Biological Efficacies Research Articles

Pyrimidine Chemistry in Crop Protection

This review aims to give an overview of the significance of pyrimidine derivatives in crop protection. The main herbicidally, fungicidally and insecticidally active pyrimidine classes are presented, together with their synthetic routes, their modes of action and their biological efficacies. Quinazolines and other bicyclic pyrimidine derivatives are also covered.

Antitumor efficacy improved by local delivery of species-specific endostatin

Conflicting results have been reported concerning the antitumor efficacy of the angiogenesis inhibitor endostatin. This may be due to differences in the biological distribution of endostatin between studies or to the varying biological efficacies of the different protein forms that were examined. To address this issue, the authors used a local delivery approach in which each tumor cell secreted endostatin, providing uniform endostatin levels throughout the tumors. This allowed a direct assessment of the biological efficacy of soluble endostatin in vivo. The authors genetically engineered BT4C gliosarcoma cells so that they would stably express and secrete either the human or murine form of endostatin. Endostatin-producing cells or mock-infected cells were implanted intracerebrally in syngeneic BD-IX rats. The antitumor efficacy of endostatin was evaluated on the basis of survival data and tumor volume comparisons. In addition, microvascular parameters were assessed. The authors confirmed the continuous release of endostatin by the BT4C cells. A magnetic resonance imaging-assisted comparison of tumor volumes revealed that local production of murine endostatin significantly inhibited tumor growth. Notably, 40% of the animals in this treatment group experienced long-term survival without histologically verifiable tumors 7 months after cell implantation. After local treatment with murine endostatin, tumor blood plasma volumes were reduced by 71%, microvessel density counts by 84%, and vascular area fractions by 75%. In contrast, human endostatin did not inhibit tumor growth significantly in this model. Centrally located regions of necrosis were present in tumors secreting both the human and the murine species-specific form of endostatin. The results suggest that endostatin inhibits tumor angiogenesis in vivo in a species-specific manner.

Effects of (R)‐ and (S)‐isomers of β‐adrenergic agonists on eosinophil response to interleukin‐5

Racemic beta2-adrenergic receptor agonists (beta2-agonists) are used frequently to treat patients with asthma. Potential differences in the biological activities and clinical efficacies among racemic beta2-agonists and their isomers are controversial, and research into these possible differences is limited. We hypothesized that the (S)- and the (R)-isomers of beta2-agonists have opposing effects on the activation of inflammatory cells. Isolated human eosinophils were pretreated with 1:1 racemic (R,S)-, (R)- or (S)-albuterol, isobutyl methylxanthine (IBMX), and stimulated with IL-5. The kinetics of superoxide production were examined by reduction of cytochrome c, and the effects of pharmacological agents on superoxide production were monitored for 180 min. (R,S)-albuterol inhibited IL-5-induced superoxide production. This inhibition was enhanced by a cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor, IBMX, and was reversed by the selective beta2-adrenergic receptor antagonist, ICI 118, 551, verifying the involvement of both cAMP and the beta2-adrenergic receptor. In addition, (R)-albuterol alone, similarly to (R,S)-albuterol, significantly inhibited IL-5-induced superoxide production up to 60 min (P<0.05, n=4), but the inhibition was lost with longer incubation. In contrast, (S)-albuterol with IBMX did not inhibit IL-5-induced superoxide production before 60 min, but it significantly enhanced IL-5-mediated superoxide production after 60 min (P<0.05, n=4). When both were present as racemic (R,S)-albuterol, the inhibitory effect of (R)-albuterol was not affected by (S)-albuterol. When incubated with IL-5-activated eosinophils, (R)-albuterol shows anti-inflammatory effects and (S)-albuterol shows pro-inflammatory effects in the presence of IBMX. The kinetics of these effects are different, and when used simultaneously, (R)-albuterol predominates. When marked usage of the (S)-isomer is anticipated, racemic (R,S)-albuterol should be used clinically with caution.

Polygonatum rhizoma affects antioxidant defense systems without changing mRNA expression in diet-induced hypercholesterolemic rabbits.

This study was conducted to determine the antioxidant effects of a Polygonatum extract compared with the major antioxidant, vitamin E, in rabbits fed a high-cholesterol diet. Rabbits were given a high-cholesterol (0.5%, wt/wt) diet with vitamin E (0.03%, wt/wt) or a Polygonatum extract (0.05%, wt/wt) for 8 weeks. The body weight gain (g/week) was only significantly increased only in the high-cholesterol-fed control group, yet the relative liver weight was significantly lower in the Polygonatum group compared with the other groups. The supplementation of vitamin E and Polygonatum extract led to an increase in the hepatic catalase (CAT) activity without any change in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. Hepatic total glutathione content was significantly higher in the Polygonatum group than in the other groups. The level of hepatic mitochondrial H(2)O(2) was significantly lower in the two supplemented groups compared with the control group, whereas the level of cytosolic H(2)O(2) was only significantly lower in the Polygonatum group than in the control group. The level of plasma thiobarbituric acid-reactive substances (TBARS) was only significantly lower in the vitamin E group, whereas the level of hepatic TBARS was slightly lower in the Polygonatum group than in the other groups. In the case of the high-density lipoprotein-related antioxidant enzyme, vitamin E supplementation produced the highest plasma paraoxonase (PON) activity compared with the other groups, although there was no difference in the hepatic PON activity among the groups. Meanwhile, the plasma vitamin E concentration was significantly higher in the vitamin E and Polygonatum groups than in the control group; however, plasma vitamin A concentration did not differ significantly between the groups. As regards the mRNA expressions of hepatic antioxidant enzymes, the vitamin E and Polygonatum extract supplementation had no effect on the SOD, CAT, GSH-Px, and PON mRNA expression. Accordingly, these results indicate that the Polygonatum extract had a positive effect on the antioxidant defense system based on decreasing the content of hepatic TBARS and hydrogen peroxide, increasing the CAT activity and total glutathione level in the liver, and sparing the plasma vitamin E. Thus, further studies on the functional components in Polygonatum extract and their biological efficacies are needed.

Modelling of fish interleukin-1 and its receptor

This paper presents original data regarding: the three-dimensional modelling of rainbow trout ( Oncorhynchus mykiss) and sea bass ( Dicentrarchus labrax) IL-1β, the modelling of trout IL-1 receptor type I (IL-1R I) and the modelling of trout IL-1β bound to its receptor. The 3D models of trout and sea bass IL-1β molecules were predicted by comparison with those already available of human and mouse, and, in both cases, a structure consisting of a beta-trefoil fold was obtained, a motif well conserved during evolution of IL-1β-related proteins. Moreover, a model for the rainbow trout IL-1 receptor alone and complexed with IL-1β was predicted and compared to the murine model. Both ligand-receptor complex models were compared with the known crystal structure of the human IL-1β/IL-1R complex. A cross-interaction of trout IL-1β with the mouse receptor was also simulated. Such analysis and the predicted interaction energies calculated from the models have helped to explain the different biological efficacies of mammalian and fish IL-1β molecules in assays based on mammalian target cells. The obtained data are discussed on the basis of evolutionary and applied perspectives.

Fluorescent Labeling of Purified β2 Adrenergic Receptor: EVIDENCE FOR LIGAND-SPECIFIC CONFORMATIONAL CHANGES

The purpose of the present study was to develop an approach to directly monitor structural changes in a G protein-coupled receptor in response to drug binding. Purified human beta 2 adrenergic receptor was covalently labeled with the cysteine-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)ethylenediamine (IANBD). IANBD is characterized by a fluorescence which is highly sensitive to the polarity of its environment. We found that the full agonist, isoproterenol, elicited a stereoselective and dose-dependent decrease in fluorescence from IANBD-labeled beta 2 receptor. The change in fluorescence could be plotted against the concentration of isoproterenol as a simple hyperbolic binding isotherm demonstrating interaction with a single binding site in the receptor. The ability of several adrenergic antagonists to reverse the response confirmed that this binding site is identical to the well described binding site in the beta 2 receptor. Comparison of the response to isoproterenol with a series of adrenergic agonists, having different biological efficacies, revealed a linear correlation between biological efficacy and the change in fluorescence. This suggests that the agonist-mediated decrease in fluorescence from IANBD-labeled beta 2 receptor is due to the same conformational change as involved in receptor activation and G protein coupling. In contrast to agonists, negative antagonists induced a small but significant increase in base-line fluorescence. Despite the small amplitude of this response, it supports the notion that antagonists by themselves may alter receptor structure. In conclusion, our data provide the first direct evidence for ligand-specific conformational changes occurring in a G protein-coupled receptor. Furthermore, the data demonstrate the potential of fluorescence spectroscopy as a tool for further delineating the molecular mechanisms of drug action at G protein-coupled receptors.

A novel bioassay for lactogenic activity: demonstration that prolactin cells differ from one another in bio- and immuno-potencies of secreted hormone.

A reverse hemolytic plaque assay for detecting casein release from individual mammary cells in culture was developed as a bioassay for PRL. Treatment with rat PRL caused dose-related increases in the percentage of mammary cells that released casein and the average size of casein plaques that formed. The assay exhibited exquisite sensitivity (156 fg rat PRL per assay slide) and could be used to evaluate the biopotency of PRL released from individual cells. By combining this "plaque bioassay" with a standard plaque assay for measuring the secretion of immunoreactive PRL, it was possible to compare the bio- and immuno-potencies of hormone released from the same pituitary cells. The results of three separate studies revealed major differences among PRL secretors in these potency estimates. Given the existence of PRL variants with different biological and immunological efficacies, these findings suggest that PRL cells differ from one another in the molecular form(s) of hormone released.

The complete sequence of the Bacillus phage ϕ 29 right early region

We have sequenced the rightmost 2216 bp of the Bacillus phage φ 29 genome. This region encompasses the right early region and completes the sequence of the φ 29 early functions. The sequence of gene 17, an early gene implicated in the replication process, is presented. From these results we predict that gene 17 encodes a 19.1-kDal protein. Further analysis of the sequence revealed five previously undetected potential genes, encoding 12.6-, 12.4-, 15.2-, 6.2- and 4.6-kDal proteins. The biological efficacies of some of these putative genes were demonstrated using an Escherichia coli in vitro transcription-translation system. We also examined the transcriptional and translational signals present on this region of the genome.

Biological Efficacy of Liquid Methionine and Methionine Hydroxy Analogue-Free Acid in Chicks

An experiment was conducted to determine biological efficacies of DL-methionine-Na (Liquimeth®) and DL-methionine hydroxy analogue (MHA)-free acid (Alimet®) relative to DL-methionine in Single Comb White Leghorn male chicks. A crystalline amino acid mixture devoid of sulfur-containing amino acids was used for the basal diet, and the test substances were added at .1, .2, and .3% levels, equivalent to DL-methionine on a molar basis, in place of cornstarch. By a slope ratio assay using chick growth as a criterion, the relative biological efficacies of DL-MHA-free acid and DL-methionine-Na to DL-methionine were found to be 70 and 113%, respectively, on a molar basis.

Synthetic alpha-adrenergic agonists are potent alpha-adrenergic blockers in human platelets.

ADRENALINE- and noradrenaline-induced aggregation of human platelets1–3 is blocked by the α-adrenergic blocking agents, dihydroergotamine, phentolamine, phenoxybenzamine and dibenamine4–6, suggesting the involvement of α-adrenergic receptors. We have demonstrated that adrenaline and noradrenaline inhibit adenylate cyclase (EC 4.6.1.1) in lysates7 and particles8 of human platelets, an effect also blocked by such α-adrenergic antagonists. Synthetic phenylethylamine and imidazoline derivatives that act as α-adrenergic agonists in various other tissues neither induced primary platelet aggregation nor inhibited adenylate cyclase when added at concentrations up to 100 µM9. α-Adrenergic receptors have been identified in human platelets by binding studies using tritiated dihydroergocryptine10,11 or dihydroergonine12,13. The sites to which the dihydrogenated ergot alkaloids were bound showed the typical pattern of an α-adrenergic receptor with respect to displacement by adrenergic agonists and antagonists. However, there have been some discrepancies between the binding affinities of some α-adrenergic agonists and their biological efficacies concerning platelet aggregation and inhibition of adenylate cyclase. The α-adrenergic agonist, clonidine, displaced dihydroergocryptine from the binding sites with similar10 or even a 10-fold higher affinity11 relative to adrenaline, but clonidine (up to 100 µM) was ineffective in inducing primary platelet aggregation and inhibition of adenylate cyclase9. Similar dissociation constants were obtained for phenylephrine and noradrenaline in binding studies12, but in contrast to the natural compound, phenylephrine (up to 100 µM) was unable to induce a biological response9. We observed similar discrepancies with methoxamine, xylometazoline and oxymetazoline12,13. Agents that undergo the primary reaction of high affinity-binding to the receptor but are unable to induce secondary reactions in the form of biological responses should act as antagonists. We have tested this hypothesis and show here that various agents that are potent α-adrenergic agonists in various other systems are α-adrenergic antagonists in human platelets with respect to adrenaline-induced aggregation and inhibition of adenylate cyclase.