Primary structure of rabbit lung uteroglobin as deduced from the nucleotide sequence of a cDNA

Double-stranded cDNA was synthesized from partially purified uteroglobin mRNA from rabbit lung. A cDNA coding for lung uteroglobin was then cloned in the plasmid pUC18 and both the nucleotide sequence and the derived amino acid sequence were determined. This allowed us to demonstrate unequivocally that uteroglobins from lung and uterus are identical proteins.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

The orientation of the distal part of the quadriceps femoris muscle as a function of the knee flexion-extension angle

Lateral view radiographs of ten autopsy knees were used to determine the orientation of the patellar ligament, patella and quadriceps tendon relative to tibia and femur at different flexion-extension angles (0-120 degrees) of the knee. The results show a linear relationship between the angle of flexion and the movement of the patellar ligament relative to the tibia and of the movement of the patella relative to tibia and femur. There is a non-linear relationship between angle of flexion and the movement of the quadriceps tendon relative to the patellar ligament, patella and femur. The angular changes between patella and patellar ligament are negligible. The complicated movements of the distal part of the quadriceps femoris muscle may significantly influence biomechanical parameters such as the forces acting at the patella and tibial tuberosity.     

2H NMR investigation of the organization and dynamics of polyisoprenols in membranes

The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells. The molecular details of how these lipid cofactors function is unknown. Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes. To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride. The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s). Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond. Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane. T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine. The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar. These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.     

Modulation of beta-adrenergic response in rat brain astrocytes by serum and hormones

Purified astrocyte cultures from neonatal rat cerebrum respond to isoproterenol, a beta-adrenergic agonist, with a transient rise in cAMP production. This astroglial property was regulated by serum, a chemically defined medium (serum-free medium plus hydrocortisone, putrescine, prostaglandin F2 alpha, insulin, and fibroblast growth factor) and epidermal growth factor. Compared to astrocytes grown in serum-supplemented medium, astrocytes grown in the chemically defined medium were nonresponsive to isoproterenol stimulation, and this difference did not appear to be due to selection of a subpopulation of cells by either medium. The data suggest that a decreased beta-adrenergic receptor number and an increased degradation of cAMP may account for the reduced response to beta-adrenergic stimulation. The nonresponsive state of astrocytes in the defined medium was reversible when the medium was replaced with serum-supplemented medium. An active substance(s) in serum was responsible for restoring the responsiveness of astrocytes. Each of the five components of the defined medium had little effect by itself; however, together they acted synergistically to desensitize astrocytes to beta-adrenergic stimulation. On the other hand, epidermal growth factor, a potent mitogen for astrocytes, was very competent by itself in reducing the cAMP response of astrocytes to beta-adrenergic stimulation. Thus purified astrocytes grown in the chemically defined medium appear to be a good model for the study of hormonal interactions and of serum factors which may modulate the beta-adrenergic response.     

Role of solute drag in intestinal transport

This study presents experiments related to the role of solvent drag and solute drag in the transmembrane movement of nonelectrolytes in a perfused rat intestine preparation. Conditions were chosen to simulate the effects of luminal hyperosmolarity on the permeability of tracer solutes. Data are presented on net water flux, transepithelial potentials, and lumen-to-blood and blood-to-lumen tracer solute movements during control electrolyte perfusion and after making the perfusate hyperosmotic. The results indicate that both solvent drag and solute drag can play significant roles in the transepithelial movement of solute and solute permeabilities in the rat ileum preparation. It is suggested that the potential roles of solvent drag and solute drag should be accounted for or considered during the characterization of the mechanisms of biological membrane function.     

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Combined detection of antigen specificity (anti-TNP) and (sub)class of intracellular antibodies

Mice and rabbits were immunized with trinitrophenyl (TNP)-conjugated keyhole limpet hemocyanin (KLH). Cells producing specific antibodies against the hapten TNP were detected in vivo in spleen and lymph nodes using a TNP--alkaline phosphatase (AP) conjugate. Using horseradish peroxidase (HRP)-conjugated anti-mouse (sub)class (IgG2A, IgG2B, IgM) antibodies and anti-rabbit class (IgG, IgM) antibodies and a double immunocytochemical staining technique for simultaneous demonstration of the enzymes AP and HRP, we were able to determine both the antigen specificity (anti-TNP) and the (sub)class of intracellular antibodies produced by individual antibody-forming cells in vivo.     

Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction

In this report we show that three mutagenized sublines of (murine) EL-4 thymoma cells can constitutively activate human and/or murine B cells via an MHC-nonrestricted cell-cell interaction. The activation signal is not by itself mitogenic but renders B cells capable of proliferating in response to interleukin 2 (IL 2). In addition, one of the mutant EL-4 sublines can constitutively respond by release of IL 2 in the presence of IL 1-containing macrophage (P388D1) supernatant. The exact relationships between these functional properties of the mutant EL-4 thymoma cells and those associated with activated normal T helper-cells remain to be established. However, the EL-4 cells provide a unique system to study in parallel murine and human B cell responses. In particular, the following observations were made during the present study. First, anti-Ig antibodies (anti-Ig) were required for B cell activation in conjunction with two EL-4 sublines acting only on murine B cells, whereas with a third subline acting on both murine and human B cells, anti-Ig was not required. Anti-Ig by itself did not lead to significant B cell activation in the absence of mutant EL-4 (or normal T) cells. Second, the growth factor-stimulated proliferation of EL-4-activated B cells, following separation of the B cells from the EL-4 cells, lasted only 2 days. These results, thus, indicate that the requirement for a surface Ig-mediated B cell activation signal depends on the quality/intensity of a direct T cell signal and that cell-cell interactions may exert a more stringent control over the growth factor responsiveness of B cells as compared with T cells.     

5S and 5.8S ribosomal RNA evolution in the suborder tetrahymenina (Ciliophora: Hymenostomatida)

Summary The nucleotide sequences of the 5S and 5.8S rRNAs of eight strains of tetrahymenine ciliates have been determined. The sequences indicate a clear distinction betweenTetrahymena paravorax and its suggested conspecificT. vorax, but leave the taxonomic distinction betweenT. vorax andT. leucophrys in doubt. The rRNA sequences of sixTetrahymena species and of three other species of the suborder Tetrahymenina have been used to deduce evolutionary schemes in which ancestral rRNA sequences and changes are proposed. These schemes suggest the predominant acceptance of GA and CT transitions in the 5S rDNA during the evolution of the suborder.     

Blockade of a motor response by cholinergic stimulation of the toad midbrain tegmentum

Application of an acid solution to the dorsal skin of conscious toads having intact nervous system induces a scratching reflex and escape movements, as well as autonomic alterations (hypertension and tachycardia) that are part of the defense response. The motor components of this response are abolished or reduced by microinjection of 60, 30, 15 or 7.5 ng carbachol into the midbrain tegmentum. The cardiovascular components, however, continue to be present, although their amplitude is reduced. The depression of the motor response is statistically significant up to 15 minutes for the 60 ng dose, up to 10 minutes for the 15 and 30 ng doses, and only up to 5 minutes for the 7.5 ng dose. The data suggest that the midbrain tegmentum may modulate the reflex motor response triggered by a noxious stimulus and also participate in the organization of the escape movements. The importance of cholinergic agents in this modulation is discussed. The persistence of the cardiovascular component of the response shows the importance of this parameter as an indicator of alert situations.