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Chronic inflammation is associated with the development of human hepatocellular carcinoma (HCC), an essentially incurable cancer. Anti-inflammatory nutraceuticals have emerged as promising candidates against HCC, yet the mechanisms through which they influence the cell signaling machinery to impose phenotypic changes remain unresolved. Herein we implemented a systems biology approach in HCC cells, based on the integration of cytokine release and phospoproteomic data from high-throughput xMAP Luminex assays to elucidate the action mode of prominent nutraceuticals in terms of topology alterations of HCC-specific signaling networks. An optimization algorithm based on SigNetTrainer, an Integer Linear Programming formulation, was applied to construct networks linking signal transduction to cytokine secretion by combining prior knowledge of protein connectivity with proteomic data. Our analysis identified the most probable target phosphoproteins of interrogated compounds and predicted translational control as a new mechanism underlying their anticytokine action. Induced alterations corroborated with inhibition of HCC-driven angiogenesis and metastasis.

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Mesenchymal stem cells (MSCs) are somatic cells with a dual capacity for self-renewal and differentiation, and diverse therapeutic applicability, both experimentally and in the clinic. These cells can be isolated from various human tissues that may differ anatomically or developmentally with relative ease. Heterogeneity due to biological origin or in vitro manipulation is, nevertheless, considerable and may equate to differences in qualitative and quantitative characteristics which can prove crucial for successful therapeutic use. With this in mind, in the present study we have evaluated the proliferation kinetics and phenotypic characteristics of MSCs derived from two abundant sources, that is, fetal umbilical cord matrix (Wharton's jelly) and adult adipose tissue (termed WJSC and ADSC, resp.) during prolonged in vitro expansion, a process necessary for obtaining cell numbers sufficient for clinical application. Our results show that WJSC are derived with relatively high efficiency and bear a substantially increased proliferation capacity whilst largely sustaining the expression of typical immunophenotypic markers, whereas ADSC exhibit a reduced proliferation potential showing typical signs of senescence at an early stage. By combining kinetic with phenotypic data we identify culture thresholds up to which both cell types maintain their stem properties, and we discuss the practical implications of their differences.

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Enzymatic synthesis of acylated derivatives of a monosaccharidic flavonoid chrysoeriol-7-O-beta-D-(3''-E-p-coumaroyl)-glucopyranoside as well as of a disaccharidic flavonoid chrysoeriol-7-[6'''-O-acetyl-beta-D-allosyl-(1-->2)-beta-D-glucopyranoside], isolated from Greek endemic plants, was performed using an immobilized Candida antarctica lipase in non-toxic organic solvents. The influence of the reaction parameters such as the molar ratio of acyl donor to flavonoid, as well as the nature of the acyl donor, on the performance of the biocatalytic process was pointed out using the acylation of naringin as a model reaction. With vinyl laurate as acyl donor, the highest conversion was observed at relatively high molar ratio (>or=10), using acetone as solvent. Lipase exhibits specificity towards primary alcohol of the glucose moiety of both flavonoid glycosides. The introduction of an acyl group into glucosylated flavonoids significantly improved their antioxidant activity towards both LDL and serum model in vitro. Furthermore, the acylated derivative of disaccharidic flavonoid increased its antimicrobial activity against two Gram-positive bacteria.

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Cell-bound lipase activity (10 pNPL units/g dry cell weight) was released when the yeast Rhodotorula glutinis was cultured in a 7-l stirred tank fermentor using palm-oil as the sole carbon source. The enzyme showed relative specificity towards medium chain organic acids since the apparent K(m) values for pNPB (p-NitroPhenyl-Butyrate) and pNPL (p-NitroPhenyl-Laurate) were equal to 2.7 and 0.7 mM, respectively. In addition, 80% of this activity could be detected on the surface of the cells. The cell-bound nature of the enzyme increased its thermal stability showing half-life times of 200 and 60 min at 50 and 60 degrees C, respectively, as well as good stability in organic solvents. Freeze-dried cell preparations were successfully used to catalyze the synthesis of fatty acid esters of butanol and heptanol in nearly anhydrous organic solvents. A conversion of 60-62% was obtained upon esterification of palmitic or oleic acid with butanol, within 96 h. The enzyme preparation was used in four consecutive batch reactions with only 10% loss of activity.

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Water-in-oil microemulsions, or reverse micelles, are being evaluated as a reaction medium for a variety of enzymatic reactions. These systems have many potential biotechnological applications. Important examples are the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. This review illustrates the biotechnological applications of microemulsions as media for bioorganic reactions. The principal focus is on lipase catalyzed processes.

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Fusarium oxysporum beta-glucosidase has been used to catalyze the production of alkyl-beta-D-glucosides from various disaccharides, based on the transglucosylation reaction, in the presence of primary, secondary and tertiary alcohols as glucosyl acceptors. Primary alcohols were found to be the best acceptors. The influence of the glucosyl donor concentration, as well as the enzyme specificity towards the cleaved glucosidic bond and the aglucone part of the donor, have also been investigated. The enzyme does not exhibit regiospecificity and seems to be unspecific towards the aglucone part. The specificity of the beta linkage has been confirmed by proton nuclear magnetic resonance (1H NMR) analysis.

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The activity of purified Pseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT). Kinetic studies showed that the reaction follows a ping-pong bi-bi mechanism with inhibition by both substrates. The apparent kinetic parameters of the reaction were found to be K(m octanol) = 310 mM, K(m lauric acid) = 78 mM, and V(max) = 250 mumol min(-1) mg(-1). The same system was used for the synthesis of mono- and diglycerides from glycerol and lauric acid, which was successful at very low w(o) values. The catalytic behavior of P. cepacia lipase was also studied in esterification reactions performed in a nonionic microemulsion system formulated by tetraethyleneglycoldodecylether (C(12)E(4)). The optimum activity was found at about w(o) = 8. The apparent values of V(max app) and K(m app) for octanol were calculated and found to be 100 mumol min(-1) mg(-1) and 76 mM, respectively. (c) 1995 John Wiley & Sons, Inc.

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Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

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Enzymic acylation of a flavonoid, rutin, with trichloroethylbutyrate (TCEB) has been performed by subtilisin protease in anhydrous pyridine solution. The addition of a hydrophobic compound on rutin is expected to change the hydrophilic/hydrophobic balance of the molecule, giving new properties to this compound. This work aimed at investigating the various cytological properties of the rutin-ester and compared them with those of the native molecule. No difference in the levels of sister chromosomes exchange (SCE) between rutin and rutin-ester treated cells at doses varying from 25 to 200 micrograms/mL was found. On the contrary impressive difference in the induced frequency of micronuclei (MN) between rutin and rutin ester treated cells was observed, for example, at a dose of 100 micrograms/mL of rutin were 3.5% MN counted, whereas for a similar dose treatment with rutin-ester a frequency of 8% of MN was found. The fact that rutin-ester is causing significantly higher levels of MN than the rutin alone can be considered as a manifestation of a higher action of the agent on the chromosome owing to its easier penetration in to the cell after its esterification.

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The kinetics of the esterification of lauric acid by (-)menthol, catalyzed by Penicillium simplicissimum lipase, was studied in water/bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping-Pong Bi--Bi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59-72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio, W(o) = [H(2)O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern.

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The activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis-(2-ethylhexyl)sulfo-succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies.

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It was found that Lactobacillus plantarum (strain BA 11) is able to synthesize sialic acids during its growth in MRS medium and that these molecules are located mainly on the surface of the bacterium. It was demonstrated also that the addition externally of N-acetylneuraminic acid in concentrations ranged from 10 to 500 microM into the culture medium, resulted to a substantial increase of the growth rate of the bacterium. Bacterial cultures in presence of added sialic acid (100 microM) for 24 hours, resulted to a two fold increase of the final bacterial mass compared to the cultures in absence of sialic acid. Maximum levels of sialic acids were observed after 48 h of bacterial growth. It was also found that neuraminic acids production was increased when Mn++ and Mg++ ions were added in the culture medium, while the addition of Co++, Ca++, Ba++, Cu++ and Ni++ had a negative effect.

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Lactate dehydrogenase and NANA-lyase were immobilized in an artificial gelantine membrane. This bienzyme system was used for continuous assay of neuraminidase activity. The K'(m) of the active membrane for lactate dehydrogenase and NANA-lyase using NADH, pyruvic acid, and N-acetylneuraminic acid as substrates were found to be 0.25mM, 0.75mM, and 2.1mM, respectively. The K(m) of soluble neuraminidase using sialyllactose as substrate was found to be 0.13 mM. The pH optimum for neuraminidase activity was 6.0. At 45 degrees C the reaction rate was higher, and no denaturation phenomena of the immobolized enzymes have been observed. This bienzyme membrane was stable for several weeks stored in the reaction buffer at 4 degrees C.

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The competition of NANA-Aldolase and Cytidine-5'-monophosphosialate-Synthase for their common substrate NANA has been studied, with, (i) the two enzymes in solution, (ii) NANA-Aldolase in solution and Cytidine-5'-monophosphosialate-Synthase immobilized in an artificial membrane (diffusion coefficient: 1.2 X 10(-3) cm2 h-1). The relation of the reaction rates for both enzyme was found 1:1 in case (i) and 2:1 in case (ii), in favor of NANA-Aldolase. These results are in agreement with the results obtained by computer simulation, where the Michaelian assumption and the diffusion effects had been considered. It was also calculated that the regulation of this branch point for the metabolic pathway of NANA is dependent on the input of NANA produced by the previous steps of the pathway and not on the concentration of CTP (second substrate of Cytidine-5'-monophosphosialate-Synthase) or the parameters controlling the diffusion of NANA. Computer simulations were performed by numerical analysis.

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Sialic acid has been assayed enzymatically by an immobilized two-enzyme system. The method includes cleavage of sialic acid to pyruvic acid by N-acetylneuraminic acid (NANA) aldolase and reduction of pyruvic acid by lactate dehydrogenase in the presence of NADH, which is followed photometrically at 349 nm. For the membrane preparation 5 units of lactate dehydrogenase and 1 unit of NANA-aldolase were used. The pH optimum of the reaction using potassium phosphate buffer was 7.0. This two-enzyme membrane remains 100% active for several weeks at 4 degrees C in the assay buffer and remains stable after performing experiments at 45 degrees C.

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The reaction between DTNB and the SH groups of N-acetylneuraminate lyase has been investigated in the presence and absence of pyruvic acid, substrate of the enzyme. It was found that DTNB inactivates N-acetylneuraminate lyase, while pyruvic acid protects the enzyme against this inactivation. When the enzyme was fully inactivated, two SH groups have reacted with DTNB. This result supports previous suggestions, that there is one cystein residue per active site responsible for enzyme activity. In the presence of SDS, approx. 6 SH groups reacted with DTNB suggesting the existence of 3 SH groups per enzyme subunit.

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The presence of acylneuraminate cytidylyltransferase has not been shown in human placenta so far. In this paper, evidence is put forward to demonstrate the presence of this enzyme in human placenta. The soluble enzyme was partially purified 30-fold using ammonium sulphate fractionation (30-60%) and DEAE Sephadex A-50 chromatography. This enzyme preparation had a specific activity of 0.75 mkat/kg protein. The pH optimum of the reaction was 9.0. The Km values for N-acetylneuraminic acid and cytidine triphosphate were 2.2 mM and 4 mM respectively. It was also found that the presence of magnesium is necessary for the enzyme activity.

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