Adrenalin and hydroxytyramine in the parotid gland venom of the toad,Bufo marinus

Preparations from the parotid gland venom of the toad Bufo marinus have been analyzed by paper chromatography for the presence of adrenalin precursors. In addition to adrenalin itself, hydroxytyramine was identified and a second compound which appeared to be an adrenalin ester—perhaps lactyl-adrenalin. Two other compounds which appeared on the chromatogram could not be identified. Neither noradrenalin nor another possible precursor, epinine, appeared to be present.     

Bioguided fractionation and isolation of natural inhibitors of advanced glycation end-products (AGEs) from Calophyllum flavoramulum

Advanced glycation end-products (AGEs) are associated with many pathogenic disorders such as Alzheimer’s disease, pathogenesis of diabetes, atherosclerosis or endothelial dysfunction leading to cardiovascular events. Clusiaceae and Calophyllaceae families are rich in compounds like polyphenols which are able to inhibit their formation and are therefore of great interest. Calophyllum flavoramulum Hend. & Wyatt-Sm., a native Malaysian plant, was selected after an anti-AGEs screening conducted on DCM and MeOH extracts from plants belonging to these aforementioned families. In a first study, bioguided fractionation of the MeOH leaf extract of C. flavoramulum afforded amentoflavone, 3-methoxy-2-hydroxyxanthone, 3,4-dihydroxy-tetrahydrofuran-3-carboxylic acid, quercitrin, 3,4-dihydroxybenzoic acid, canophyllol and apetalactone. Amentoflavone and 3-methoxy-2-hydroxyxanthone were found to be very potent (IC₅₀ = 0.05 and 0.06 mM respectively), while anti-AGEs activities of quercitrin and 3,4-dihydroxybenzoic acid appeared as moderately strong (IC₅₀ = 0.5 mM). In a second study, a systematic phytochemical study of the cyclohexane, DCM and EtOAc extracts obtained from the same plant was conducted to isolate the following products: flavoramulone, 6-deoxyjacareubin, rheediachromenoxanthone, 2,3-dihydroamentoflavone and benzoic acid. 3,4-Dihydroxy-tetrahydrofuran-3-carboxylic acid and flavoramulone were isolated for the first time and their structures were identified by means of IR, MS and NMR spectrometries.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.     

Degradation of Phenanthrene and Anthracene by Cell Suspensions of Mycobacterium sp. Strain PYR-1

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2′-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.     

Lunularic acid and related compounds in liverworts,algae and Hydrangea

Lunularic acid and lunularin were detected in 76 species of hepatics, but not in any of the Anthrocerotales or Algae examined. Lunularic acid, lunularin, 3,4′-dihydroxystilbene and a glycoside of lunularic acid were also identified in extracts of Hydrangea macrophylla roots, together with hydrangenol, hydrangeic acid and their glucosides.     

Microbial metabolism of phenolic amines: degradation of dl-synephrine by an unidentified arthrobacter.

Microorganisms capable of degrading dl-synephrine were isolated from soil of Citrus gardens by enrichment culture, with dl-synephrine as the sole source of carbon and nitrogen. An organism which appears to be an arthrobacter, but which cannot be identified with any of the presently recognized species was predominant in these isolates. It was found to metabolize synephrine by a pathway involving p-hydroxyphenylacetaldehyde, p-hydroxyphenylacetic acid, and 3,4-dihydroxyphenylacetic acid as intermediates. Some of the enzymes of this pathway were demonstrated in cell-free extracts. An aromatic oxygenase, which could also be readily obtained in a cell-free system, was found to degrade 3,4-dihydroxyphenylacetic acid by meta cleavage.     

Alternative routes of aromatic catabolism in Pseudomonas acidovorans and Pseudomonas putida: gallic acid as a substrate and inhibitor of dioxygenases.

When 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) was added to Pseudomonase acidovorans growing at the expense of succinate, enzymes required for degrading homoprotocatechuate to pyruvate and succinate semialdehyde were strongly induced. These enzymes were effectively absent from cell extracts of the organism grown with 4-hydroxyphenylacetic acid, and this substrate was metabolized by the catabolic enzymes of the homogentisate pathway. Two separate ring-fission dioxygenases for 3,4,5-trihydroxybenzoic acid (gallic acid) were present in cell extracts of Pseudomonas putida when grown with syringic acid, and gallate was degraded by reactions associated with meta fission. One of the two gallate dioxygenases also attacked 3-O-methylgallic acid; the other, which did not, was induced when cells were exposed to gallate. This organism possessed ortho fission enzymes, including protocatechuate 3,4-dioxygenase (EC 1.13.11.3) and cis,cis-carboxymuconate-lactonizing enzyme (EC 5.5.1.2), after induction with 3,4-dihydroxybenzoic acid (protocatechuic acid). Gallate was a substrate for protocatechuate 3,4-dioxygenase, with a Vmax about 3% of that of protocatechuate and with an apparent Km slightly lower. Gallate was a powerful competitive inhibitor of protocatechuate oxidation.     

A Study of Mimosine Toxicity in Plants

Mimosine and 3,4-dihydroxypyridine are inhibitory to the growthof mung bean seedlings. Mimosine inhibition could be reversedby tyrosine or nicotinic acid, but it was reversed by pyridoxalphosphate or ferrous ions. Inhibition due to dihydroxypyridinealso could be reversed by ferrous ions, but not by pyridoxalphosphate. The possible mechanisms of growth inhibition arediscussed. An enzyme is present in extracts of Leucaena seedlings thatdegrades mimosine stoichiometrically to 3,4-dihydroxypyridine,pyruvate, and ammonia. An enzymic breakdown of mimosine is alsocatalysed by extracts of mung bean seedlings. The occurencein Leucaena of dichrostachinic acid and a C-S-Iyase which degradesit to a thiol derivative, pyruvate, and ammonia were described;the latter enzyme was shown to be distinct from the enzyme-splittingmimosine. The substrate specificity, pH optimum, co-factor requirements,and inhibitors of this mimosine C-N-Iyase were investigated.     

Establishing a novel biosynthetic pathway for the production of 3,4-dihydroxybutyric acid from xylose in Escherichia coli

3-Hydroxy-γ-butyrolactone (3HBL) is an attractive building block owing to its broad applications in pharmaceutical industry. Currently, 3HBL is commercially produced by chemical routes using petro-derived carbohydrates, which involves hazardous materials and harsh processing conditions. Only one biosynthetic pathway has been reported for synthesis of 3HBL and its hydrolyzed form 3,4-dihydroxybutyric acid (3,4-DHBA) using glucose and glycolic acid as the substrates and coenzyme A as the activator, which involves multiple steps (>10 steps) and suffers from low productivity and yield. Here we established a novel five-step biosynthetic pathway for 3,4-DHBA generation from D-xylose based on the non-phosphorylative D-xylose metabolism, which led to efficient production of 3,4-DHBA in Escherichia coli. Pathway optimization by incorporation of efficient enzymes for each step and host strain engineering by knocking out competing pathways enabled 1.27 g/L 3,4-DHBA produced in shake flasks, which is the highest titer reported so far. The novel pathway established in engineered E. coli strain demonstrates a new route for 3,4-DHBA biosynthesis from xylose, and this engineered pathway has great potential for industrial biomanufacturing of 3,4-DHBA and 3HBL.     

O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase

Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.     

[3H]Indole-3-Acetyl-myo-Inositol Hydrolysis by Extracts of Zea mays L. Vegetative Tissue

[³H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37°C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as α-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other fraction enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.     

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.     

p-Hydroxyphenylacetic Acid Metabolism in Pseudomonas putida F6

Pseudomonas putida F6 was found to metabolize p-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde. Cell extracts of P. putida F6 catalyze the NAD(P)H-independent hydroxylation of p-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage.     

Characterization of quillaja bark extracts and evaluation of their purity using liquid chromatography–high resolution mass spectrometry

In the course of development of semi-preparative liquid chromatographic methods for the isolation of individual quillaja saponins from Quillaja saponaria (L.), some commercially available quillaja bark extracts revealed a distinctive and characteristic pattern of additional peaks in the chromatogram that could not be attributed to saponins commonly present in quillaja. To identify these peaks, analytical procedures based on HPLC coupled with high resolution MS detection were optimized which allowed the identification of the additional saponins Mi saponin A, Mi saponin B, Mi saponin C, madhucoside A and madhucoside B. These compounds are known to be the main saponins of the Indian plant Madhuca longifolia (L.). Tandem MS experiments were performed for the unambiguous assignment of the sapogenin. Madhuca saponins yielded a characteristic fragment of protobassic acid, whereas quillaja saponins showed a fragment of quillaic acid as expected. In addition, samples from madhuca seed kernels were analysed to verify the origin of the characteristic chromatographic peak pattern observed frequently in commercially available quillaja bark extracts.     

Metabolic actions of vasopressin, glucagon and adrenalin in the intact rat.

Metabolic effects of vasopressin, glucagan and adrenalin were compared, in intact rats, especially in regard to time courses of effects. Hyperglycaemia was transient in response to vasopressin, prolonged following adrenalin, and, suprisingly, was not discernible after glucagon, except in response to a very large dose. Vasopressin decreased and adrenalin increased, the plasma free fatty acid concentration; both hormones decreased the triacylglycerol level. Muscle glycogen concentrations, measured in heart, diaphragm and skeletal muscle, exhibited small changes, with complex time courses, following hormone administration. Vasopressin brought about a rapid but transient activation of heaptic glycogen phosphorylase which resembled that due to adrenalin. The activation by glucagon of phosphorylase was greater and more prolonged, despite the absence of hyperglycaemia. In response to vasopressin, there was in increase in plasma insulin. Incorporation of 14C from [14C]glucose into glycogen or fatty acids was not influenced by vasopressin. Taken together, these results may be explained by rapid metabolic action of vasopressin on hepatic glycogenolysis, whereas adrenalin has multiple prolonged actions.     

N-malonyltransferases from peanut

Three distinct N-malonyltransferases were purified from peanut seedlings, accepting either anthranilic acid, D-tryptophan, or 3,4-dichloroaniline, respectively, as a substrate. Partially purified malonyl-CoA:D-tryptophan malonyltransferase also catalyzed the formation of the corresponding malonic acid conjugate when 1-aminocyclopropane-1-carboxylic acid was employed as a substrate. These N-malonyltransferases were clearly distinguished from several O-malonyltransferase activities also present in the same seedlings. N-Malonic acid conjugates have been previously isolated from peanut either as a natural constituent or after feeding with xenobiotics. By analogy to the results reported with cultured parsley cells, multiple malonyltransferases in peanut may have a role in vacuolar transport. Crude extracts of young peanut seedlings were incapable of hydrolyzing the respective N-malonic acid conjugates. However, dialyzed extracts of older plants released malonic acid from malonyl-1-aminocyclopropane-1-carboxylic acid but not from malonyl-3,4-dichloroaniline, suggesting that some N-malonic acid conjugates may be metabolized in plants which are approaching senescence.     

The use of boric and benzeneboronic acids in the partial acetonation of monosaccharides

The preparation of mono-O-isopropylidene derivatives and mono-O-isopropylidene benzeneboronates of monosaccharides in one step is described, together with their p.m.r. and mass-spectral characteristics. In particular, the use of boric acid in the synthesis of the new acetal 1,2-O-isopropylidene-β-L-arabinopyranose (8) is described, together with improved procedures for the preparation of 2,3-O-isopropylidene-D-mannofuranose (5) and 3,4-O-isopropylidene-L-arabinopyranose (10). The use of boric acid in the partial hydrolysis of 1,2:3,4-di-O-isopropylidene-β-L-arabinopyranose to give the 1,2-acetal is reported.     

Metabolic actions of vasopressin,glucagon and adrenalin in the intact rat

Metabolic effects of vasopressin, glucagon and adrenalin were compared, in intact rats, especially in regard to time courses of effects.Hyperglycaemia was transient in response to vasopressin, prolonged following adrenalin, and, surprisingly, was not discernible after glucagon, except in response to a very large dose. Vasopressin decreased and adrenalin increased, the plasma free fatty acid concentration; both hormones decreased the triacylglycerol level. Muscle glycogen concentrations, measured in heart, diaphragm and skeletal muscle, exhibited small changes, with complex time courses, following hormone administration. Vasopressin brought about a rapid but transient activation of hepatic glycogen phosphorylase which resembled that due to adrenalin. The activation by glucagon of phosphorylase was greater and more prolonged, despite the absence of hyperglycaemia. In response to vasopressin, there was an increase in plasma insulin. Incorporation of ¹⁴C from [¹⁴C] glucose into glycogen or fatty acids was not influenced by vasopressin. Taken together, these results may be explained by rapid metabolic action of vasopressin on hepatic glycogenolysis, whereas adrenalin has multiple prolonged actions.     

Phylloflavan,a characteristic constituent of Phyllocladus species

Selective visualization of 2D TLC of extracts of Phyllocladus species with vanillin—hydrochloric acid showed a characteristic pattern of constituents attributable to catechin, epicatechin and phylloflavan, a new flavanoid compound. Optical rotation measurements showed that while catechin and epicatechin are of the normal type with the 2R configurations, the flavan moiety in phylloflavan is of the opposite 2S configuration. Spectroscopic data of the new compound and its hydrolysis products showed phylloflavan to be ent-epicatechin-3-δ-(3,4-dihydroxyphenyl)-β-hydroxypentanoate.     

The molecular mechanism by which adrenalin inhibits glycogen synthesis.

Improved methodology was used to establish that the phosphorylation of a serine located 10 residues from the N-terminus of glycogen synthase (N10) increases from 0.12 mol.mol-1 to 0.54 mol.mol-1 in vivo in response to adrenalin. The only 'N10 kinase' detected in muscle extracts was casein kinase-1 (CK1), although its activity was unaffected by injection of adrenalin in vivo or by incubation with cyclic-AMP-dependent protein kinase and MgATP in vitro. Prior phosphorylation of the serine residue N7 by phosphorylase kinase increased sixfold the rate of phosphorylation of glycogen synthase by CK1, and altered the specificity of CK1 so that it phosphorylated the serine residue N10 specifically. Stoichiometric phosphorylation of N7 decreased the activity ratio (+/- glucose 6-phosphate) of glycogen synthase from 0.80 to 0.45, and subsequent phosphorylation of N10 to 0.8 mol.mol-1 produced a further decrease to 0.17, demonstrating that N10 phosphorylation inhibits glycogen synthase. The major 'N10 phosphatase' in skeletal muscle extracts was identified as the glycogen-associated form of protein phosphatase-1 (PP1G), accounting for approximately 75% of the N10 phosphatase activity in the extracts and about 90% of the activity in isolated glycogen particles. Phosphorylation of N10, after prior phosphorylation of N7, decreased the rate of dephosphorylation of N7. These results, in conjunction with previous findings, establish that adrenalin inhibits glycogen synthase by increasing the phosphorylation of N7, N10 and three further serines located 30, 34 and 38 residues from the start of the C-terminal CNBr peptide (termed the region C30-C38). They also indicate that increased phosphorylation of N10, the region C30-C38, and perhaps N7, is initiated through the inhibition of PP1G by adrenalin, which results from phosphorylation of its glycogen-targetting subunit by cyclic-AMP-dependent protein kinase [Hubbard, M.J. & Cohen, P. (1989) Eur. J. Biochem. 186, 711-716]. The conclusion that direct phosphorylation of glycogen synthase by cyclic-AMP-dependent protein kinase makes little contribution to inhibition by adrenalin, is at variance with the teachings of the major textbooks of biochemistry.