Stimulation of interferon production]

Data on the effect of some factors on interferon production in vitro are presented The kinetics of interferon synthesis in response to superinduction was similar to the respective curve of the effect of UV-radiation on the cell. Possible similarity in the effect of these factors on the mechanisms controlling interferon production is noted. An increase in interferon synthesis under the effect of ascorbic acid in cells of chick embryo fibroblast and L-929 was found. Combined use of the inductors provided an increase in the tests of interferon.     

Stimulation by glutamine and proline of HGF production in hepatic stellate cells

Amino acids regulate cellular functions in a variety of cell types. Most notably, leucine stimulates protein production through the mammalian target of rapamycin (mTOR)-dependent signaling pathway. We investigated the effect of amino acids on hepatocyte growth factor (HGF) production. Treatment with glutamine and proline, as well as leucine, increased HGF levels in the culture medium of a rat hepatic stellate cell clone in a dose-dependent manner. Up-regulation of phosphorylation of 70 kDa ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 was not apparent in the cells after treatment with glutamine or proline. When rats received injections of glutamine or proline, hepatic and circulating HGF levels increased and peaked around 12 h after treatment. Glutamine and proline may have the potential to stimulate HGF production but the mechanism underlying this stimulation seems not to be through the mTOR-dependent signaling pathway.     

Stimulation of hepatic cholesterol biosynthesis by oleic acid

Livers from normal fed male rats were perfused $\text{in vitro}$ with an erythrocyte-free, bloodless medium containing serum albumin (3%), and glucose (100 mg %). Oleic acid (663 μmoles) bound to albumin, or albumin alone, was infused at a constant rate. Biosynthesis of cholesterol was evaluated by incorporation of radioactivity from ³H₂O. Oleic acid stimulated output of cholesterol (1.60 ± 0.08 SEM vs 1.18 ± 0.04 μmoles/g) but did not change the concentration of cholesterol in the liver or hepatic microsomes. Incorporation of ³H into cholesterol was stimulated by oleate; dpm per μmole cholesterol/dpm per μg atom H was 3.94 ± 0.33, 3.46 ± 0.32, and 4.46 ± 0.37 in the total cholesterol of liver, perfusate, and microsomes, respectively, when oleate was infused. Corresponding values when oleate was not infused were 1.71 ± 0.23, 1.62 ± 0.20, and 2.09 ± 0.26, respectively (P<0.001 in all cases). It is suggested that the stimulation of biosynthesis of cholesterol by oleate results from the obligatory requirement of cholesterol, as a moiety of the very low density lipoprotein, for the secretion of triglyceride by the liver.     

Stimulation of hepatic glycogenolysis by acetylglyceryl ether phosphorylcholine

1-O-Alkyl-2-acetyl-sn-glyceryl 3-phosphorylcholine or acetylglyceryl ether phosphorylcholine (AGEPC) stimulated glycogenolysis in perfused livers from fed rats at concentrations as low as 10(-11) M. At the lower AGEPC concentrations, e.g. 2 X 10(-10) M, a single transient phase of enhanced hepatic glucose output was elicited upon infusion of this agonist. At higher concentrations, e.g. 2 X 10(-8) M, a sharp transient spike of glucose output was observed, followed by a stable elevated steady state rate of glucose output until the AGEPC infusion was terminated. Increased rates of lactate and acetoacetate output and a diminished hepatic oxygen consumption were characteristic of the response of the livers to AGEPC at 2 X 10(-10) M. Neither alpha- nor beta-adrenergic antagonists blocked the glycogenolytic response of AGEPC. Repeated infusion of AGEPC led to homologous desensitization of the response, but the response of the liver to the alpha-adrenergic agonist, phenylephrine, or to glucagon, subsequent to AGEPC stimulation, was unaffected. Increasing the period of perfusion between successive additions of AGEPC, from 7 to 30 min, resulted in an increased glycogenolytic response to this agonist. When the perfusate calcium concentration was reduced from 1.25 to 0.05 mM, the glycogenolytic response to AGEPC was markedly diminished; calcium efflux from the liver following stimulation with AGEPC was not observed. The data presented in this study illustrate a potent agonist effect of AGEPC on the glycogenolytic system in the rat liver.     

Stimulation of hepatic triglyceride synthesis and secretion by clofibrate

Isolated hepatocytes prepared from rat and squirrel monkey livers were used to explore the mechanism of action of clofibrate, a hypolipidemic agent in current use. Addition of sodium clofibrate to cells suspended in Hanks medium stimulated the conversion of [1-¹⁴C]palmitate into esterified lipids and to ¹⁴CO₂. This agent also promoted the incorporation of [2-³H]glycerol into cellular lipids when fatty acids were present in the incubation medium. Triglycerides were the major lipid class increased by the drug. Sodium clofibrate enhanced the discharge of labeled lipids into the medium from liver cells prelabeled with [2-³H]glycerol. These data suggest that clofibrate does not lower plasma triglyceride levels by interference with hepatic triglyceride production or secretion.     

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.     

T-kinin release from T-kininogen by rat-submaxillary-gland endopeptidase K

Submaxillary gland extracts have been fractionated to characterize the enzyme responsible for the T-kininogenase activity previously reported in this tissue [Damas, J. & Adam, A. (1985) Mol. Physiol 8, 307-316] and to know whether this activity could be of physiological relevance, since no enzyme reacting in catalytic amounts has been described so far to be able to release a vasoactive peptide from T-kininogen. The purified enzyme, provisionally called endopeptidase K, has an apparent Mr of 27,000 when not reduced prior to analysis but 21,000 after reduction and an acidic pI of 4.3 +/- 0.1. Antigenically, it is not related to tissue kallikrein. Upon incubation with purified T-kininogen it may induce a complete liberation of T-kinin from the precursor provided it is added in stoichiometric amounts. However, in parallel with the liberation of immunoreactive kinin, a proteolysis of T-kininogen is observed which is not restricted to the site of insertion of T-kinin as would be expected using a specific kininogenase. In agreement with these results, no change of the mean blood pressure was observed upon injection of endopeptidase K into the circulation of normal rats even if the amount of injected enzyme was up to ten times that required for tissue kallikrein to induce a significant fall in blood pressure. However, in spite of the large proteolysis induced by incubation with stoichiometric amounts of endopeptidase K, the total papain inhibiting capacity of T-kininogen as well as the value of the apparent inhibition constant, Ki, with this proteinase remained unchanged. Proteolytic fragments which retain cysteine-proteinase-inhibiting activity may therefore be released from T-kininogen by endopeptidase K more easily than immunoreactive kinin, thus emphasizing a prominent function of proteinase inhibitor or of proteinase inhibitor precursor for this molecule.     

Stimulation of in vitro immunoglobulin production by interferon-alpha

The effect of various natural and recombinant DNA-derived human interferon-alpha (IFN-alpha) on immunoglobulin (Ig) production by human B cells was investigated. The cell populations examined included peripheral blood mononuclear cells (PBMC) and highly purified B cell and helper T cell populations obtained by negative selection by using monoclonal antibodies and a fluorescence-activated cell sorter. In the presence of all forms of IFN-alpha tested, IgG and IgM production by PBMC increased twofold to fourfold. This increase was noted in the absence of pokeweed mitogen (PWM), was not affected by depletion of monocytes, required that IFN-alpha was present early in the culture period, and reached maximal levels around 500 U/ml IFN-alpha. Both IgG and IgM production were affected, but the magnitude of the IgM response was greater. The augmentation of Ig production was noted with the recombinant DNA-derived subtype, IFN-alpha F, two analogs, IFN-alpha Con1 and IFN-alpha Con2, as well as with buffy-coat-derived (leukocyte) IFN-alpha. The recombinant DNA-derived forms of IFN-alpha appeared to differ in their ability to augment Ig production. In the presence of PWM, IFN-alpha Con1 failed to increase Ig production by PBMC. In contrast to these results with PBMC, IFN-alpha Con1 increased the Ig production of purified B cells 10- to 20-fold in the presence of PWM. This increase reached maximal levels around 500 U/ml IFN-alpha Con1. Although purified B cells responded to IFN-alpha and PWM, maximal responses occurred in the presence of low numbers of helper T cells. Cell dilution experiments suggested that the effect observed with purified B cells was the result of the interaction of B cells with residual cells, e.g., helper T cells, remaining in the preparations.