Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Reappraisal of osmium tetroxide and OTAN histochemical reactions

Summary Osmium tetroxide and OTAN histochemical reactions have been reappraised following recent publications in this journal by Ellbder and Lojda. Provided that the standard OTAN reaction is used on frozen sections of conventional thickness, unsaturated hydrophilic polar lipids are stained in an orange or red shade while unsaturated hydrophobic non-polar lipids are stained brown-black or black. We have confirmed the anomaly — pointed out by Elleder and Lojda — that the polar lipid reaction of the atherosclerotic plaque is essentially extinguised by acetone. Solubility and blockading methods provide circumstantial evidence that Elleder and Lojda's assumed OsO₄-protein reactions are due to the lipid moiety of a lipoprotein complex.     

A quantitative histochemical study of 5′-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol

Summary 5-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein—Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5mm AMP, 10mm magnesium chloride, 7.2mm lead nitrate, 0.1m Tris—maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8m. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4mm in pericentral zones and 0.8mm in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.     

Aminopeptidase A is angiotensinase A

Summary Quantitative histochemical measurements of aminopeptidase A (APA; E.C.3.4.11.7) were done kinetically in the kidney glomeruli of rat and mouse with an instrumental setup consisting of a microdensitometer and a computer-supported morphometric system. The histochemical demonstration of APA was carried out using the simultaneous azo coupling technique (purest-grade Fast Blue B as coupling agent and -l-glutamic acid-4-methoxy-2-naphthylamide as substrate). The methodological studies show that APA activity is calcium-ion-dependent and increases linearly with the thickness of the tissue section (3–12 m) and that the time-course of APA activity as determined by linear regression is linear only for the first 1 to 2 min of the reaction. — Kinetic measurements indicate a 40% decrease in APA activities when -l-glutamic acid-4-methoxy-2-naphthylamide (-l-Glu-MNA) is replaced by -l-aspartic acid-4-methoxy-2-naphthylamide. When -l-Glu-MNA is replaced with l-alanine-4-methoxy-2-naphthylamide, which is a substrate of aminopeptidase M (APM) only very low reaction rates are measurable (about 1.4% of those with -l-Glu-MNA). 100 and 130 mM NaCl in the incubation medium increase APA activities by approximately 16%–17%. — To clarify the functional importance of APA in the kidney, their activities were measured under the influence of angiotensins. The glomerulus was selected as the measuring site, for besides APA it contains no APM or other peptidases that could degrade angiotensins (the glomerular dipeptidyl peptidase IV is not inhibited by angiotensin II). Using the Lineweaver-Burk plot, we determined a K _m of 0.16 mM for the APA in rat glomeruli and 0.14 mM in mouse glomeruli. The V _max in mouse glomeruli is 1.6 times higher than in rat glomeruli. Ang iotensin I, II and III competitively inhibit APA in the rat and mouse glomeruli. — With quantitative histochemical techniques it was possible to show that APA is equivalent to angiotensinase A (splitting off the N-terminal aspartic acid from angiotensin I and II).Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Polycarnitine—a new biomaterial

The natural product l-carnitine is—due to its biotechnological accessibility and specific properties—on the way to becoming an attractive biobased bulk product. l-Carnitine is a natural betaine with vitamin properties. Carnitine is an essential part of the fatty acid metabolism of human beings and animals. Carnitine was first isolated in 1905 from meat extract and important recent developments include the biosyntheses of l-carnitine from l-lysine or -butyrobetaine. Our synthesis routes are designed to maintain the primary structure and specific properties of carnitine, such as hydrophilicity and stiffening effects for polymeric structures and applications. l-Carnitine is converted via lactonization or olefinization into polymerizable basic molecules. The properties and the applications of carnitine polymers are described.This revised version was published online in February 2005 with text corrections in the subsection Biotechnological production of L-carnitine of the Introduction section.     

Proton transfer in and polarizability of hydrogen bonds in proteins. Tyrosine-lysine and glutamic acid-lysine hydrogen bonds — IR investigations

The OH N O^– H⁺N hydrogen bonds formed between tyrosine and lysine, and between glutamic acid and lysine residues are studied by infrared spectroscopy considering the following systems: (l-lys)_n + phenol, copoly (l-lys, l-tyr)_n, (l-lys)_n + (l-tyr)_n and (l-lys)_n + (l-glu)_n. The phenol-lysine hydrogen bonds are largely symmetrical in the average if the pK_a of the protonated lysine is 2.2 units larger than that of the phenols. In the case of the hydrogen bonds between tyrosine and lysine residues in copoly (l-lys, l-tyr)_n and (l-lys)_n + (l-tyr)_n, the weight of the proton limiting structure OH N is 80–90%, and that of the polar O^– H⁺N structure 10–20%. Double minimum proton potentials occur but the proton is preferentially present at the tyrosine residues. In the (l-lys)_n + (l-glu)_n system, the protons are present at the lysine residues. Thus, these hydrogen bonds have very large dipole moments (about 10 D). With the lysine-phenole hydrogen bonds, hydration shifts the proton transfer equilibrium a little in favour of the polar proton limiting structure O^– H⁺N. These hydrogen bonds are broken to a large extent, however, when only about 3 water molecules are present per lysine residue. When less water is present, as in the copoly (l-lys, l-tyr)_n and (l-lys)_n + (l-tyr)_n systems, these hydrogen bonds are, however, formed quantitatively. Thus — as discussed in this paper — the tyrosine-lysine hydrogen bonds can participate in proton conducting hydrogen bonded systems — as, for instance, present in bacteriorhodopsin — performing the proton transport through hydrophobic regions of biological membranes.     

Glutamate-like immunoreactivity revealed in rat olfactory bulb,hippocampus and cerebellum by monoclonal antibody and sensitive staining method

Summary Although there is good evidence favoring l-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. — The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The cross-reactivity of anti-glutamate mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA. — Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.     

l-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons

In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitorl-nitroarginine (l-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e.,l-NNA-dependent NO synthesis in these neurons. However,l-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 M-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration and final reaction product generated. Preincubation with the NOS-specific inhibitorl-NNA in glycine-NaOH buffer (pH 8.5–9.5) but notl-nitroarginine methyl ester (l-NAME) revealed a concentration-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of al-NNA sensitive NADPH-d activity. Blocking withl-NNA (100 M-10 mM) was prevented by excessl-arginine (10–100 mM), suggesting competitive binding sites. NADPH-d staining was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus, in formaldehyde-fixed nervous tissue both DPI andl-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-dependent conversion of nitroblue tetrazolium to formazan.Presented in the Workshop Detection of NO-synthases at the XXXVI Symposium of the Society for Histochemistry on Oxy Radicals, 20–23 September 1994, Heidelberg, Germany     

Beitrag zur Kenntnis der tschechoslowakischen Cnidion venosi-Wiesen

Zusammenfassung In der vorliegenden Arbeit wird die Charakteristik und das Verbreitungsareal der Überschwemmungswiesen des Verbandes Cnidion venosiBal.-Tul. 1965 näher behandelt. Es handelt sich um einen kontinentalen Verband, dessen zusammenhängendes Areal in Mitteleuropa einerseits in Ostdeutschland, anderseits in Südmähren, Österreich und Nordost-Kroatien ausklingt. Den tschechoslowakischen Cnidion venosi-Wiesen: Lathyrus paluster — Gratiola officinalis-Ass.Bal.-Tul. 1963, Gratiola officinalis — Carex praecox-suzae-Ass.Bal.-Tul. 1963, Cnidium dubium — Viola elatior-Ass.K. Walther Mscr. inR. Tüxen 1954, Cnidium dubium — Viola pumila-Ass.Korneck 1962 und Serratula tinctoria — Plantago altissima-Ass.Ilijani 1967, wurde ein besonderes Kapitel gewidmet (Seite 202–205). Ihre Zusammensetzung ist auch aus Tabelle II ersichtlich.

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Summary The characteristics and area of the flood meadows of the alliance Cnidion venosiBal.-Tul. 1965 are treated extensively. This is a continental alliance, whose area is delimited in Central-Europe on the one side in eastern Germany, on the other side in southern Moravia, Austria and north-east Croatia.A special chapter treats the types of meadows which in Czechoslovakia belong to this alliance.

     

Evidence for the participation of disulfide and sulfhydril groups in the specific binding of [3H]prazosin in cerebral cortex

The tritiated ₁ antagonist prazosin [³H]PRZ binds specifically and with high affinity to postsynaptic adrenoceptors in membrane preparations from cerebral cortex. Since adrenoceptors are of protein nature, it was of interest of investigate the possible role of disulfide (—SS—) and sulfhydril (—SH) groups in the binding of [³H]PRZ. Pretreatment of the membranes with the disulfide and sulfhydryl reactivesdl0Dithiothreitol,l-Dithiothreitol, Dithioerythritol or 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB), alone or in combination with the alkylating agent N-Methylmaleimide (NMM), decreased specific [³HPRZ binding, with minor changes in the non-specific counts. Saturation experiments revealed that all these reagents reduced the affinity of the binding site for [³H]PRZ, as judged by theK _d 25°C, but only the alkylating agent NMM and the oxydizing reagent DTNB produced in addition to the increase inK _d, a decrease of the maximum binding capacity (B _max). The present results provide evidence for a participation of—SS—and/or—SH groups in the recognition site of the ₁-adrenoceptor of cerebral cortex.     

Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine by entrapped ACV-synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase fromStreptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45–46% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g^–1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.     

Die Wirkung von Phytochrom und Actinomycin D auf die Chlorophyll a-Synthese von Senfkeimlingen (Sinapis alba L.)

Zusammenfassung Eine Vorbestrahlung der etiolierten Senfkeimlinge mit 4 Std Dunkelrot bewirkt eine Steigerung der Chlorophyll a-Synthese im Weißlicht. — Andererseits wird die Chlorophyll a-Synthese bereits durch relativ niedrige Actinomycin D-Konzentrationen gehemmt oder verzögert. — Die hemmende Wirkung von Actinomycin D ist — ausgedrückt in Prozent Hemmung — mit und ohne Vorbelichtung genau dieselbe. Auch die übrigen Daten deuten darauf hin, daß Actinomycin D nicht auf die Protochlorophyllsynthese als solche wirkt, sondern vielmehr die Synthese bestimmter Strukturproteine in den Plastiden beeinträchtigt. —Die Daten der Arbeit werden genphysiologisch gedeutet. Der wesentliche Punkt dabei ist, daß aktive Gene (z.B. jene, welche die Enzyme der bekanntlich auch im Dunkeln ablaufenden Protochlorophyllsynthese codieren) relativ unempfindlich gegenüber Actinomycin D sind; potentiell aktive Gene hingegen (z.B. jene, die einige nur im Licht entstehende spezifische Strukturproteine der Plastiden codieren) scheinen sehr viel empfindlicher gegenüber Actinomycin D zu sein. Diese Schlüsse stehen im Einklang mit einer früher geäußerten Hypothese (Lange und Mohr, 1965) und mit den Daten von Schopfer (1966) über die Regulation der Ascorbatsynthese im Senfkeimling durch Phytochrom und Actinomycin D.

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The action of phytochrome and actinomycin D on chlorophyll a formation in mustard seedlings (Sinapis alba L.)

Summary In the mustard seedling chlorophyll a synthesis under white light is enhanced by a pretreatment with far-red which maintains a low but virtually stationary concentration of active phytochrome (=P₇₃₀) (Fig. 1) during the period of irradiation (4 hours). — On the other hand chlorophyll a synthesis is inhibited or delayed by relatively low concentrations of Actinomycin D(=Act) (Fig. 2)The inhibitory action of Act (on a percent basis) is exactly the same with and without a far-red pre-irradiation (Fig. 3). Act in relatively low doses (5 or 10 g/ml) greatly extends the lag-phase of chlorophyll synthesis; however, these doses do not influence the effect of the far-red pretreatment on the rate of chlorophyll synthesis when it finally takes place (Fig.4,5,6). The data presented in this paper indicate that Act does not inhibit protochlorophyll synthesis as such; we have rather to conclude that Act inhibits the de novo synthesis of some specific structural proteins which are prerequisites of chlorophyll accumulation and maintenance in the plastids (Table 1). Synthesis of these structural proteins seems to be under the control of phytochrome too.It is concluded that those genes which are already in function are relatively resistant to Act (e. g. those genes which are needed for protochlorophyll synthesis) whereas potentially active genes (e. g. those which code some specific structural proteins of the plastids) are very sensitive to Act. —A similar conclusion has been reached in an earlier paper in connection with phytochrome-induced antocyanin synthesis (Lange and Mohr, 1965). Our argumentation is further supported by Schopfer's data on control of ascorbate synthesis in the mustard seedling by phytochrome and Act (Schopfer, 1966).

     

Cytochemical evaluation of the guard procedure a regressive staining method for demonstrating chromosomal basic proteins

Summary Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for sex chromatin display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are differentiated in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, condensed chromatin can be stained preferentially.Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 — probably due to extraction of some histone fractions — and the least amount of dye was bound in Bouin's-fixed chromatin — probably due to blockage of arginine residues by picric acid.The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl.The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.     

Physical and mapping properties of distant linkages between genetic markers in transformation of Bacillus subtilis

Summary DNA purified by a procedure based on phenol extraction contained many intact linkages, i.e. between genetic markers showing less than 20% cotransfer. The molecular weight of this DNA, as revealed by zone centrifugation, varied between 4.5×10⁷ and 2.5×10⁸. Linkages with cotransfer frequencies greater than 10% were found to consist of not more than 6.0×10⁷ daltons of DNA, while one linkage showing only 2% cotransfer was provisionally estimated to be about 1.5×10⁸ daltons. With the aim of extending the transformation map of B. subtilis, 16 mutations for nutritional requirements, not suspected—on the basis of the phenotypes involved—to be linked to any other markers, were examined for linkage with each other and to markers on the existing map. Three new pairs of distantly linked markers were found, but no linkage to any location on the known map.The study of the linkage properties of markers which Oishi, Yoshikawa and Sueoka localized on their replication map suggests that some of these markers may have been misplaced.     

Über die Wirkung von Kaliumjodid auf die lichtstimulierte endogene Atmung von Algen

Summary The endogenous respiration of an achlorophyllous mutant of Chlorella vulgaris is enhanced by small amounts of blue light. The action spectrum for this effect shows two peaks at 460 and 375 m, which points to a flavin or a carotenoid in cis configuration as the likely photoreceptor responsible. Weber's (1950) observation that in vitro potassium iodide (KI) quenches the fluorescence of riboflavin was employed to distinguish between these two pigments. — KI in concentrations from 0.033 to 0.50 M lowers the oxygen uptake in blue light increasingly (Fig. 1, Table 1), but not specifically: KI inhibits the respiration of exogenous glucose even more (Fig. 2). Furthermore neither the inhibition of endogenous nor that of exogenous respiration is iodide-specific; a decrease in both of them takes place with KNO₃ of corresponding concentrations, too (Table 2). The somewhat smaller inhibition with KNO₃ compared to that with KI fits the known observation that iodide has a greater inhibiting effect on metabolic reactions of plant cells than nitrate (Hewitt and Nicholas, 1963). — Finally it was observed that irradiation with blue light (>366 to <550 m) of the iodide solutions used liberates some iodine (Fig. 3). Since the promoting effect of iodine on the isomerisation of carotenoids is well documented (Zechmeister, 1962), it might be impossible to determine whether a flavin or a cis-carotenoid participates in a blue light specific reaction by using an iodide solution, as has been done several times recently.

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Herrn Prof. Dr. R. Harder zum 80. Geburtstag gewidmet.     

The ultrastructural immunocytochemical localization of superoxide dismutase in the amphibian urinary bladder: Effect of aldosterone

Summary Transmission electron microscopy and immunohistochemistry, the latter employing the avidin—biotin—peroxidase (ABC complex) technique, were utilized to localize copper—zinc superoxide dismutase (CuZn—SOD) enzyme activity in the epithelial cells of the toad urinary bladder mucosa. This scavenger enzyme catalyses the dismutation (reduction—oxidation) of the superoxide anion (O₂ ^–), a toxic free radical generated during normal cellular respiration. In unstimulated epithelial cells, enzyme activity was seen in the cytosol of granular, mitochondrial-rich and goblet cells. The basal cells were generally devoid of enzyme activity. In addition to the cytosol, SOD activity was also seen in association with the apical plasma membrane of the epithelial cells. In the presence of the steroid hormone aldosterone (10^–7 m, 30 min—6 h), CuZn—SOD activity was markedly increased along the luminal mucosal membrane of granular, mitochondrial-rich and goblet cells. This increase was seen as early as 30 min after the addition of hormone, and as long as 6 h after treatment. The cytosolic reaction was usually decreased or absent under these conditions. From the data presented, it appears that CuZn—SOD is involved in electrolyte (sodium) transport in the epithelial cells of the toad urinary bladder. The latter may involve hormone-induced alterations in luminal cell membrane structure and chemistry.To whom reprint requests should be addressed.     

Unexpected enhancement of β-lactam antibiotic formation inStreptomyces clavuligerus by very high concentrations of exogenous lysine

l-Lysine is known to stimulate production of -lactam antibiotics byStreptomyces clavuligerus via provision of the lysine breakdown product,l--aminoadipic acid, which is a limiting precursor. Previous investigations utilized levels of 10–20 mMl-lysine as an addition to chemically-defined media resulting in 50–100% improvement in antibiotic production. We were surprised to note that as the concentration was further increased, the organism responded by producing even higher titers of antibiotics. The optimum concentration of 100 mMl-lysine yielded an approximate 500% increase in production with only minor effects on growth.dl- andd-lysine also exerted enhancements suggesting the presence of a lysine racemase or some other route fromd-lysine tol--aminoadipate in this organism;d-lysine was considerably less potent thandl- orl-lysine.Participant in the MIT Undergraduate Research Opportunities Programs (UROP)     

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     

Der Lichteinfluss auf die Haarbildung am Hypokotyl vonSinapis alba L.

Summary In an earlier paper (Mohr 1957) it was described that the formation of anthocyanin and the inhibition of lengthening of the hypocotyl of dark grown seedlings (Sinapis alba) is governed by the action of two photomorphogenic action systems. The one system is the well known reversible red far-red reaction system (low energy reaction), the other one is a high energy reaction system which can be called — in reference to the action peaks in the far-red and in the blue — blue far-red reaction system. The chemical nature of the absorbing pigments is still unknown.In the present paper another photomorphogenic response of the young dark grown seedlings ofSinapis alba, the light dependent formation of unicellular hairs from epidermal cells of the hypocotyl, has been investigated. It has been shown that this response is also governed by these two reaction systems. These systems have been physiologically separated. Experiments have been presented which evaluate the importance of the assumption that the red absorbing pigment of the reversible red far-red pigment system is reformed from a precursor when the pigment present before irradiation is transformed into the far-red absorbing pigment by an irradiation with red.

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Mit 3 Textabbildungen     

Effect of administration of 5-hydroxytryptophan and an inhibitor ofl-aromatic amino acid decarboxylase on glucose metabolism in rat brain

The effect of 5-hydroxytryptophan (5-HTP)—the precursor of serotonin (5-hydroxytryptamine, 5-HT)—and of an inhibitor,N-(dl-seryl)-N-(2,3,4-trihydroxybenzyl)hydrazine (Ro4-4602), ofl-aromatic amino acid decarboxylase on the metabolism of glucose to amino acids in brain tissue was investigated. Labeled glucose (20 Ci, 0.24 mg in 0.2 ml 0.9% saline) was injected intravenously into fed rats pretreated with Ro4-4602 (50 mg/kg intraperitoneally) either alone or in combination with 5-HTP (30 mg/kg intravenously) or with the appropriate vehicle. After the injection of Ro4-4602 plus 5-HTP, the concentrations of 5-HT and 5-HTP in brain were increased, but the increase of 5-HTP that Ro4-4602 slightly inhibits the reaction of decarboxylation in the brain, although at the dose used the drug is usually considered to act only peripherally. After administration of Ro4-4602 alone or combined with 5-HTP, the concentration of glucose in plasma was not significantly increased. However, the concentration of glucose in brain was markedly increased with such treatments. The administration of Ro4-4602 alone or combined with 5-HTP reduced the flux of¹⁴C from labeled glucose to amino acids in brain. The concentrations of amino acids in brain were little changed by these treatments.