Antigen-binding receptors on T cells from long-term MLR. evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (VH) determinants strongly inhibited vesicle binding by both primary and long-term MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesicle-binding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectivelly blocked with the anti-VH reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.     

Guanosine 5′-triphosphate and guanosine 5′-[βγ-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase

1. GTP, but not p[NH]ppG (guanosine 5′-[βγ-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, ¹²⁵I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and ¹²⁵I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.     

Structural studies of the haemophilus influenzae type f capsular polysaccharide

The structure of the Haemophilus influenzae type f capsular polysaccharide was studied by chemical and nuclear magnetic resonance spectroscopic techniques. The repeating unit of the polysaccharide was found to be

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Regulation of RNA synthesis in yeast III

Summary Centrifugal elutriation was used to separate cells in different stages of the cell cycle from a culture of Saccharomyces cerevisiae in balanced exponential growth. The rate of DNA and RNA synthesis was determined using a pulse-long-term label technique that is capable of distinguishing between exponential, linear, and periodic variations in the rate of synthesis through the cell cycle. It was found that while the rate of DNA synthesis varies periodically through the cell cycle, the rate of synthesis of mRNA, rRNA, and tRNA increases exponentially through the cell cycle. The implications of these findings for the control of RNA synthesis are discussed.     

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K⁺ > Rb⁺ > (Na⁺ = Li⁺). Calcium and Mg²⁺ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO₄³⁻ > NO₃⁻ > Cl⁻. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca²⁺ ions, or to effects on K⁺ uptake. A mixture of Na⁺ and K⁺ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na⁺ plus K⁺. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.     

Permeability of the liver cell membrane to quinolinate.

Quinolinate was taken up by both rat and guinea-pig liver cells. Equilibrium was reached after approx. 20 min with rat cells, but guinea-pig cells had not achieved a steady state after 60 min. There was no evidence to suggest that quinolinate is rapidly metabolized by either species. The concentrations of quinolinate attained in rat and guinea-pig cells after short periods of incubation with 0.5 mM-quinolinate did not inhibit gluconeogenesis. These results raise further doubts as to the mechanism of quinolinate action in liver.     

Preparation and characterization of isolated parenchymal cells from guinea pig liver

1. A method is described for the preparation of isolated cells from guinea pig liver. This involved perfusion in situ, in the non-physiological direction, with collagenase. 2. The cell yield was 20--30%, comparable with those from the livers of other species. 3. The ratio of lactate dehydrogenase to glutamate dehydrogenase in the cells was similar to that in vivo, indicating that there was negligible leakage of cytoplasmic enzymes. 4. The concentrations of K+ and adenine nucleotides were initially lower than in the perfused liver; normal values were obtained on incubation, particularly in the presence of substrate. 5. The L-lactate: pyruvate ratio is 16:1, close to established values. The total beta-hydroxybutyrate: acetoacetate ratio indicates that the mitochondrial redox state is more oxidised than in the perfused liver, but the intracellular ratio is similar to that of the intact liver. 6. Rates of gluconeogenesis and ureogenesis, are within the physiological range. Maximal gluconeogeneis from L-lactate was preceded by a lag period. L-lysine stimulated glucose production from L-lactate but did not abolish the lag phase. 7. The effects of aminooxyacetate and octanoate on L-lactate gluconeogenesis were similar to those in the perfused liver.     

beta-Adrenergic receptor induction in HeLa cells: synergistic effect of 5-azacytidine and butyrate

³H-Labeled leukotriene C₃ was efficiently taken up by the isolated, perfused rat liver and excreted into the bile. The isolated, perfused kidney eliminated leukotriene C₃ from the perfusate slower and excreted only a fraction of the radioactivity into the urine. Isolated hepatic, intestinal and renal cells also took up leukotriene C₃, the renal cells being the most effective in accumulating the label. Anthglutin, an inhibitor of γ-glutamyl transferase, decreased the uptake by kidney cells but had no effect on the uptake by the other cell types. In liver cells, the uptake rate was sensitive to temperature and to cellular ATP content. Chromatographic analyses indicated that renal cells metabolized leukotriene C₃ more rapidly than hepatic and intestinal cells. Leukotriene D₃ and E₃ were formed during the incubations with kidney cells, whereas intestinal cells produced mainly more polar metabolites.     

Interleukin-5, interleukin-3, and granulocyte-macrophage colony-stimulating factor cross-compete for binding to cell surface receptors on human eosinophils.

Human interleukin (IL)-5 receptors were characterized by means of binding studies using bioactive 125I-labeled IL-5. Of purified primary myeloid cells, eosinophils and basophils but not neutrophils or monocytes expressed surface receptors for IL-5. Binding studies showed that eosinophils expressed a single class of high affinity receptors (Ka = 1.2 x 10(10) M-1) with the number of receptors being small (less than 1000 receptors/cell) and varying between individuals. Among several cell lines examined only HL-60 cells showed detectable IL-5 receptors which were small in numbers (200 receptors/cell) and also bound 125I-IL-5 with high affinity. The binding of IL-5 was rapid at 37 degrees C while requiring several hours to reach equilibrium at 4 degrees C. Specificity studies revealed that the two other human eosinophilopoietic cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited the binding of 125I-IL-5 to eosinophils. No competition was observed by other eosinophil activating or nonactivating cytokines. The inhibition of 125I-IL-5 binding by IL-3 and GM-CSF was partial up to a concentration of competitor of 10(-7) M with GM-CSF consistently being the stronger competitor. Converse experiments using IL-5 as a competitor revealed that this cytokine inhibited the binding of 125I-IL-3 and of 125I-GM-CSF in some but not all the individuals tested, perhaps reflecting eosinophil heterogeneity in vivo. Cross-linking experiments on HL-60 cells demonstrated two IL-5-containing complexes of Mr 150,000 and Mr 80,000 both of which were inhibited by GM-CSF. The competition between IL-5, IL-3, and GM-CSF on the surface of mature eosinophils may represent a unifying mechanism that may help explain the common biological effects of these three eosinophilopoietic cytokines on eosinophil function. This unique pattern of competition may also be beneficial to the host by preventing excessive eosinophil stimulation.