Activation of striatal tyrosine hydroxylase by neurocatin,a neuroregulator from mammalian brain

Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of dopamine synthesis in striatal rat synaptosomes. Incubation of intact synaptosomes with neurocatin caused an increase in the rate of dopamine synthesis measured by accumulation of DOPA. The increase is rapid (within two minutes) and dependent on the concentration of added neurocatin. The stimulatory effect of neurocatin on dopamine synthesis occurred only in intact synaptosomes and was almost completely abolished by lysis of the synaptosomes with Triton X-100 or sonification prior to neurocatin addition. The kinetic parameters of tyrosine hydroxylase were measured in lysates prepared from synaptosomes preincubated with neurocatin. These showed that with increasing neurocatin concentration there was an increase in V_max with no significant change in K_M for the pteridine cofactor, compared to control. Activation of tyrosine hydroxylase by neurocatin is at least partially caused by a receptor mediated increase in phosphorylation of the enzyme. Protein kinase C and protein kinase II may be involved in this process.     

Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes

Crude striatum synaptosomes (P₂ fraction) from Fisher 344 female rats were incubated in the presence of ADP-chelated Fe³⁺ (0.5–50 M) and ascorbate (250 M). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by¹⁴CO₂ evolution froml-[1-¹⁴C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe³⁺/ascorbate was found with 50% inhibition occurring at 2.5 M Fe³⁺ concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe³⁺ in the absence of exogenous ascorbate was effective only above 25 M. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P₂ fractions using sucrose gradients. Membrane microviscosity of the purifled synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe³⁺/ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulhydryl content. It is concluded that ironinduced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Morphometric and autoradiographic analysis of crude synaptosomal preparations from rat cerebral cortex

Morphometric and autoradiographic studies have been made of a crude synaptosomal preparation, which has been used extensively for membrane transport studies. When filters are used to separate membrane bound structures from incubation medium, the structures which survive filtration are those that are entrapped within the matrix of the filter structure. The population of membrane bound structures differs when one compares pellets of the preparation to sections of loaded 0.45 and 0.65 m pore size filters. Both the relative numbers of synaptosomes, mitochondria, and other membrane bound structures (OMBS) and the mean size of each of the structures differ for pellet, 0.45 m, and 0.65 m filters. The percentage of total membrane bound volume attributable to synaptosomes increases from 28 in the crude preparation to 40 in 0.45 m filters and 61 in 0.65 m filters. The total volume of synaptosomes entrapped by differing pore size filters roughly correlates with the amount of substrate uptake. Neither mitochondrial volume nor the volume of other membrane bound structures was found to correlate with uptake. These results indicated that only the synaptosomes contribute measurably to this function. Autoradiographic studies confirm this conclusion. EM autoradiography following loading of the synaptosomal preparation with tritiated glutamate or GABA showed about 81% of the grains to be associated with synaptosomes. It is concluded that crude synaptosomal preparations may be used without further purification for membrane transport studies with unambiguous results.     

The Effect of Phenylalanine on DOPA Synthesis in PC12 Cells

DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (~55 pmol/min/mg protein for tyrosine; ~40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of ~10 M for either amino acid. The K_m for either amino acid was about 1 M (medium concentration). At tyrosine concentrations above 30 M, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (300 M). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the K_m for either amino acid is 20–30 M; maximal synthesis occurred at 120–140 M. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 M), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 M). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It was found to be able to grow in the presence of micromolar molecular oxygen (O₂). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O₂ to hydrogen peroxide (H₂O₂), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent V_max value of 37 mol NADH oxidized min^–1 mg^–1 protein. Apparent K_m values for NADH and O₂ were determined to be 7.5 M and 85 M, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85°C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H₂O₂ produced by the NADH oxidase to H₂O. It seems possible that O₂ can be reduced to H₂O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O₂.     

Inhibition of benzo[a]pyrene-induced cytotoxicity and cytochrome P450 1A activity by dietary flavonoids in human liver cell model: structure-activity relationship

Inhibition of benzo[a]pyrene (B[a]P)-induced cytotoxicity and cytochrome p450 1A (CYP 1A) activity by flavonoids (1–100 M) was examined in terms of the structure-activity relationship in the human liver-derived cell model (HepG2). Two hydroxyl groups in the 5- and 7-position of flavonoids were essential to inhibit B[a]P-induced cytotoxicity. Generally, flavones (IC₅₀; 5.0–17.2 M) were more potent than the corresponding flavonols (IC₅₀; 42.7–131.8 M), and flavonoids such as apigenin (IC₅₀; 7.2 M) were more active than the corresponding isoflavonoids, genistein (IC₅₀; 61.7 M). The planar structure of flavone proved to be important in inhibiting B[a]P-induced toxicity and CYP 1A activity. The inhibitory effect of flavonoids on B[a]P-induced CYP 1A activity was correlated well with the inhibition of B[a]P-induced cytotoxicity (r=0.635, p<;0.01).     

Diaminobutyric acid: A tool for discriminating between carrier-mediated and non-carrier-mediated release of GABA from synaptosomes?

The carrier-mediated transport of GABA in rat brain synaptosomes was strongly and permanently inhibited byl-2,4-diaminobutyric acid (DAB). In order to discriminate between carrier-mediated and non-carrier-mediated release of [³H]GABA, synaptosomes prelabeled with 0.5 M [³H]GABA in the presence of 100 M DAB, or with 0.2 M [³H]GABA without DAB, were superfused in conditions stimulating the release of [³H]GABA. Only the release elicited by unlabeled GABA or DAB (by homo- and heteroexchange, respectively) was strongly inhibited in DAB-pretreated synaptosomes. The spontaneous release and the release induced by 56 mM KCl in the presence of CaCl₂, by the ionophore A23187, by ouabain, by lack of K⁺, or by purified black widow spider toxin were unaffected or only barely decreased in DAB-treated synaptosomes, and therefore do not seem to be mediated by the DAB-blocked GABA carrier.     

Toxic responses of germinating pollen and soybeans to aflatoxins

Aflatoxin producing strains of Aspergillus grow on soybeans thereby contaminating the latter through secretion of the toxin. Investigations dealing with either soybean seed germination or intact seedling growth responses to aflatoxin (B₁) are lacking. Similarly, a possible interaction of aflatoxins with phosphate in the germination and elongation of both soybeans and pollen as well as roots of the former and tubes of the latter has not been examined. Imbibition of Glycine max, cv. Essex seeds for 18 hours in solutions containing 0.38, 2.90, 5.80 or 11.60 g/ml (AFB₁) yielded% germination inhibitions of 5, 20, 40 and 80, respectively. By 36 hours these were 6, 4, 13 and 19 % for the same toxin concentration series. At 140 hours attached root elongation was inhibited 26, 35 and 50 % for 2.90, 5.80 and 11.60 g/ml AFB₁. No effect was noted at 0.38 g/ml AFB₁. Incubation of excised roots in medium containing 3.0 mM KH₂PO₄ stimulated their elongation 3.2 fold. Addition of 33.28 g/ml mixed aflatoxins together with KH₂PO₄ resulted in only a 1.5 fold stimulation. When KH₂PO₄ was added to a culture medium lacking AFB₁, Lilium longiflorum, cv. Ace pollen germination was enhanced 50%. Withholding KH₂PO₂ but supplying AFB₁ did not markedly affect germination. However, supplementing the medium with KH₂PO₄ while simultaneously adding AFB₁ did not inhibit germination at 5 and 10 g/ml but caused 27.3 and 45.1 % declines at 25 and 30 g/ml. In the absence of KH₂PO₄ AFB₁ stimulated pollen tube elongation 7.5, 14.3, 16.5 and 13.2 % at 5, 10, 15 and 20 g/ml but 30 g/ml inhibited it 11.1%. In contrast, tube elongation was suppressed at all AFB₁ concentrations (maximum 36.1% at 30 g/ml) tested upon KH₂PO₄ addition. Results derived from germinating pollen in medium supplemented with KH₂PO₄ or NaH₂PO₄ indicate that the phosphate anion does not preferentially promote aflatoxin-induced inhibition of tube elongation.Aided by grant IN-127 from the American Cancer Society to W.V. Dashek and funds from the Departments of Biology, West Virginia University and Virginia Commonwealth University and the West Virginia University Foundation.     

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

The Effect of Neurocatin on Protein Phosphorylation in Striatal Synaptosomes from Rat Brain

Abstract: Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of ～80 and ～60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60-kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to ～7.5 ng/100 μl of suspension), incorporation of ³²P orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin >7.5 ng/100 μl caused progressive decrease in incorporation of ³²P into many synaptosomal proteins; by a concentration of neurocatin of ～45 ng/100 μ/l, the level of ³²P incorporation into many proteins was ≤70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of ～7.5 ng/100 μl of neurocatin, increased incorporation of 32P into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times >2.0 min, showed progressive decrease in ³²P incorporation. Removing extrasynaptosomal Ca²⁺ with EGTA attenuated the increased ³²P incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin-induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of ³²P incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on the plasma membrane.     

Aluminium impacts elements of the phosphoinositide signalling pathway in neuroblastoma cells

Inositol phosphate formation was examined in aluminium-treated murine neuroblastoma cells labelled with [³H]-myoinositol. Employing fluoride-stimulated intact cells, aluminium (0.2M to 1 mM) reduced inositol phosphate formation in a dose-dependent manner. In digitonin-permeabilized cells, stimulated with nonhydrolyzable GTP[S], inositol phosphate formation was also inhibited by increasing aluminium doses; the IC₅₀ value was about 20M aluminium, while the inositol phosphate level was reduced 2.5 to 3 fold by 50M aluminium. The inhibitory effect of aluminium (50M) could not be reversed by increasing GTP[S] concentrations up to 500M. Prechelation of aluminium to citrate or EGTA completely abolished the aluminium-triggered inhibition of fluoride-stimulated inositol phosphate formation in intact cells, but had little effect on the inhibition of permeabilized cells stimulated with GTP[S]. In neuroblastoma cells phosphoinositide hydrolysis could be evoked either through a pathway involving the Mg²⁺/guanine nucleotide binding (G_p) protein, or via a pathway operative in the presence of high intracellular Ca²⁺ concentrations. In the Mg²⁺/G_p protein-mediated pathway, formation of inositol triphosphate, IP₃, inositol diphosphate, IP₂, and inositol monophosphate, IP, was apparently inhibited by aluminium in an interdependent manner. As to the Ca²⁺-mediated pathway, aluminium application mainly diminished the release of IP₃. Following interiorization, aluminium thus acts upon elements critical for phosphoinositide-associated signal transduction. An aluminium target apparently resides on the G_p protein. Phosphatidylinositol-4,5-diphosphate-specific phospholipase C probably harbours a second aluminium target.     

Exposure to Ebselen Changes Glutamate Uptake and Release by Rat Brain Synaptosomes

We investigated effects of Ebselen, diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ on [³H]glutamate uptake and release by brain synaptosomes. Ebselen after acute exposure inhibited K⁺-stimulated [³H]glutamate release by brain synaptosomes. (PhSe)₂ and (PhTe)₂ did not change [³H]glutamate release by brain synaptosomes. Ebselen, (PhSe)₂ and (PhTe)₂ had no significantly effects on [³H]glutamate uptake after acute exposure. In vitro, Ebselen (100 M) inhibited [³H]glutamate release and uptake. (PhSe)₂ had no significant effect, while (PhTe)₂ (100 M) inhibited [³H]glutamate uptake by brain synaptosomes. In vitro, (PhSe)₂, (PhTe)₂ and Ebselen caused a significant inhibition of [³H]glutamate uptake by brain synaptic vesicles in vitro. The results demonstrated that organochalcogenides have a rather complex effect on glutamate homeostasis depending on the compound and the schedule of exposition. We propose that the neuroprotective action of Ebselen can be related, in addition to its glutathione peroxidase-like and antilipoperoxidative activity, to a direct interaction with the glutamatergic system by reducing K^ï-evoked glutamate release.     

Endogenous Adenosine Modulation of 22Na Uptake by Rat Brain Synaptosomes

To evaluate if endogenous extracellular adenosine influences sodium channel activity in nerve terminals, we investigated how manipulations of extracellular adenosine levels influence ²²Na uptake by rat brain synaptosomes stimulated with veratridine (VT). To decrease extracellular adenosine levels, adenosine deaminase (ADA) that converts adenosine into an inactive metabolite was used. To increase extracellular adenosine levels, we used the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), as well as the inhibitor of adenosine transport, nitrobenzylthioinosine (NBTI). ADA (0.1–5U/ml) caused an excitatory effect on ²²Na uptake stimulated by veratridine, which was abolished in the presence of the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 25M). Both the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1–10M) and the adenosine deaminase inhibitor EHNA (10–25M) inhibited ²²Na uptake by rat brain synaptosomes. It is suggested that adenosine is tonically inhibiting sodium uptake by rat brain synaptosomes.     

Die Kopfregeneration vonLineus sanguineus Rathke(Nemertini) unter dem Einfluβ von Actinomycin-D. Chemomorphologische Befunde

Zusammenfassung Isolierte Körperteile vonLineus sanguineus wurden in Seewasser gehalten, das unterschiedliche Konzentrationen (1,9, 0,95 und 0,48 g/ml) von Actinomycin-D enthielt. Nach 4, 5, 6 und 10 Tagen wurden die Regenerate histologisch und histochemisch (Glykogen, Glucose-6-phosphat-Dehydrogenase und RNS) untersucht. In Abhängigkeit von der Actinomycin-D-Konzentration werden bestimmte Phasen der Regeneration gehemmt; dabei sind Proliferationsprozesse weniger empfindlich als Differenzierungsprozesse. Histochemisch werden bei Hemmung der wachstumsaktiven Areale die im Normalfall zu beobachtenden stofflichen Merkmale: Glykogeneinlagerung und nachfolgender Anstieg der G-6-P-DH-Aktivität und der RNS-Konzentration vermißt.

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The head regeneration ofLineus sanguineus Rathke (Nemertini) under the influence of actinomycin-D. Chemomorphological data

Summary Fragments ofLineus sanguineus were incubated in seawater, which contained different concentrations of actinomycin-D (1.9 g/ml, 0.95 g/ml and 0.48 g/ml). After 4, 5, 6 and 10 days the regenerates were studied by histological and histochemical methods (glycogen, glucose-6-phosphate-dehydrogenase and RNA). Corresponding to actinomycin-D concentration certain phases of regeneration are inhibited. The phases of proliferation are less sensitive to actinomycin-D than the phases of differentiation. If morphogenetic areas are inhibited, the normally demonstrable histochemical characteristics: accumulation of glycogen and later, the increase of G-6-P-DH activity and RNA concentration do not appear.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Ability of bisbenzylputrescine and propanediamine analogs to modulate the intracellular polyamine pool of P388D1 cells

The effects of a series of bisbenzyldiamine analogs have been tested on P388D1 cell line in vitro. Their effects on cell growth, polyamine oxidase (PAO) activity and intracellular polyamine content were determined. The cytotoxicity tests were performed in culture medium supplemented with 100 mol/L aminoguanidine (I), 100 mol/L aminoguanidine and 100 mol/L N,N-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527) (II), and finally 100 mol/L aminoguanidine and 200 mol/L D,L-difluoromethylornithine (DFMO) (III). The IC50 values under conditions I and III were similar, suggesting that inhibition of ornithine decarboxylase by DFMO did not affect the biological effect of our derivatives. Spermine and spermidine remained nontoxic in conditions I and III. However in the condition II, the toxicity of all tested compounds (excepted spermidine) was increased, suggesting that the inhibition of cellular PAO increased their toxicity.The enzymatic test of PAO showed that at high doses inhibition of this enzyme by putrescine analogs occurred, while the N-methylated propanediamine derivative increased the enzyme activity; however, these results do not correlate with cytotoxicity tests. When these derivatives were incubated for 48 h with the cells, all of them increased the cell content in putrescine (160%) and spermine (145%) and decreased the spermidine content (75%) without any modification of the total amount of polyamine.The correlation between the cytotoxic results and the intracellular polyamine determination shows that the increase in spermine content along with the inhibition of retroconverting PAO enzyme increases the toxic effect of tested compounds (including spermine), suggesting that spermine toxicity is more important in the absence of intracellular oxidation processes.     

Effects of Lead on Adenylate Cyclase Activity in Rat Cerebral Cortex

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC₅₀ = 2.5 ± 0.1 M). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 M Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 M) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 M), lead (3 M) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 M). In membranes preincubated with 10 M lead, the addition of 10 M Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 M lead + 10 M Gpp(NH)p, Gpp(NH)p (10 M) or forskolin (10 M) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.     

A peroxisomal glycolate oxidase in the algaMougeotia

Microbodies of the algaMougeotia were isolated in a linear sucrose gradient. The organelles, which moved to the density 1.24 g cm^–3, contained about 70% of the glycolate oxidase (EC 1.1.3.1) found in this alga. The enzyme oxidized glycolate, utilizing either oxygen or 2,6-dichlorophenolindophenol (DCPIP) as the electron acceptor. L-Lactate was an alternate substrate; almost no D-lactate was utilized. In the presence of O₂, a K_m of 415 M was determined for glycolate, whereas the K_m for L-lactate was about 5,000 M. In the presence of DCPIP, lower concentrations of glycolate and L-lactate were sufficient to obtain the highest rates of enzyme activity.Abbreviations DCPIP 2,6-dichlorophenolindophenol Supported by the Deutsche Forschungsgemeinschaft     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.