Onset of Inflammation With Ischemia: Implications for Donor Lung Preservation and Transplant Survival.

Lungs stored ahead of transplant surgery experience ischemia. Pulmonary ischemia differs from ischemia in the systemic organs in that stop of blood flow in the lung leads to loss of shear alone because the lung parenchyma does not rely on blood flow for its cellular oxygen requirements. Our earlier studies on the ischemia-induced mechanosignaling cascade showed that the pulmonary endothelium responds to stop of flow by production of reactive oxygen species (ROS). We hypothesized that ROS produced in this way led to induction of proinflammatory mediators. In this study, we used lungs or cells subjected to various periods of storage and evaluated the induction of several proinflammatory mediators. Isolated murine, porcine and human lungs in situ showed increased expression of cellular adhesion molecules; the damage-associated molecular pattern protein high-mobility group box 1 and the corresponding pattern recognition receptor, called the receptor for advanced glycation end products; and induction stabilization and translocation of hypoxia-inducible factor 1α and its downstream effector VEGFA, all of which are participants in inflammation. We concluded that signaling with lung preservation drives expression of inflammatory mediators that potentially predispose the donor lung to an inflammatory response after transplant.

Noncovalent adducts of poly(ethylene glycols) with proteins.

A new method of preparation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumine) under high pressure has been developed. The involvement of polymer in the complexes was proved using (3)H-labeled PEG. The composition of the complexes (the number of polymer chains per one ChT molecule) depends on the molecular mass of PEG and decreases with the increase in molecular mass from 300 to 4000, whereas the portion of the protein (wt %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt %. The kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical with the corresponding values for the native ChT. According to the data obtained by the method of circular dichroism, the enzyme in the complexes fully retains its secondary structure. The steric availability of PEG polymer chains in the complexes was evaluated by their complexation with alpha-cyclodextrin (CyD) or polymer derivatives of beta-CyD modified with PEG (PEG-beta-CyD). In contrast to free PEG, only part of PEG polymer chains ( approximately 10%) interact with alpha-CyD. Thus, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule located far from the active site and results in the sufficient decrease in the availability of polymer chains. The complexes between PEG chains in PEG-protein adducts and PEG-beta-CyD may be considered as a novel type of dendritic structures.

[Supramolecular complexes of polyethylene glycol-alpha-Chymotrypsin covalent and noncovalent adducts with polyoxyethylene-modified cyclo dextrins]. / Supramolekuliarnte kompleksy kovalentnykh i nekovalentnykh adduktov poliétilenglikol'-alpha-khimotripsina s tsiklodekstrinami, modifitsi rovannymi poliétilenglikolem.

Complexes of covalent and noncovalent adducts of polyethylene glycol (PEG) and alpha-chymotrypsin (ChT), PEG-ChT, were generated in the presence of beta-cyclodextrin derivatives of polyoxyethylene (beta CD-PEO), and their thermal stability was studied. The covalent [PEG-ChT]c conjugates were obtained by chemical modification of the protein amino groups with the monoaldehyde derivatives of monomethoxypolyethylene glycol. The noncovalent [PEG-ChT]n complexes were obtained by the treatment of ChT-PEG mixtures with increasing pressure (1.1-400 MPa). Supramolecular structures resulting from complex formation between PEG chains of the PEG-ChT adducts (PEGad) and beta CD-PEO were studied. The decrease in the rate constant of the slow stage of ChT thermal inactivation in PEG-ChT adducts (k2) can serve as confirmation of complex formation between beta CD-PEO and PEGad. The stoichiometric composition of our supramolecular structures was determined from the k2 dependence on the molar ratio of beta CD-PEO to PEGad. It was shown that each polymeric chain in the [PEG-ChT]c conjugates forms an inclusion complex with beta CD-PEO, whereas only half of the PEGad polymeric chains participate in the formation of supramolecular structures in the case of [PEG-ChT]n complexes. Although covalent and noncovalent PEG-ChT adducts of the same composition significantly differ in their thermal stability, the maximal values of the k2 rate constants for [PEG-ChT]c and [PEG-ChT]n adducts in the triple system attainable at the (beta CD-PEO) to (PEGad) ratio corresponding to the stoichiometry of the resulting ternary systems are practically the same (k2 = 0.007 c-1 at 45 degrees C in 0.02 M Tris-HCl buffer solution, pH 8.0). Structures for the supramolecular dendrite-like ensembles formed upon the interaction of covalent and noncovalent PEG-ChT adducts with beta CD-PEO were suggested.

[Enzymic activity of polymer-protein complexes in water-miscible organic solvents]. / Fermentativnaia aktivnost' kompleksov polimer-belok v smeshivaiushch ikhsia s vodoi organicheskikh rastvoriteliakh.

The enzymic activity of noncovalent complexes of alpha-chymotrypsin with polyethylene glycol and a block-copolymer of polyethylene oxide and polypropylene oxide (proxanol) was studied in aqueous-organic media. It was shown that complex formation activated the enzyme in media with a high content of the organic solvent, whereas in systems containing more than 50% water the enzymic activity of complexes was the same as that of the native enzyme. The activation in polyethylene glycol-containing complexes was greater than in complexes with proxanol of the same molecular mass.

Using hydrated reversed micelles to evaluate the dimensions of polymer-protein adducts.

Hydrolysis of N-trans-cynnamoylimidazole catalyzed by conjugates and complexes of alpha-chymotrypsin (ChT) with poly(ethylene glycol) (PEG) of different molecular mass (from 300 to 5000 daltons) was studied in the system of the hydrated reversed micelles of aerosol OT (AOT) in octane at 25 degrees C. The plot of the deacylation constant k3 for PEG--ChT conjugates and complexes versus the degree of hydration of reversed micelles (w0 = [H2O]/[AOT]) was studied. These plots are bell-shaped with maxima shifted to higher degrees of micelle hydration compared to the corresponding value of the shift for ChT. As for PEG--ChT conjugates, the value of the shift of w0 increases with increasing of molecular mass of the attached PEG and/or with the number of polymer chains per ChT molecule. Another picture was observed for PEG--ChT complexes for which the position of the maximum on k3 versusw0 curves was practically the same for all compounds. The values of the thickness of the polymer layer for PEG--ChT conjugates and complexes were calculated. Thus, polymer chains in conjugates placed in hydrated micelles are highly packed, whereas in the case of complexes they form a flat layer on the surface of the protein.

Noncovalent complexes between poly(ethylene glycol) and proteins.

A new method of formation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumin) under high pressure has been developed. The existence of polymer in complexes was proved using 3H-labeled PEG. Complexes between PEG and ChT were studied in detail. It was shown that the composition of complexes (the number of polymer chains per ChT molecule) depends on the molecular mass of PEG and decreases with the increase of molecular mass from 300 to 4000. At the same time, the portion of the protein (wt. %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt. %. It was shown that kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical to the corresponding values for the native ChT. The conformational properties of ChT in complexes were studied by circular dichroism. It was shown that the enzyme in complexes fully retains its secondary structure. The estimation of steric availability of PEG polymer chains in complexes was evaluated by the complexation with alpha-cyclodextrin (CyD). It was shown that in contrast to free PEG, only part (approximately 10%) of PEG polymer chains in PEG--ChT complexes participate in the complexation with CyD. Hence, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule in a region far from the active site of the enzyme and results in the significant decrease in the mobility of polymer chains.

Application of the method of thermal denaturation for investigation of alpha-chymotrypsin adducts with poly.

The thermostability of conjugates, non-covalent complexes and mixtures of alpha-chymotrypsin (alpha-ChT) with poly(alkylene oxides)--poly(ethylene glycol) (PEG) with molecular mass of 1.9 kD and diblock copolymers of ethylene and propylene oxides (proxanols)--has been investigated. It was shown that the addition of PEG in concentration up to 2 wt. % to the solution of alpha-ChT did not affect the rate of the enzyme thermoinactivation. Meanwhile the addition of proxanol in the same concentration resulted in twofold decrease in the rate constant for the slow inactivation step, k2. Even more pronounced decrease in the thermoinactivation rate was observed for alpha-ChT--proxanol complexes obtained by heating or under the action of high pressure. The general tendency in the behavior of complexes of both types was the decrease in the k2 constant as the temperature or pressure used for complex preparation increased. The highest stabilizing effect was observed for complex obtained by heating up to 52 degreesC and containing maximal number of polymer chains (molar ratio proxanol/alpha-ChT was 10). For this complex fourfold decrease in the k2 value was observed. Covalent attachment of PEG or proxanol to enzyme gives maximal stabilizing effect with up to tenfold decrease in the k2 value. The investigation of the thermal denaturation kinetics of alpha-ChT and its adducts with poly(alkylene oxides) by means of fluorescence spectroscopy has shown that the presence of polymer chains practically does not affect the rate of protein denaturation registered by the decrease in the intensity of protein fluorescence. The polymer chains, probably, diminish the rate of melting of the active site-containing region of the protein molecule. At the same time, the overall denaturation rate is independent of the presence of polymer chains in the vicinity of the protein globule.

[Complex formation between alpha-chymotrypsin and block copolymers based on ethylene and propylene oxide, induced by high pressure]. / Kompleksoobrazovanie mezhdu alpha-khimotripsinom i blok-sopolimerami na osnove okisi étilena i okisi propilena, indutsiruemoe deistviem vysokikh davlenii.

A new method of formation of non-covalent adducts based on an amphiphilic diblock copolymer of ethylene and propylene oxides with molecular mass of 2 kDa and alpha-chymotrypsin (ChT) under high pressure, has been developed. The composition of the complexes corresponds to seven polymer molecules per one ChT molecule in the pressure range of 1.1 to 400 MPa. The complexes fully retain the catalytic activity. Kinetic constants (Km and kcat) for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester catalyzed by the complexes are identical with the corresponding values for native ChT. Analysis of kinetics of thermal inactivation of the complexes revealed that the constant of the rate of the slow inactivation step is markedly lower than for ChT.

[Noncovalent complexes between alpha-chymotrypsin and block copolymers from ethylene and propylene oxides]. / Nekovalentnye kompleksy mezhdu alpha-khimotripsinom i blok-sopolimerom oksidov étilena i propilena.

The ability of alpha-chymotrypsin to form complexes with amphiphilic block copolymer of ethylene oxide and propylene oxide upon heating up to 44-60 degrees C has been demonstrated for the first time. Depending on temperature and the initial component ratio, some complexes were obtained which varied in both composition and enzymatic activity. With a rise in the complexation temperature, the polymer content in the complex increased, while the enzymatic activity of the complex decreases. The complexes are very stable in water, but dissociate in 8 M urea and are characterized by enhanced thermal stability as compared with the original enzyme. It is assumed that both hydrophobic interactions and hydrogen bonds between the components are involved in the complex formation.