Probing a membrane matrix regulating hormone action: I. The molecular length of effective lipids

Auxin-induced pea (Pisum sativum) stem section elongation is enhanced at levels of 3 to 40 micromolar by six new classes of alkane derivatives additional to those described earlier, providing that length of their molecules exceeds 20 A. Increasingly longer homologous series of alkyl chlorides, bromides and iodides, alkyl benzenes, dialkyl ethers, and sulfides show a logarithmically linear increase in specific activity above this length, reaching an optimum near 28 to 30 Å. Longer dialkyl ethers and sulfides are less effective, while steroids, or alkanes with substituents at both ends, are ineffective.     

Utilization of 1,3-Dioxolanes in the Synthesis of α-branched Alkyl and Aryl 9-[2-(Phosphonomethoxy)Ethyl]Purines and Study of the Influence of α-branched Substitution for Potential Biological Activity

Syntheses of α-branched alkyl and aryl substituted 9-[2-(phosphonomethoxy)ethyl]purines from substituted 1,3-dioxolanes have been developed. Key synthetic precursors, α-substituted dialkyl [(2-hydroxyethoxy)methyl]phosphonates were prepared via Lewis acid mediated cleavage of 1,3-dioxolanes followed by reaction with dialkyl or trialkyl phosphites. The best preparative yields were achieved under conditions utilizing tin tetrachloride as Lewis acid and triisopropyl phosphite. Attachment of purine bases to dialkyl [(2-hydroxyethoxy)methyl]phosphonates was performed by Mitsunobu reaction. Final α-branched 9-[2-(phosphonomethoxy)ethyl]purines were tested for antiviral, cytostatic and antiparasitic activity, the latter one determined as inhibitory activity towards Plasmodium falciparum enzyme hypoxanthine-guanine-xanthine phosphoribosyltransfesase. In most cases biological activity was only marginal.     

Activation of histamine secretion from rat mast cells by phosphatidylserine analogues resistant to phospholipase A action

In order to elucidate how phosphatidylserine enhances histamine release induced by concanavalin A from rat mast cells, two phosphatidylserine analogues were synthesized and their effects were examined. A dialkyl analogue of phosphatidylserine, both ester bonds of which are replaced by ether bonds, potentiated histamine secretion from mast cells stimulated by concanavalin A, although it was much less active than natural phosphatidylserine. An alkyl analogue of phosphatidylserine, in which an ether bond is present at the 1 position, showed almost the same low activity as the dialkyl analogue. It is suggested that deacylation of the incorporated phosphatidylserine by phospholipase A is not required for potentiation by phosphatidylserine and the existence of an ester bond at position 1 is important for phosphatidylserine to enhance concanavalin A-induced histamine secretion from mast cells.     

Factors influencing the rate of `aging' of a series of alkyl methylphosphonyl-acetylcholinesterases

1. The progressive development of resistance to reactivation by an oxime (;aging') shown by a series of alkyl methylphosphonyl-acetylcholinesterases is slow when the alkyl group is a primary alcohol, whether or not the carbon chain is branched, but is much more rapid if the alkyl group is a secondary or cyclic alcohol. 2. Aging is accelerated by increase of temperature or decrease of pH. 3. Aging is inhibited by the quaternary amine N-methylpyridinium iodide. 4. The results are discussed in relation to the role played by aging in the therapy of poisoning by organophosphorus compounds.     

Occurrence of dialkyl ether phospholipids in Stigmatella aurantiaca DW4.

We investigated the lipid composition of vegetative cells of Stigmatella aurantiaca. Four phospholipids were isolated and identified: phosphatidylethanolamine as the main component, phosphatidylglycerol, lysophosphatidylethanolamine in an exceptionally large amount (17%), and phosphatidylinositol (18 to 25%), rare in procaryotic cells. This composition did not change significantly during growth. The fatty acids of total lipids were found to be rather similar to those of other strains of myxobacteria; the main fatty acids found were unsaturated and branched. We noted a different fatty acid pattern for each phospholipid. The presence of unusual alkyl ether linkages, established by chemical hydrolysis and infrared spectroscopy, was unexpected in these bacteria. Diacyl ester, dialkyl ether, and monoacyl-monoalkyl structures were shown in phosphatidylethanolamine and phosphatidylglycerol. Lysophosphatidylethanolamine was essentially a monoacyl form, whereas phosphatidylinositol was a unique dialkyl ether phospholipid.     

Mechanism of pH-independent Cleavage and Isomerization of 3′-Ribonucleotide Phosphodiesters

Abstract

The detailed mechanisms of pH-independent cleavage and isomerization reactions of the phosphodiester bond are evaluated by examining kinetics of hydrolytic reactions of selected mono- and dialkyl esters of 3′-uridylic acid. The mechanistic choices are made by comparing LFER's between reaction rates and acidity of the alkyl group of phosphodi- and triesters.     

Glutathione S-aryl transferase in the metabolism of parathion and its analogs

A soluble enzyme (glutathione S-aryl transferase) which converts parathion and related insecticidal organophosphorus triesters to S-$\text{p}$-nitrophenylglutathione and the corresponding dialkyl phosphorothioic or phosphoric acid has been identified and assayed in vertebrate liver. The activity of this enzyme can be differentiated from that of the analogous glutathione S-alkyl transferase also present in rat tissues. Its relationship to other known glutathione S-aryl transferases remains to be established but considerable differences in optimum pH have been observed.     

Concentration effect of hydrophilic ionic liquids on the enzymatic activity of Candida antarctica lipase B

A systematic study of the effects of hydrophilic ionic liquids concentration and nature (alkyl chain length and type of anion) on the activity of Candida antarctica lipase B is here reported. The increase in the concentration of the studied ionic liquids is shown to cause a decrease of the enzyme activity, but the effect is dependent on the ionic liquid used. This behavior is partially due to the ionic liquid impact on the thermodynamic water activity, but direct interactions between the hydrophilic ionic liquid and the enzyme are also disclosed. Cations with longer alkyl chains decrease the enzyme activity by obstruction of its non-polar active site, while direct interactions established between the enzyme and the anions, dominated by dispersion forces and hydrogen-bonding, contribute also for the loss of activity observed.     

The oxidative cleavability of protein cross-linking reagents containing organoselenium bridges

The intensive use of cleavable cross-linking reagents to study macromolecular biological interactions has shown a demand for optimizing these reagents in such a way that the involved macromolecules remain intact. The present work focuses on the development of selenium linkers that are cleavable by mild oxidation. The efficiency of cross-linking and subsequent cross-linker cleavage with a new series of such homo- or heterobifunctional cross-linking reagents have been tested in a simple model system, consisting of albumin and cytochrome c. Resultant, or residual, covalent complex formation is examined by SDS-polyacrylamide gel electrophoresis. From this work it can be concluded that diallyl selenides are readily cleaved by mild oxidation, whereas dialkyl selenides and benzyl alkyl selenides can only be cleaved when the alkyl part of the selenide has an electron-withdrawing group next to the beta-carbon from selenium.     

Ionic liquids as refolding additives: N'-alkyl and N'-(omega-hydroxyalkyl) N-methylimidazolium chlorides

The purpose of this work was to investigate the influence of a series of N'-alkyl and N'-(omega-hydroxy-alkyl)-N-methylimidazolium chlorides on the renaturation of two model proteins, namely hen egg white lysozyme and the single-chain antibody fragment ScFvOx. All tested ionic liquids acted as refolding enhancers, with varying efficacies and efficiencies. The results of the refolding screening could be interpreted by taking into account the effect of the studied ionic liquids on protein aggregation, together with the systematic variations of their influence on the stability of native proteins in solution. More hydrophobic imidazolium cations carrying longer alkyl chains were increasingly destabilizing, while terminal hydroxylation of the alkyl chain made the salts more compatible with protein stability. The studied ionic liquids can be classified as preferentially bound, slightly to moderately chaotropic cosolvents for proteins.     

Design,synthesis, in silico studies and in vitro evaluation of isatin-pyridine oximes hybrids as novel acetylcholinesterase reactivators

Organophosphorus poisoning caused by some pesticides and nerve agents is a life-threating condition that must be swiftly addressed to avoid casualties. Despite the availability of medical countermeasures, the clinically available compounds lack a broad spectrum, are not effective towards all organophosphorus toxins, and have poor pharmacokinetics properties to allow them crossing the blood-brain barrier, hampering cholinesterase reactivation at the central nervous system. In this work, we designed and synthesised novel isatin derivatives, linked to a pyridinium 4-oxime moiety by an alkyl chain with improved calculated properties, and tested their reactivation potency against paraoxon- and NEMP-inhibited acetylcholinesterase in comparison to the standard antidote pralidoxime. Our results showed that these compounds displayed comparable in vitro reactivation also pointed by the in silico studies, suggesting that they are promising compounds to tackle organophosphorus poisoning.     

Stabilization of alpha-chymotrypsin by ionic liquids in transesterification reactions.

Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.     

Parathion utilization by bacterial symbionts in a chemostat.

A continuous-culture device was used to select and enrich for microorganisms, from sewage and agricultural runoff, that were capable of using the organophosphorus insecticide parathion as a sole growth substrate. Parathion was dissimilated by the highly acclimated symbiotic activities of Pseudomonas stutzeri, which non-oxidatively and cometabolically hydrolyzed the parathion to ionic diethyl thiophosphate and p-nitrophenol, and P. aeruginosa, which utilized the p-nitrophenol as a sole carbon and energy source. Ionic diethyl thiophosphate was found to be inert to any transformations. Methyl parathion was dissimilated in an analogous way. The device functioned as a chemostat with parathion as the growth-limiting nutrient, and extraordinarily high dissimilation rates were attained for parathion (8 g/liter per day) and for p-nitrophenol (7 g/liter per day). This is the first report of parathion utilization by a defined microbial culture and by symbiotic microbial attack and of dissimilation of an organophosphorus pesticide in a chemostat.     

Parathion utilization by bacterial symbionts in a chemostat.

A continuous-culture device was used to select and enrich for microorganisms, from sewage and agricultural runoff, that were capable of using the organophosphorus insecticide parathion as a sole growth substrate. Parathion was dissimilated by the highly acclimated symbiotic activities of Pseudomonas stutzeri, which non-oxidatively and cometabolically hydrolyzed the parathion to ionic diethyl thiophosphate and p-nitrophenol, and P. aeruginosa, which utilized the p-nitrophenol as a sole carbon and energy source. Ionic diethyl thiophosphate was found to be inert to any transformations. Methyl parathion was dissimilated in an analogous way. The device functioned as a chemostat with parathion as the growth-limiting nutrient, and extraordinarily high dissimilation rates were attained for parathion (8 g/liter per day) and for p-nitrophenol (7 g/liter per day). This is the first report of parathion utilization by a defined microbial culture and by symbiotic microbial attack and of dissimilation of an organophosphorus pesticide in a chemostat.     

The alkyl and alk-1-enyl glycerols in the liver of rats fed long-chain alcohols or alkyl glycerols

Dietary long-chain alcohols and alkyl glycerols, including polyunsaturated compounds, are incorporated into the alkyl and alk-1-enyl moieties of the ionic alkoxylipids of rat liver, whereas polyunsaturated fatty acids are not.     

Phosphate and soil binding: factors limiting bacterial degradation of ionic phosphorus-containing pesticide metabolites.

Soils that had a high binding capacity for inorganic orthophosphate (Pi) had reduced capacities to bind ionic alkyl phosphorus compounds. Only ionic methylphosphonate (MPn) and ionic phenylphosphonate exhibited moderate binding. Pseudomonas testosteroni used either MPn or Pi as a sole phosphorus source and exhibited diauxic utilization of MPn and Pi. The utilization of MPn was suppressed in the presence of Pi. This suppression was abolished by a Pi-binding soil. The soil did not have a significant effect on the maximum rate of degradation of either MPn or the poorly bound ionic O-isopropyl methylphosphonate, whereas the amount of MPn (but not the amount of O-isopropyl methylphosphonate) metabolized was reduced in the presence of soil     

Phosphate and soil binding: factors limiting bacterial degradation of ionic phosphorus-containing pesticide metabolites.

Soils that had a high binding capacity for inorganic orthophosphate (Pi) had reduced capacities to bind ionic alkyl phosphorus compounds. Only ionic methylphosphonate (MPn) and ionic phenylphosphonate exhibited moderate binding. Pseudomonas testosteroni used either MPn or Pi as a sole phosphorus source and exhibited diauxic utilization of MPn and Pi. The utilization of MPn was suppressed in the presence of Pi. This suppression was abolished by a Pi-binding soil. The soil did not have a significant effect on the maximum rate of degradation of either MPn or the poorly bound ionic O-isopropyl methylphosphonate, whereas the amount of MPn (but not the amount of O-isopropyl methylphosphonate) metabolized was reduced in the presence of soil     

Desulfuration of Dialkyl Thiophosphoric Acids by a Pseudomonad

A strain of Pseudomonas acidovorans used the organophosphorus pesticide breakdown products, ionic O,O-diethyl phosphorothioate and ionic O,O-diethyl phosphorodithioate, as sulfur sources. The growth yields from the thiophosphates and sulfate were 3.6 to 4.1 kg of protein per mol of sulfur. Elemental sulfur and sulfide also served as sulfur sources but gave lower growth yields.     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

Stereoselective synthesis of (1-alkoxyalkyl) alpha- and beta-D-glucopyranosiduronates (acetal-glucopyranosiduronates): a new approach to specific cytostatics for the treatment of cancer

The reaction of methyl 2,3,4,6-tetra-O-acetyl-1-O-trimethylsilyl-beta- and -alpha-D-glucopyranuronate severally with the dimethyl or diethyl acetals of formaldehyde, bromoacetaldehyde, propionaldehyde, 3-benzyloxypropionaldehyde, 5-carboxypentanal, and 2-bromohexanal in the presence of catalytic amounts of trimethylsilyl trifluoromethanesulfonate at -78 degrees gave the corresponding (1-alkoxyalkyl) alpha- and beta-glycosides (acetal-glucopyranosiduronates) with retention of configuration at C-1 in yields of 41-91%. Instead of the dialkyl acetals, the corresponding aldehydes and alkyl trimethylsilyl ether can be used. Deacetylation gave the corresponding methyl (acetal-beta- and -alpha-D-glucopyranosid)uronates in good yield. De-esterification of methyl [(1R)-1-methoxybutyl beta-D-glucopyranosid]uronate with esterase gave the acetal-beta-D-glucopyranosiduronic acid which was an excellent substrate for beta-D-glucuronidase.