Improved paclitaxel accumulation in cell suspension cultures of Taxus chinensis by brassinolide

When brassinolide was added at 17 ng l^–1 to Taxus chinensis cell suspension cultures, the paclitaxel content was increased by over 100% to 580 g g^–1 dry weight. Brassinolide may therefore be a novel regulator of paclitaxel biosynthesis.     

Substituent-dependent, positive and negative modulation of Bombyx mori adenylate cyclase by synthetic octopamine/tyramine analogues

Octopamine (OCT)/tyramine (TYR) analogues, mainly including p- and beta-substituted phenylethylamines, were prepared as probes for the ligand-binding site(s) of adenylate cyclase-coupled OCT or TYR receptors, and were examined for their effects on cAMP production in the head membranes of Bombyx mori larvae. Small structural changes in OCT and TYR proved to lead to three types of OCT/TYR analogues: (1) compounds that increase the cAMP level by themselves and also depress OCT-stimulated cAMP production, (2) compounds that do not stimulate cAMP production by themselves but inhibit OCT-stimulated cAMP production, and (3) compounds that are not active in either the stimulation of cAMP production or the inhibition of OCT-stimulated cAMP production. Tyramine, which belongs to the second group, also inhibited the basal level of cAMP production at high concentrations. The data indicate that two biogenic amine systems that positively and negatively regulate the level of the second messenger cAMP are present in the head part of B. mori larvae. This finding points to the necessity of separately evaluating the positive and negative regulatory effects in order to quantitatively understand the structure-activity relationships of OCT receptor ligands. Arch.     

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.     

Liquid chromatography-tandem mass spectrometry identification of metabolites of three phenylcarboxyl derivatives of the 5-HT(1A) antagonist,N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl) trans-4-fluorocyclohexanecarboxamide (FCWAY), produced by human and rat hepatocytes

We have previously described fluorine-18 radiolabeled FCWAY [N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl) trans-4-fluorocyclohexanecarboxamide] as a high affinity ligand for imaging the 5-HT(1A) receptor in vivo. In a search for radiopharmaceuticals with unique imaging applications using positron emission tomography (PET), we have also developed three new phenylcarboxamide analogues of FCWAY. Two of these analogues were generated by replacing the fluorocyclohexane carboxylic acid with fluorobenzoic acid (FBWAY) or with 3-methyl-4-fluorobenzoic acid (MeFBWAY). The final analogue was generated by replacing the pyridyl group with a pyrimidyl group and the fluorocyclohexane carboxylate with fluorobenzoic acid (FPWAY). We evaluated the metabolic profile of these compounds using either human or rat hepatocytes to produce metabolites and LC-MS/MS to identify these metabolites. We also compared the metabolic rate of these compounds in human or rat hepatocytes. These in vitro metabolism studies indicate that hydrolysis of the amide linkage was the major metabolic pathway for FPWAY and FBWAY in human hepatocytes, whereas aromatic oxidation is the major metabolic pathway for MeFBWAY. The comparative metabolic rate in human hepatocytes was FPWAY>FBWAY>MeFBWAY. In rat hepatocytes, aromatic oxidation was the major metabolic pathway for all three analogs and the rate of this process was similar for all of the analogues. These in vitro metabolic studies demonstrated species differences prior to the acquisition and interpretation of in vivo results.     

Synthesis and Characterization of N,N-Dichlorinated Amino Acids: Taurine,Homotaurine, GABA and L-Leucine

Epilepsy, trauma and other circumstances leading to hyperexcitable conditions in the CNS tend neurochemically to be associated with excessive stimulated release of glutamic acid and/or a failure of GABA modulated inhibition. Somewhat to a lesser extent, taurine and its homologue homotaurine, have also been shown to antagonize the excitatory actions of glutamic acid. Here we report the successful synthesis and isolation in pure form of N,N-dichlorinated GABA, taurine, homotaurine and leucine. These compounds are much more lipophilic than their parent compounds and may therefore more readily penetrate the blood-brain barrier systems into the neural tissue, where they can be easily dechlorinated. Very preliminary biological testing shows that this may indeed occur. The synthesis and purification methodology will likely also be applicable to a number of other amino acids as well as certain peptides or selected proteins.     

Some molecular and enzymatic properties of a homogeneous preparation of thiaminase I purified from carp liver

A homogeneous preparation of thiaminase I (thiamine:base 2-methyl-4-aminopyrimidine-5-methenyl transferase, EC 2.5.1.2) was obtained from carp liver, for the first time from a nonbacterial source. Its molecular mass was 55 kDa by gel filtration and by SDS—PAGE regardless the presence of the reducing agent, indicating that the native enzyme consists of a single polypeptide chain. The determined sequence of 20 residues at the N-terminal of carp thiaminase I seemed to be unique. The enzyme was tested for ability to decompose a number of thiamine analogues. Even very extensive modifications of the thiazolium fragment were well tolerated, but around the pyrimidine fragment the active center seemed to exert steric restrictions against 1 (N)- and 2 (C)- atoms, while the 4-amino group and untouched 6-carbon atom were absolutely essential for the enzyme action. Numerous nucleophiles could be used by the enzyme as cosubstrates, aniline, pyridine, and 2-mercaptoethanol being the best among compounds tested. Protein chemical modification experiments indicated that histidine residues, carboxyl groups, and sulfhydryl groups may play specific roles in the thiaminase I-catalyzed reaction. Like in the bacterial enzyme, a sulfhydryl group may be a catalytically critical active-site nucleophile. The histidine residues and carboxyl groups may be essential for thiamine binding to the active site.     

Survival, fecundity and fertility of Bactrocera oleae, as affected by amino acid analogues

The effect of twenty five amino acid analogues at various concentrations upon the adult olive fruit fly Bactrocera oleae Gmelin (Diptera: Tephritidae), was tested. Insect survival was significantly shortened by the following amino acid analogues: (in parentheses are indicated the antagonized amino acids) D-cycloserine (alanine), L-glutamic acid--hydrazide (glutamine), DL-allyl-glycine (cysteine), L-canavanine (arginine), L-methionine-DL-sulfoximine (methionine) and 3,4-dehydro-DL-proline (proline). Fecundity was significantly reduced by the same analogues plus aminoethanesulfonic acid (glycine), taurine (alanine), L-norvaline (leucine), a-methyl-DL-serine (serine), DL-hydroxyglutamic acid (glutamic acid), (S)-2-(aminoethyl)-L-cysteine (lysine), a-methyl-DL-methionine (methionine) and a-methyl-DL-histidine (histidine). All the above amino acid analogues also depressed egg-hatching with the exception of taurine, DL-hydroxyglutamic acid, DL-allyl glycine, a-methyl-DL-methionine and a-methyl-DL-histidine. Finally, y-glutamyl-p-nitroanilide (glutamic acid), crotyl-glycine (methionine), DL-7-azatryptophan and 5-methyl-DL-tryptophan (tryptophan), DL-1,2,4 triazole-3-alanine (histidine) and DL-pipecolic acid (proline) did not affect any of the parameters tested.     

In vitro stem elongation of sweet pepper in media containing 24-epi-brassinolide

In vitro regeneration of sweet pepper (Capsicum annuum L. cvs Jupiter and Pimiento Perfection) has been performed via direct organogenesis. The resulting shoot-buds were placed on media containing 24-epi-brassinolide (EBR) 0.1 μM, a plant steroid lactone, in the presence or absence of zeatin 9.1 μM plus GA3 5.2 μM for further stem elongation. Different responses to these treatments were recorded depending upon the protocols used and the genotypes tested. It appears that EBR does not always act directly on stem elongation but may be an elicitor and/or an enhancer of elongation in concert with endogenous and other exogenously added growth regulators. Elongated shoots were easily rooted with alpha-naphtalenacetic acid 0.5 μM (0.1 mgl-1) and transfered to soil, and following acclimation were taken to maturity in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.