Effects of calcium on the activities of cytosolic antioxidative enzymes and IAA oxidase during in vitro adventitious rooting of mung bean seedlings

The effects of Ca²⁺ on antioxidative enzymes and indole-3-acetic acid (IAA) oxidase during adventitious rooting were investigated in mung bean (Vigna radiata). CaCl₂ significantly promoted the formation and growth of adventitious roots. EGTA (a Ca²⁺ chelator) or ruthenium red (a Ca²⁺-channel blocker) significantly inhibited root formation and growth, but these inhibitory effects could be partially reversed by CaCl₂. Furthermore, inclusion of 5 mM CaCl₂ significantly increased superoxide dismutase (SOD) activity by 10% at 3 h and catalase (CAT) activity by an average of 29.6% at each time point. CaCl₂ decreased peroxidase (POD) activity by 9.4% and 21% at 12 and 24 h, respectively, and ascorbate peroxidase (APX) activity by an average of 13.9% at each time point. These CaCl₂-induced changes in enzymatic activities were similar to changes caused by indole-3-butyric acid (IBA). Treatment with EGTA or ruthenium red decreased SOD activity by an average of 18.4% and 15.2%, respectively; POD activity by 27.4% and 57.6%, respectively; APX activity by 10.3% and 15.6%, respectively; and CAT activity by 19.3% and 5.2%, respectively, when compared with CaCl₂. In addition, CaCl₂ increased IAA oxidase activity by an average of 5.5% beginning at 6 h, whereas EGTA significantly decreased IAA oxidase activity by 29.2%, 22.9%, and 13.5% at 6, 9, and 12 h, respectively. The inhibitory effects of EGTA could be partially suppressed by addition of CaCl₂. These results imply that the stimulative effect of Ca²⁺ on adventitious rooting is partially related to Ca²⁺-induced changes in the activities of antioxidative enzymes and IAA oxidase.     

Increases in peroxide formation by the Photosystem II oxygen evolving reactions upon removal of the extrinsic 16, 22 and 33 kDa proteins are reversed by CaCl2 addition

This communication introduces a new spectrophotometric assay for the detection of peroxide generated by Photosystem II (PS II) under steady state illumination in the presence of an electron acceptor. The assay is based on the formation of an indamine dye in a horseradish peroxidase coupled reaction between 3-(dimethylamino)benzoic acid and 3-methyl-2-benzothiazolinone hydrazone. Using this assay, we found that as the O₂ evolution activity of PS II-enriched membrane fragments is decreased by treatments which cause the dissociation of the 33 and/or 23 and 16 kDa extrinsic proteins (i.e., CaCl₂-washing, NaCl-washing, lauroylcholine-treatment and ethylene glycol-treatment), light-induced peroxide formation increases. Both the losses of O₂ evolution and increases in peroxide formation seen under these conditions are reversed by CaCl₂ addition, indicating that the two activities originate from the water-splitting site. However, the increased rates of peroxide formation do not quantitatively match the losses in O₂ evolution activity. We suggest that a rapid consumption of the peroxide takes place via a catalase/peroxidase activity at the water-splitting site which competes with both the O₂ evolution and peroxide formation reactions. The observed peroxide formation is interpreted as arising from enhanced water accessibility to the catalytic site upon perturbation of the extrinsic proteins which then leads to alternate water oxidation side reactions.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichloro)-1,1-dimethylurea - DCPIP 1,6-dichlorophenolindophenol - DMAB 3-(dimethylamino)benzoic acid - DMBQ 2,6-dimethyl-p-benzoquinone - DPC diphenylcarbazide - HEPES 4-(2-hydroxyethyl)-1-piperazinesulfonic acid - HMD HRP, MBTH, DMAB - HRP horseradish peroxidase - LCC lauroylcholine chloride - MBTH 3-methyl-2-benzothiazolinone hydrazone - MES 4-morpholinoethanesulfonic acid     

Selenium inhibition of avian fatty acid synthetase complex

Injection of 0.48 or 0.72 mg of selenium/100 g body weight (as Na₂SeO₃) into 3-week-old chicks depressed hepatic activity of fatty acid synthetase compared with saline-injected controls. In in vitro experiments with fatty acid synthetase purified to homogeneity, Na₂SeO₃ was a competitive inhibitor (K_i = ca. 70 μM). Dithiothreitol (DTT) at low concentrations increased the inhibition of the enzyme by Na₂SeO₃. At higher DTT concentrations the potentiating effect of DTT on selenium inhibition of the enzyme disappeared. At still higher DTT concentrations, selenium inhibition of fatty acid synthetase was partically relieved. If DTT and Na₂SeO₃ (2 : 1 molar ratio, respectively) in inhibitory concentrations, were reacted together prior to addition to enzyme and substrate, no inhibition was observed. Potentiation of selenium inhibition of fatty acid synthetase was observed with 2-mercaptoethanol but not with ascorbate. Several organic seleno-compounds were not inhibitory. The data suggest that selenium inhibits fatty acid synthetase by reversible bonding to the sulfhydryl (SH) groups (possibly at the active sites for acetyl-CoA and/or malonyl-CoA binding) of the enzyme. Selenotrisulfide formation involving selenium and the SH groups from the enzyme and thiol compounds is advanced as a possible explanation for the interaction among Se, DTT and enzyme observed in these experiments.     

Enzymatic ethylene formation from 1-aminocyclopropane-1-carboxylic acid by manganese,a protein fraction and a cofactor of etiolated pea shoots

The cofactor of enzymatic, 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation was concentrated on cation exchange columns. When chelators of cations were added to the homogenates, cofactor activity was lost. Cofactor fractions were partly resistant to oxidation at 600° C. Mn²⁺ substituted for the cofactor in ethylene formation from 1-aminocyclopropane-1-carboxylic acid by a protein fraction isolated from etiolated pea shoots. In addition, Mn²⁺ enhanced the stimulatory effect of the concentrated cofactor. The elution volume for the cofactor on a Sephadex G-25 column was lower than that of MnCl₂. In paper electrophoresis the cofactor migrated to the cathode at pH 10.8 and 2.2. The R_F of cofactor on cellulose plates developed in butanol: acetic acid: H₂O was 0.4. After cellulose chromatography, cofactor activity had to be reconstituted by the addition of MnCl₂. Chelators, anti-oxidants, and catalase were inhibitors of Mn²⁺-cofactor-dependent ethylene formation. The protein necessary for 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation in vitro was seperated from 95–98% of the total protein in homogenates by DE-52 cellulose chromatography and (NH₄)₂SO₄-fractionation.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EDTA ethylenediaminetetraacetic acid - DDTC diethyldithiocarbamate     

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E₂ and F_2α which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of ³H-arachidonic acid labelled phospholipids, releasing free ³H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases ³H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-³H]inositol labelled phospholipids. Phospholipase A₂ (PLA₂), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, ³H-arachidonic acid labelled phospholipid breakdown, and ³H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitve) whereas the formation of inositol phosphates from myo-[2-³H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA₂ and PLD, but not by activation of phosphatidylinositol-specific PLC. © 1993 Wiley-Liss, Inc.     

Enzymatic aspects of the reduction of microsomal glycerolipid biosynthesis after perfusion of the liver with dibutyryl adenosine-3',5'-monophosphate.

Livers from fed male rats were perfused in a nonrecycling system for 60 min with a medium containing 100 mg/dl glucose, 3 g/dl bovine serum albumin, and ~0.5 mm oleic acid, with or without 20 μm dibutyryl cyclic adenosine-3′,5′-monophosphate (Bt₂cAMP). At the termination of the experiment, microsomes were isolated from these livers. In agreement with data reported previously, Bt₂cAMP decreased output of triacylglycerol, but stimulated ketogenesis and output of glucose; uptake of free fatty acid was unaffected by the nucleotide. Perfusion with Bt₂AMP decreased the biosynthesis of triacylglycerol, diacylglycerol, and phosphatidate from sn-[U-¹⁴C]glycerol-3-phosphate by microsomes isolated from these livers. Perfusion with Bt₂cAMP also decreased incorporation of sn-glycerol-3-phosphate into phosphatidate by microsomes isolated from the livers, when the microsomes were incubated with NaF to inhibit phosphatidate phosphohydrolase, and when fatty acid, coenzyme A and ATP were replaced by the acyl coenzyme A derivative; the formation of phosphatidate under these conditions was used as an estimate of the activity of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). However, the activities of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20), measured with microsomal bound substrate, were increased by Bt₂cAMP. These data have been interpreted to mean that Bt₂cAMP inhibits hepatic microsomal synthesis of triacylglycerol at a step prior to the formation of phosphatidate, presumably at the glycerophosphate acyltransferase (EC 2.3.1.15) step(s).     

Effects of all CIS-5, 8,11,14,17 eicosapentaenoic acid and PGH3 on platelet aggregation

In human platelet-rich plasma (PRP) eicosapentaenoic acid (EPA) inhibited platelet aggregation induced by a stable analogue of PGH₂ (U46619), arachidonic acid, collagen or ADP. EPA was more potent than oleic, linoleic, α-linolenic or γ-linolenic acids. In aspirin-treated platelets, aggregation induced by U46619 was inhibited to a similar extent by arachidonic acid or by EPA over a range of concentrations of 0.05–0.3 mM. EPA incubated with PRP did not induce the generation of a thromboxane (TXA)-like activity; indeed it prevented the formation of TXA₂ induced by arachidonic acid or by collagen. The anti-aggregatory activity of EPA was not influenced by inhibitors of cyclo-oxygenase and lipoxygenase. The anti-aggregatory action of EPA may be caused by a rapid occupancy by EPA of TXA₂/PGH₂ “receptors” on platelet membrane as well as by a slower displacement of arachidonic acid from platelet phospholipids by chemically unchanged molecules of EPA.Not all samples of PRP were irreversibly aggregated by PGH₂, but in those that were, PGH₃ also induced an immediate dose-dependent but reversible aggregation. After a 4 min incubation of non-aggregating doses of PGH₂ or PGH₃ (100–300 nM) with PRP a stable anti-aggregatory compound was detected. The inhibitory activity produced from PGH₃ was apparently more potent (ca 10 times) than that obtained from PGH₂. The anti-aggregating compounds were identified by TLC and GLC-MS as PGD₂ and PGD₃. The apparent difference of potency between PGD₂ and PGD₃ was attributed to the concurrent production of PGE₂ and PGE₃. PGE₂ prevented the inhibitory effect of PGD₂ whereas PGE₃ did not affect the activity of PGD₃.It is concluded that one of the reasons for the low incidence of myocardial infarction in Eskimos could be that the pro-aggregatory arachidonic acid is replaced in their phospholipids by the anti-aggregatory EPA.     

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: Evaluation of the roles of phospholipases C and D

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In³²P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar¹]angiotensin II was shown to rapidly induce formation of³²P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as³²P- or³H-phosphatidylethanol was produced when cells labeled with [³²P]H₃PO₄ or 1-O-[1,2-³H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar¹]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca²⁺, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar¹]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar¹]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar¹]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar¹]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such asde novo synthesis, may contribute to [Sar¹]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar¹]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity. These results suggest that phosphatidic acid may function as an intracellular second messenger of angiotensin II in cardiac fibroblasts and may contribute to the mitogenic action of this hormone on these cells. (Mol Cell Biochem141: 135–143, 1994)Abbreviations DAG diacylglycerol - DMSO dimethyl sulfoxide - lysoPC 1-O-hexadecyl-2-lyso-sn-glycero-3-phosphocholine - NRCF newborn rat cardiac fibroblasts - PA phosphatidic acid - PAPase phosphatidic acid phosphohydrolase - PC phosphatidylcholine - PEt phosphatidylethanol - PI phosphatidylinositol - PL (labeled) phospholipids - PLC phospholipase C - PLD phospholipase D Drs. G. W. Booz and M. M. Taher contributed equally to the work described here.     

Volatile C6-Aldehyde Formation via Hydroperoxides from C18-Unsaturated Fatty Acids in Etiolated Alfalfa and Cucumber Seedlings

1. Etiolated seedlings of alfalfa and cucumber evolved n-hexanal from linoleic acid and cis-3-hexenal and trans-2-hexenal from linolenic acid when they were homogenized.

2. The activities for n-hexanal formation from linoleic acid, lipoxygenase and hydro-peroxide lyase were maximum in dry seeds and 1~2 day-old etiolated seedlings of alfalfa, and in 6~7 day-old etiolated seedlings of cucumber.

3. n-Hexanal was produced from linoleic acid and 13-hydroperoxylinoleic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. cis-3-Hexenal and trans-2-hexenal were produced from linolenic acid and 13-hydroperoxylinolenic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. But these extracts, particulariy cucumber one, showed a high isomerizing activity from cis-3-hexenal to trans-2-hexenal.

4. When the C₈-aldehydes were produced from linoleic acid and linolenic acid by the crude extracts, formation of hydroperoxides of these C₁₈-fatty acids was observed.

5. When 9-hydroperoxylinoleic acid was used as a substrate, trans-2-nonenal was produced by the cucumber homogenate but not by the alfalfa homogenate.

6. As the enzymes concerned with C₆-aldehyde formation, lipoxygenase was partially purified from alfalfa and cucumber seedlings and hydroperoxide lyase, from cucumber seedlings. Lipoxygenase was found in a soluble fraction, but hydroperoxide lyase was in a membrane bound form. Alfalfa lipoxygenase catalyzed formation of 9- and 13-hydroperoxylinoleic acid (35: 65) from linoleic acid and cucumber one, mainly 13-hydroperoxylinoleic acid formation. Alfalfa hydroperoxide lyase catalyzed n-hexanal formation from 13-hydroperoxylinoleic acid, but cucumber one catalyzed formation of n-hexanal and trans-2-nonenal from 13- and 9-hydroperoxylinoleic acid, respectively.

7. From the above results, the biosynthetic pathway for C₆-aldehyde formation in etiolated alfalfa and cucumber seedlings is established that C₆-aldehydes (n-hexanal, cis-3-hexenal and trans-2-hexenal) are produced from linoleic acid and linolenic acid via their 13-hydroperoxides by lipoxygenase and hydroperoxide lyase.     

Characterization of Ribonuclease Activity of Three S-Allele-Associated Proteins of Petunia inflata

Three S-allele-associated proteins (S-proteins) of Petunia inflata, a species with gametophytic self-incompatibility, were previously found to share sequence similarity with two fungal ribonucleases, RNase T₂ and RNase Rh. In this study, the S-proteins from P. inflata plants of S₁S₂ and S₂S₃ genotypes were purified to homogeneity by gel filtration and cation-exchange chromatography, and their enzymatic properties were characterized. The three S-proteins (S₁, S₂, and S₃), with pairwise sequence identity ranging from 73.1 to 80.5%, were similar in most of the enzymatic properties characterized. The ribonuclease activity had a pH optimum of 7.0 and a temperature optimum of 50°C. Diethylpyrocarbonate at 1 millimolar almost completely abolished the ribonuclease activity; cupric sulfate and zinc sulfate at 1 millimolar reduced the ribonuclease activity of the three S-proteins by 50 to 75%. EDTA and RNasin had no inhibitory effect. All three S-proteins hydrolyzed polycytidylic acid preferentially, but varied in their nucleolytic activity toward polyadenylic acid and polyuridylic acid.     

A Brominating and Hydroxylating Peroxidase from the Red Alga Cystoclonium purpureum

A particulate enzyme displaying peroxidase activity has been extracted from the red alga Cystoclonium purpureum (Huds.) Batt. The enzyme preparation was shown to contain ferri-protoporphyrin IX through its formation of formic acid hemochromogen with absorption maxima at 399, 402, 555 and 600 nm. The preparation catalyses the formation of 3-bromo-p-hydroxybenzyl alcohol from p-hydroxybenzyl alcohol in the presence of H₂O₂ and NaBr at pH = 5.4. The formation of 3-bromo-p-hydroxybenzyl alcohol was measured by gas chromato-graphy-mass spectrometry. At pH = 6.7, 4,5-di-hydroxybenzyl alcohol was formed. Addition of homogentisic acid stimulated the formation of dihydroxybenzyl alcohol and suppressed the brominating reaction. Iodide inhibits the enzyme. The results are consistent with a two-site model of the enzyme.     

Development of novel lithocholic acid derivatives as vitamin D receptor agonists

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D₃, 1α,25-dihydroxyvitamin D₃ (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells. Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6 bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated derivatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis of the complex of VDR ligand-binding domain (LBD) with 6.     

Characterization of prostaglandin synthesis in the housefly,Musca domestica (L.)

The in vitro formation of prostaglandins (PG) was examined in the housefly Musca domestica. PG synthetase activity was detected in homogenates of whole insects and in head and thorax, abdomen, ovary and male reproductive tissues. Studies to determine the sub-cellular localization of PG synthetase indicated that the microsomal fraction contained the highest activity. Products obtained from radiolabeled arachidonic acid (20:4) and 8,11,14-eicosatrienoic acid (20:3, n-6) were PGE₂ and PGE₁, respectively, with lower amounts of the PGF series also present. In microsomal preparations from whole insects and reproductive tissues from both males and females, 20:3(n-6) was 2–2.5 times more efficiently converted to PG than was 20:4. Non-steroidal anti-inflammatory drugs (NSAID) fed to houseflies did not inhibit PG production from 20:4, whereas when they were included in microsomal preparations at high levels, they inhibited PG synthesis.     

Synthesis of 3-O-methylgallic acid a powerful antioxidant by electrochemical conversion of syringic acid

Background

A kinetic study of the electrochemical oxidation of syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) by cyclic voltammetry at treated gold disk was combined with results of electrolyses at Ta/PbO₂ anode in order to convert it into potentially high-added-value product.

Methods

The electrochemical oxidation of syringic acid was carried out in order to convert this compound to 3-O-methylgallic acid. This latter was identified by mass spectrophotometry using LC-MS/MS apparatus. The 3-O-methylgallic acid synthesis was controlled by cyclic volammetry, Ortho-diphenolicdeterminations and DPPH radical-scavenging activity.

Results

The proposed mechanism is based on the hypothesis of a bielectronic discharge of syringic acid molecule under free and adsorbed form involving two intermediate cation mesomers. Hydrolysis of the more stable of this last one leads to the formation of the 3,4-dihydroxy-5-methoxybenzoic acid (3-O-methylgallic acid) as a major product. The latter aromatic compound was synthesized by anodic oxidation of syringic acid at PbO2 electrode. The cyclic voltammogram of the electrolysis bath of syringic acid shows that the anodic peak potential of 3-O-methylgallic acid was lower (E_pa = 128 mV) than that of SA (E_pa = 320 mV). And the strongest antiradical activity was detected when the 3-O-methylgallic acid concentration was higher".

Conclusion

The electrochemical oxidation using PbO₂ anode is a rapid, simple and efficient method tool for a conversion of SA into 3-O-methylgallic acid, a potent antioxidant derivative

General Significance

The electrochemical process consists in a simple transformation of the syringic acid into 3-O-methylgallic acid having a better antioxidant capacity. This result has been justified by cyclic voltametry which shows that anodic peak of 3-O-methylgallic acid is reversible. Furthermore, its potential is lower than that of the irreversible anodic peak of syringic acid to 3-O-methylgallic acid.     

Stimulation of the oxidative decarboxylation of indole-3-acetic acid in citrus tissues by ethylene

Ethylene has been shown to stimulate the degradation of indole-3-acetic acid (IAA) in citrus leaf tissues via the oxidative decarboxylation pathway, resulting in the accumulation of indole-3-carboxylic acid (ICA). Preliminary data indicated that ethylene stimulates only the first step of this pathway, i.e. the decarboxylation of IAA which leads to the formation of indole-3-methanol. The effect of ethylene seems to be a specific one since 2,5-norbornadiene, an ethylene action inhibitor, significantly inhibited the stimulation of IAA decarboxylation by ethylene. It has long been suggested that peroxidase or a specific form of the peroxidase complex (`IAA oxidase') catalyse this step. However, we did not observe a clear effect of ethylene on the peroxidase system. An alternative possibility, that the stimulatory effect of ethylene on IAA catabolism results from increased formation of hydrogen peroxide (H₂O₂), a co-factor for peroxidase activity, was verified by direct measurements of H₂O₂ in the tissues or by assaying the activity of gluthathione reductase, which has been shown to be induced by oxygen species. This possibility is further supported by the observations showing that IAA decarboxylation in control tissues was enhanced to the level detected in ethylene-treated tissues by application of H₂O₂.     

IBA-induced changes in antioxidant enzymes during adventitious rooting in mung bean seedlings: The role of H2O2

The changes in antioxidant enzyme activity during the induction of adventitious roots in mung bean seedlings treated with Indole-3-butyric acid (IBA), hydrogen peroxide (H₂O₂), ascorbic acid (ASA) and diphenylene iodonium (DPI) were investigated. As compared with the controls, treatments of seedlings with 10 μM IBA significantly decreased POD activity by 55% and 49.6% at 3 h and 12 h of incubation, respectively, and significantly increased by 49.8% at 36 h of incubation; treatments of seedlings with 10 mM H₂O₂ significantly decreased POD activity by 42%, 60%, 39% and 38% at 3 h, 12 h, 24 h and 48 h of incubation, respectively, the changes in POD activity were coincident with those in IBA-treated seedlings during the 0–12 h incubation period; treatments of seedlings with 2 mM ASA significantly decreased APX activities by 27% only at 3 h of incubation, the varying trend of POD activity was similar to incubation with water; 10 μM DPI treatments significantly decreased POD activity by 42%, 40%, 54% and 28% at 3 h, 6 h, 12 h and 48 h of treatment, respectively. CAT activities remained at relatively stable levels and no major changes occurred from 0 h to 48 h during the incubation phase of adventitious rooting. The results may imply that CAT, an H₂O₂-metabolizing enzyme, is inactivated by H₂O₂ during the formation of adventitious roots. As compared with the controls, IBA treatments significantly decreased APX activities by 48%, 53% and 66% at 3 h, 9 h and 12 h of treatment, respectively; H₂O₂ treatments significantly decreased APX activities by 59%, 51% and 57% at 3 h, 12 h and 36 h of incubation, respectively; ASA treatments significantly decreased APX activities by 37% only at 3 h of incubation; DPI treatments significantly decreased APX activities by 54%, 53% and 63% at 3 h, 6 h and 12 h of incubation, respectively, and significantly increased APX activity by 106% at 24 h. These results indicated that the influence of IBA, H₂O₂, ASA and DPI on the changes in APX activity were the same as on the changes in POD activity. Furthermore, similar trends in the changes of APX activity and POD activity were observed during the induction and initiation rooting phase. This finding implies that APX and POD serve the same functions, possibly related to the level of H₂O₂, during the formation of adventitious roots. The early decrease of POD and APX activities in the initiation phase of IBA- and H₂O₂-treated seedlings may be one mechanism underlying the IBA- and H₂O₂-mediated facilitation of adventitious rooting.     

Mechanism of action of suprofen, a new peripheral analgesic, as demonstrated by its effects on several nociceptive mediators

Suprofen is a new potent, orally effective non-narcotic analgesic agent having a potent inhibitory action on prostaglandin (PG) biosynthesis. Recent experiments have shown that suprofen inhibits uterine hyperactivity induced by the physiological substances, arachidonic acid, bradykinin (BK) and PGF_2α. The present study explores the possibility that the analgesic activity of suprofen may involve multiple mechanisms of interaction with PGs, inhibiting synthesis at low doses and with higher doses possibly directly interacting with PGs and other physiological mediators of nociception at a common site. Experiments in mice have shown that suprofen antagonizes abdominal stretching induced by the physiological precursor of PG release, arachidonic acid (ED₅₀ = 0.07 mg/kg, p.p.), and by the nociceptive agents acetylcholine (ACh) (ED₅₀ = 1.7 mg/kg, p.o), BK (ED₅₀ = 65 mg/kg, p.o.) acetic acid (HAC) (H⁺ ion; ED₅₀ = mg/kg, p.o), and PGE₂, itself (ED₅₀ = 20.2 mg/kg, i.p.). In rabbits, i.a. administered suprofen (ED₅₀ = 0.98 mg/kg) blocked the reflex discharge of spinal sensory neurons evoked by BK (2 to 8 μg, i.a). The analgesic activity of suprofen may involve multiple mechanism of interaction with PGs and other mediators, including BK; suprofen blocks the nociceptive actions of PGs by inhibiting their formation, via the cyclooxygenase pathway, and possibly at PG sites of action, probably at peripheral nerve endings.     

Anti-Leishmanial activity of homo- and heteroleptic bismuth(III) carboxylates

Bismuth(III) complexes of NSAIDs (Non-Steroidal Anti Inflammatory Drugs) and substituted benzoic acids (o-methoxybenzoic acid, m-methoxybenzoic acid, o-nitrobenzoic acid, 3,5-diacetamidobenzoic acid, and 5-[(R/S)-2,3-dihydroxypropyl carbamoyl]-2-pyridine carboxylic acid) have been synthesised and fully characterised. Two new bis-carboxylato bismuth complexes have been characterised by single crystal X-ray diffraction, namely [PhBi(o-MeOC₆H₄CO₂)₂(bipy)]·0.5EtOH (bipy = 2,2'-bipyridine) and [PhBi(C₉H₁₁N₂O₃CO₂)₂(H₂O)]·6H₂O. All compounds were tested against the parasite Leishmania major promastigotes for their anti-Leishmanial activity and were further assessed for their toxicity to mammalian cells. The NSAID free acids and their bismuth derivatives show negligible anti-Leishmanial activity at concentrations 1.95 to 250 μg/mL against the promastigotes of L. major whereas in the case of mammalian cells only bismuth complexes of naproxen and mefenamic acid have significant effect at concentration ≥ 250 μg/mL. The bismuth(III) complexes of substituted benzoic acids show significant anti-Leishmanial activity against the promastigotes of L. major V121 at very low concentrations while their respective free carboxylic acids show no effective activity. However, the bismuth compounds inhibit the growth of the mammalian cells at all concentrations studied (1.95 to 500 μg/mL) following 48 h incubation. The comparatively low toxicity of BiCl₃ and Bi(NO₃)₃, suggests that overall toxicity of bismuth complexes towards the parasite is both ligand and metal dependent.     

Biodegradation of commercial linear alkyl benzenes byNocardia amarae

Laboratory degradation studies of two indigeneously produced linear alkyl benzenes byNocardia amarae MB-11 isolated from soil showed an overall degradation of linear alkyl benzenes isomers to the extent of 57–70%. Degradation of 2-phenyl isomers of linear alkyl benzenes was complete and faster than that of other phenyl position (C3–C7) isomers which were degraded to the extent of 40–72% only. Length of alkyl side chains (C₁₀–C₁₄) had little or no impact on the degradation pattern. Major metabolities detected were 2-, 3-and 4-phenyl butyric acids, phenyl acetic acid and cis, cis-muconic acid. Minor metabolites weretrans-cinnamic acid, 4-phenyl 3-butenoic acid and 3-phenyl pentanoic acid along with two unidentified hydroxy acids. On the basis of the formation pattern of these metabolities, three catabolic pathways of linear alkyl benzenes isomers inNocardia amarae MB-11 were postulated. All the phenyl position (C2–C7) isomers of C₁₀, C₁₂, and C₁₄ linear alkyl benzenes along with 3-phenyl and 5-phenyl isomers of C₁₁ and C₁₃ linear alkyl benzenes were degraded viacis,cis-muconic acid pathway. Other phenyl position isomers of C₁₁ and C₁₃ linear alkyl benzenes with phenyl substitution at even number carbon atoms were principally degraded via phenyl acetic acid pathway whiletrans-cinnamic acid formation provided a minor pathway     

Ethylene inhibitors enhance in vitro root formation on faba bean shoots regenerated on medium containing thidiazuron

The possible involvement of ethylene in in vitrorooting of faba bean (Vicia faba L.) shootsregenerated on medium containing thidiazuron wasinvestigated. The effects of the ethylene precursor1-aminocyclopropane-1-carboxylic acid (ACC) and threeethylene inhibitors, silver nitrate (AgNO₃),acetyl salicylic acid (ASA) and cobalt chloride(CoCl₂) on root formation were tested in vitrousing TDZ-induced shoots of faba bean accession 760.ACC inhibited root formation. In contrast, ethyleneinhibitors promoted root formation, AgNO₃ at theappropriate concentrations enhanced root emergence andincreased root number per shoot, root growth rate, androot length. Both CoCl₂ and ASA at theappropriate concentrations increased rootingefficiency. These promotive effects may result from areduction in ethylene concentration or inhibition ofethylene action. The results offer a new approach toimprove the rooting efficiency of TDZ-induced shootsof faba bean and possibly of other plant species.