles composed of binary mixtures of bovine brain phosphatidylserine (PS) and acyl-chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The effect of protein on lipid melting behavior and order has been monitored with differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). The phase diagram for PS/DPPC-d62 is consistent with that previously reported for PS/DPPC (Stewart et al. (1979) Biochim. Biophys. Acta 556, 1-16) and indicates that acyl chain perdeuteration does not greatly alter the lipid mixing characteristics. The use of deuterated lipid allows the examination of lipid order by FT-IR of each lipid component in the binary mixtures as well as in the ternary (lipid/lipid/protein) systems. Addition of glycophorin to a 30:70 PS/DPPC-d62 binary lipid mixture results in a preferential glycophorin/PS interaction leading to bulk lipid enriched in DPPC-d62. This is revealed in two ways: first, through cooperative calorimetric transitions increased in temperature from the binary lipid system and second, through FT-IR melting curves of the DPPC-d62 component which shows transitions increased in both onset and completion temperatures in the presence of protein. In addition, non-cooperative melting events are observed at temperatures below the onset of phase separation. The FT-IR data are used to assign these non-cooperative events to the melting of the PS component. For the 50:50 lipid mixture with protein, two transitions are observed in the DSC experiments. The IR results indicate that both lipid components are involved with the lower temperature event.     

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.     

Iontophoretic injection of lucifer yellow CH into zygotes and blastomeres of the hydroidHydractinia echinata

The dye Lucifer yellow CH was iontophoresed into recently fertilized eggs and early blastomeres ofHydractinia echinata. Iontophoresis was carried out on the stage of an inverted microscope in order to follow filling of the injected cells by short pulses of epifluorescent illumination. Lucifer yellow proved to be nontoxic and development in embryos with injected blastomeres proceeded normally. When zygotes were injected all the cells of the forming embryo contained dye. When one of the first two blastomeres was injected all the progeny of the injected cell also contained dye. Dye-coupling between injected and uninjected blastomeres did not occur in two-cell embryos nor between descendants of either line. Development of Lucifer-yellow-filled blastomeres or zygotes could be stopped by blue light irradiation. In a number of injected cells, the dye tended to accumulate forming brightly shining spots. The dye did not penetrate the nuclear envelope of injected cells.     

Wives and Warriors: Women and the Military in the United States and Canada

Wives and Warriors: Women and the Military in the United States and Canada. Laurie Weinstein and Christie C. White. eds. Westport, CT: Bergin and Garvey, 1997. 252 pp.