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.     

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.     

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.     

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.