Peripheral T cell receptor gamma delta variable gene repertoire maps to the T cell receptor loci and is influenced by positive selection.

Although the mechanisms that determine TCR-alpha beta V gene repertoire are well studied, the genetic influences involved in TCR-gamma delta repertoire development are unclear. Unlike the TCR-gamma delta populations that localize in epithelial tissues, the circulating peripheral TCR-gamma delta V region repertoire is quite diverse. Previous studies have shown that three TCR-gamma chains and at least six TCR-V delta genes are expressed by splenic TCR-gamma delta cells. However, the relative frequency of individual gamma delta subsets among genetically diverse mice has not been determined. Therefore, the repertoire of TCR-gamma delta cells was examined using anti-TCR V region specific mAb against V gamma 2 and V delta 4 on TCR-gamma delta + cells from total splenocytes. We found that there was a strain-specific variation in TCR-gamma delta usage. The frequency of V gamma 2 expression in different strains varied from 54 to 12%, and the frequency of V delta 4 expression in different strains varied from 38 to 10%. However, the level of V delta 4 and V gamma 2 expression for an individual strain was highly consistent from experiment to experiment. F1 analysis between parental strains that differed in relative frequency of either V gamma 2+ or V delta 4+ cells revealed that high expression was genetically dominant, suggesting that positive selection events play a major role in the peripheral gamma delta repertoire. Variations in the levels of V gamma 2+ cells and V delta 4+ cells was not associated with Mls or MHC haplotype. Analysis of recombinant inbred strains revealed that high V delta 4 expression mapped to the TCR-gamma locus, while high V gamma 2 expression was influenced by the TCR-delta locus. Back-cross analysis confirmed that the TCR loci dominantly influenced the level of V delta 4+ cells and V gamma 2+ cells; however, there was clear evidence that multiple genes affect the TCR-gamma delta repertoire.     

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.     

The effect of gyrase inhibitors and cyclic AMP on induction and glucose repression of the 6-hydroxy-nicotine oxidases in Arthrobacter oxidans

The induction by d,l-nicotine of the enantiozymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase in Archrobacter oxidans was differently affected by the inhibitors of Escherichia coli gyrase, novobiocin and nalidixic acid. These compounds inhibited 6-hydroxy-L-nicotine oxidase induction slightly, but led to an increase in the level of 6-hydroxy-D-nicotine oxidase activity. Furthermore, the specific repression by glucose of 6-hydroxy-D-nicotine oxidase synthesis was not abolished by the addition of cAMP but by that of novobiocin.Abbreviations 6-HDNO 6-hydroxy-D-nicotine oxidase - 6-HLNO 6-hydroxy-L-nicotine oxidase - cAMP cyclic 3,5-adenosine monophosphate - Enzymes Adenylate cyclase - ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1) - cAMP-phosphodiesterase 3:5-cyclic-nucleotide 5-nucleotido-hydrolase (EC 3.1.4.17) - DNA gyrase DNA topoisomerase II (EC 5.99) - DNA polymerase deoxynucleosidetriphosphate: DNA desoxynucleotidyl-transferase (EC 2.7.7.7) - 6-hydroxy-L-nicotine oxidase 6-hydroxy-L-nicotine: oxygen oxidoreductase (EC 1.5.3.5) - 6-hydroxy-D-nicotine oxidase 6-hydroxy-D-nicotine: oxygen oxidoreductase (EC 1.5.3.6) - -lactamase penicillin amido--lactamhydrolase (EC 3.5.2.6) - nicotine dehydrogenase nicotine: (acceptor)6-oxidoreductase (hydroxylating) (EC 1.5.99.4)     

Superoxide release by zymosan-stimulated rat Kupffer cells in vitro

Kupffer cells were isolated from pronase-perfused rat livers and were maintained as a monolayer culture in a state of high purity and viability. Immediately after contact with zymosan particles, O2 uptake of the Kupffer cells increased fivefold; about 50% of the net oxygen consumed was accounted for as superoxide released into the medium. Concomitantly, a transient burst of luminol-dependent chemiluminescence, an increased activity of NAD(P)H oxidase and a stimulation of the flow of glucose through the hexose monophosphate shunt were observed. Chemiluminescence and O2- production were almost completely inhibited by superoxide dismutase and iodoacetate. Zymosan-induced chemiluminescence was not inhibited in the presence of the non-penetrating thiol reagents, 5,5'-dithio-bis-2-nitrobenzoate and iodoacetyl-sepharose. Iodoacetate acted on the cytosolic glucose-6-phosphate dehydrogenase rather than on NAD(P)H oxidase of the cell membrane.     

Properties and biosynthetic connection of the nucleotide pyrophosphatases of rat liver plasma membrane and endoplasmic reticulum

The detergent-solubilized nucleotide pyrophosphatases of the rat liver plasma membrane and endoplasmic reticulum fractions were purified by lectin affinity chromatography. They have the same molecular mass of 148 000 dalton; their catalytic properties are also very similar and correspond to those of the trypsin-solubilized activities from the same membrane preparations. Pulse-chase experiments on isolated perfused livers using [3H]leucine indicated different labelling kinetics of the proteins isolated from plasma membrane and endoplasmic reticulum. The plasma membrane enzyme became only slightly labelled in the presence of 100 microM vinblastine. The data support a precursor-product relationship of the nucleotide pyrophosphatases from endoplasmic reticulum and plasma membrane.     

Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.     

Influence of thyroid hormones on neuronal death and differentiation in larval Rana pipiens.

The sphingid moth, Manduca sexta, typically passes through five larval instars, a pupal, and an adult stage. The larval labial glands secrete silk in the first instar and a viscous lubricant in the fifth. During metamorphosis the glands develop into salivary organs which produce an invertase-rich secretion. In normal development, the uniform population of cells in the duct of the larval gland transforms into the four sequentially arranged regions of secretory and conductive cells of the adult gland. In order to determine when competence to form the adult gland is established, fragments of labial gland ducts from first through fifth instar larvae were implanted into pupae. These gland fragments underwent metamorphosis with their hosts, passing through the same developmental phases. Glands from as early as the first instar were competent to form histologically and functionally normal adult regions. In later instars, transplants of measured fragments demonstrated that larval cells were programmed in situ to develop into the four adult cell types.     

Hormonal regulation of gap junction differentiation

Thin-section, tracer, and freeze-cleave experiments on hypophysectomized Rana pipiens larvae reveal that gap junctions form between differentiating ependymoglial cells in response to thyroid hormone. These junctions assemble in large particle-free areas of the plasma membrane known as formation plaques. Between 20 and 40 h after hormone application, formation plaque area increases approximately 26-fold while gap junction area rises about 20-fold. The differentiation of these junctions requires the synthesis of new protein and probably RNA as well. On the basis of inhibitor experiments, it can be reported that formation plaques develop at about 16-20 h after hormone treatment and stages in the construction of gap junctions appear 4-8 h later. These studies suggest that gap junction subunits are synthesized and inserted into formation plaque membrane during the differentiation of the anuran ependymoglial cells.