Derivatives of Benzotetrazine-1,3-dioxide Are New NO-donors, Activators of Soluble Guanylate Cyclase, and Inhibitors of Platelet Aggregation

The ability of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides to generate nitric oxide (NO) and activate soluble guanylate cyclase was investigated. All of these compounds were found to be thiol dependent NO-donors and guanylate cyclase activators. The maximal stimulatory effect of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides was observed at 10 M concentration and the activity increase was 4.5-, 15.0-, and 8.2-fold in the presence of 20 M dithiothreitol and 11.3-, 31.6-, and 20.5-fold, respectively, in the presence of added glutathione (100 M). The NO-dependent mechanism of benzotetrazine-1,3-dioxide nitroderivative-induced activation of soluble guanylate cyclase (in the presence of 100 M glutathione) was confirmed by the inhibition (by 78%) of 7-nitrobenzotetrazine-1,3-dioxide (10 M)-stimulated guanylate cyclase activity in the presence of the NO-scavenger-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Carboxy-PTIO, 50 M) and by the inhibition with 1H-[1,2,4 ]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 0.3 M) of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides (10 M)-stimulated guanylate cyclase by 34, 69, and 39%, respectively. All compounds used inhibited ADP-induced aggregation of human platelets with IC ₅₀ of 10.0, 1.3, and 2.0 M for 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides, respectively. A clearly defined correlation was established between the ability of the compounds to generate NO, activate soluble guanylate cyclase, and inhibit platelet aggregation.     

The interrelationship of somatostatin and guanylate cyclase activity

Summary Somatostatin has been shown to inhibit the release of various polypeptide hormones including insulin, glucagon, gastrin, thyroid stimulating hormone, and growth hormone. The mechanism by which somatostatin inhibits the release of these various polypeptide hormones has not been fully eluciadated. It has been reported that somatostatin increases the level of the second messenger cyclic GMP in rat brain and in the anterior pituitary gland. The present investigation was designed to determine if these responses seen in the anterior pituitary gland and brain were due to activation of guanylate cyclase GTP-pyrophosphate lyase (cyclizing), E.C.4.6.1.2., the enzyme that catalyzes the formation of cyclic GMP. Somatostatin at a concentration of 2 pm enhanced guanylate cyclase activity two-fold in rat cerebrum and anterior pituitary gland. This enhancement of guanylate cyclase activity was also seen in rat liver, pancreas, stomach, and small intestine at the same concentration of somatostatin. Increasing the concentration of somatostatin to 20 m, caused a marked inhibition of guanylate cyclase activity in all these tissues. Dose-response curves done on gastric guanylate cyclase activity revealed that over a concentration range of 2 pm to 0.2 m, somatostatin had a stimulatory effect on guanylate cyclase activity while at concentrations above 10 m somatostatin was inhibitory to guanylate cyclase activity. The biphasic pattern of enhancement of guanylate cyclase activity at lower concentrations of somatostatin and inhibition at higher concentrations may help to explain some of the discrepancies seen with previous investigations with somatostatin, hormone release, and cyclic nucleotide metabolism.     

Effect of manganese on zinc-65 absorption by rice seedlings and its translocation within the plants

Summary Effect of manganese on absorption and translocation of zinc by rice seedlings was studied in a nutrient solution using radioactive zinc (Zn⁶⁵). With increase in manganese application, zinc uptake decreased in roots, but increased in shoots with an application upto 10 M manganese. Reduction in zinc absorption was more at low rates of zinc application. However, translocation of zinc from roots to shoots increased with manganese application upto 20 M but the increase was more pronounced with an application of 2M zinc only.     

On the manganese content of the egg ofOryzias latipes and its changes during the early development

Summary The content of manganese of the egg ofOryzias latipes was determined by atomic absorption spectrophotometry and activation analysis. The amount of manganese in the unfertilized egg was 0.51 mg and increased remarkably in one hour after fertilization. Although the increasing rate became gradually lower in further stages, the content of manganese amounted to 2.61 mg in the embryo after gastrulation. Tracer-experiments showed that the egg accumulated manganese positively from the surrounding medium during the short time after fertilization.     

Intracellular distribution of biotin-14COOH in rat liver

Biotin clearance, its distribution in liver and liver fractions after intravenous administration of 5 Ci/100 g body weight (21.55 g) of biotin-¹⁴COOH in normal and biotin-deficient rats are reported. In the biotin deficient animal there is a more rapid disappearance of the labeled biotin from the blood stream. Biotin-¹⁴COOH incorporation in the liver of the deficient rat is more rapid and larger than the incorporation in normal rat liver. Almost all the biotin recovered from liver homogenate is found in the mitochondria and in the pH 5.2 cytosol fraction; whereas in the microsomes only a very small amount is present. The intracellular distribution of biotin is in agreement with its known metabolic roles.     

Influence of unsaturated fatty acids on the production of tumour necrosis factor and interleukin-6 by rat peritoneal macrophages

The effect of individual unsaturated fatty acids on the release of tumour necrosis factor (TNF) and interleukin 6 (IL6) was investigated in thioglycollate — induced rat peritoneal macrophages. The intracellular mechanisms associated with the changes of cytokine production in response to fatty acids were also studied. Incubation of macrophages with 100 M docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) increased TNF (21% and 15% respectively) and IL6 (69% and 40% respectively) production. Linoleic acid (LA) diminished TNF production by 16%. At 100 M oleic acid (OA), LA and EPA concentration an increase in macrophage adenylate cyclase activity (110%, 72% and 39% respectively) and a decrease (14%) in the presence of DHA was observed. PGE₂ production in the presence of 100 M DHA was reduced by 36%, whereas in the presence of 100 M LA an increase (75%) was observed. Phospholipase A₂ (PLA₂) activity was also found to be modified in the presence of EPA and DHA at 50 M (20% and 60% respectively) and 100 M (34% and 62% respectively) concentrations. The activities of both protein kinase A (PKA) and protein kinase C (PKC) were effected by the different fatty acids. At 50 M all fatty acids suppressed PKA activity except OA which enhanced PKA activity by 14%. At 100 M fatty acid concentration, EPA suppressed PKA activity by 40%. PKC activity was enhanced by LA and OA, by 18% and 21% respectively. However, at 100 M EPA and DHA, PKC activity was suppressed by 37% and 17% respectively, whereas PKC activity was enhanced by 146% in the presence of 100 M LA. These results show for the first time that unsaturated fatty acids have an effect on macrophage PLA₂ activity and that PGE₂ may be a potent modulator of IL6 production. From these studies it is tempting to speculate that macrophage TNF and IL6 release may, in part, occur via a PKC and PKA independent pathway and that PLA₂ activity and PGE₂ concentration are inversely related to production of TNF and IL6.     

Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases

The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1–0.5 M) in the absence of Ca²⁺. This increase was not significantly altered by the presence of diisopropylfluorophsophate (DPF;2.5 mM)—although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 M) additively enhanced the dithiothreitol (DTT; 1.0 mM)—increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 M) enhanced the effect of Ca²⁺ (10 M) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca²⁺ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 M) was significantly decreased by the presence of calpastatin (24 g/ml), an inhibitor of Ca²⁺-activated neutral protease (calpain). Now, regucalcin (0.25 M) increased about 7-fold the activity ofm-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.     

Regional and subcellular distribution of taurine-synthesizing enzymes in the rat central nervous system

The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94–99 mol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44–51 mol/h/g; cerebellum, 71 mol/h/g; pons-medula and spinal cord, 94 and 60 mol/h/g, respectively. The distribution of CSD activity expressed as mol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14–21 mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8–13 mol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, colliculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.     

Rhizogenesis and somatic embryogenesis in calli from wild olive (Olea europaea var. sylvestris (Miller) Lehr) mature zygotic embryos

In calli from distal and proximal cotyledon segments and from radicles of mature wild olive zygotic embryos, rhizogenesis prevailed during the first and somatic embryogenesis during the second 30 day subculture period. Rhizogenesis was enhanced by low IBA (0.5 and 2.5 M) and 5.0 M 2iP. By reducing by half the media salt concentrations and by inserting a 21 day dark period rhizogenesis was also enhanced. Somatic embryogenesis was inhibited by both 2iP and IBA at concentrations higher than 5.0 M, but was not influenced by lower salt concentrations or by inserting the dark period.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - 2iP N⁶-[2-isopentenyl]adenine - BA 6-benzylaminopurine - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA naphthaleneacetic acid - OM Olive medium [4] - OMc OM medium in which OM macroelements were replaced with the ones proposed by Bourgin & Nitsch [2] - OMe OM salts of quarter strength, with half strength vitamin mixture (except biotin which added in full strength), with 5 M 2iP and 20 g l^-1 sucrose [2] - OMr OM salts half strength containing 5 M IBA and 20 g l^-1 sucrose [2]     

Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis

Cultures of Haematococcus pluvialis were exposed to mutagens like u.v. and EMS (ethyl methanesulphonate). The results showed that the survival rate decreased with the increase in u.v. exposure time and increase in EMS concentration. These mutants were further screened using inhibitors of the carotenoid biosynthetic pathway viz. diphenylamine (15–90 M), nicotine (160–320 M) and compactin (1.5–3.0 M). The mutants thus obtained showed early enhanced (2.2–3.2-fold) astaxanthin accumulation and also exhibited higher lycopene cyclase activity.     

Rat osseous plate alkaline phosphatase: mechanism of action of manganese ions

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by manganese ions in a similar way as by zinc ions. For concentrations up to 1.0 nm, the enzyme was stimulated by manganese ions, showing site-site interactions (n = 2.2). However, larger concentrations (> 0.1 m) were inhibitory. Manganese ions could play the role of zinc ions stimulating the enzyme synergistically in the presence of magnesium ions (K _d = 7.2 m; V = 1005.5 U mg^–1). Manganese ions could also play the role of magnesium ions, stimulating the enzyme synergistically in the presence of zinc ions (K _d = 2.2 m; V = 1036.7 U mg^–1). However, manganese ions could not substitute for zinc and magnesium at the same time since ion assymetry is necessary for full activity of the enzyme. A steady-state kinetic model for the modulation of enzyme activity by manganese ions is proposed.     

Measurements of manganese in thylakoids of Sinapis alba grown under high-light and low-light conditions

The manganese content of thylakoids and tissues was measured in leaves grown under high- and low-light conditions. Especially when grown in a nutrient medium enriched in manganese (20 M), the thylakoids contained large amounts of manganese, which could be removed by EDTA washing without impairment of the Hill reaction. The unremovable content of manganese was almost the same in thylakoids from plants grown in nutrient media of normal (2 M) and reduced (0.2 M) manganese content. Up to this limit of manganese content, Hill activity did not seem to be impaired. 1.2 atoms Mn per 100 molecules chlorophyll were found in low-light thylakoids and 1.6 atoms Mn in high-light thylakoids. This is similar to the behaviour of other electron transport components, the number of which is also decreased under low-light conditions. However, the decrease in the manganese content is not as striking as the decrease in, for example, the cytochrome f and ferredoxin content. This may be attributed to an invariable pool of manganese which is not involved in the oxygen evolving system. Alternatively, if all of our measured manganese is involved in electron transport to PS II, this could indicate that in low-light chloroplasts the ratio of PS II/PS I components may be somewhat increased.

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Zusammenfassung Der Mangangehalt von Thylakoiden und Gewebe aus Starklicht- und Schwachlichtblättern wurde untersucht. Besonders bei Pflanzen, welche unter erhöhtem Manganangebot (20 M) angezogen wurden, besaßen die Thylakoide sehr viel Mangan, welches durch Waschen mit EDTA entfernt werden konnte, ohne die Hill-Aktivität zu beeinträchtigen. In Thylakoiden aus Pflanzen, welche unter normalem (2 M) und reduziertem (0,2 M) Manganangebot gewachsen waren, unterschied sich der nicht entfernbare Mangangehalt nicht sehr. Dies scheint die untere Grenze des Mangangehalts zu sein, bis zu welchem die Hill-Aktivität noch nicht beeinträchtigt wird. Schwachlicht-Thylakoide besitzen 1,2 Atome Mn pro 100 Chlorophyllmoleküle, während Starklicht-Thylakoide 1,6 Atome Mn pro 100 Chlorophyllmoleküle enthalten. Dies gleicht dem Verhalten anderer Komponenten des Elektronentransports, welche ebenfalls im Starklicht vermehrt vorkommen. Die Unterschiede im Mangangehalt sind jedoch geringer als die Unterschiede im Gehalt von z.B. Cytochrom f und Ferredoxin. Dies könnte auf einen konstanten Anteil von Mangan zurückzuführen sein, welcher nicht am wasserspaltenden System beteiligt ist. Wenn jedoch das gesamte gemessene Mangan am Elektronentransport zum PS II beteiligt ist, könnte dies ein Hinweis sein, daß in Schwachlicht-Chloroplasten sich das Verhältnis der PS II-/PS I-Komponenten etwas vergrößert.

     

Chromate tolerance in strains of Rhodosporidium toruloides modulated by thiosulfate and sulfur amino acids

Cr(VI) tolerance was studied in four strains of Rhodosporidium toruloides and compared with that of a fifth strain, DBVPG 6662, isolated from metallurgical wastes and known to be Cr(VI) resistant. Tolerance was studied in relation to different species of sulfur (sulfates, thiosulfates, methionine, cysteine) at different concentrations. Djenkolic acid, a poor source of sulfur and an activator of sulfate transport, was also considered. In synthetic medium all strains except the Cr(VI)-resistant one started to be inhibited by 10 g ml^– (0.2 mm) Cr(VI) as K₂Cr₂O₇. DBVPG 6662 was inhibited by 100 g ml^– (2.0 mm) Cr(VI). In Yeast Nitrogen Base without amino acids (minimal medium), supplemented with varying concentrations of chromate, all Cr(VI)-sensitive strains accumulated concentrations of total chromium (from 0.8 to 1.0 g mg^– cell dry wt) after 18 h of incubation at 28 °C. In minimal medium supplemented with 10 g ml^– Cr(VI), the addition of sulfate did not significantly improve the yeast growth. Cysteine at m levels increased tolerance up to 10 g ml^–, whereas methionine only reduced the Cr(VI) toxicity in the strain DBVPG 6739. Additions of djenkolic acid resulted in increased Cr(VI) sensitivity in all strains. The best inorganic sulfur species for conferring high tolerance was thiosulfate at concentrations up to 1 mm. In all cases increased Cr(VI) tolerance was due to a significantly reduced uptake in the oxyanion by the cells and not to the chemical reduction of Cr(VI) to Cr(III) by sulfur compounds.     

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes

The association of [¹²⁵I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 M Ca²⁺, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 M Ca²⁺. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)-1]^–1. Arrhenius-type plots of [(r_o/r)-1]^–1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 M Ca²⁺. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F^–) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.     

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes

The effect of regucalcin, a regulatory protein of Ca²⁺ signaling, on guanosine-5-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca²⁺ (25–100 M) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni, and Mn) with 50 M. The activatory effect of calcium (50 M) was significantly decreased by calmodulin (2.5 and 5 g/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1–0.5 M) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 M). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca²⁺ in rat liver plasma membranes.     

Peptides from the N-terminus of the atrial natriuretic factor prohormone enhance guanylate cyclase activity and increase cyclic GMP levels in a wide variety of tissues

The 98 amino acid (a. a.) N-terminus of the 126 a. a. atrial natriuretic factor (ANF) prohormone contains three peptides consisting of a. a. 1–30 (proANF 1–30), a. a. 31–67 (proANF 31–67) and a. a. 79–98 (proANF 79–98) with blood pressure lowering, sodium and/or potassium excreting properties similar to atrial natriuretic factor (a. a. 99–126, C-terminus of prohormone). ProANF 1–30 and proANF 31–67 have separate and distinct receptors from ANF in both vasculature and in the kidney to help mediate the above effects. At the cellular level proANFs 1–30, 31–67, and 79–98 as well as ANF's effects are mediated by enhancement of the guanylate cyclase (EC 4.6.1.2) — cyclic GMP system in vasculature and in the kidney. These peptides from the N-terminus of the ANF prohormone circulate normally in man and in all animal species tested. The object of the present investigation was to determine if these peptides have the ability to enhance either guanylate cyclase and/or adenylate cyclase in a variety of other tissues in addition to kidney and vasculature. ProANF 1–30, proANF 31–67, proANF 79–98, and ANF all increased rat lung, liver, heart and testes, but not spleen, particulate guanylate cyclase 2- to 3-fold at their 100 nM concentrations. Dose response curves revealed that maximal stimulation of particulate guanylate cyclase activity by these newly discovered peptides was at their 1 M concentrations, with no further increase in activity above their 1 M concentrations. Half-maximal (EC₅₀) enhancement of particulate guanylate cyclase occurred at 0.15 ± 0.01, 0.3 ± 0.02, 0.5 ± 0.03, and 0.9 ± 0.03 nM for proANF 1–30, proANF 31–67, proANF 79–98 and ANF, respectively. ProANFs 1–30, 31–67, 79–98, and 99–126 (i.e., ANF) each increased cyclic GMP but not cyclic AMP levels in tissue slices of liver, lung, small intestine, heart, and testes. None of these peptides enhanced either adenylate cyclase or the soluble 100,000 G form of guanylate cyclase. The ability of these N-terminal peptides to enhance particulate guanylate cyclase activity in a wide variety of tissues suggests that they may have effects in a much wider variety of tissues than presently thought.     

Effect of sulfur supply on sulfate uptake,and alkaline sulfatase activity in free-living and symbiotic bradyrhizobia

The effect of sulfur limitation on sulfate transport and metabolism was studied in four bradyrhizobia strains using sulfur-limited and sulfur-excess chemostat cultures. Characteristics of bradyrhizobia associated with sulfurlimitation were determined and these parameters used to bioassay the sulfur status of bacteroids in nodules on sulfur adequate or sulfur deficient soybean and peanut plants. Sulfur-limited cells took up sulfate 16- to 100-fold faster than sulfur-rich cells. The sulfate-uptake system appeared similar in all strains with apparent K _m values ranging from 3.1 M to 20 M sulfate with maximum activities between 1.6 and 10 nmol·min^-1·mg^-1 protein of cells. Sulfate-limited cells of all strains derepressed the enzyme alkaline sulfatase in parallel with the derepression of the sulfate transport system. Similarly, the initial enzyme of sulfate assimilation (ATP sulfurylase) was fully derepressed in sulfur-limited cultures. Bacteroids isolated from sulfur adequate and sulfur deficient soybean and peanut possessed very limited sulfate uptake activity and low levels of activity of ATP sulfurylase as well as lacking alkaline sulfatase activity. These results indicate bacteriods have access to adequate sulfur to meet their requirements even when the host plant is sulfur-deficient.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide     

Sisal plant regeneration via organogenesis

Callus was initiated from in vitro grown immature leaf and ex vitro grown mature leaf and rhizome explants of Agave sisalana Perr. ex. Engelm, on MS medium containing 2,4-D (9.05 M) and kinetin (4.6 M) or 2,4-D (9.05 M), kinetin (4.6 M) and CH (1000 mg l^–1) or mod. MS (NH₄NO₃, 1500 mg l^–1) containing 2,4-D (9.05 M) and kinetin (4.6 M). Light was essential for callus formation which, however, was different in three types of explants on three different media compositions. Increasing NH₄ ⁺had a negative impact while addition of CH had a positive impact on callus formation. Shoot regeneration from callus from CH-supplemented medium only was achieved for rhizome and immature leaf tissues. The highest rate of regeneration was obtained with BA (26.6 M) as the sole hormone. Shoot buds g^–1 callus varied according to BA concentrations. Shoot proliferation rate increased on half-strength MS medium containing BA (8.9 M). Microshoots developed on MS medium containing BA (2.22 M) and GA₃ (1.44 M) and finally rooted on MS medium containing IAA (11.42 M). Acclimatized rooted plantlets are growing satisfactorily in ex vitro. This is the first report on plant regeneration via organogenesis of A. sisalana.     

Nonselective Inhibition of Monoamine Oxidases A and B by Activators of Soluble Guanylate Cyclase

Several activators of soluble guanylate cyclase were investigated as potential inhibitors of rat liver mitochondrial monoamine oxidases (MAO) A and B. They all fitted into the previously designed molds of substrate–inhibitor binding sites of these enzymes. However, only two of them, NO donors (7-nitro-benzotetrazine-1,3-dioxide (7-NBTDO) and benzodifuroxan), caused nonselective inhibition of MAO A and MAO B with IC ₅₀ values of 1.3-1.6 and 6.3-6.8 M, respectively. The inhibitory effect on both MAO A and MAO B was reduced by mitochondria wash suggesting reversible mode of the enzyme inhibition. There was no correlation between potency of MAO inhibition and activation of human platelet soluble guanylate cyclase. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) had no effect on the manifestation of MAO inhibition by benzodifuroxan and 7-NBTDO; however, at 50 M concentration carboxy-PTIO caused potent inhibition of MAO A with minor effect on MAO B activity. The data suggest that nonselective inhibition of MAO A and MAO B by benzodifuroxan and 7-NBTDO can be attributed to the properties of the chemical structures of these compounds. The results of the present study demonstrate a real possibility for the development of a new generation of effective reversible nonselective MAO inhibitors exhibiting equal inhibitory activity with respect to both MAO A and MAO B.     

Ultrastructural changes in the mitochondria of brown adipose cells during the hibernation cycle of Citellus lateralis

Summary The mitochondrial structure in the brown adipose cells of the golden mantled squirrel, Citellus lateralis, was examined throughout the year in biopsy samples. The mitochondria showed remarkable and apparently reversible changes in size and internal structure related to the physiologic activity of the animal. In the active animal the size of the largest mitochondria was 2.4 m × 1.5 m; during hibernation it increased to 7 m × 2.5 m; and during arousal it reached 11.2m × 5.3 m. The cristae of the mitochondria in the brown adipose cells of the animals in hibernation phase formed loops, whorls and mesh-like interconnections. During the arousal phase they underwent further configurational changes. The most remarkable structure was associated with mitochondria of most unusual proportions which by dissolution gave rise to a new generation. This was a common finding during arousal but did not occur in any other phase of the hibernation cycle. The new mitochondria were virtually indistinguishable from those of brown adipose cells of any active animal.Supported by a grant from the Medical Research Council of CanadaThe author is grateful to colleagues, Dr. G. Dempster and Dr. W.A. Spencer, for many valuable suggestions in the course of the work