Modifications of sphingomyelin and phosphatidylcholine metabolism by tricyclic antidepressants and phenothiazines

Phenothiazines and tricyclic antidepressants, when added to culture medium, gave rise in several types of cells (C6 rat glioma cells and human fibroblasts), to a decrease in lysosomal sphingomyelinase activity. The effect of chlorpromazine and desipramine was dose dependent, and was observed after 3 hours of incubation with the drugs at concentrations ranging between 1 and 10 microM. In C6 glioma cell cultures, the decrease in sphingomyelinase activity was related to the clinical effectiveness of phenothiazines, tricyclic antidepressants and derivatives. Incorporation of (choline-14C) sphingomyelin showed that the metabolic pathway implying the synthesis of phosphatidylcholine from the hydrolysis of sphingomyelin and/or transfer of phosphorylcholine to phosphatidylcholine was also partially reduced.     

Regulation of major acute-phase plasma proteins by hepatocyte- stimulating factors of human squamous carcinoma cells

Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.     

Genetics and evolution of the acute phase proteins in mice

Summary In mammals, a number of liver-derived plasma proteins, termed acute phase reactants, are induced during an inflammation response. We have studied genetic variation in the structure and expression of several of these proteins in a variety of inbred and wild-derived mice. In a genetic cross, electrophoretic polymorphisms for the two ₁-acid glycoproteins, AGP-1 and AGP-2, co-segregated in 58 backcross progeny, indicating that either a single gene or two tightly-linked genes on chromosome 4 encode the AGPs. In the same backcross, segregation of variation in haptoglobin structure showed that the gene encoding this acute phase reactant is on chromosome 8. Structural variation in serum amyloid A correlated with restriction fragment length polymorphisms in the Saa gene determined by Taylor and Rowe (1984). Analysis of a number of highly diverged species of mice indicated that AGP expression has undergone considerable modification during evolution of the Mus genus; this is associated with alterations in Agp gene organization, which may include species-specific amplification and/or deletion events.     

N-Acetylneuraminic acid storage disease

Summary Increased amounts of free sialic acid were found in body fluids, leukocytes, cultured fibroblasts, and liver tissue of a four-year-old boy with mental retardation, ataxia, and clinical and radiologic findings of a mild mucopolysaccharidosis. A diagnosis of Salla disease was made though in contrast to earlier reports, recurrent upper respiratory infections and hepatosplenomegaly were present already in infancy, and skeletal abnormalities of dysostosis multiplex were found in early childhood. Free sialic acid in the urine was identified as N-acetylneuraminic acid by ¹H-NMR spectroscopy. Sialidase activities were normal. Increased amounts of bound sialic acid were found in liver and cultured fibroblasts and were attributed to an intracellular inhibition of sialyloligosaccharide-degrading neuraminidase by excessive amounts of free neuraminic acid. The molecular basis of N-acetylneuraminic acid storage disease is unknown but may be related to a defective transport mechanism preventing neuraminic acid from leaving the lysosomal compartment.Abbreviations Ganglioside GD 1a IV³NeuAc,II³Neu5Ac-GgOse₄Cer - sialidase neuraminidase, EC 3.2.1.18 This paper is dedicated to Professor Günter Quadbeck on the occasion of his 70th birthday     

Glucose starvation leads in rat hepatoma cells to partially N-glycosylated glycoproteins including alpha 1-acid glycoproteins. Identification by endoglycolytic digestions in polyacrylamide gels

Within minutes of glucose starvation confluent monolayers of rat hepatoma cells synthesize glycoproteins, including alpha 1-acid glycoprotein, which appear on two-dimensional gels as size heterogeneous spot series. The longer the period of glucose starvation the more the production of the glycoproteins is shifted toward smaller molecular weight forms. To compare these forms with the corresponding glycoproteins synthesized either in a cell-free system or by nonstarved cells, a mapping of the N-glycan was done by endo-beta-N-acetylglucosaminidase digestion within a polyacrylamide gel. Glycoproteins from glucose-starved cells contain a reduced number of N-glycans which belong to both the endo H-sensitive and resistant type. The decrease of N-glycosylation may be correlated with the accumulation of truncated lipid-bound oligosaccharides, for the gel chromatography of the oligosaccharides released from the lipid and protein fractions of glucose-starved cells revealed a drastic reduction in their size. Most of the lipid-linked oligosaccharides synthesized during glucose starvation are resistant to endo H digestion. Under conditions of limiting glycosylation we were able to show by glycopeptide analysis, that in the case of alpha 1-acid glycoprotein, N-glycans are added randomly to the 6 possible N-glycosylation sites. Furthermore, non- or partially N-glycosylated proteins do not acquire additional oligosaccharide units after restoration of glucose although the proteins can undergo secondary modification and, in the case of the secretory proteins, can be exported.     

Preferential degradation of the terminal carbohydrate moiety of membrane glycoproteins in rat hepatoma cells and after transfer to the membranes of mouse fibroblasts

Glycoproteins in the plasma membrane of rat hepatoma cells were labeled at their externally exposed tyrosine residues with 131I and at their galactose and sialic acid residues with 3H. The degradation of both isotopes in the total cell protein fraction, in glycoproteins purified by concanavalin A, and in glycoproteins separated on two-dimensional gels was determined. Similarly, the total cellular membrane glycoproteins were metabolically labeled with [35S]methionine and [3H]fucose. The fate of both incorporated labels was followed by lectin chromatography or by precipitation of the proteins with specific antibodies followed by electrophoretic gel separation. In both labeling experiments, the carbohydrate markers were lost from the ligand- recognized fraction with similar kinetics as from the total cell protein fraction. In some glycoprotein species which were separated by two-dimensional gel electrophoresis, the polypeptide portion exhibited up to a twofold slower rate of degradation relative to that of the carbohydrate moiety. This difference is most pronounced in carbohydrate- rich glycoproteins. To corroborate this finding, double-labeled membrane glycoproteins were incorporated into reconstituted phospholipid vesicles which were then transferred via fusion into the plasma membrane of mouse fibroblasts. Both the polypeptide and carbohydrate moieties of the transferred membrane glycoproteins were degraded with the same relative kinetics as in the original hepatoma cells. The rate of degradation is mostly a function of the structural properties of the membrane components as shown by the preservation of metabolically stable fucogangliosides of Reuber H-35 hepatoma cells transferred onto the fibroblasts. The technique of insertion of membrane components into the plasma membrane of another cell should assist in the elucidation of the exact route and mechanism of membrane protein destruction.     

Metabolic Relations between Methylxanthines and Methyluric Acids in Coffea L

Metabolism of purine alkaloids in the leaves of Coffea dewevrei De Wild et Durand var excelsa Chev, Coffea liberica Bull ex Hiern and Coffea abeokutae Cramer was studied by analyzing leaf discs collected during vegetative development and by feeding the following radioactive tracers: [¹⁴C]theobromine, [¹⁴C]caffeine, and [¹⁴C]theacrine (1,3,7,9-tetramethyluric acid). Their principal metabolites were quantitatively and qualitatively determined. All three species convert the precursors to the same radioactive products, and proceed through the same four maturity stages characterized by the alkaloid accumulation pattern and by a particular transformation potency: (stage 1) young plant accumulating caffeine, transforms theobromine to caffeine; (stage 2) caffeine is gradually replaced by theacrine, theobromine and caffeine are converted to theacrine; (stage 3) theacrine disappears whereas liberine (O(2), 1,9-thrimethyluric acid) accumulates, theacrine is metabolized to liberine; (stage 4) branched-out plant containing liberine but no theacrine, caffeine is converted rapidly to liberine via theacrine. Methylliberine (O(2),1,7,9-tetramethyluric acid), presumably the direct precursor of liberine, is occasionally found in low concentrations at stage 3 and 4.

The collective term `liberio-excelsoid' introduced by geneticists for the numerous races or species of Pachycoffea is in accordance with the phytochemical equality found in this work.

     

Zur Problematik der Chromosomenautoradiographie

The distribution of silver grains on the chromosomes of group G was studied in 216 mitoses from five patients with Down's syndrome after incubation with tritated thymidine in order to test the replication pattern of these autosomes. The different labelling densities over the chromosomes were determined by comparative estimations of the blackened areas and subdivided into four degrees: very strongly labelled, strongly labelled, weakly labelled and unlabelled. The 56 combinations for five G chromosomes and four labelling densitives were divided into three groups: group A, with three strong and two weak labelling types, group B, with two strong and three weak labelling types, and group C, with the remaining combinations. Thereby 51 (23.6%) mitoses were counted in six combinations of group A, 31 (14.4%) in six combinations of group B, and 134 (62.0%) in 44 combinations of group C. In order to test the possibility of identifying the G chromosomes based on a different DNA replication pattern the findings were subjected to a statistical analysis. The expected values were calculated for the different combinations of labelling degrees and were compared with the observed values. This method being used, the question whether the extra chromosome is replicating late or early compared with the remaining G chromosomes could not be answered with sufficient certainty, because P_A=1, P_B=0 could not be assumed on our figures. We conclude from these findings that the termination phases for the chromosomes 21 as well as 22 lie close together and that apparently the autoradiographic representation of the replication pattern can be influenced by various factors. The influencing factors can only be understood in connection with the problems of quantitative autoradiography. They are subdivided into three groups and discussed in this way: A. Factors due to the physiology of the genetic material: 1. Duration of the DNA-synthesis and the G₂-period. 2. Chromosomal replication pattern. 3. The relation of ³H-Thymidin and unlabelled thymidine in the newly synthesized DNA. B. Factors due to the autoradiographic and cytologic method: 1. Statistics of the radioactive disintegrations. 2. -self-absorption. 3. Coincidence. 4. Effectivity. 5. Back-ground. 6. Fading. 7. Condensation of the chromosomes. 8. Cytologic preparation. 9. Thickness of the emulsion layer. 10. Duration of exposition and developping. C. Factors due to the method of evaluation: 1. Accuracy of counting. 2. Deficient analysis of the results. After considering these factors it seems clear that the autoradiographic technique is not capable to demonstrate subtle differencies of the DNA replication. Thus the autoradiographic identification of chromosomes remains questionable if the termination phases of DNA replication lie close together as this seems the case for the chromosomes 21 and 22.

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Studie im Rahmen der Assoziation Hämatologie EURATOM — GSF.