Saponins from the roots of Zygophyllum gaetulum and their effects on electrically-stimulated guinea-pig ileum

A new ursanolide, 3beta-O-alpha-L-rhamnopyranosyl-(1->2)-O-alpha-L-arabinopyranosyl-(1->2)-beta-D-glucopyranosyl)oxy]-ursan-20beta,28-olide, zygophyloside N (1), and three known quinovic acid glycosides were isolated from the methanolic extract of the roots of Zygophyllum gaetulum. The crude extract, some fractions and 3-O-beta-D-glucopyranosyl quinovic acid 28-beta-D-glucopyranosyl ester, significantly and dose-dependently, are able to reduce the electrically-induced contractions of isolated guinea-pig ileum. The structure of compound (1) was determined, using 1D and 2D NMR techniques.     

Purinoreceptors are involved in the control of acute morphine withdrawal.

The effects exerted by P1 and P2 purinoceptor agonists and antagonists on the acute opiate withdrawal induced by morphine were investigated in vitro. Following a 4 min in vitro exposure to morphine, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The P1 purinoceptor agonist, adenosine, was able dose-dependently to reduce morphine withdrawal whereas alpha,beta-methylene ATP (APCPP), a P2 purinoceptor agonist, increased morphine withdrawal. Caffeine, a P1 purinoceptor antagonist, was able significantly and in a concentration dependent manner to increase morphine withdrawal whereas quinidine, a P2 receptor antagonist, reduced it. The results of our experiments indicate that both P1 and P2 purinoceptor agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the purinergic system and opioid withdrawal.     

Mutation of glutamic acid 103 of toluene o-xylene monooxygenase as a means to control the catabolic efficiency of a recombinant upper pathway for degradation of methylated aromatic compounds

Toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of Pseudomonas stutzeri OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and o-xylene. For example, the sequential monooxygenation of o-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the P. stutzeri meta pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from o-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from o-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.     

Vascular effects of caffeic acid phenethyl ester (CAPE) on isolated rat thoracic aorta

This study was aimed to investigate the vascular activity of caffeic acid phenethylester (CAPE), one of the major components of honeybee propolis. Experiments were performed on rat thoracic aortic rings, mounted in an isolated organ bath and connected to an isometric force transducer. The effect of CAPE (0.1-300 microM) was evaluated on tissue pre-contracted with phenylephrine (PE, 1 microM) or with KCl (100 mM). In another set of experiments, tissue was incubated with CAPE (1-100 microM) and responses to PE (0.01-3 microM) or KCl (60 mM) were evaluated. The effect of CAPE on cytosolic Ca(2+) concentration in aortic smooth muscle cells stimulated with PE or KCl was also evaluated. CAPE (0.1-300 microM) caused a concentration-dependent relaxation (pEC(50) 4.99 +/- 0.19; Emax 100.75 +/- 1.65%; n = 4) of tissue pre-contracted with PE that was reduced by endothelium removal or by incubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM). CAPE also relaxed KCl-precontracted tissue (pEC(50) 4.40 +/- 0.08; n = 4). CAPE inhibited contractile responses to PE or to KCl, and also inhibited the contractile response to PE obtained in a Ca(2+)-free medium. In addition, CAPE inhibited the increase in cytosolic Ca(2+) concentration triggered by stimulation of aortic smooth muscle cells with PE or KCl. Our results demonstrate a vascular activity for CAPE, that is only partially dependent on nitric oxide. Indeed, at high concentrations, CAPE vasorelaxant effect occurs also in absence of endothelium and it is likely due to an inhibitory effect on calcium movements through cell membranes.     

The role of calcium in eicosanoid production induced by ricinoleic acid or the calcium ionophore A23187

Rat isolated intestine incubated in Krebs solution converted exogenous [¹⁴C]-arachidonic acid into products that chromatographed with prostaglandins, leukotriene B₄ and 5-hydroxy-eicosatetraenoic acid. Accumulation of these products was increased by the laxative ricinoleic acid (0.34 mM) or the calcium ionophore A23187 (7.6μM). In the presence of the calcium antagonists TMB-8 (0.43μM). or verapamil (0.2μM) the mean effects of ricinoleic acid or the calcium ionophore were smaller. Stimulation of arachidonic acid metabolism by ricinoleic acid therefore seems likely to involve a calcium-dependent mechanism.     

Conformational aspects of drug-DNA interactions: Studies on anthracycline antibiotics and psoralen derivatives

The interaction of anticancer agents, analogues of adriamycin and of photo-chemotherapeutic compounds of the psoralen structural type with DNA was investigated using spectroscopic, hydrodynamic and chiroptical techniques. The nucleic acid may undergo conformational changes from the B form to more compact structures as a result of the binding process to charged compounds. Different complex geometries adopted byvarious drugs were observed and discussed in terms of intercalation into the polynucleotide double helix and orientation of the ligand in the base-pair pocket. The binding of chemotherapeutic agents to functionally organized DNA was also studied. Lower binding affinities and modified spectral responsesareindicativeofdifferent drug-DNA complexation patternsinthiscase. The results of these studies allow a better understanding of drug-nucleic acid interactions at a molecular level.     

Solid-state conformations of aminosuccinyl peptides: crystal structure of tert-butyloxycarbonyl-L-leucyl-L-aminosuccinyl-L-phenylalaninami de

The protected tripeptide tert-butyloxycarbonyl-L-leucyl-L-aminosuccinyl-L-phenylalaninamide crystallizes in the orthorhombic space group P2(1)2(1)2(1), with a = 6.214(3), b = 12.832(3), c = 33.094(4) A, Z = 4. The structure was solved by direct methods using MULTAN 80 and refined to an R value of 0.055 for 1458 reflections. The bond lengths and angles are in good agreement with the standard values. The peptide backbone adopts a type II' beta-bend conformation with a weak intramolecular hydrogen bond between the CO group of the leucyl residue and the C-terminal NH2 group. In agreement with previous studies, this structure confirms the high propensity of aminosuccinyl peptides to adopt a type II' beta-bend conformation. The role of this conformation in relation to the deamidation process in proteins is also discussed.     

Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A.

Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.     

Changes in dopamine uptake and developmental effects of dopamine receptor inactivation in the sea urchin

[³H]-dopamine ([³H]-DA) uptake was measured in the presence or absence of the catecholamine uptake inhibitor nomifensine in both unfertilized and fertilized eggs. Specific [³H]-DA uptake depended on time and [³H]-DA concentration; it was high in unfertilized eggs, declined 20–30 min after fertilization, and rose again during cleavage. Irreversible inactivation of dopamine receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in a complete loss of sensitivity of egg adenylate cyclase to dopamine stimulation. In fertilized eggs treated with EEDQ for 1 hr, restoration of adenylate cyclase activity sensitive to dopamine stimulation could be observed 4 hr after the end of treatment, thus suggesting the appearance of new dopamine receptors in cleaving eggs. Short-term EEDQ treatment on unfertilized eggs, although not impairing fertilization, resulted in cleavage inhibition; the same treatment carried out soon after fertilization, on the other hand, elicited no effect on development. On the contrary, in embryos subjected to continuous treatment with EEDQ, development was impaired independent of the stage at which the treatment was started. © 1995 Wiley-Liss, Inc.