Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells.

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.     

Degradation of long chain alkanes by bacilli

Summary Among 14 different types of bacillus and 100 isolates from soil samples, no bacillus was able to assimilate C₁₄-C₁₈ alkanes as sole carbon source. In contrast to these results, five types of bacillus were substantially able to oxidize alkanes in the presence of other carbon sources.All five strains tested formed relatively high amounts of secondary alcohol in contrast to low amounts of ketones and traces of primary alcohols with chain length equivalent to the n-alkane in the substrate, showing that the degradation pathway in bacilli is mainly subterminal.This conclusion is also supported by comparison of the extracellular fatty acids of cultures of bacilli grown on n-tridecane and on n-tetradecane supplemented with other carbon sources and on the same carbon sources without alkane.     

Ribosomally-synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds

Owing to their exceptional stability and favourable pharmacokinetic properties, plant-derived cyclic peptides have recently attracted significant attention in the field of peptide-based drug design. This article describes the three major classes of ribosomally-synthesised plant peptides – the cyclotides, the PawS-derived peptides and the orbitides – and reviews their applications as leads or scaffolds in drug design. These ribosomally-produced peptides have a range of biological activities, including anti-HIV, cytotoxic and immunomodulatory activity. In addition, recent interest has focused on their use as scaffolds to stabilise bioactive peptide sequences, thereby enhancing their biopharmaceutical properties. There are now more than 30 published papers on such ‘grafting’ applications, most of which have been reported only in the last few years, and several such studies have reported in vivo activity of orally delivered cyclic peptides. In this article, we describe approaches to the synthesis of cyclic peptides and their pharmaceutically-grafted derivatives as well as outlining their biosynthetic routes. Finally, we describe possible bioproduction routes for pharmaceutically active cyclic peptides, involving plants and plant suspension cultures.     

Degradation pathways from n-tridecane to α, ω-tridecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-tridecanedioic acid via n-tridecanoic acid and via ,-tridecanediol from n-tridecane in the mutant S 76 of Candida tropicalis was studied. It was found that resting cells of S 76 produced ,-tridecanediol from n-tridecane.With n-tridecanol as substrate, the ,-diol could also be detected. The mutant S 76 was able to produce ,-tridecanedioic acid using either n-tridecanol or n-tridecanoic acid as the sole carbon source. Quantitative changes in the concentration of -hydroxy tridecanoic acid and other intermediates were recorded during the formation of ,-dioic acid.The results confirm the existence of two metabolic pathways mentioned above in the course of ,-dioic acid formation from odd n-alkane in the mutant S 76 of C. tropicalis.     

Regulation der mikrobiellen Alkanoxidation mit Hinblick auf die Produktbildung

Catabolic pathways of long-chain n-alkanes in the range of C₈ to C₁₈ are demonstrated and results of investigation about the regulation of monoterminal oxidation are given:

• – Enzymes of monoterminal alkane oxidation usually are inducible
• – Several intermediates of alkane oxidation can inhibit the primary oxidation of concerned alkane
• –Substances, e.g. glucose and glycerol, which ordinary don't be developed in catabolic alkane reactions, in many cases have an inhibitory effect on alkane oxidation

The regulation of catabolic pathways has a great influence on the formation of specific products This influence is demonstrated examplarily at the production of biotin, fatty acids and citric acid.     

Regulation der fettsäurebildung bei der mikrobiellen alkanoxidation

At the microbial oxidation of long-chain alkanes such monocarbonic acids at a monoterminal oxidation are formed which correspond to the chain length of the alkanes. At diterminal oxidation the corresponding dioic acids are produced. Under the influence of cerulenin a cerulenin a direct incorporation of fatty acids into cell lipids increases. Undecanoic acid cannot be metabolized byMortierella isabellina. It causes an inhibition of alkane oxidation. A main effect of undecanoic acid is an inhibition of the elongation of other fatty acids. The de novo fatty acid biosynthesis has not been inhibited by undecanoic acid.     

Die Resistenz von Dekalin gegenüber mikrobiellem Abbau

Zusammenfassung Es gelang nicht, Dekalin durch mehr als 50 verschiedene Flavobacterium-, Micrococcus-Arthrobacter- und Corynebacterium-Stämme, sowie Stämmen, die aus 250 Bodenproben isoliert worden waren, zu oxidieren. Auch eine Cooxidation des Dekalins war nicht möglich. Mit verschiedenen gaschromatographischen Methoden gelang es nicht, irgendwelche Abbauprodukte festzustellen.

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Resistance of decalin against microbiological oxidation

Summary We succeeded not in oxidizing decalin by more than 50 different strains of Flavobacterium, Micrococcus, Arthrobacter and Corynebacterium, as well as by strains, which were isolated from 250 samples of soil. A cooxidation of decalin was not possible also. We were not able to determine any products of decomposition with various gaschromatographical methods.

     

Inhibition of Low pH-induced Elongation in Avena Coleoptiles by Abscisic Acid

An angular position-sensing transducer was used to make continuous measurements of acid-induced elongation of Avena sativa coleoptile segments. Elongation rates at pH 4.5 (5 mm succinate buffer) were about 5-fold greater than those at pH 6.0. Buffered 0.1 mm abscisic acid produced a partial decrease of the growth rate. Pretreatments with abscisic acid buffered at pH 6.0 usually caused a further reduction of the elongation response when the coleoptile segments were subsequently placed in buffer at pH 4.5 containing abscisic acid. Abscisic acid did not completely prevent the pH effect in any of these experiments, and the brief latent period of the pH response was not affected by abscisic acid treatments. At pH 4.5, where the inhibitory effect of ABA was maximum, low pH-induced elongation was also inhibited by KCN and HgCl₂. These results suggest that pH-(4.5) induced elongation in this system may be dependent on some metabolic processes and that abscisic acid-induced inhibition of this elongation may involve an interaction with these processes.     

The Intrinsic Electrophysiological Characteristics of Fly Lobula Plate Tangential Cells: II. Active Membrane Properties

The voltage-gated currents in the fly lobula plate tangential cellswere examined using the switched electrode voltage clamp technique. InCH cells, two currents were identified (Figs. 1, 2): a slow calciuminward current and a delayed rectifying, noninactivating potassiumoutward current. HS and VS cells appear to possess similar currentsto CH cells, but in addition, exhibit a fast-activating sodium inwardcurrent and a sodium-activated potassium outward current(Figs. 3, 4). While the delayed rectifying potassium current in allthree cell classes is responsible for the observed outwardrectification described previously (Borst and Haag, 1996), the sodiuminward current produces the fast and irregular spikelikedepolarizations found in HS and VS cells but not in CH cells: Whenthe sodium current is blocked by either TTX or intracellular QX314,no more action potentials can be elicited in HS cells undercurrent-clamp conditions (Fig. 5). As is demonstrated in HS cells,space clamp conditions are sufficient to suppress synapticallyinduced action potentials (Fig. 6).The currents described above were incorporated with the appropriatecharacteristics into compartmental models of the cells (Figs. 7, 8).The anatomical and electrically passive membrane parameters of thesecells were determined in a preceding paper (Borst and Haag,1996). After fitting the current parameters to the voltage-clamp data(Fig. 9), the model cells qualitatively mimicked the fly tangentialcells under current clamp conditions in response to current injection(Fig. 10). The simulations demonstrated that the electricalcompactness seen in the HS and VS cells, either in passive models orin active models during continuous hyperpolarization, decreasedsignificantly in the active models during continuous depolarization(Fig. 11). Active HS models reproduce the frequency-dependentamplification of current injected into their axon (Fig. 12).     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.