Previous studies of the oligoacyllysyl (OAK) series acyl-lysyl-lysyl-aminoacyl-lysine-amide, suggested their utility towards generating robust linear lipopeptide-like alternatives to antibiotics, although to date, none exhibited potent broad-spectrum bactericidal activity. To follow up on this premise, we produced a new analog (C14KKc12K) and investigated its properties in various media. Mechanistic studies suggest that C14KKc12K uses a non-specific membrane-disruptive mode of action for rapidly reducing viability of Gram-negative bacteria (GNB) similarly to polymyxin B (PMB), a cyclic lipopeptide used as last resort antibiotic. Indeed, C14KKc12K displayed similar affinity for lipopolysaccharides and induced cell permeabilization associated with rapid massive membrane depolarization. Unlike PMB however, C14KKc12K was also bactericidal to Gram-positive bacteria (GPB) at or near the minimal inhibitory concentration (MIC), as assessed against a multispecies panel of >50 strains, displaying MIC50 at 3 and 6 µM, respectively for GPB and GNB. C14KKc12K retained activity in human saliva, reducing the viability of cultivable oral microflora by >99% within two minutes of exposure, albeit at higher concentrations, which, nonetheless, were similar to the commercial gold standard, chlorhexidine. This equipotent bactericidal activity was also observed in pre-formed biofilms of Streptococcus mutans, a major periodontal pathogen. Such compounds therefore, may be useful for eradication of challenging poly-microbial infections.

Kappa-casein (κCN) and beta-casein (βCN) are disordered proteins present in mammalian milk. In vitro, βCN self-assembles into core-shell micelles. κCN self assembles into similar micelles, as well as into amyloid-like fibrils. Recent studies indicate that fibrillization can be suppressed by mixing βCN and κCN, but the mechanism of fibril inhibition has not been identified. Examining the interactions of native and reduced kappa-caseins (N-κCN and R-κCN) with βCN, we expose a competition between two different self-assembly processes: micellization and fibrillization. Quite surprisingly, however, we find significant qualitative and quantitative differences in the self-assembly between the native and reduced κCN forms. Specifically, thermodynamic analysis reveals exothermic demicellization for βCN and its mixtures with R-κCN, as opposed to endothermic demicellization of N-κCN and its mixtures with βCN at the same temperature. Furthermore, with time, R-κCN/βCN mixtures undergo phase separation into pure βCN micelles and R-κCN fibrils, while in the N-κCN/βCN mixtures fibril formation is considerably delayed and mixed micelles persist for longer periods of time. Fibrils formed in N-κCN/βCN mixtures are shorter and more flexible than those formed in R-κCN/βCN systems. Interestingly, in the N-κCN/βCN mixtures, the sugar moieties of N-κCN oligomers seem to organize on the mixed micelles surface in a manner similar to the organization of κCN in milk casein micelles.

Supramolecular chiral assemblies of R(-) and S(+) 2-butanol, in their neat form or when dissolved in their nonchiral isomer isobutanol, were evaluated by isothermal titration calorimetry (ITC) ensuing mixing. Dilution of 0.5 M solution of R(-) 2-butanol in isobutanol into the latter liberated heat of several calories per mole, which was approximately double than that obtained in parallel dilutions of S(+) 2-butanol in isobutanol. The ITC dilution profiles indicated an estimate of about 100 isobutanol solvent molecules surrounding each of the 2-butanol enantiomers, presumably arranged in chiral configurations, with different adopted order between the isomers. Mixings of neat R and S 2-butanol were followed by endothermic ITC profiles, indicating that, in racemic 2-butanol, both the supramolecular order and the intermolecular binding energies are lower than in each of the neat chiral isomers. The diversion from symmetrical ITC patterns in these mixings indicated again a subtle difference in molecular organization between the neat enantiomers. It should be noted that the presence of impurities, α-pinene and teterhydrofuran, at a level totaling 0.5%, did not influence the ITC heat flow profiles. The findings of this study demonstrate for the first time that chiral solutes in organic solvents are expected to acquire asymmetric solvent envelopes that may be different between the enantiomers, thus broadening this phenomenon beyond the previously demonstrated cases in aqueous solutions.

Amphiphilic block copolymers and mixtures of amphiphiles find broad applications in numerous technologies, including pharma, food, cosmetic and detergency. Here we report on the interactions between a biological charged diblock copolymer, β-casein, and a synthetic uncharged triblock copolymer, Lutrol F-127 (EO(101)PO(56)EO(101)), on their mixed micellization characteristics and the micelles' structure and morphology. Isothermal titration calorimetry (ITC) experiments indicate that mixed micelles form when Lutrol is added to monomeric as well as to assembled β-casein. The main driving force for the mixed micellization is the hydrophobic interactions. Above β-casein CMC, strong perturbations caused by penetration of the hydrophobic oxypropylene sections of Lutrol into the protein micellar core lead to disintegration of the micelles and reformation of mixed Lutrol/β-casein micelles. The negative enthalpy of micelle formation (ΔH) and cooperativity increase with raising β-casein concentration in solution. ζ-potential measurements show that Lutrol interacts with the protein micelles to form mixed micelles even below its critical micellization temperature (CMT). They further indicate that Lutrol effectively masks the protein charges, probably by forming a coating layer of the ethyleneoxide rich chains. Small-angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM) indicate relatively small changes in the oblate micellar shape, but do show swelling along the small axis of β-casein micelles in the presence of Lutrol, thereby confirming the formation of mixed micelles.

beta-Casein is an intrinsically unstructured amphiphilic protein that self-assembles into micelles at neutral pH. This paper reports that beta-casein self-organizes into micelles also under acidic conditions. The protein association behavior and micelle characteristics at pH 2.6, well below the pl, are presented. The pH was found to strongly affect the micelle shape and dimensions. Cryogenic transmission electron microscopy (cryo-TEM) experiments revealed disk-like micelles of 20-25 nm in length and similar to 3.5 nm in height in acidic conditions. An aggregation number of 6 was determined by sedimentation equilibrium under these conditions. Isothermal titration calorimetry experiments verified the association below the pl and allowed determination of the micellization enthalpy, the critical micellar concentration, and the micellization relative cooperativity (MR). Small-angle X-ray scattering results at concentrations below the critical micellization concentration (CIVIC) suggest that the monomeric protein is likely in a premolten globule state at low pH. Calculations of the protein charge at acidic and neutral pH reveal a similar high net charge but considerable differences in the charge distribution along the protein backbone. Overall the results show that beta-casein is amphiphilic at low pH, but the distribution of charge along the protein chain creates packing constraints that affect the micelle organization, leading at concentrations above the CIVIC to the formation of disk micelles.

Truncation and acylation were combined to investigate the broad-spectrum bactericidal and hemolytic peptide S4(1–15). Substitution of up to seven residues with dodecanoic acid (C12) gradually led to specific antipseudomonal activity: out of 40 bacterial strains tested in vitro, C12-S4(8–15) displayed similar minimal inhibitory concentrations (MICs) as S4(1–15) against Pseudomonas aeruginosa sp. (identical MIC90) but was practically inactive against most other bacteria or erythrocytes. Surface plasmon resonance and isothermal titration calorimetry experiments revealed the binding properties of S4(1–15) to be consistent with its nonselective activities, while discriminative activities of C12-S4(8–15) correlated with high binding affinity to a membrane containing pseudomonal lipopolysaccharides and with lower affinities to membranes containing nonpseudomonal lipopolysaccharides or cholesterol. Various mechanistic studies failed to detect significant differences in secondary structure, bactericidal kinetics, or ability to perturb the cytoplasmic membrane, pointing to a similar mode of action.

The assumption that the thermal effect (heating) is the sole factor that should be considered when a microwave source is applied has been debated by many reports, often claiming that athermal (non-thermal) effects exist as well. Such effects are claimed to change the chemical, biochemical, or the physical behaviour of some systems while the temperature and all other parameters remain unaltered. The possibility of an athermal effect was tested in a number of chemical, biological and physical systems in a very well controlled, high radiation intensity system (2.45GHz, up to 1000W/kg, with continuous radiation up to 48h). The systems that were tested included: Maillard reaction, protein denaturation and polymer solubility, mutagenesis of bacteria, mutarotation equilibrium of α/β-d-glucose, and saturation solubility of NaCl. All data failed to show any significant athermal effects. The results of this study are in contrast to what has been previously reported for some of the tested systems.

A decoction of leaves and unripe fruits prepared from C. aronia syn. azarolus (L) (Rosaceae), the indigenous Israeli hawthorn, is used to treat diabetes in Arab traditional medicine. There is a growing body of evidence reporting the presence of systemic oxidative stress in insulin-dependent and non-insulin dependent diabetes. We have already reported that this decoction has antioxidant properties in vitro. In this report, we extended these in vitro observations to study its antioxidant action in the streptozotocin (STZ)-induced diabetic rat in which we measured its effect on plasma and tissue indices of oxidative stress, as well as blood glucose levels. In an experiment lasting 24 days, 30 diabetic and 30 control rats were each subdivided into three separate groups. Group 1 rats drank tap water, group 2 rats drank the decoction containing 50 mg grounded dried plant/mL and group 3 rats drank the decoction containing 350 mg grounded dried plant/mL. Upon completion of the treatment period, the rats were anesthetized and blood glucose was measured using a drop of blood obtained by tail vein puncture. The abdomen was opened and aortic blood was withdrawn for measurement of the plasma levels of lipid peroxides, glutathione, cholesterol and triglycerides and the livers were harvested for determination of the extent of lipid peroxidation. The results of these experiments show that a decoction prepared from the leaves and unripe fruits of C. aronia normalizes plasma lipid peroxide levels and lowers blood glucose levels in diabetic rats. These findings indicate that the therapeutic benefit of the decoction could be due to these two actions, thereby providing a basis for its use in treating diabetes in traditional Arab medicine.

The association behavior, critical micellization concentration (CMC), and enthalpy of demicellization (DeltaHdemic) of bovine beta-casein were studied, for the first time by isothermal titration calorimetry, in a pH 7.0 phosphate buffer with 0.1 ionic strength and in pure water. In the buffer solutions, the CMC decreased asymptotically from 0.15 to 0.006 mM as the temperature was raised from 16 to 45 degrees C. DeltaHdemic decreased with increasing temperature between 16 and 28 degrees C but increased from 28 to 45 degrees C. Thermodynamic analysis below 30 degrees C is consistent with the Kegeles shell model, which suggests a stepwise association process. At higher temperatures, this model exhibits limitations, and the micellization becomes much more cooperative. The CMC values in water, measured between 17 and 28 degrees C, decreased with increasing temperature and, expectedly, were higher than those found in the buffer solutions. beta-Casein micelles were visualized and characterized, for the first time in their hydrated state, using advanced digital-imaging cryogenic transmission electron microscopy. The images revealed small, oblate micelles, about approximately 13 nm in diameter. The micelles shape and dimensions remained nearly constant in the temperature range of 24-35 degrees C.

Teucrium polium L. (Lamiaceae) (RDC 1117) is a medicinal plant whose species have been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, tonic, antipyretic, antispasmodic and cholagogic properties. The therapeutic benefit of medicinal plants is often attributed to their antioxidant properties. We previously reported that an aqueous extract of the leaves and stems of this plant could inhibit iron-induced lipid peroxidation in rat liver homogenate at concentrations that were not toxic to cultured hepatic cells. Others have reported that organic extracts of the aerial components of this plant could inhibit oxidative processes. Against this background, we felt further investigation on the antioxidant action of the extract of T. polium prepared according to traditional Arab medicine was warranted. Accordingly, we assessed (i) its ability to inhibit (a) oxidation of β-carotene, (b) 2,2′-azobis(2-amidinopropan) dihydrochloride (AAPH)-induced plasma oxidation and (c) iron-induced lipid peroxidation in rat liver homogenates; (ii) to scavenge the superoxide ($${\hbox{ O }}_{2}^{\bullet -}$$) radical and the hydroxyl radical (OH•); (iii) its effects on the enzyme xanthine oxidase activity; (iv) its capacity to bind iron; and (v) its effect on cell glutathione (GSH) homeostasis in cultured Hep G2 cells. We found that the extract (i) inhibited (a) oxidation of β-carotene, (b) AAPH-induced plasma oxidation (c) Fe2+-induced lipid peroxidation in rat liver homogenates (IC50 = 7 ± 2 μg ml−1); (ii) scavenged $${\hbox{ O }}_{2}^{\bullet -}$$(IC50 = 12 ± 3 μg ml−1) and OH• (IC50 = 66 ± 20 μg ml−1); (iii) binds iron (IC50 = 79 ± 17 μg ml−1); and (iv) tended to increase intracellular GSH levels resulting in a decrease in the GSSG/GSH ratio. These results demonstrate that the extract prepared from the T. polium possesses antioxidant activity in vitro . Further investigations are needed to verify whether this antioxidant effect occurs in vivo .

In response to increased popularity and greater demand for medicinal plants, a number of conservation groups are recommending that wild medicinal plants be brought into cultivation systems. We collected four medicinal herbs Cichorium pumilum, Eryngium creticum, Pistacia palaestina and Teucrium polium used in traditional Arab medicine for greenhouse cultivation to assess the effects of different fertilization regimes on their growth and antioxidant activity. Wild seedlings were collected and fertilized with either 100% Hoagland solution, 50% Hoagland solution, 20% Hoagland solution or irrigated with tap water. Plant height was measured and the number of green leaves and branches counted weekly. Thereafter, the aboveground parts of plants were harvested for preparing a water-soluble powder extracts of which antioxidant activity was measured by their ability to suppress the oxidation of β-carotene. Of the fertilization regimes, we found either 20 or 50% Hoagland solution produced the most consistent response of the plant growth parameters. All powders prepared from the four wild growing plants inhibited oxidation of β-carotene. Increasing the amount of fertilizer caused a significant concentration-dependent increase in antioxidant activity of the cultivated T. polium compared with the wild type. In contrast, increasing the amount of fertilizer caused a significant concentration-dependent reduction in the antioxidant activity of powders prepared from the cultivated E. creticum when compared with wild plants. Our results showed that cultivation success should not rely solely on parameters of growth but should incorporate assessment related to indices of therapeutic potential.

Relating thermodynamic parameters to structural and biochemical data allows a better understanding of substrate binding and its contribution to catalysis. The analysis of the binding of carbohydrates to proteins or enzymes is a special challenge because of the multiple interactions and forces involved. Isothermal titration calorimetry (ITC) provides a direct measure of binding enthalpy (DeltaHa) and allows the determination of the binding constant (free energy), entropy, and stoichiometry. In this study, we used ITC to elucidate the binding thermodynamics of xylosaccharides for two xylanases of family 10 isolated from Geobacillus stearothermophilus T-6. The change in the heat capacity of binding (DeltaCp = DeltaH/DeltaT) for xylosaccharides differing in one sugar unit was determined by using ITC measurements at different temperatures. Because hydrophobic stacking interactions are associated with negative DeltaCp, the data allow us to predict the substrate binding preference in the binding subsites based on the crystal structure of the enzyme. The proposed positional binding preference was consistent with mutants lacking aromatic binding residues at different subsites and was also supported by tryptophan fluorescence analysis.

Bio-imprinting of lipases with fatty acids was shown to be a feasible, effective method for obtaining highly active enzymes in organic solvents. The increase in activity was dependent on the enzyme type, the solvent type and the imprint molecule itself. A correlation between the initial activity of caprylic acid-imprinted Candida rugosa lipase (CRL), and solvent hydrophobicity was observed. In addition, the combination of bio-imprinting with adsorption onto an inert support such as celite, proved to be a powerful technique for obtaining an even more active and stable enzyme preparations. In the case of lipase from Pseudomonas sp., the increase in activity resulting from bio-imprinting with caprylic acid and immobilization onto celite, was 20-fold. Porcine pancreatic lipase (PPL), treated in the same manner, retained 70% of its initial activity at the end of 20 consecutive reaction cycles, compared to only 20% residual activity for the non-treated control.

A fused protein consisting of a cellulose-binding domain (CBD) and horseradish peroxidase (HRP) was bound to cellulose beads and evaluated in aqueous-organic solvent systems. The CBD–HRP fusion protein containing two different functionalities, a catalytic domain and a binding domain, preserved both capabilities in this non-conventional environment. A six-fold increase in the half-life of the enzyme in buffer resulted from immobilization onto cellulose via CBD. The immobilized enzyme was also more stable than the native enzyme in increasing concentrations of acetone (0–92%). There was a general decrease in activity as the solvent concentration in the mixture increased (in all solvent types: THF, acetone, acetonitrile and ethanol). However, the immobilized enzyme was at all times more active than the soluble enzyme forms. The thermostability of the enzyme in buffer, at 40–60°C, was also improved by immobilization. The soluble CBD–HRP fusion protein exhibited greater stability (both to organic solvents and temperature), but lower activity, in comparison with the native HRP. This work demonstrates for the first time the use of a cellulose-bound CBD-enzyme as a catalyst in aqueous-organic solvent media.

To the Editor: About 9000 Trilucent soybean oil–filled breast implants (SOBIs) (LipoMatrix Inc, Neuchâtel, Switzerland) were implanted in about 5000 patients in Britain and 700 in Switzerland. In the United States, about 200 women were enrolled in a clinical study approved by the Food and Drug Administration to evaluate the safety and effectiveness of these implants. Reports indicating a high rupture rate of SOBIs and possible long-term adverse effects resulting from chemical breakdown of the filler prompted the British Medical Devices Agency (MDA) to advise against the further use of SOBIs in March 1999. In June 2000, after new data regarding high concentrations of potentially genotoxic and carcinogenic components from the breakdown of soybean oil filler, the MDA issued a hazard notice recommending that all women with SOBIs have them removed. This recommendation was adopted by the European Committee on Quality Assurance and Medical Devices in Plastic …

An efficient two-step enzymatic process for production of (R)- and (S)-ethyl-3-hydroxybutyrate (HEB), two important chiral intermediates for the pharmaceutical market, was developed and scaled-up to a multikilogram scale. Both enantiomers were obtained at 99% chemical purity and over 96% enantiomeric excess, with a total process yield of 73%. The first reaction involved a solvent-free acetylation of racemic HEB with vinylacetate for the production of (S)-HEB. In the second reaction, (R)-enriched ethyl-3-acetoxybutyrate (AEB) was subjected to alcoholysis with ethanol to derive optically pure (R)-HEB. Immobilized Candida antarctica lipase B (CALB) was employed in both stages, with high productivity and selectivity. The type of butyric acid ester influenced the enantioselectivity of the enzyme. Thus, extending the ester alkyl chain from ethyl to octyl resulted in a decrease in enantiomeric excess, whereas using bulky groups such as benzyl or t-butyl, improved the enantioselectivity of the enzyme. A stirred reactor was found unsuitable for large-scale production due to attrition of the enzyme particles and, therefore, a batchwise loop reactor system was used for bench-scale production. The immobilized enzyme was confined to a column and the reactants were circulated through the enzyme bed until the targeted conversion was reached. The desired products were separated from the reaction mixture in each of the two stages by fractional distillation. The main features of the process are the exclusion of solvent (thus ensuring high process throughput), and the use of the same enzyme for both the acetylation and the alcoholysis steps. Kilogram quantities of (S)-HEB and (R)-HEB were effectively prepared using this unit, which can be easily scaled-up to produce industrial quantities.