Semantics and Conversations for an Agent Communication Language

We address the issues of semantics and conversations for agent communication languages and the Knowledge Query Manipulation Language (KQML) in particular. Based on ideas from speech act theory, we present a semantic description for KQML that associates ``cognitive'' states of the agent with the use of the language's primitives (performatives). We have used this approach to describe the semantics for the whole set of reserved KQML performatives. Building on the semantics, we devise the conversation policies, i.e., a formal description of how KQML performatives may be combined into KQML exchanges (conversations), using a Definite Clause Grammar. Our research offers methods for a speech act theory-based semantic description of a language of communication acts and for the specification of the protocols associated with these acts. Languages of communication acts address the issue of communication among software applications at a level of abstraction that is useful to the emerging software agents paradigm.Comment: Also in in "Readings in Agents", Michael Huhns and Munindar Singh (eds), Morgan Kaufmann Publishers, In

The Use of Therapeutic Cloning in Transplantations: An Aristotelian Perspective

Therapeutic cloning for transplantations is one of the most complex issues in Bioethics. This is because it involves two of the most controversial subjects in today's medicine: cloning and transplantations. Although therapeutic cloning is a modern issue in the scope of transplantations, we consider Aristotle's contribution to be of high importance. On a first level, we will describe the modem phenomenon of therapeutic cloning, concerning the medical technique itself, its aim, its use in transplantations and its core legal and political aspects. On a second level, the depth of Aristotelian concepts of nature, art and their interrelationship will enlighten crucial aspects of our discussion, concerning human health and medicine. On a third level, Aristotle's concept of matter will constitute the basis for the solution of the ethical problems of transplantation through therapeutic cloning. On a fourth level, the Aristotelian concept of practical wisdom, considered as a part of Politics, will provide us with practical guidance as to how decision-making and legislation should be utilised to support the appropriate use of therapeutic cloning; thus, it will become clear that it is a moral-law and moral-politic issue. Finally, we will address the moral objections to therapeutic cloning, establishing some fundamental elements for the building of consensus between different countries on this subject

Synthesis and Application of Dye-Ligand Affinity Adsorbents

Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity towards a large number of proteins. They are commercially available, are inexpensive, and can easily be immobilized. Important factors that contribute to the successful operation of a dye-ligand chromatography include adsorbent properties, such as matrix type and ligand concentration, the buffer conditions used in the adsorption and elution stages, and contacting parameters like flow rate and column geometry. In general, with dye-ligand affinity chromatography, the specificity is provided by the adsorption and elution conditions employed in a particular purification, and these must often be worked out by trial and error. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a dye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris crude extract on Cibacron Blue 3GA-Sepharose is given as an example

Protein engineering of glutathione transferase for the development of optical biosensor to detect xenobiotics

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible enzymes that play essential role in detoxification and degradation of toxic compounds, including pesticides. The purpose of the present study is the development of an optical enzyme biosensor based on GSTs, for the detection and determination of pesticides in environmental samples. Protein engineering was used for the creation of a GST variant with higher selectivity towards pesticides. cDNA libraries were created from Phaseolus vulgaris and Glycine max stressed plants using degenerated primers and reverse transcription-PCR. Large diversity in GST genes was accomplished employing directed evolution through DNA shuffling of a mixture of GST genes from P. vulgaris and G. max stressed plants. The shuffled library of chimaeric GST genes was cloned in E. coli expression plasmid. Screening of the library led to the isolation of a novel GST enzyme that displays both glutathione transferase and glutathione peroxidase activities. The enzyme was purified by affinity chromatography and characterized by kinetic analysis towards 20 different substrates and 66 different pesticides. The results showed that the organoclorine insecticides and strobilurins (fungicides) are strong inhibitors of the enzyme. The specificity of the enzyme towards pesticides was further improved using site-saturation mutagenesis at position Phe117. The mutant Phe117Ile displays 5-fold higher catalytic efficiency and selectivity towards organochlorine insecticides. Therefore, the mutant GSTPhe117Ile was used for the development of an optical biosensor. The enzyme was immobilized in alkoxysilane (TEOS/PTMOS) sol-gel system in the presence of the pH indicators bromocresol purple (acidic) and phenol red (basic). The bioactive material exhibits linearity in the range of 0.625-30 μΜ α-endosulfan (pH=4-7) at 562 nm and was used for the development of an analytical method for the determination of α-endosulfan in environmental samples

Cloning and characterization of herbicide-degrading glutathione transferases from cicer arietinum

GSTs are multifunctional enzymes that catalyze the conjugation of glutathione (GSH) to reactive electrophiles. These electrophiles are diverse and include important endogenous compounds, as well as xenobiotic chemicals, therefore GSTs play an important role in stress tolerance and herbicide detoxification. GSTs are usually active as a dimer of 24–29 kDa subunits. Each monomer of dimeric GSTs contains a G-site, at the N-terminal, capable of binding the GSH substrate and an H-site, at the C-terminal, that has xenobiotic compound-binding capabilities. Different classes of herbicides such as triazines, thiocarbamates, chloroacetanilides, diphenylethers, and aryloxyphenoxypropionates can be metabolized by GSTs. Herbicide tolerance in plants is based primarily on the differential ability of plant species to detoxify a herbicide, with the formation of a herbicide-GSH conjugate in the resistant but not in the susceptible species. The plant-specific phi and tau GSTs are primarily responsible for herbicide detoxification, showing class specificity in substrate preference. In present work, we report the cloning, kinetic and structural characterization of three members of the GST family from Cicer arietinum leaves (CaGSTs)

Cytochrome P450 102A2 Catalyzes Efficient Oxidation of Sodium Dodecyl Sulphate: A Molecular Tool for Remediation

Bacterial cytochrome P450s (CYPs) constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. In the present work we report the characterization of CYP102A2 from B. subtilis with a focus on its substrate specificity. CYP102A2 is more active in oxidation of sodium dodecyl sulphate (SDS) than any other characterized CYP. The effect of SDS and NADPH concentration on reaction rate showed nonhyperbolic and hyperbolic dependence, respectively. The enzyme was found to exhibit a bell-shaped curve for plots of activity versus pH, over pH values 5.9–8.5. The rate of SDS oxidation reached the maximum value approximately at pH 7.2 and the pH transition observed controlled by two pKas in the acidic (pKa = 6.7 ± 0.08) and basic (pKa = 7.3 ± 0.06) pH range. The results are discussed in relation to the future biotechnology applications of CYPs

Cloning and Characterization of a Biotic-Stress-Inducible Glutathione Transferase from Phaseolus vulgaris

Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its Km for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris

Molecular and catalytic characterization of the herbicide-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris

The Use of Highly Specific GSTs towards the Development of Stress Tolerant Transgenic Plants (pp. 263-274)

Glutathione transferases (GSTs) belong to a super-family of multifunctional proteins. GSTs play a key role in cellular detoxification from xenobiotic substances like herbicides, secondary metabolites and toxic degradation products resulting from oxidative stress and cellular metabolism. Furthermore, environmental conditions generate oxidative stress, the products of which have to be detoxified by plants. It is anticipated that environmental stresses will worsen over the following years due to climate change. Hence, plants must adapt rapidly to the new environmental conditions in order to both survive and satisfy the constantly increasing human demand for agricultural products. Genetic engineering has been successfully used to develop plants resistant to stresses and, having taken all the necessary precautions, could offer a solution as it can help to develop plants with desirable traits in a short period of time. We present here the use of GST isoenzymes in the development of transgenic plants. Although transgenic plants over-expressing various GSTs have been used for “in planta” evaluation of the enzymes used in response to different stresses, the results show that GSTs could be of great value for generating stress tolerant plants. However, the literature is limited and more studies should be performed in order to exploit their full potential

Inhibition of human glutathione transferases by pesticides: Development of a simple analytical assay for the quantification of pesticides in water

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II cellular detoxification mechanism. Here, screening of the inhibition potency of a wide range of pesticides toward selected human GST isoenzymes (hGSTA1-1, hGSTP1-1, hGSTT2-2 and hGSTO1-1) was carried out. hGSTA1-1 was found more susceptible to inhibition by pesticides than other isoenzymes. The insecticides dieldrin and spiromesifen were identified as potent reversible inhibitors toward hGSTA1- 1 with IC50 values equal to 17.9 ± 1.7 M and 12.1 ± 3.4 M, respectively. Based on in silico docking analysis and kinetic inhibition studies it was concluded that dieldrin and spiromesifen bind specifically to the enzyme presumably at a distinct position that partially overlaps with both the G- and H-site. The ability of dieldrin and spiromesifen to inhibit hGSTA1-1 activity was exploited for the development of analytical quantification assays for these two pesticides. Linear calibration curves were obtained for dieldrin and spiromesifen, with useful concentration in the range of 0–10 M. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of these pesticides in real water samples without sample preparation steps