Thermoreversible gelation in aqueous binary solvents of chemically modified agarose

The thermoreversible gelation of chemically modified agarose has been studied in aqueous binary solvents (dimethyl sulfoxide and a series of formamide) by differential calorimetry, mechanical testing, and small-angle neutron scattering. The temperature–composition phase diagrams have been established. It is concluded that gelation is promoted by the formation of ternary complexes modified agarose/water/cosolvent, wherein the cosolvent mediates the interaction between chains through the formation of electrostatic interactions.     

Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments.

An extracellular lipase from the low-water-tolerant bacterium P. aeruginosa YS-7 was produced, purified, and characterized with respect to its functional properties in aqueous solutions and organic solvents. The enzyme was partially released from the cells during fermentation in defined medium with 5% (wt/vol) soybean oil. Approximately one-half of the total culture activity remained in solution after removal of cells. More than 95% of the activity was found in culture supernatant after mild detergent treatment (10 mM sodium deoxycholate) or after shifting the carbon source during the fermentation from triglyceride to a free fatty acid. The enzyme was recovered from an acetone precipitate of the whole culture and purified by hydrophobic interaction chromatography, yielding a preparation having a specific activity of about 1,300 mumol of fatty acid mg-1 h-1. The lipase (molecular size, approximately 40 kDa) hydrolyzes a variety of fatty acid esters and has an optimum pH of about 7. The enzyme retained its full activity at 20 to 55 degrees C, even after prolonged exposure (more than 30 days) to different concentrations of water-miscible organic solvents such as alcohols, glycols, pyridine, acetonitrile, dimethyl formamide, and dimethyl sulfoxide. The hydrolysis of 4-nitrophenyl laurate ester and of triglyceride emulsified in water was slightly accelerated with increasing concentrations of alcohols and glycols up to about 20% but was abolished with a further increase in alcohol concentration or in the presence of acetonitrile. In contrast, the rate of hydrolysis of these substrates in concentrated solutions of dimethyl formamide or dimethyl sulfoxide was markedly increased, by more than twofold and more than fivefold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments.

An extracellular lipase from the low-water-tolerant bacterium P. aeruginosa YS-7 was produced, purified, and characterized with respect to its functional properties in aqueous solutions and organic solvents. The enzyme was partially released from the cells during fermentation in defined medium with 5% (wt/vol) soybean oil. Approximately one-half of the total culture activity remained in solution after removal of cells. More than 95% of the activity was found in culture supernatant after mild detergent treatment (10 mM sodium deoxycholate) or after shifting the carbon source during the fermentation from triglyceride to a free fatty acid. The enzyme was recovered from an acetone precipitate of the whole culture and purified by hydrophobic interaction chromatography, yielding a preparation having a specific activity of about 1,300 mumol of fatty acid mg-1 h-1. The lipase (molecular size, approximately 40 kDa) hydrolyzes a variety of fatty acid esters and has an optimum pH of about 7. The enzyme retained its full activity at 20 to 55 degrees C, even after prolonged exposure (more than 30 days) to different concentrations of water-miscible organic solvents such as alcohols, glycols, pyridine, acetonitrile, dimethyl formamide, and dimethyl sulfoxide. The hydrolysis of 4-nitrophenyl laurate ester and of triglyceride emulsified in water was slightly accelerated with increasing concentrations of alcohols and glycols up to about 20% but was abolished with a further increase in alcohol concentration or in the presence of acetonitrile. In contrast, the rate of hydrolysis of these substrates in concentrated solutions of dimethyl formamide or dimethyl sulfoxide was markedly increased, by more than twofold and more than fivefold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prediction of penicillin V acylase stability in water-organic co-solvent monophasic systems as a function of solvent composition

Hydrolytic activity of penicillin V acylase (EC 3.5.1.11) can be improved by using organic cosolvents in monophasic systems. However, the addition of these solvents may result in loss of stability of the enzyme. The thermal stability of penicillin V acylase from Streptomyces lavendulae in water-organic cosolvent monophasic systems depends on the nature of the organic solvent and its concentration in the media. The threshold solvent concentration (at which half enzymatic activity is displayed) is related to the denaturing capacity of the solvent. We found out linear correlations between the free energy of denaturation at 40 degrees C and the concentration of the solvent in the media. On one hand, those solvents with logP values lower than -1.8 have a protective effect that is enhanced when its concentration is increased in the medium. On the other hand, those solvents with logP values higher than -1.8 have a denaturing effect: the higher this value and concentration, the more deleterious. Deactivation constants of PVA at 40 degrees C can be predicted in any monophasic system containing a water-miscible solvent.     

Differences in the capacity of male odours to affect investigatory behaviour and different urinary marking patterns in two strains of mice,selectively bred for high and low aggressiveness.

The investigatory behaviour of male and female mice on sawdust soiled by male mice with different levels of aggressiveness was studied in two experiments. To investigate whether high and low aggressive males show different urinary marking patterns, a third experiment was set up. The strains TA(Turku Aggressive) and TNA (Turku Nonaggressive) have been developed by selective breeding. The TA-soiled bedding discouraged investigation by male mice, while females avoided TNA-soiled areas. Also the urine marking patterns differed between the high aggressive TA and low aggressive TNA males. The results indicate that the urine marking behaviour and the odour communication system in the TA and TNA males correlate with their hereditarily determined disposition for aggressive behaviour.     

Effect of amides and ureas on the reversible gelation of arachin

The effect of formamide and urea and their amino-substituted derivatives dimethyl formamide and tetramethyl urea (at 1 m level) on thermal denaturation and protein protein interactions (at pH 3.6) that led to gelation of arachin were studied by gel melting temperature, electrophoresis, u.v. difference and fluorescence spectral measurements. Melting temperature and electrophoretic measurements showed that formamide and urea decreased the heat-induced protein-protein interactions while their methyl derivatives had the opposite effect. Melting temperature measurements also revealed a decrease in both -ΔH_bonding and -ΔS_bonding in the presence of formamide and urea while their methyl derivatives increased these thermodynamic parameters. In both the cases urea and tetramethyl urea had a greater effect on changing both the thermodynamic parameters compared with formamide and dimethyl formamide respectively. U.v. difference and fluorescence spectral measurements suggested that addition of formamide, urea and their methyl derivatives at 1 m level to orachin at pH 3.6 and room temperature induced unfolding. Addition of these compounds to the heated arachin solution at the same pH also promoted the thermal denaturation of the protein. The effectiveness followed the order tetramethyl urea > urea > dimethyl formamide > formamide. The promotive effect of formamide and urea on thermal denaturation and their preventive effect on the protein-protein interactions of arachin could be due to their favourable interaction with interpeptide hydrogen bonds. On the other hand, the promotive effect of dimethyl formamide and tetramethyl urea on the thermal denaturation of the protein may be due to their solubilization effect on the intraprotein hydrophobic interactions. The increase in protein-protein interactions in the presence of these compounds could be due to an increase in interprotein hydrogen bonding. This hypothesis of the mechanism of the additives on the heat-induced protein-protein interactions at pH 3.6 is consistent with the measured thermodynamic parameters of gelation.     

Synthesis of lovastatin with immobilized Candida rugosa lipase in organic solvents: Effects of reaction conditions on initial rates

Lipase from Candida rugosa immobilized on a nylon support has been used to synthesize lovastatin, a drug which lowers serum cholesterol levels, by the regioselective acylation of a diol lactone precursor with 2-methylbutyric acid in mixtures of organic solvents. Analogs of lovastatin having a different side chain were also obtained through this method by reacting the diol substrate with different carboxylic acids. The selection of reaction conditions that maximize the initial reaction rate is investigated. Since the diol substrate has very low solubility in non-polar solvents, reaction solvents consisting of mixtures of hexane with a different, more polar cosolvent are considered. For each of the cosolvent mixtures studied, the reaction rate is maximum for an intermediate percentage of cosolvent in hexane. With total concentrations of the diol lactone in the range 6.25-12.5 mM, maximum initial rates correspond approximately to those cosolvent concentrations that permit a complete solubilization of the substrate. At higher cosolvent concentrations, lower rates are obtained. When considering the same dissolved substrate concentration, the reaction rate was found to increase with increasing values of logP(mix) and decreasing values of the dielectric constant, when varying the composition of a binary solvent mixture. However, when comparing different cosolvents, no general trend with respect to these properties was observed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56:671-680, 1997.     

Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies

Effect of six organic solvents—methanol, ethanol, propanol, dimethyl sulphoxide (DMSO), N,N-dimethyl formamide (DMF), and glycerol on the conformation and interaction of catalase and anticatalase antibodies were studied with the aim of identifying the solvents in which antigen–antibody interactions are strong. The antigen binding activity of the antibodies in the various organic solvents increased in the following order: ethanol < methanol < no organic solvent < propanol < DMSO < DMF < glycerol. The structure of both the antibody and the antigen molecule was affected significantly in 40% concentration of the organic solvents used in this study. Catalase activity was inhibited in DMSO. However, the enzyme was activated in DMF upto about 50% of its concentration.     

Amyloid Fibrillation of Insulin under Water-Limited Conditions

Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation.     

Amyloid Fibrillation of Insulin under Water-Limited Conditions

Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation.     

Inhibitory effect of organic solvents on the mutagenicity of N-nitrosodialkylamines in Salmonella

The influence of organic solvents on the mutagenicity of 11 N-nitrosamines was examined in Salmonella typhimurium TA100 using the Ames's liquid incubation assay in the presence of rat-liver S9. The mutagenic activities of N-nitrosodimethylamine, N-nitrosodiethylamine, 6 oxidative derivatives of N-nitrosopropylamine and N-nitroso-2,6-dimethylmorpholine were considerably decreased by addition of dimethyl sulfoxide, dimethyl formamide, acetone, 95% ethanol or acetonitrile, which are recommended for use as solvents in the assay by Ames's group, to the incubation mixture. The mutagenic activities of N-nitrosodipropylamine and N-nitrosodibutylamine, which are barely soluble in water, were also suppressed by increasing concentrations of dimethyl sulfoxide. These organic solvents did not appear to exert their influence by desmutagenic and antimutagenic actions. In contrast, the recoveries of unmetabolized carcinogens from preincubation mixtures and from agar plates were significantly higher in the presence of organic solvents than in their absence. The results indicate that the inhibitory effect is a result of interference with the process of metabolic activation by liver S9.     

Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides.

N-acyloxy-N-alkoxybenzamides are mutagenic in TA100 without the need for metabolic activation with S9. Electronic effects of substituents on both the benzamide ring in N-acetoxy-N-butoxybenzamides or the benzyloxy ring in N-acetoxy-N-benzyloxybenzamides do not influence mutagenicity levels. For N-benzoyloxy-N-benzyloxybenzamides, mutagenicity levels are inversely related to the electron-withdrawing effect of substituents on the benzoyloxy leaving group. Since reactivities increase with increasing electron-withdrawing effects, mutagenicity correlates with stability rather than reactivity of these mutagens. Hydrophobicity is the dominant factor controlling mutagenicity levels and data for all mutagens correlate with computed logP values with a lower dependence (h=0.22) than that recorded for indirect mutagens (h=1.0), except where a sterically demanding p-tert-butyl substituent or a naphthyl group is present. N-acetoxy-N-butoxynaphthamide exhibits a much higher level of mutagenicity than predicted by its logP value and activity may be ascribed to an intercalative binding process with DNA rather than straightforward hydrophobic binding in the major or minor groove. Since these are direct-acting mutagens, structural factors influence binding and reactivity towards DNA.     

Some properties of erythrocuprein treated by organic solvents.

The effect of various types of organic solvents on the properties of bovine erythrocuprein was studied. Three organic solvents were found in which the protein is soluble, these were: dimethyl sulfoxide, formamide and N-methylformamide. It was shown that in formamide and dimethyl sulfoxide media the protein possees superoxide dismutase activity, but in N-methylformamide the protein has negligible activity. In organic solvents the substrate (superoxide radical) and solvated electron result in reduction of the protein copper. At high concentrations of superoxide radical or solvated electrons an inactivation of protein and stabilization of superoxide radicals was noted. The stabilization is most pronounced in N-methylformamide. The protein that is reduced by the radical or the solvated electron may be reoxidized by molecular oxygen, the latter being reduced to the superoxide radical.     

The osmoprotectants glycine and its methyl derivatives prevent the thermal inactivation of protective antigen of Bacillus anthracis

Protective antigen (PA) is the main immunogenic constituent of all vaccines against anthrax. It is known to lose its biological activity even at 37 degrees C. Its thermolabile nature has, thus, remained a cause of concern as even transient exposure of the vaccine to higher temperature could compromise its efficacy. Various types of cosolvent excipients have been used to stabilize a number of proteins with variable success. However, no comprehensive and systematic study to stabilize anthrax PA molecule using this approach has ever been undertaken. We have carried out a systematic study on the effect of osmoprotectants like glycine and its methyl derivatives, sarcosine, dimethylglycine, and betaine, on the thermostability of PA. The thermal stability of PA was found to be highly sensitive to pH with maxima at pH 7.9. All the cosolvent additives used were able to enhance the thermal stability of PA as inferred from an increase in T(1/2) values, the temperature at which 50% activity was retained during short-term incubation. Glycine was found to be the best stabilizer, while the ability of its methyl derivatives to stabilize PA decreased with an increase in the number of substituted methyl groups suggesting perturbation of hydrophobic interactions. On extended incubation at 40 degrees C the half-life of PA thermal inactivation increased more than four times in the presence of glycine. Thus, glycine could be used as an effective stabilizer to enhance the shelf life of recombinant vaccine against anthrax.     

Temporin A and its retro-analogues: synthesis, conformational analysis and antimicrobial activities.

Temporin A (TA) is a hydrophobic peptide isolated from the skin of the European red frog Rana temporaria. Strong antimicrobial activity against gram-positive cocci and Candida, as well as its small molecular weight (10-13 aa residues), makes TA an interesting antimicrobial compound. However, its synthesis is rather problematic. Here, the synthesis of two retro-analogues of TA--retro-TA and (6-1)(7-13)-TA--is reported. The synthesis of retro-TA was performed without any problems, while during the synthesis of (6-1)(7-13)-TA problems similar to those encountered during the synthesis of TA were faced. Antimicrobial assays showed minimal inhibitory concentration (MIC) values of retro-TA to be, in most cases, only one dilution higher than those of original TA, but still remained relatively low. An analysis of the circular dichroism spectra of the peptides shows that TA and (6-1)(7-13)-TA adopt an alpha-helical structure in a hydrophobic environment, while retro-TA forms mainly unordered conformation under both hydrophobic and hydrophilic conditions. One can postulate that differences in conformation of the peptide chain might be responsible for the lower antimicrobial activity of retro-TA as compared to that of the parent molecule. In any case, retro-TA can be interesting owing to its simple and nonproblematic synthesis.     

One pot synthesis of pyrimidine and bispyrimidine derivatives and their evaluation for anti-inflammatory and analgesic activities

A number of pyrimidine derivatives (1-10) have been synthesized by condensation of 4-isothiocyanato-4-methylpentan-2-one with furfurylamine, histamine, 1-(3-aminopropyl)imidazole, 1-(3-aminopropyl)-2-pyrrolidinone, 2-aminobenzonitrile and 3-isothiocyanatobutanal with 1-(3-aminopropyl)-2-pyrrolidinone and 2-hydrazinopyridine under different reaction conditions. Various bispyrimidine derivatives (11-15) were obtained by condensation of 4-isothiocyanato-4-methylpentan-2-one with 2,4,8,10-tetraoxaspiro[5,5]undecane3,9-dipropamine (11'), 1,4-bis(3-aminopropyl)piperazine (13'), 3,5-diamino 1,2,4-triazole (15') and 3-isothiocyanatobutanal with 2,4,8,10-tetraoxaspiro[5,5]undecane 3,9-dipropamine, 1,4-bis(3-aminopropyl)piperazine. All these compounds were characterized by correct FT-IR, (1)H NMR, MS and elemental analysis. These compounds were screened for anti-inflammatory and analgesic activities. Anti-inflammatory activity of 3 is comparable while analgesic activity was found to be better than that of standard drug.     

Polyethylene glycol-modified enzymes trap water on their surface and exert enzymic activity in organic solvents

Summary Polyethylene glycol-modified enzymes dissolved and had high enzymic activity in organic solvents. A trace amount of water was found to be necessary for the activity. It was reasoned that the amphipathic polymer covalently attached to enzymes kept water molecules around them. This was supported by findings that : (1) high enzymic activity was found in water- immiscible solvents, whereas activity was never observed in water-miscible solvents; (2) enzymic activity was inhibited by increasing the concentration of dimethyl sulfoxide in benzene; (3) activity of lipase was inhibited by a water-miscible alcohol substrate, but was steadily elevated by increasing the concentration of a water-immiscible alcohol substrate; (4) water was not absorbed from benzene solution containing a modified enzyme by molecular sieves, while it was easily absorbed in the presence of a water-miscible organic solvent, dimethyl sulfoxide.     

Antibacterial activities of temporin A analogs

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL-NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and -resistant Staphylococcus aureus as well as vancomycin-resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N-terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all-D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non-chiral interactions with membrane lipids.     

Effect of additives on the thermostability ofBacillus stearothermophilus α-amylase

The effect of additives on the thermostability ofBacillus stearothermophilus -amylase was determined. Polyols, dimethyl formamide, and dimethyl sulfoxide all increased the half life of the enzyme approximately 2-fold when tested at a 10% (w/v) addition. These results suggest that the enzyme's structure is stabilized against thermal denaturation through ionic interactions. Addition of dextran or polyvinyl alcohol (hydrophilic polymers which increase the viscosity of the solution) had a slight positive effect on enzyme stability while addition of polyethylene glycol or polyvinylpyrrolidone (hydrophobic polymers which increase the viscosity of the solution) resulted in a 2-fold decrease in enzyme half life.