Effect of the Compositions of Sample and Polymer Sorbents on the Extraction of Volatile Compounds by Solid-Phase Microextraction

A comparative study was performed by solid phase microextraction and capillary gas chromatography to establish the ability of four polymer sorbents of different compositions to extract and concentrate volatile organic compounds from the gas phase above an aqueous solution. All polymer sorbents sorbed nonpolar monoterpene hydrocarbons via a cooperative mechanism with almost equal and high efficiency. Sorbents based on polymethyl disiloxane and its mixture with divinylbenzene were more effective in extracting acetates and sesquiterpenes. As the concentration of these compounds in the gas phase increased, their binding by sorbents decreased. It was found that the determination of polar compounds depended on the presence of a solvent in the system. Compounds that are highly soluble in water (alcohols, ketones, etc.) had low coefficients of distribution between gas and water phases. Consequently, their sorption to any of the polymer sorbents was negligible. In the absence of the solvent, the degree of their extraction from the gas phase above the sample was high. It was shown that the actual composition of compounds in the initial mixture of essential oils could significantly differ from their composition in the gas phase. This method is convenient and informative for the purpose of studying the composition of volatile compounds in the gas phase that determine the flavor of the product.     

Conservativeness of residues in nonpolar nuclei of microbial ribonucleases]

We present the application of a new method for analysis of nonpolar structure of proteins. A detailed analysis of the composition and properties of nonpolar nuclei and microclusters of microbial ribonucleases with known sequence have been carried out on the basis of 3D-structure of RNase Pbi and that of RNase Ti. It has been shown that all residues in nonpolar nuclei have high homology, about 95% for proteins with an identical scheme of S-S bridges and about 75% for nonidentical scheme of S-S bridges. The stability of nonpolar nuclei, conservation of their composition and their position in the protein globule allows one to assume that they play an important functional role in protein structure and possibly can be considered as independent structural elements of 3D-structure of a protein.     

Mechanical spectroscopy of karaya gum—alginate mixed dispersions

The rheological properties of blends obtained by mixing alginate and karaya gum dispersions were described from oscillation, shear and creep experiments using a controlled stress rheometer. The mechanical spectra shown by such mixtures were strongly modified when compared with those of each of the blend components.

It was found that the viscoelastic behaviour of mixtures was strongly affected by the age of the karaya gum. Contrary to that which is observed with fresh karaya gum, maximum synergy was observed with a mixture containing 75% aged karaya gum. It is suggested that mutual incompatibility between the two polysaccharides could explain the formation of a mixed network.     

Ribosome binding to the membranes of Candida albicans]

The in vitro binding of free ribosomes to the membranes of polyenoic antibiotics-sensitive and resistant strains of Candida albicans, considerably differing in the composition of their sterol components, was studied. It was shown that the extent and type of membrane-ribosome interaction is similar in both cases. The dependence of binding on ribosome concentration in the incubation mixture is double-phase, as was shown for different membrane fractions of both sensitive and resistant strains. The role of lipid membrane components, particularly that of sterols in the membrane-ribosome interactions is discussed.     

Anionic phospholipids are essential for alpha-helix formation of the signal peptide of prePhoE upon interaction with phospholipid vesicles.

The conformational consequences of the interaction of the PhoE signal peptide with bilayers of different types of phospholipids was investigated using circular dichroism. It was found that interaction of the signal peptide with anionic phospholipid vesicles of dioleoylphosphatidylglycerol and dioleoylphosphatidylserine results in induction of high amounts of alpha-helical structure of 70% and 57%, respectively. Upon addition of the signal peptide to cardiolipin vesicles, less but still significant alpha-helical structure was induced (29%). In contrast, no alpha-helix formation was observed upon the interaction of the signal peptide with zwitterionic dioleoylphosphatidylcholine vesicles. In bilayers of dioleoylphosphatidylcholine with dioleoylphosphatidylglycerol, it was shown that in the presence of 100 mM NaCl a minimum amount of 50% of negatively charged lipid was required for induction of the maximal percentage of alpha-helix, whereas in the absence of salt a minimum amount of 35% of negatively charged lipid was necessary. Induction of alpha-helix structure appeared to be correlated with functionality, since, in a less functional analogue of the PhoE signal peptide, the PhoE-[Asp-19,20] signal peptide, less alpha-helix was induced than in the wild-type PhoE signal peptide. It is proposed that the interaction with anionic phospholipids is essential for a functional conformation of the PhoE signal sequence during protein translocation.     

In vitro assessment of antimicrobial activity of carvacrol, thymol and cinnamaldehyde towards Salmonella serotype Typhimurium DT104: effects of pig diets and emulsification in hydrocolloids

AIMS: To determine the effect of pig diets in vitro on the antimicrobial activity of carvacrol, thymol and cinnamaldehyde, and to identify an emulsifier/stabilizer that can stabilize the essential oil (EO) components in aqueous solution and retain their antimicrobial activity in the presence of the diets. METHODS AND RESULTS: Emulsification of essential oil components with hydrocolloid solution was achieved by blending with a Polytron. Antimicrobial activity was measured through in vitro assays to determine the inhibition of bacterial growth by measuring the optical density at 600 nm or plating on nutrition agar after incubation of the mixtures of an EO component with the culture of Salmonella serotype Typhimurium DT104 in the presence or absence of pig diets. The results generated through the in vitro assays indicated that pig diets were able to abolish the antimicrobial activity of EOs. Xanthan, fenugreek and yellow mustard gums were the best in forming stable emulsions of five different EO components among ten different plant polysaccharides and surfactants examined. Emulsification of all the EO components in the fenugreek gum solution did not alter their antimicrobial activity. However, the antimicrobial activity of geraniol was significantly reduced when emulsified with other polysaccharides and surfactants. Both fenugreek and xanthan gum solutions were unable to protect the antimicrobial activity of carvacrol and thymol when mixed with the diets. Although cinnamaldehyde required no emulsification, but a high concentration (equivalent to at least three times of minimum bactericidal concentration for cinnamon oil) to inhibit Salmonella growth significantly in the presence of the diets, emulsification in fenugreek gum appeared to be essential for cinnamaldehyde solution to retain its antimicrobial activity during storage. CONCLUSIONS: The diets for newly weaned pigs were a significant factor limiting the antimicrobial activity of EOs and their components. Cinnamaldehyde required a high concentration to retain its antimicrobial activity in the diets, in addition to its requirement for emulsification to stabilize its activity during the storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay with the diets used in this study for measuring the antimicrobial activity can be used in vitro for rapid and effective screening of potential antimicrobials for swine production. This study has identified polysaccharides that are able to stabilize EO component solutions. It has also identified cinnamaldehyde for further in vivo studies that may have potential in future application in controlling Salmonella and possibly other enteric pathogens in swine production.     

Insulin-Like Growth Factor I (IGF-I) Binding in Skeletal Muscle of Lamprey Lampetra fluviatilis Predominates over Insulin Binding and Increase in the Course of Pre-Spawning Migration

Binding of ¹²⁵I-insulin and ¹²⁵I-IGF-I to partially purified receptors of lamprey skeletal muscles was studied during pre-pawning migration. It has been shown that throughout this whole period the IGF-I binding to skeletal muscle predominates over the insulin binding. Besides, a certain time dynamics was observed: the insulin binding rose since October to reach maximum in February–March, then it decreased to a minimum level in May; the IGF-I binding also increased: it rose statistically significantly in March compared to October, became maximal in April, and then decreased to a minimum. The dynamics of the receptor IGF-I binding has been shown to depend on changes of receptor affinity, whereas the change of the insulin binding was determined by binding capacity (the number of binding sites). Highly specific IGF-I receptors of the lamprey skeletal muscle bound insulin with an affinity about 1% from that of IGF-I, while insulin receptors had identical affinity for the insulin and IGF-I binding. Both peptides, insulin and IGF-I, activated autophosphorylation of beta-subunits in their receptors. The increase of the IGF-I binding from October to April could be a factor that maintains a high functional activity of lamprey skeletal muscles in the course of the pre-pawning migration. It is suggested that IGF-I promotes maintaining this activity due to its property of inhibiting apoptosis.     

Influence of Guar Gum on the In Vitro Starch Digestibility—Rheological and Microstructural Characteristics

The present study investigates the effect of guar gum on the digestibility of a waxy maize starch in vitro under simulated gastric and intestinal conditions. A detailed rheology and confocal scanning laser microscopy of the digesta were performed in order to study the possible mechanisms of interactions involved during in vitro hydrolysis of starch. No starch hydrolysis was observed under simulated gastric conditions, whereas more than 90% hydrolysis was observed at the end of digestion under simulated intestinal conditions. In the presence of guar gum, the starch hydrolysis was reduced by nearly 25% during the first 10 min and by 15% at the end of in vitro intestinal digestion. The rheological behavior of the digesta was significantly affected in the presence of the gum. The viscosity of digesta decreased during intestinal digestion; however, the extent of decrease was quite low in the presence of guar gum. The consistency index increased and flow behavior index of digesta decreased in the presence of gum after 1 min of intestinal digestion. All the samples (digested or undigested) displayed a pseudoplastic behavior as their apparent viscosity (η _a) decreased with an increase in shear rate. A negative correlation between the starch hydrolysis (%) and storage modulus for the starch sample without gum and a positive correlation for the starch sample with gum were found. Large granule remnants observed through confocal micrographs have shown that the solubilization of starch granule remnants during in vitro digestion is significantly reduced in the presence of gum.     

Time-resolved fluorescence spectroscopy of human adenosine deaminase: effects of enzyme inhibitors on protein conformation

Adenosine deaminase, a purine salvage enzyme essential for immune competence, was studied by time-resolved fluorescence spectroscopy. The heterogeneous emission from this four-tryptophan protein was separated into three lifetime components: tau 1 = 1 ns and tau 2 = 2.2 ns an emission maximum at about 330 nm and tau 3 = 6.3 ns with emission maximum at about 340 nm. Solvent accessibility of the tryptophan emission was probed with polar and nonpolar fluorescence quenchers. Acrylamide, iodide, and trichloroethanol quenched emission from all three components. Acrylamide quenching caused a blue shift in the decay-associated spectrum of component 3. The ground-state analogue enzyme inhibitor purine riboside quenched emission associated with component 2 whereas the transition-state analogue inhibitor deoxycoformycin quenched emission from both components 2 and 3. The quenching due to inhibitor binding had no effect on the lifetimes or emission maxima of the decay-associated spectra. These observations can be explained by a simple model of four tryptophan environments. Quenching studies of the enzyme-inhibitor complexes indicate that adenosine deaminase undergoes different protein conformation changes upon binding of ground- and transition-state analogue inhibitors. The results are consistent with localized structural alterations in the enzyme.     

Chemical composition,intraspecies variation and seasonal variation in essential oils of Calendula arvensis L.

The essential oil composition of Calendula arvensis was established for the first time using GC and GC/MS. Eighty-five essential oil components were identified, which accounted for 90.3 g/100 g of essential oil. The oil contained a high concentration of sesquiterpenes, of which δ-cadinene and α-cadinol were the main components. The chemical composition of 25 Corsican C. arvensis oils was analyzed to determine intraspecies variation in essential oil composition. A matrix linking essential oil composition to sample location was composed to identify relationships between concentrations of volatile samples and the geographical origins of samples. Two main groups of compounds were identified according to the amount of sesquiterpenic compounds (hydrocarbons and alcohols) and soil characteristics. Seasonal variation (winter vs. spring) in the concentrations of two major compounds during the flowering period was observed.     

Gum ghatti: A promising polysaccharide for pharmaceutical applications

Gum ghatti (Anogeissus latifolia) or Indian gum is a complex non-starch polysaccharide. It has been widely employed in food, pharmaceuticals, paper and other industries primarily due to its excellent emulsification and thickening property. Other applications of gum ghatti are inadequately investigated owing to lack of information on it. Researchers in the recent years have shown a great interest in exploring its molecular structure and functional properties. This article is aimed at discussing the structural features, functional properties and applications of gum ghatti with an emphasis on its pharmaceutical potential.     

Equilibrium shift by target DNA substrates for determination of DNA binding ligands

An equilibrium containing the thiol derivative of Hoechst33258 (Ht-SH), glutathione (G-SH), and the corresponding homo and hetero disulfides was shifted by the addition of the duplex DNA. It was shown from the analysis of the components that the hetero disulfide Ht-SS-G increased by binding with the DNA (CA14) with an A(3)T(3) binding motif for the structure of Hoechst33258, and that the different equilibrium shift was observed in the presence of CT14 with no A(3)T(3) binding motif.     

Ligand-induced structuring of polyreactive immunoglobulins

The influence of several hydrophobic compounds with different structures on the binding of native and chaotropically modified bovine immunoglobulins to native and denatured nonspecific protein immobilized on the surface is researched. It is shown that the character of binding depends on the amount and mutual location of hydrophobic nuclei in the effectors structure. The authors observed moderate suppression of binding or absence of the expressed effect while using one-nucleus effector. The influence of compounds with two distanced benzene nuclei differs qualitatively from the influence of the compound with two condensate ones, described earlier: it was observed that the stimulation level of the binding depends on structural-functional condition of immunoglobulin and immobilized protein. In case of the effector with three spaced hydrophobic nuclei the stimulating effect is much more expressed (3-4 times higher). The concentration dependence of the ligand-induced effects is demonstrated. It is supposed, that stimulative influence of effectors with spaced hydrophobic nuclei is caused by two different processes. The first one is the formation of highly binding center as the result of ligand-induced structuring of immunoglobulins. The other one is the competition for the center formed between dissolved polynuclear effector and ligand groups of aminoacids hydrophobic residues statistically formed on the surface of immobilized protein.     

Regulation of ovarian steroidogenesis. The disparity between 125I-labelled choriogonadotropin binding cyclic adenosine 3',5'-monophosphate formation and progesterone synthesis in the rat ovary.

The binding of 125I-labelled human choriogonadotropin, formation of cyclic adenosine 3',5'-monophosphate (cyclic AMP), and synthesis of progesterone were examined in ovarian cells from immature rats. Collagenase dispersed ovarian cells were found to respond specifically to lutropin-like activity. The equilibrium dissociation constant (Kd) for the binding of 125I-for the binding of 125I-labelled choriogonadotropin was 1.7-10(-10) M. Progesterone synthesis was increased at least 40-fold and cyclic AMP formation 10-fold in response to maximum hormonal stimulation. The concentration of choriogonadotropin which stimulated progesterone synthesis maximally in Eagle's minimum essential medium--0.1% gelatin (2 ng/ml), resulted in minimal (less than 30% of maximum) increases in cyclic AMP accumulation and hormone binding. Similarly, binding of choriogonadotropin was not saturated at a hormone concentration (50 ng/ml) that stimulated maximal cyclic AMP formation. These results are consistent with the existence of receptor reserve in the ovarian cell. A marked shift in the dose vs. response relationship for progesterone synthesis occurred when fetal calf serum was used to supplement Eagle's minimum essential medium, however. Under these experimental conditions, progesterone synthesis reached a maximum at a hormone concentration of the same order of magnitude as did cyclic AMP formation. It is concluded that the degree of spare receptor effect observed may depend not only on an absolute amount of excess receptor, but also on the readiness of the system to respond in a given fashion.     

Inactivation of three monohydroxybenzoate mono-oxygenases from Rhodococcus erythropolis: The role of an arginine residue in the substrate-binding domain of p-hydroxybenzoate 3-hydroxylase

Abstract p-Hydroxybenzoate 3-hydroxylase from Rhodococcus erythropolis was inactivated by 2,3-butanedione (BD), phenylglyoxal (PGO), and other chemical reagents. p -Hydroxybenzoate and NADH protected the enzyme from inactivation by BD. Judging from the amino acid composition of BD-treated enzyme in the presence and absence of p -hydroxybenzoate, one essential arginine residue in substrate-binding domain of the enzyme was shown to be essential to the binding of p -hydrozybenzoate to the enzyme. Salicylate 5-hydroxylase and m -hydroxybenzoate 6-hydroxylase from R. erythropolis were hardly inactivated. Neither of these two enzymes was considered to have a functional arginine residue required for interaction with the substrate.     

Hydrophobicity at the surface of proteins

A new method is presented to quantitatively estimate and graphically display the propensity of nonpolar groups to bind at the surface of proteins. It is based on the calculation of the binding energy, i.e., van der Waals interaction plus protein electrostatic desolvation, of a nonpolar probe sphere rolled over the protein surface, and on the color coding of this quantity on a smooth molecular surface (hydrophobicity map). The method is validated on ten protein-ligand complexes and is shown to distinguish precisely where polar and nonpolar groups preferentially bind. Comparisons with existing approaches, like the display of the electrostatic potential or the curvature, illustrate the advantages and the better predictive power of the present method. Hydrophobicity maps will play an important role in the characterization of binding sites for the large number of proteins emerging from the genome projects and structure modeling approaches.     

An essential histidine residue for the activity of UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was completely inactivated by diethylpyrocarbonate following pseudo-first order reaction kinetics. The pH profile of diethylpyrocarbonate inhibition and reversal of inhibition by hydroxylamine suggested specific modification of histidyl residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of 1 essential histidine residue to be responsible for loss in catalytic activity of yeast epimerase. No major structural change in the quarternary structure was observed in the modified enzyme as shown by the identical elution pattern on a calibrated Sephacryl 200 column and association of coenzyme NAD to the apoenzyme. Failure of the substrates to afford any protection against diethylpyrocarbonate inactivation indicated the absence of the essential histidyl residue at the substrate binding region of the active site. Unlike the case of native enzyme, sodium borohydride failed to reduce the pyridine moiety of the coenzyme in the diethylpyrocarbonate-modified enzyme. This indicated the presence of the essential histidyl residue in close proximity to the coenzyme binding region of the active site. The abolition of energy transfer phenomenon between the tryptophan and coenzyme fluorophore on complete inactivation by diethylpyrocarbonate without any loss of protein or coenzyme fluorescence are also added evidences in this direction.     

Chemotypes in Achillea collina based on sesquiterpene lactone profile

The lactone profile of six origins of Achillea collina growing in Bulgaria was studied and significant variability was observed. The reasons for the differences in the lactone composition are discussed. Twenty-five components in total were isolated and identified, while the presence of ten lactones was proved by intensive TLC analysis in comparison with reference compounds. The structures of the components 17, 20, 25-30 were established by spectroscopic methods. The structure of 7, a cyclization product of 6, was also discussed. The anti-inflammatory activity of some extracts, fractions and individual compounds was tested in vitro by determining the inhibitory effects on induced human neutrophils.     

De novo formation of desmosomes in cultured cells upon transfection of genes encoding specific desmosomal components

Desmosomes are cell junctions and cytoskeleton-anchoring structures of epithelia, the myocardium, and dendritic reticulum cells of lymphatic follicles whose major components are known. Using cultured HT-1080 SL-1 fibrosarcoma-derived cells and transfection of cDNAs encoding specific desmosomal components, we have determined a minimum ensemble of proteins sufficient to introduce de novo structures, which, by morphology and functional competence, are indistinguishable from authentic desmosomes. In a more refined analysis, the influence of the desmosomal proteins desmoplakin (Dp), plakoglobin (Pg), and plakophilin 2 (Pp2) on the lateral clustering of the desmosomal transmembrane-glycoprotein desmoglein 2 (Dsg) was examined. We found that for efficient clustering of desmoglein 2 and desmosome structure formation, all three major plaque proteins-desmoplakin, plakoglobin, and plakophilin 2- were necessary. Furthermore, in this cell model, plakophilin 2 was capable of directing desmoplakin to adhaerens junctions (AJ), whereas plakoglobin was crucial for the segregation of desmosomal and AJ components. These results are discussed with respect to the variability in cell junction composition observed in various nonepithelial tissues.     

Extracellular matrix components in atherosclerotic arteries of Apo E/LDL receptor deficient mice: an immunohistochemical study

During accelerated vascular remodeling such as in atherosclerosis, the composition of the extracellular matrix becomes altered. The matrix components of the diseased artery influence cellular processes such as adhesion, migration and proliferation. Furthermore, in atherosclerosis, the inability of the cells within the lesion to produce a mechanically stable matrix may lead to plaque rupture. In this immunohistochemical study of atherosclerotic mice aorta, we have reviewed the presence of ECM components with roles in maintaining tissue structure and function. These components include osteopontin and COMP as well as the leucine rich repeats proteins decorin, PRELP, and fibromodulin. Immunohistochemistry demonstrated presence of osteopontin, COMP, decorin, PRELP and fibromodulin in lesion areas of ApoE/LDLr deficient mice. Some advanced lesions exhibited areas of cartilage-like morphology and were shown to represent cartilage by their content of the cartilage specific proteins collagen II and aggrecan. The results suggest that cartilage-associated cell/collagen binding ECM proteins may be involved in the pathogenesis of atherosclerosis.