Regulation of voltage-gated Ca channels by lipids

Great skepticism has surrounded the question of whether modulation of voltage-gated Ca²⁺ channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acid's carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in Ca_V2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP₂), a palmitoylated accessory subunit (β_2a) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M₁ muscarinic receptors may stimulate multiple lipases to break down the PIP₂ associated with VGCCs and leave PIP₂'s freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs.     

Changes in responsiveness to ethylene and gibberellin during corolla expansion ofIpomoea nil

Corolla expansion inIpomoea nil appears to be triggered by changes in gibberellin concentration and ethylene production during development. We investigated the role of responsiveness to GA and ethylene in corolla expansion. The effects of growth regulators applied in vitro were measured as a change in area of corolla segments from younger (15–17 mm) and older (18–20 mm) whole corollas. Applied gibberellic acid (GA₃) significantly (p < 0.05) promoted growth in the younger segments but was less effective in the older segments. Moreover, applications of the GA biosynthesis inhibitors, PP333 (paclobutrazol) AMO₁₆₁₈ (2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride), chlorocholine chloride, and tetcyclasis had little effect on younger segments but inhibited growth of older segments. The older corollas have apparently synthesized and accumulated enough GA-like substances to become less responsive to additional applied GA₃. The amount of growth induced by applied or endogenous GA depended on the amount of ethylene simultaneously produced in the tissue. The younger corollas rapidly produced ethylene from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) and did not respond to applied ACC whereas the older corollas naturally produced much less ethylene and were significantly (p < 0.05) inhibited by applied ACC. When ethylene production was inhibited by applying aminoethoxyvinylglycine (AVG), growth was promoted in all segments. However, only the growth of the younger segments was further stimulated by simultaneously applied AVG and GA₃ over the GA₃ control. Thus the differential responses of segments from 15- to 20-mm long corollas to applied growth regulators reflect developmental changes in responsiveness of the developing corolla. The change in responsiveness is attributed in part to the changes in production of endogenous growth regulators and to the effect of one endogenous plant growth regulator (PGR) on the responsiveness of the corolla to another PGR.     

Protoplast formation and reversion in Actinomadurae

Summary The optimum conditions for efficient protoplast formation and regeneration in Actinomadura species have been determined. Effective protoplast formation in A. madurae, A. salmonea and strain 2AMI was accomplished using a growth medium containing 1.5% (w/v) glycine, harvested after 3 days growth. A combination of 3 cell wall lytic enzymes was essential for maximal conversion. Using surface spread cultures on a hypertonic regeneration medium, a high efficiency (60%) of regeneration was achieved. Cephamycin C production in strain 2AMI was unaffected.     

Kinetic evidence for differential agonist and antagonist binding to bovine hippocampal synaptic membrane opioid receptors

To examine the kinetics of opioid receptor binding, the agonists [D-Ala2-D-Leu5]enkephalin (DADL) and [D-Ala2-MePhe4-Gly-ol5]enkephalin (DAGO) and the antagonists diprenorphine and naltrexone were used with bovine hippocampal synaptic plasma membranes. By computer modeling of equilibrium binding displacement curves utilizing the LIGAND program, we found opioid peptides bind with high affinity to single populations of synaptic plasma membranes receptors, whereas opiate alkaloids bind to multiple sites. Initial kinetic experiments revealed that agonist rates of association were radioligand concentration-independent. Pseudo first-order rate constants for DADL, DAGO, diprenorphine, and naltrexone association were estimated to be 5.63 X 10(5), 5.08 X 10(5), 4.60 X 10(6), and 2.3 X 10(6) mol-1 X s-1, respectively. After preincubation of 0.2-1 nM radioligand for variable time intervals, dissociation was initiated by addition of 1 microM unlabeled ligand. If saturation binding was achieved before dissociation was initiated, then nearly monophasic dissociation of DADL, DAGO, and diprenorphine and a biphasic off-rate for naltrexone were observed. When association times were reduced to pre-equilibrium intervals, the kinetics of dissociation of agonists became biphasic and association time-dependent, but that for antagonists did not change significantly. Comparisons by both graphical methods and computerized nonlinear regression analyses of rate constants revealed that the fraction of the rapid component of agonist dissociation decreases and that of the slow component is elevated with increasing receptor occupancy. In the presence of 100 mM NaCl, DADL dissociation became association time-independent. These data are consistent with the idea that the Na+ effect is brought about by a change of receptor to an antagonist-like conformation. On the basis of both association and dissociation kinetic data, opioid agonists appear to interact in a multistep process in which a rapid, reversible association is followed by the formation of a more tightly bound complex.     

The levels of ecdysteroids in uninjured and leg-autotomized nymphs of Blattella germanica (L.)

The levels of ecdysteroids in control and leg-autotomized first-instar nymphs of Blattella germanica were determined by radioimmunoassay from hatching to the time of the first ecdysis. Uninjured nymphs showed a distinct release of ecdysteroids half-way through the stadium, and this resulted in the commencement of the moult cycle which formed the cuticle of the second instar. Cockroaches which had legs autotomized at 48 h after hatching (i.e. before the control ecydsteroid release) had their instar duration increased by that time period. Releases of ecdysteroids and events of the moulting cycle were also postponed by the 48 h period. The titre of ecdysteroids in injured animals was double that of controls. Nymphs were also autotomized at 96 h (i.e. after the normal release of ecdysteroids) but no changes in instar duration, ecdysteroid releases, or events of the moult cycle were recorded. The effects of injury, prothoracicotropic hormone activity and ecdysteroid release are discussed.     

Competition between electron acceptors in photosynthesis: Regulation of the malate valve during CO2 fixation and nitrite reduction

For maximal rates of CO₂ assimilation in isolated intact spinach chloroplasts the generation of the adequate NADPH/ATP ratio is achieved either by cyclic electron flow around photosystem I or by linear electron transport to oxaloacetate, nitrite or oxygen (Mehler-reaction). The interrelationships between these poising mechanisms turn out to be strictly hierarchical. In the presence of antimycin A, an inhibitor of ferredoxin-dependent cyclic electron transport, the reduction of both, oxaloacetate and nitrite, but not that of oxygen restores CO₂ fixation. When oxaloacetate and nitrite are added at low concentrations simultaneously during steady-state CO₂ fixation, the reduction of nitrite is clearly preferred over the reduction of oxaloacetate, but CO₂ fixation is not influenced. Nitrite reduction is not decreased upon addition of oxaloacetate, but vice versa. This is due to the regulation of NADP-malate dehydrogenase activation by electron pressure via the ferredoxin/thioredoxin system on the one hand, and by the NADPH/(NADP+NADPH) ratio (anabolic reduction charge, ARC) on the other hand. Thus the closing of the malate valve prevents drainage of reducing equivalents from the chloroplast (1) when a low ARC indicates a high demand for NADPH in the stroma and (2) when nitrite reduction reduces the electron pressure at ferredoxin. The malate valve is opened when cyclic electron transport is inhibited by antimycin A. Under these conditions the rate of malate formation is higher than in the absence of the inhibitor even in the presence of oxaloacetate, thus indicating that the regulation of the malate valve functions at various redox states of the acceptor side of Photosystem I.Abbreviations ARC anabolic reduction charge (NADPH/(NADP+NADPH)) - Chl chlorophyll - DTT dithiothreitol; Fd-ferredoxin - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetate - PS photosystem - qN non-photochemical quenching - qP photochemical quenching - _E quantum efficiency of PS II Dedicated to Prof. Dr. Hans Walter Heldt on the occasion of his 60th birthday.     

Interaction between fatty-acid and starch synthesis in isolated amyloplasts from cauliflower floral buds

The interaction of fatty-acid synthesis with starch synthesis has been studied in intact amyloplasts isolated from floral buds of cauliflower (Brassica oleracea L.). These amyloplasts perform acetate-dependent fatty acid synthesis at maximum rates only at high external ATP concentrations. Neither pyruvate nor malate inhibit acetate-dependent fatty-acid synthesis. In contrast, acetate is inhibitory to the low pyruvate-dependent fatty acid synthesis. These observations indicate that neither pyruvate nor malate are used as natural precursors of fatty-acid synthesis. In contrast to fatty-acid synthesis, the rate of glucose-6-phosphate-dependent starch synthesis is already saturated in the presence of much lower ATP concentrations. Rising rates of starch synthesis influence negatively the process of acetate-dependent fatty acid synthesis. This inhibition appears to occur under both limiting and saturating concentrations of external ATP, indicating that the rate of ATP uptake is limiting when both biochemical pathways are active. The rate of starch synthesis is modulated specifically by the concentration of 3-phosphoglycerate in the incubation medium. This observation leads to the conclusion that the activity of ADP-glucose pyrophosphorylase is of primary importance for the control of both, starch and fatty-acid synthesis. Using the modified approach of Kacser and Burns (1973; Symp. Soc. Exp. Biol.27, 65–104) we have quantified the contribution of the rate of starch synthesis to the control of the metabolic flux through fatty-acid synthesis.Abbreviations ADPGlc-PPase ADPglucose pyrophosphorylase - Glc6P glucose-6-phosphate - PGA 3-phosphoglyceric acid     

Über die Verwendung der reversibel polymeren Metachromasie von Pseudoisocyanin zur Gewebsfärbung

Zusammenfassung 1. Pseudoisocyanin gibt mit den dicht gelagerten elektronegativen Gruppen von Mukopolysacchariden in Geweben und Lösungen, wie auch mit synthetischen Produkten mit linear angeordneten elektronegativen Gruppen in Lösung wie z. B. Polyäthylensulfosäuren eine metachromatische Reaktion mit der charakteristischen langwelligen Bande (vgl.Scheibe u.Schauer 1958). Die elektronegativen Gruppen binden die Farbstoffmoleküle elektrostatisch und bilden die Gruppierung des reversiblen Polymerisates.2. Die metachromatische Reaktion mit der reversibel polymeren Bande läßt sich in Gewebsschnitten deutlich demonstrieren. Das Farbstoffpolymerisat absorbiert in Lösung bei der gleichen Wellenlänge wie im Gewebe, wodurch die Gleichheit der Vorgänge im Gewebe und in Lösung bewiesen ist.3. Das Pseudoisocyanin erscheint für die Darstellung von Mukopolysacchariden besonders geeignet, da nach früheren Arbeiten (Scheibe 1938,Zimmermann u.Scheibe 1956) schon eine monomolekulare Schicht die reversibel polymere Bande und damit die Metachromasie beobachtbar macht. Ferner sind bei Betrachtung der mit Pseudoisocyanin gefärbten Schnitte im monochromatischen Licht bei der Wellenlänge der polymeren Absorption Spuren von Mukopolysacchariden noch deutlich zu erkennen, die bei Betrachtung im weißen Licht unauffällig bleiben.4. An Hand einiger Beispiele (Mastzellen, Knorpelgewebe, hyalinisiertes Bindegewebe) wird die Verwendungsmöglichkeit in der Histochemie gezeigt.

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Summary 1. Pseudoisocyanin interacts with densly positioned electronegative groups of mucopolysaccharides in tissues and in solutions in the same way as it interacts with linear positioned electronegative groups of synthetic products in solution (for instance polyaethylensulfoacids). The metachromasia, which is due to this reaction of pseudoisocyanin with mucopolysaccharides shows a characteristic wave-band 5727 Å (Scheibe undSchauer 1958). The dye is bound electrostatically by the electronegative groups in form of a reversible polymerisate.2. The metachromatic reaction with the reversible polymerisate has been demonstrated in tissue-sections. The polymerisate with the dyestuff is shown to adsorb light at the same wavelength in tissues as in solutions. This finding confirms the identity of the reaction in tissues and in solutions.3. Pseudoisocyanin seems to be especially suited for the detection of mucopolysaccharides, for even a monomolecular layer of dyestuff allows the observation of the reversible polymeric band and therefore shows metachromasia. Further, after staining with pseudoisocyanin even small trans of mucopolysac charides which are not visible in the white light can be demonstrated by means of monochromatic light at the wave-length of the polymer absorption.4. As shown by staining mastcells, cartilage-tissue, hyaliniced connectivetissue, pseudoisocyanin seems to be of use for appliance in histochemistry.

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Mit 4 Textabbildungen     

A serotype-specific epitope of dengue virus 1 identified by phage displayed random peptide library

Abstract From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885–890 of polyprotein or residues 111–116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide.