Studies on the relationship between glycolysis, lipogenesis, gluconeogenesis, and pyruvate kinase activity of rat and chicken hepatocytes.

Chicken hepatocytes synthesize glucose and fatty acids at rates which are faster than rat hepatocytes. The former also consume exogenous lactate and pyruvate at a much faster rate and, in contrast to rat hepatocytes, do not accumulate large quantities of lactate and pyruvate by aerobic glycolysis. α-Cyano-4-hydroxycinnamate, an inhibitor of pyruvate transport, causes lactate and pyruvate accumulation by chicken hepatocytes. Glucagon and N⁶,O²′-dibutyryl adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) convert pyruvate kinase (EC 2.7.1.40) of rat hepatocytes to a less active form. This effect explains, in part, inhibition of glycolysis, inhibition of lipogenesis, stimulation of gluconeogenesis, and inhibition of the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment by these compounds. In contrast, pyruvate kinase of chicken hepatocytes is refractory to inhibition by glucagon or dibutyryl cyclic AMP. Rat liver is known to have predominantly the type L isozyme of pyruvate kinase and chicken liver predominantly the type K. Thus, only the type L isozyme appears subject to interconversion between active and inactive forms by a cyclic AMP-dependent, phosphorylation-dephos-phorylation mechanism. This explains why the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment of chicken hepatocytes is insensitive to cyclic AMP. However, glucagon and dibutyryl cyclic AMP inhibit net glucose utilization, inhibit fatty acid synthesis, inhibit lactate and pyruvate accumulation in the presence of α-cyano-4-hydroxycinnamate, and stimulate gluconeogenesis from lactate and dihydroxyacetone by chicken hepatocytes. Thus, a site of action of cyclic AMP distinct from pyruvate kinase must exist in the glycolytic-gluconeogenic pathway of chicken liver.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. III. Cholera enterotoxin induces lymph node cells to release soluble factor(s) which enhance(s) antibody synthesis by antigen-treated lymph node cells.

Unprimed or KLH-primed rabbit lymph node cells were pulsed with cholera enterotoxin or KLH for 2 hr and washed. KLH-treated LNC were mixed with equal numbers of CT-treated LNC or boiled CT-treated LNC. Cocultivation of CT-treated LNC with KLH-treated cells resulted in at least a 100% increase in antibody synthesis compared to control cultures. Delaying cocultivation for 24 hr reduced enhancement to 25%. Thus it appears that an early event—before 24 hr—is involved in CT enhancement. Using ¹²⁵I-CT, it was shown that these effects were not due to CT carry-over. When KLH- and CT-pulsed LNC were cultured in chambers separated by polycarbonate membranes (0.2- to 0.4-μm pore size) antibody production was enhanced 50–80%. Supernates of CT-treated LNC also enhanced antibody production by KLH-treated LNC. These results suggest that CT triggers the release of soluble factor(s) which enhance(s) antibody synthesis by antigen-primed and antigen-challenged LNC.     

A sterile leakproof plastic vial for cell cryopreservation in liquid nitrogen: Application to parasitic protozoa

Polyethylene bulbs derived from sterile transfer pipets were evaluated as leak-proof vials for cryopreservation of parasitic protozoa. The bulbs were easily frozen and stored in conventional liquid nitrogen refrigeration equipment and were less expensive and safer to handle than plastic screw-capped cryovials. The easily testable heat seal of the polyethylene bulb never failed during longterm immersion in liquid nitrogen, and dispensing of thawed material was simple and safe. Good survival of L. donovani (80%), T. rhodesiense (95%), and C. fasciculata (95%) was obtained after cryopreservation in polyethylene bulbs or plastic screw-capped vials. The polyethylene bulb was considered an excellent container for the safe cryopreservation of pathogens and other biological material.     

Serum testosterone concentration and penile tumescence changes in men

Serum testosterone concentration of 24 human males was correlated with penile diameter changes in response to erotic stimuli. Mean testosterone concentration was significantly and negatively correlated with latency to maximum tumescence and it is hoped that this finding will shed light on the psychophysiological mechanisms involved in normal and impaired erectile function.     

A rapid column chromatographic method for the isolation of catechol-type siderophores.

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.     

Vitamin B12 and the macromolecular composition of Euglena. I. Kinetic analysis of the cell cycle and chloroplast replication

The kinetics of cell division, chloroplast replication and mean DNA/cell of cultures progressing through B₁₂ deficiency do not follow smooth curves, but contain transient plateaus which are consistent mathematically with the hypothesis that one portion of the Euglena cell cycle, the S phase, is differentially extended under B₁₂ deficiency. A computer simulation of the B₁₂-deficient cultures was capable of duplicating the division kinetics of the actual culture. Chloroplast replication in B₁₂-deficient cells is not directly affected by B₁₂ deficiency, but is a function of the division kinetics of the cells. The chloroplasts continue to replicate initially at a high rate after the cells have entered B₁₂ deficiency, and then follow the kinetics of the cells.     

1H-N.M.R. spectra of glycosaminoglycan monomers and dimers in solution in methyl sulphoxide and water

¹H-N.m.r. spectra of glycosaminoglycuronan monomers and dimers in solution in methyl sulphoxide-d₆ have been investigated; N-H and O-H resonances were observed and partially assigned. Their temperature-dependence suggests hydrogen-bonding to the solvent, with the notable exception of that of HO-4 Of sodium D-gluctironate, which was consistently downfield and relatively temperature-insensitive. The concentration-dependence ofthis signal indicates that the corresponding hydroxyl group is involved in the formation of a dimer. Signals for N-H and O-H were observed for aqueous solutions, especially at subzero temperatures.     

Separation of enzymatic activities in Dictyostelium discoideum by high-pressure gel permeation chromatography.

High-pressure gel permeation chromatography was used to separate the cyclic AMP phosphodiesterase and ATP pyrophosphohydrolase activities of Dictyostelium discoideum. Two types of column packings, with different functional groups on the silica-bonded carbon side chains, were used to separate the two activities in approximately the same amount of time and with the same elution pattern. Recovery of both activities was enhanced when acetate, rather than sulfate, was the mobile phase. This recovery of activity following chromatography at high pressure demonstrates that high-pressure gel permeation chromatography can be used for the purification of enzymes.     

A simplified assay for phosphoenolpyruvate.

A new method for analysis of phosphoenolpyruvate has been developed. The assay is based upon the stoichiometric conversion of ADP to ATP by the enzyme pyruvate kinase in the presence of variable amounts of PEP, and subsequent measurement of the ATP with a luciferin-luciferase preparation.     

Cholinephosphophotransferase activities in early rat embryos and their associated placentas.

CDP-Choline:1,2-diglycerolcholinephosphotransferase (EC 2.7.8.2, cholinephosphotransferase) activities were determined in subcellular fractions prepared from rat embryos, placentas, or yolk sacs obtained on the fourteenth day of gestation. It was found that, in all of the tissues studied, cholinephosphotransferase activity (1) copurified with NADPH-cytochrome c reductase activity (EC 1.6.2.4), (2) was maximal around pH 8.0; (3) was stimulated by MgCl₂, exogenous diolein, and cytidine diphosphocholine (CDP-choline); and (4) was highest in homogenates of placentas, lowest in those of embryos, and intermediate in those of yolk sacs. These data substantiate, for the first time, that the early mammalian (rat) embryo, placenta, and yolk sac have the ability to synthesize phospholipids de novo.     

In vitro and in vivo induction of effector T cells mediating DTH responses to a protein and a synthetic polypeptide antigen.

We have developed an in vitro system for the activation of T cells in order to get a better insight into the genetic and molecular mechanisms involved in the generation of delayed-type hypersensitivity (DTH) effector T cells. Low doses of fowl γ-globulin (FγG) as well as the synthetic polypeptide (T,G)-A-L were bound to splenic adherent cells and served as immunogens for the in vitro sensitization of lymphocytes. In parallel, (T,G)-A-L-specific T cells were activated in vivo in irradiated recipient mice. The ability of the in vitro- and in vivo-activated cells to mediate DTH responses was determined in naive recipient mice by the radioisotopic ear assay. Twenty to thirty × 10⁶ “educated” cells were sufficient to elicit significant DTH responses. Irradiation of the spleen cells prior to their transfer resulted in higher responses. The DTH reactivity was transferable by nylon wool-enriched T cells but not by a Thy 1.2-depleted population indicating the T-cell dependency of the response. The in vitro and in vivo antigen-activated T-cell population exhibited also helper-cell activity as determined by their cooperation with B cells in adoptive transfer experiments.     

Inhibition of tropoelastin secretion by incorporation of DL-3,4-dehydroproline

Cells isolated from embryonic chick aorta were incubated in suspension culture with labeled amino acids and proline analogs. Incorporation of 4-cis-hydroxy-l-proline inhibited the secretion of labeled procollagen but not tropoelastin, while incorporation of dl-3,4-dehydroproline inhibited the secretion of both proteins and caused them to accumulate intracellularly. Protein synthesis did not appear to be significantly diminished during the 2-h incubation period. Incorporation of dl-3,4-dehydroproline may alter the conformation of tropoelastin leading to abnormal intracellular processing and a decreased rate of secretion.     

The effects of cell density and starvation on early developmental events in Dictyostelium discoideum

We show that removal of yeast extract and trypticase from growth medium is sufficient for induction of several key events which occur during the early stages of Dictyostelium differentiation: run-off of polysomes, the earliest known change in macromolecular metabolism; appearance of the cell surface cAMP receptor; and aggregation itself. Starvation of glucose has little effect on these parameters. These results are consistent with those of other investigators who showed that starvation only of amino acids will induce other activities associated with cAMP-mediated cell signaling and cell-cell adhesion. We show, in contrast, that other factors are involved in the increase in the relative rates of synthesis of three polypeptides very early in differentiation: actin, and two proteins (“45-min” proteins) which are synthesized only during the period of 45–90 min. The induction of synthesis of these three proteins and presumably, of their mRNAs, is not the result of starvation for glucose or amino acids but is the result of plating cells at high density. The increases in the synthesis of these proteins are dependent on the density at which cells are plated and do not occur at a density 75-fold lower than the density used in standard experiments. Cells growing at high density or near stationary phase do not show the induction of increased synthesis of actin or the “45-min” proteins. These experiments suggest that these early developmental changes may be dependent on a threshold level of a diffusible factor excreted early in development.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.     

Induction of male-typical aggression by androgens but not by estrogens in adult female mice

Ovariectomized adult CF-1 female mice were implanted with silastic capsules containing either testosterone (T), dihydrotestosterone (DHT), methyltrienolone (R1881), estradiol (E2), diethylstilbestrol (DES), or oil vehicle and were tested for aggressive behavior. The androgenic treatments (T, DHT, R1881) were highly effective in promoting male-like aggression while the estrogens (DES, E2) were completely ineffective. Subsequent receptor-binding studies confirmed assumptions about the specificity of DES, DHT, and R1881 binding to estrogen and androgen receptors in mouse hypothalamus.     

A new class of model glycolipids: synthesis, characterization, and interaction with lectins.

A method is described for preparing model glycolipids by linking aldobionic acids to an alkylamine through an amide bond. These compounds may be rapidly prepared in large quantities. The glycolipids precipitate specifically with lectins. Precipitation occurs at glycolipid concentrations just above their critical micelle concentration.     

T-cell mediated suppression in the MRL mouse

MRL/lpr mice possess an autosomal recessive gene, lpr, which is associated with lymphoproliferation and acceleration of autoimmune disease. Lymphoproliferation has been ascribed to a single gene defect predominantly affecting the T-lymphocyte component of the immune system. MRL/++ mice do not possess the lpr autosomal recessive mutation and do not develop early lymphadenopathy. T-lymphocyte functional activity was studied in these mice using the polyclonal T-cell mitogens PHA and Con A. Our results indicated a significant suppression of the spleen and lymph node response of MRL/lpr mice to these polyclonal mitogens as compared to the MRL/++ response noted as early as 6 weeks of age. In addition, there was a progressive decline in the MRL/lpr spleen and lymph node cell mitogenic responses with increasing age. Spleen and lymph node cells from 20-week MRL/lpr mice were also relatively unresponsive in the mixed lymphocyte culture reaction as compared to cells from MRL/ ++ or BALB/c mice. The in vitro proliferative response of the MRL mice was further examined with respect to possible accessory cell modulation by both macrophages and T cells. It was found that in 20-week MRL/lpr lymph nodes a significant degree of suppression of lymphocyte proliferation could be mediated by the MRL/lpr T cell. Increased lymphocyte proliferation to a mitogenic signal could only be demonstrated in those MRL/lpr mice 3 weeks of age.     

Occurrence of two extracellular acidic polysaccharides as products of Rhizobium meliloti 201

The extracellular polysaccharide of Rhizobium meliloti 201 consists of two acidic polysaccharides, APS-I and APS-II. APS-I is composed of D-glucose, D-mannose and D-glucuronic acid in a molar ratio of 3:3:2, whereas APS-II is composed of D-glucose, D-galactose, D-mannose and pyruvic acid in a molar ratio of 4:3:2:1.APS-II was separated from the extracellular polysaccharide preparation by hydrolysing APS-I to its octasaccharide repeating unit with a specific enzyme. APS-I and APS-II were also separated by treatment with cetylpyridinium chloride and by paper electrophoresis of the depyruvylated polysaccharide.