Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-buffered media (-log [Ca²⁺] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca²⁺]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca²⁺] in M. scalaris that was nearly independent of the external [Ca²⁺] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca²⁺ from intracellular stores. Increased cytoplasmic [Ca²⁺] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca²⁺-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.Abbreviations EGTA ethylene glycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - indo-1 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,Ntetraacetic acid - pCa log [Ca²⁺] - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - x_G geometric mean Dedicated to Professor Wolfgang Haupt on the occasion of his 70th birthdayThis paper is part of the Ph.D. thesis of U. Russ at the Justus-Liebig-Universitat Giessen (FRG). Part of this work has been presented at a meeting on Calcium and intracellular signalling in plants in Plymouth, UK, Dec. 1990We are indebted to Dr. G. Seibold and Dipl. Phys. H. Weintraut for their advice on the technique of microspectrofluorometry and for allowing access to the microspectrophotometric facilities in the Strahlenzentrum der Justus-Liebig-Universität, Giessen, FRG. We thank Mrs. A. Quanz for reliable culture of the algae and evaluation of the videotapes. Bay-K8644 was a generous gift of Bayer AG, Wuppertal, FRG. U. russ was supported by a scholarship according to the Hessisches Graduierten Förderungsgesetz. This work was supported by the Deutsche Forschungsgemeinschaft.     

Characterization of maltose biosynthesis from α-d-glucose-1-phosphate in Spinacia oleracea. L.

The de novo synthesis of maltose in spinach (Spinacia oleracea L.) was shown to be catalyzed by a maltose synthase, which converts two molecules of -d-glucose-1-phosphate (-G1P) (K_m 1.5 mmol l^-1) to maltose and 2 orthophosphate (Pi). This enzyme was purified 203-fold by fractionated ammonium sulfate precipitation and by column chromatography on Sepharose 6B. The addition of -G1P (15 mmol l^-1) to the isolation buffer is required to stabilize the enzyme activity during the extraction and purification procedure. Molecular weight determination by gel filtration yielded a value of 95,000. -Gluconolactone, ATP and Pi are competitive inhibitors toward the substrate -G1P. The maltose synthase catalyzes an exchange of the phosphate group of -G1P with [³²P] orthophosphate; this transfer reaction suggests that the synthesis of maltose occurs via a glucose-enzyme in a double displacement reaction. The physiological role of this enzyme as a starch initiator system is discussed.Abbreviations Fru fructose - Glc glucose - -G1P -d-glucose-1-phosphate - -G1P -d-glucose-1-phosphate - G6P d-glucose-6-phosphate This enzyme is tentatively called maltose synthase in this publication     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

Ecto-ATPase/phosphatase activity in the olfactory sensilla of the spiny lobster,Panulirus argus: localization and characterization

Summary Electrophysiological studies have shown that the olfactory organ (antennule) of the spiny lobster, Panulirus argus, has chemoreceptors that are selectively excited by adenine nucleotides in seawater. Biochemical studies have revealed that these same nucleotides can be rapidly dephosphorylated by ectoenzymes associated with the olfactory sensilla (aesthetascs). In this study the distribution of ecto-ATPase/phosphatase activity within aesthetascs was determined cytochemically and the nature of the adenine-nucleotide dephosphorylating activity was dissected biochemically. Cytochemically, the distribution of ATP-dephosphorylating activity was similar to that shown previously for AMP and -glycerol phosphate; i.e., cerium phosphate reaction product was specifically localized to the transitional zone where the sensory dendrites develop cilia and branch to form the outer dendritic segments. Unlike the dephosphorylation of AMP and -glycerol phosphate, Mg²⁺ or Ca²⁺ was required for ecto-ATPase/phosphatase activity. Biochemical measures of both AMP-and ATP-dephosphorylating activity within aesthetascs corroborated the cytochemical evidence that these activities are localized to the transitional zone. A major portion of the AMP dephosphorylation (about 67%) derives from nonspecific alkaline phosphatase activity that is insensitive to levamisole and L-bromotetramisole. In contrast, nonspecific phosphatase activity accounted for a much smaller part of the ATP dephosphorylation (about 15%). Ectoenzymatic activity in the transitional zone may be an important means of removing excitatory/inhibitory nucleotides from this region.Abbreviations ADP Adenosine 5-diphosphate - AMP adenosine 5-monophosphate - AMPCP , -methylene ADP - ASW artificial seawater - ATP adenosine 5-triphosphate - -GP -glycerol phosphate - EM electron microscopy     

Studies of the action of ceramide-like substances (d- andl-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

We have studied the effects ofD-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and itsL-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found thatD-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOse₃Cer and GbOse₄Cer. It also directly inhibited the activity of UDP-glucose:ceramide 1 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer 1 4 galactosyltransferase (GalT-2). In contrast,L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse₃Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse₄Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited byD-PDMP and stimulated byL-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding toD-PDMP and, in the case of GbOse₄Cer synthase, byL-PDMP as well. The stimulatory effects ofL-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.Abbreviations GalT-2 UDP-galactose:GlcCer -galactosyltransferase - GbOse₃Cer Gal1 4Gal1 GlcCer - GbOse₄Cer GalNAc1 3Gal1 4Gal1 GlcCer - GlcCer glucosylceramide - GlcT-1 UDP-glucose:ceramide -glucosyltransferase - GSLs glycosphingolipids - LacCer lactosylceramide - PDMP threo-1-phenyl-2-decanolyamino-3-morpholino-1-propanol     

Regulation of proline catabolism in Pseudomonas aeruginosa PAO

Mutants of Pseudomonas aeruginosa deficient in the utilization of l-proline as the only carbon and nitrogen source have been found to be defective either in proline dehydrogenase activity or in both proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities of the bifunctional proline degradative enzyme. The latter type of mutants was unable to utilize l-ornithine, indicating that a single ¹-pyrroline-5-carboxylate dehydrogenase activity is involved in the degradation of ornithine and proline. Proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities were strongly and coordinately induced by proline. It was excluded that ¹-pyrroline-5-carboxylate acted as an inducer of the bifunctional enzyme and it was shown that the low level induction observed during growth on ornithine was due to the intracellular formation of proline. The formation of the proline degradative enzyme was shown to be subject to catabolite repression by citrate and nitrogen control.Abbreviations EMS Ethylmethane sulfonate - NG N-methyl-N-nitro-N-nitrosoguanidine - P Minimal medium P - Pro-DH Proline dehydro-genase - P5C ¹-Pyrroline-5-carboxylate - P5C-DH ¹-Pyrroline-5-carboxylate dehydrogenase     

The effect of some glycolytic intermediates and long-chain acyl-CoA esters on rat skeletal muscle mitochondrial α-glycerophosphate dehydrogenase

Summary -Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: acceptor oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from rat skeletal muscle has been studied. The pH optimum of the enzyme activity was about 7.4 and the apparent K_m value for DL--glycerophosphate was approxinately 1.6mm. The activity of this enzyme was found to be inhibited by DL-glyceraldehyde-3-phosphate, phosphoenolpyruvate and 3-phosphoglycerate in a competitive manner: the apparent K_i values at pH 7.4 being 0.3mm, 1.5mm and 4.0mm respectively. The enzyme was found to be more sensitive to phosphoenolpyruvate at pH 7.0 than 7.6.The activity of -glycerophosphate dehydrogenase in rat skeletal muscle was also inhibited by palmitoyl-CoA and stearoyl-CoA in a competitive manner. The K_i values being about 9.0 m for both metabolites. This inhibition was partly reversed by Ca²⁺ and Mg²⁺ ions. Palmitoylcarnitine also exerted inhibitory effect on -glycerophosphate dehydrogenase activity but palmitate, carnitine and CoA added alone was without effect. It is proposed that the activity of -glycerophosphate dehydrogenase in rat skeletal muscle mitochondria may be controlled by changes of the cytosolic levels of some glycolytic intermediates and long-chain acyl-CoA esters. These results are discussed with respect to the regulation of -glycerophosphate shuttle activity in skeletal muscle.     

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides

A number ofD-glutamyl andL-aspartyl dipeptides, glutathione, -D-glutamylglycine and -D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, theL enanthiomorphs of -glutamyl peptides were more potent displacers than -D-glutamylglycine and-taurine but the latter were more specific for the quisqualate type of receptors. -L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas -L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. -L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent -L-glutamyl peptides (glutathione, -L-glutamyl-L-glutamate,-L-aspartate, and-glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

Chronic administration of a nitric oxide synthase inhibitor,Nω-nitro-l-arginine,and drug-induced increase in cerebellar cyclic GMP in vivo

N-nitro-l-arginine (N^G-nitro-l-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N-nitro-l-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), andl-glutamic acid- (400 g/10 l/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increase induced by harmaline, picrotoxin, andl-glutamic acid was attentuated in N-nitro-l-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist,l-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N-nitro-l-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N-nitro-l-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.Part of this work was presented at the Experimental Biology 93 FASEB Meeting at New Orleans, March 1993.     

Interactions of γ-L-glutamyltaurine with excitatory aminoacidergic neurotransmission

The in vitro effects of -L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with -D-glutamyltaurine as reference. -L-Glutamyltaurine enhanced the K⁺-stimulated release of [³H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). -L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than -D-glutamyltaurine. The basal influx of Ca²⁺ into cultured cerebellar granular cells was not affected by 1 mM -L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg²⁺ (0.1 mM) and Mg²⁺-free media. The glutamate-evoked increase in free intracellular Ca²⁺ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM -L-giutamyltaurine. We propose that -L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Cation effects on the conformations of muscle and non-muscle α-actinins

We examined the effects of changing KCl concentration on the secondary structures of -actinins using circular dichroism (CD), 1,1-bis(4-anilino) naphthalene-5,5-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mm KH₂PO₄, pH 7.5, increasing KCl from 0 to 120 mm led to decreases in -helix conformation for brain, platelet and heart -actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mm KCl, 0.65 mm calcium produced small changes in the CD spectra of both brain and platelet -actinin, but had no effect on heart -actinin. bisANS fluorescence of all three -actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mm KCl increasing concentrations of Ca²⁺ or Mg²⁺ did not have significant effects on the bisANS fluorescence of any -actinin. Digestion of brain, platelet and heart -actinins with -chymotrypsin showed an increase of proteolytic susceptibility in 120 mm KCl. These experiments also showed that increasing the concentration of Ca²⁺ or Mg²⁺ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mm KCl. The results suggest that -actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different -actinins.     

Use of mitochondrial inhibitors to differentiate kinetic properties of the ATP-dependent Ca2+ uptake system in synaptic membranes

Synaptosomal membranes accumulate 3–6 times more Ca²⁺ in the presence of ATP (50–1000 M) than basal Ca²⁺ accumulation (-ATP). The location of this Ca²⁺ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca²⁺ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 g/ml), sodium azide (100 M) and dinitrophenol (100 M) differentiate mitochondrial from nonmitochondrial Ca²⁺ accumulation under conditions that are [Ca²⁺]- and ATP-dependent. In the presence of low concentrations of ATP (<150 M) and Ca _free ²⁺ (2.5 or 6.8 M), Ca²⁺ accumulation occurs as one process in both lysed synaptosomal membranes and purified synaptic plasma membranes in the presence and/or absence of MI. When ATP levels are increased (>200 M), the Ca²⁺ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca _free ²⁺ =2.5 M. When Ca _free ²⁺ is increased to 6.8 M, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca²⁺ accumulating mechanisms in synaptosomal membranes depend on both [Ca²⁺] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca²⁺ uptake may be a viable mechanism for the regulation of synaptosomal Ca²⁺ levels.     

Die optischen Übertragungseigenschaften der Komplexaugen von Drosophila

Summary The transfer properties of the optical system in the arthropod compound eye are determined by the interommatidial angle , influencing the resolving power, and by the width of the visual fields of single ommatidia , influencing the response at high spatial frequencies of brightness distributions in the object space. The energy transfer/ receptor is proportional to ( )² and decreases with in-inreasing approximation of the perfect-imaging condition: gD 0; 0. However, a value > 0 has to be maintained in order to overcome the threshold of nervous excitation at a certain minimum-brightness level. Theoretical treatment yields /=0.62 to 0.88 as the corresponding optimum-imaging relation. The actual ratio can be derived from measurements of the optomotor reactions to the movement of periodic brightness patterns. The approximate value 0.76 is obtained from the fruitfly Drosophila with normal and mutant eye pigmentation. As a result, the parameters of this imaging system are found to be established in a way that enables optimum performance at sufficient illumination. An dieser Stelle möchte ich Dr. W. Reichardt für sein eingehendes Interesse und manche anregende Diskussion über die Sehvorgänge im Komplexauge meinen Dank sagen. Dr. K. Kirschfeld verdanke ich ebenfalls wertvolle Hinweise. Herrn E. Freiberg bin ich für die Anfertigung der Abbildungen sehr verbunden.     

Effects of local anesthetics on phospholipid topology and dopamine uptake and release in rat brain synaptosomes

Summary The effects of local anesthetics on the topology of aminophospholipids and on the release and uptake of dopamine in rat brain synaptosomes have been examined. A metabolically intact preparation of synaptosomes was prepared which maintains aminophospholipid asymmetry and the capacity for sodium-driven uptake and depolarization-dependent release of dopamine. Incubation of synaptosomes with local anesthetics at 37°C induced perturbations in the topology of aminophospholipids as determined by their reactivities to the covalent probe trinitrobenzenesulfonic acid. The reaction of trinitrobenzenesulfonate with phosphatidylethanolamine and phosphatidylserine was inhibited 10–20% by low concentrations of tetracaine (1–100 m) and enhanced by high concentrations (0.3–1.0mm). Other local anesthetics showed a similar biphasic effect with a potency order of dibucaine>tetracaine>lidocaineprocaine. K⁺-stimulated, Ca²⁺-dependent release of [³H]dopamine was inhibited significantly at low concentrations of tetracaine (1–10 m) but enhanced at higher concentrations (0.1–1.0mm). Dibucaine and procaine had a similar biphasic effect on the dopamine release. For each of the local anesthetics tested, the inhibition of the reaction of phosphatidylethanolamine and phosphatidylserine with trinitrobenzenesulfonate occurred at concentrations which were shown also to inhibit the release of [³H]dopamine. Local anesthetics were shown to inhibit uptake of [³H]dopamine with a potency order which reflects their potency in producing anesthesia. The inhibition of dopamine uptake by dibucaine, tetracaine, lidocaine, or procaine was characterized by inhibitory constants (K _I ) of 1.8±0.4 m, 27±5 m, 190 m and 0.5mm, respectively.Abbreviations TNBS 2,4,6-trinitrobenzene sulfonate - PE phosphatidylethanolamine - PS phosphatidylserine - ESR electron spin resonance - TLC thin-layer chromatography - DA dopamine     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Variations in the rate of synthesis of beta and beta'' RNA polymerase polypeptides under the influence of certain factors.

Summary In merodiploid cells containing a double dose of structural genes of RNA polymerase subunits-rpoBand rpoC-the rate of and subunits synthesis is 2 times higher than in haploidcells. Missence mutation rpoC1 (tsX) alters polypeptide and inducesthe and subunits synthesis at increased rate, particularly at a nonpermissive temperature. When rpoBCoperon carrying mutation rpoC1 is duplicated no dosage effect is observed. In the rpoC ⁺/rpoC1 heterodiploid the rpoC1 mutation does not significantly accelerate RNA polymerase subunits synthesis i.e. is recessive with respect to rpoC ⁺ Rifampicin causes 6-fold stimulation of RNA polymerase subunits synthesis in a sensitive wild-type strain. The rpoC1 mutation itself accelerates the synthesis of these subunits 3-fold. In the presence of rifampicin the mutant strain produces 13–22-fold faster as compared to wild-type strain without the drug. Thus, the effects of rifampicin and the mutation are multiplied suggesting that these factors act independently. Similar data have also been obtained with rifampicin-treated cells of rpoB22 (ts22) amber-mutant.After UV-irradiation of cells and synthesis is depressed much stronger than the total protein synthesis. Infection with a transducing phage rif ^d-47 which carries rpoB gene provokes a higher rate of synthesis. When pre-irradiated cells (500 erg/mm²) are infected with this phage, the rate of synthesis grows 20-fold compared to irradiated, non-infected cells and 6.5-fold compared to intact cells.The data are discussed in terms of the possible regulatory mechanisms of RNA polymerase subunit synthesis.     

Amiloride and calcium effect on the outer barrier of the frog skin

Summary Amiloride (0.1mm) as well as Ca⁺⁺ (10mm) inhibit Na⁺ transport across frog skin by blocking Na⁺ entrance across the outer barrier of the epithelium. The inhibition produced by amiloride consists of an early and a late phase which together account for almost a total inhibition of the short-circuit current (SCC). The analysis of the time course indicates that the two phases are due to the inhibition of superficially and deeply located Na sites, respectively. Ca⁺⁺, instead, only blocks a fraction of the SCC, and this fraction seems to correspond to the inhibition of the same population of Na sites blocked by the late phase of amiloride effect. The location of the two populations of Na sites as well as the possible relationship between them are discussed in terms of maturation of the outermost cell layer.