Glycerolipid formation and PGE2 and PGF2α production of rat renal papillae in vitro, the effects of urea

The glycerolipid production by rat renal papillary slices varied inversely with the urea concentration (0a–1660 mM) whether the production was measured as labelling of the glycerol backbone from glucose or as incorporation of labelled arachidonic acid and palmitic acid. The rate of phospholipid formation was most dependent on medium urea concentrations in the range between 0 and 1100 mM. The production of prostaglandins PGE₂ and PGF_2α, measured radioimmunologically or by an isotope derivative method was in the same range inversely related to the production of glycerolipids and chain elongations. The effect of urea on prostaglandin formation is probably indirectly caused by the inhibition of the phospholipid formation and chain elongation, since the effect was abolished by 1% defatted albumin in the medium. The data suggest that the level of free arachidonic acid within the cells is controlled to an important extent by glycerolipid formation and chain elongation.     

Polyclonal antibodies against iduronate 2-sulphate sulphatase from human urine

Low-density lipoproteins (density = 1.019-1.063 g/ml) were isolated in 10 subjects with type V hyperlipoproteinemia by ultracentrifugation in a zonal rotor under rate flotation conditions. Plasma LDL concentrations in these patients were extremely reduced, as well as being heterogeneous, and two different subclasses consisting of LDL2 (density = 1.019-1.045 g/ml) and LDL3 (density = 1.045-1.063 g/ml) were observed. LDL2 and LDL3 have similar electrophoretic mobilities in beta position in agarose gel, and their diameters, calculated from gel filtration studies, were inversely proportional to their densities. LDL2 and LDL3 have a mean hydrated density of 1.034 and 1.054 g/ml, respectively. In comparison with normal LDL2, the LDL2 and LDL3 of hypertriglyceridemic subjects are particularly rich in triacylglycerols and poor in cholesteryl esters and free cholesterol, while they have an increasing amount of proteins. The protein moiety is composed almost exclusively of apolipoprotein B-100 in IDL, LDL2 and LDL3 ; in addition, IDL also contain apolipoprotein C peptides. This characterization of LDL heterogeneity in type V hyperlipoproteinemia should be considered in interpreting kinetic data in human normal and pathological lipid metabolism and in evaluating the atherogenic risk of hypertriglyceridemia.     

Bicarbonate/chloride antiport in Vero cells: I. Evidence for both sodium-linked and sodium-independent exchange

The effect of bicarbonate on the ability of cells to regulate the internal pH after acid and alkali loads was studied. In the presence of Na+, the normalization of the internal pH after acid loads occurred more rapidly in the presence than in the absence of bicarbonate. DIDS (4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid) strongly inhibited the pH increase, whereas amiloride inhibited it to a lesser extent. The Na+-linked, bicarbonate-dependent pHi increase after an acid load was strongly reduced in cells depleted of Cl-. When cells were transferred to gluconate or mannitol balanced buffers containing bicarbonate, there was a rapid alkalinization of the cytosol, apparently due to influx of bicarbonate induced by chloride efflux. When the internal pH was below 7.0, the pH increase was much more rapid in the presence than in the absence of Na+, whereas at higher internal pH, there was no measurable effect of Na+. The ability of the cells to reduce the internal pH after an alkali load was increased in the presence of bicarbonate. The data indicate that both Na+-linked and Na+-independent bicarbonate/chloride exchange occur in Vero cells.     

Fatty acid-binding to erythrocyte ghost membranes and transmembrane movement

Summary Palmitate binding to human erythrocyte ghost membranes has been investigated with ghost preparations suspended in 0.2% albumin solutions. Free unbound palmitate in the extracellular water phase was measured in equilibrium studies using albumin-filled acid loaded ghosts as small semipermeable bags. The apparent dissociation constant of binding to the membrane is 13.5 nM and the binding capacity 19 nmoles per 7.2 × 109 cells.The 0°C exchange efflux kinetics of palmitate from albumin-filled ghosts is described by a model, which provides estimates of the rate constant of membrane transfer, k₃ = 0.024 s^–1, independent of the molar ratio of palmitate to albumin () and of a mean dissociation rate constant of the palmitate-albumin complex, k₁ = 0.0015 s^–1 at 0.2, allowing for a heterogeneity of the palmitate binding to albumin.The values of a third kinetically determined dependent model constant, Q, the ratio of palmitate bound to the membrane inner surface to palmitate on intracellular albumin, are not different from the Q values obtained by equilibrium experiments.The temperature dependences of k₁ and k₃ in the interval 0°C to 15°C give activation energies of 96 and 103 kJ/mole, respectively. The 0°C exchange efflux increases about 2 fold in response to a rise of pH from 6 to 9. The results suggest a carrier mediated palmitate flux at low with a V_max about 2 pmoles min^–1 cm^–2 at 0°C pH 7.3.     

The role of fluorinated pyrimidine analogues in the induction of the in vitro expression of the fragile X chromosome

Summary The modes of action of 5-fluoro-2-deoxyuridine (FdUrd) and 5-fluoro-2-deoxycytidine (FdCyd) were studied in PHA-stimulated lymphocytes from normal volunteer donors and a fragile X patient. In both cell types, FdUrd and FdCyd inhibited cell proliferation at concentrations of 3x10^-8 M. Thymidylate synthetase was identified as the decisive target for the action of both FdUrd and FdCyd, as judged from the following observations: First, addition of thymidine to the culture medium was able to counteract both FdUrd and FdCyd toxicities, whereas addition of dCyd had no observable effect. Second, inhibition of the in situ thymidylate synthetase activity measured as an increase in the level of [³H]-dThd incorporation coincided with the inhibition of cell proliferation. Third, the inhibition of the thymidylate synthetase-dependent incorporation of [³H]-dUrd into newly synthesized DNA coincided with the inhibition of cell proliferation. The effects of FdUrd and FdCyd on the in vitro expression of fragile site Xq27 of fragile X chromosomes was shown to be based on the depletion of the intracellular pool of thymidine-5-monophosphate (dTMP), as fudged from the following observations: First, both the FdUrd- and FdCyd-dependent induction of site Xq27 coincided with the antiproliferative effects of the respective fluoropyrimidines. Second, addition of thymidine (dThd) to the culture medium both prevented the expression of site Xq27 and neutralized the cytotoxicity of FdUrd and of FdCyd. On the basis of these findings, we provide further evidence for the concept that the fragile X site is located in an AT-rich region.     

Phytochrom-induzierte Enzymbildung (Phenylalanindesaminase), ein schnell ablaufender Prozeß

Zusammenfassung Bei Erstbelichtung des Senfkeimlings mit Dauer-Dunkelrot tritt der P₇₃₀-abhängige Anstieg der Phenylalanindesaminase-Aktivität erst mit einer lag-Phase von 1,5 Std ein (Abb. 3, 4 unten). Bei einer Zweitbelichtung (Programm: Erstbelichtung — längere Dunkelperiode — Zweitbelichtung) fehlt die lag-Phase (Abb. 4, oben). Die Enzymaktivität steigt sofort linear an. Da der Anstieg der Enzymaktivität wahrscheinlich auf eine de novo Synthese von RNS und Enzymprotein zurückzuführen ist (Tabelle), so erscheint der Schluß berechtigt, daß P₇₃₀ sehr rasch eine differentielle Genaktivierung mit anschließender Enzymsynthese bewirken kann, falls die Gene der Aktivierung durch P₇₃₀ zugänglich sind. Die relativ lange lag-Phase nach Einsetzen der Erstbelichtung benötigt das P₇₃₀ offenbar dazu, die potentiell aktiven Gene (P₇₃₀) für das P₇₃₀ zugänglich zu machen. Das Problem der primären lag-Phase ist in einer vorangegangenen Arbeit zur P₇₃₀-abhängigen Anthocyansynthese ausführlich diskutiert worden (vgl. Lange, Bienger und Mohr, 1967).

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Phytochrome-mediated enzyme formation (Phenylalanine deaminase) as a rapid process

Summary In previous papers we have reported (Mohr and Durst, 1966a, b) that synthesis of phenylalanine deaminase (EC 4.3.1.5), an important enzyme of phenolic metabolism, can be stimulated by the physiologically active phytochrome (=P₇₃₀) in the mustard seedling. The data of the present paper suggest that induction of this enzyme is a rapid process if the gene in question is easily accessible for the activating action of P₇₃₀.The seedlings were irradiated with continuous standard far-red light. Longtime irradiation with far-red will maintain a low but virtually constant level of P₇₃₀ in the seedling over an extended period of time. At the moment when the far-red light is turned off the action of P₇₃₀ will virtually cease. — Fig. 3 and Fig. 4, lower part, show the kinetics of enzyme induction by P₇₃₀ in an etiolated seedling. The initial (or primary) lag-phase after the onset of far-red is 1.5 hours. If, however, a seedling which has been pre-irradiated with 12 hours of far-red is kept in darkness for 6 hours and is then re-irradiated with far-red no lag-phase for the action of the second irradiation can be found. Enzyme activity increases immediately after the onset of far-red. Since the action of the second irradiation as measured by increase of enzyme activity can be inhibited by relatively low doses of Puromycin and Cycloheximide (table) we conclude that the re-appearance of P₇₃₀ leads to de novo synthesis of enzyme protein. — Application of Actinomycin D (10 g/ml) only partially inhibits the action of the second irradiation as measured by increase of enzyme activity. This finding was to be expected. In preceding papers (e.g. Mohr and Bienger, 1967) it has been concluded that genes which have once been activated by P₇₃₀ remain less sensitive towards Actinomycin D even when P₇₃₀ has disappeared. Taking into account all available data the conclusion seems to be justified that the induction of enzyme synthesis by P₇₃₀ (i.e. differential gene activation followed by enzyme synthesis) is a rapid process if the genes are accessible for the action of P₇₃₀. The relatively long initial lag-phase (1.5 hours) is needed to make the potentially active genes (P₇₃₀) accessible for the action of P₇₃₀. The problem of how the initial lag-phase can be understood has been dealt with more in detail in a previous paper on phytochrome-mediated anthocyanin synthesis (Lange, Bienger and Mohr, 1967).

     

Blütenbildung von Pinguicula lusitanica in vitro durch Fütterung mit Pollen

Zusammenfassung Pinguicula lusitanica wurde in vitro auf stickstoff- und phorphorfreiem Mineralsalzagar kultiviert; jede Pflanze stand für sich in einem Erlenmeyerkolben.Als die Pflanzen 8 Wochen alt waren, wurden 20 Exemplare innerhalb von 5 Wochen viermal mit Pinuspollen gefüttet. 20 gleichgroße und gleichalte dienten als Kontrollen.Durch die Fütterung steigerte sich die Blattzahl und der Durchmesser der Blattrosette, die Blätter wurden intensiver grün und alterten langsamer.Vor allem wurde durch die Pollenfütterung die Blütenbildung ausgelöst. Schon nach der 2. Fütterung traten die ersten Knospen auf, eine Woche darauf blühten bereits 70% und noch vor der letzten Fütterung alle gefütterten Pflanzen, von den ungefütterten dagegen keine einzige. In dem anschließenden halben Jahr entwickelten die 20 gefütterten Pflanzen im ganzen 127 Blüten; die größte Blütenzahl einer Einzelpflanze was 14. Die nichtgefütterten Pflanzen waren auch jetzt noch rein vegetativ.

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Flowering of in vitro cultures of Pinguicula lusitanica after feeding with pinus pollen

Summary Plants of Pinguicula lusitanica were grown in individual Erlenmeyer flasks on an inorganic agar medium containing no nitrogen or phosphorus. After 8 weeks of culture, twenty of the plants were fed Pinus pollen 4 times over a period of 5 weeks.As a result of the feeding, the number of leaves as well as the diameters of the rosettes were increased. The leaves became turned a deeper green and aged more slowly.The most spectacular effect of the pollen feeding was an initiation of flowering. The first buds were already visible after the second feeding. All of the treated plants flowered before the last feeding, whereas none of the untreated plants flowered. During the following 6 months, the treated plants developed 127 flowers, the largest number on a single specimen being 14. Even after this period of time the untreated plants remained vegetative.

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