Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

1H NMR studies at 360 Mhz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI)

In the ¹H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformations are discussed.     

Xanthones et C-glucosides flavoniques du genre Gentiana (section Cyclostigma)

Xanthones with a uniform 1,3,7,8-oxidation pattern, the C-glycosides isoorientin and isovitexin, and in some cases mangiferin, were isolated from the aerial parts of G. nivalis, G. brachyphylla, G. favrati, G. rostani, G. utriculosa and G. schleicheri Kunz. The distribution of these compounds within the section Cyclostigma is given. Comparison of phenolic patterns in other sections of Gentiana is made.     

Zur Struktur von Reaktionszentren in phototrophen Bakterien: eine Standortbestimmung

Es ist gelungen, bakterielle Reaktionszentren-Komplexe (RC) ohne Lichtsammlerpigmente zu isolieren. Unsere Gruppe bearbeitet zur Zeit die Isolierung und Charakterisierung von Reaktionszentren aus Rhodospirillaceen. Bei phototrophen Bakterien sind 2 bis 4% des Bacteriochlorophylls mit einem Proteinkomplex assoziiert, in welchem sie durch aktinisches Licht oxidierbar sind und sich im Dunkeln wieder zurückreduzieren (P₈₇₀). Am besten untersucht sind RC-Komplexe von Rhodopseudomonas spheroides, R-26. Diese Mutante weist einen geringen Gehalt an Karotinoiden auf. Die RC-Komplexe werden mittels mild wirkender Detergenzien aus der Chromatophorenmembran solubilisiert. Sie enthalten je Mol RC 2 Mole BPhäo a, 4 Mole Bchl a, 1 Mol nichthämartig gebundenes Fe und 1 bis 2 Mole Ubichinon, die mit einem hydrophoben Trägerprotein assoziiert sind. Der Proteinanteil hat ein MW von etwa 7,5 10⁴ Dalton und ist aus drei verschiedenen Untereinheiten aufgebaut. Die größte der drei Untereinheiten kann vom Komplex wegdissoziieren, während die Pigmente an die beiden leichteren Untereinheiten gebunden bleiben und teilweise photochemische Aktivität bewahren. Die primäre, photochemische Reaktion ist ein lichtinduzierter Ladungstrennungsvorgang bei dem die RC-Bchl-Moleküle ein Elektron an einen Elektronenakzeptor abgeben. Als primäre Elektronenakzeptoren werden heute drei Möglichkeiten in Erwägung gezogen: Nichthäm-Eisen, Chinone und Ferrochinon-Komplexe. Es scheint, daß verschiedene Mikroorganismen ungleiche primäre Elektronenakzeptoren aufweisen. Energieübertragung in photosynthetischen Bakterienmembranen ist sehr wirkungsvoll. Weniger als 5% der absorbierten Photonen gehen als Fluoreszenzenergie verloren; der Rest führt zu Ladungstrennungen und Elektronentransport. Der hohe Wirkungsgrad muß durch charakteristische Strukturen, Umgebungsbedingungen und Interaktionen erklärt werden können. Interaktionen der Pigmentmoleküle untereinander, mit der Umgebung in der Chromatophorenmembran und mit den Trägerproteinen werden diskutiert als Standortbestimmung zur Beantwortung der Frage: Was macht die Bchl-Mole-küle des Reaktionszentrums geeignet, als Zentren des photochemischen Ladungstrennungsprozesses zu wirken? Ich danke den Mitarbeitern der Photosynthesegruppe Zürich für die kritischen Diskussionsbeiträge während des Kolloquiums über “Photosynthetische Membranen” an unserem Institut. Unsere Arbeit wird unterstützt durch den Schweizerischen Nationalfonds, NF-3.156-0.73, und die Fritz Hoffmann-La Roche-Stiftung.     

Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division

Summary The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.Some of these results were presented at the IX Congress of the International Society of Developmental Biologists in Basle, Switzerland, August 28–September 1, 1981     

Prostaglandin F2a inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F_2a (PGF_2a. The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF₂. A dose-dependent inhibition by PGF_2a (0.5–50 μM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165–165 μM). The stimulation by epinephrine on progesterone production was inhibited by PGF_2a (5 μM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF_2a can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF_2a shows in this respect the same agedependent inhibitory pattern as in relation to LH stimulation.     

Establishment of a germ-line competent C57BL/6 embryonic stem cell line

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.     

The design,synthesis and study of siderophore-antibiotic conjugates Siderophore mediated drug transport

Summary The use of conjugates of microbial iron chelators (siderophores) and antibiotics for illicit transport of antibiotics into cells is a potentially powerful method for the rational design of therapeutic agents. The structural complexity of most natural siderophores has impeded progress in this area. Described here are the design, syntheses and preliminary biological studies of several siderophore--lactam antibiotic conjugates. Both hydroxamic-acid-based and catechol-based conjugates with and without amino acid spacers to carbacephalosporins were synthesized and demonstrated to be effective inhibitors ofEscherichia coli X580. Mutant selection was noted for each class of conjugates. Mutants selected from exposure of theE. coli to the hydroxamate conjugates were susceptible to the catechol conjugates and vice versa. Combinations of hydroxamate-and catechol-carbacephalosporin conjugates were most effective inhibitors ofE. coli X580.     

Coordinate Regulation of the Cyclic AMP System with Firing Rate and Expression of Tyrosine Hydroxylase in the Rat Locus Coeruleus: Effects of Chronic Stress and Drug Treatments

Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP-dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP- and forskolin-stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP-dependent protein kinase activity in the LC. 6-Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP-dependent protein kinase activity in this brain region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of external carbonic anhydrase in light-dependent alkalization byFucus serratus L. andLaminaria saccharina (L.) Lamour. (Phaeophyta)

It has been proposed that many marine macroalgae are able to utilize HCO ₃ ^– for photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae,Fucus serratus L. andLaminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCO ₃ ^– to OH^– and CO₂; CO₂ is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake.Abbreviations AZ acetazolamide - CA carbonic anhydrase - CAext extracellular carbonic anhydrase - C_i inorganic carbon - DBS dextran-bound sulfonamide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PPFD photosynthetic photon flux density This study was carried out with financial support by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Trygger's Fund for Scientific Research (Sweden), SJFR (Swedish Council for Forestry and Agricultural Research) and CICYT (Spain). Z. Ramazanov is an invited professor of Ministerio de Educación y Ciencia, Spain.