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BACKGROUND: Ischemia-reperfusion injury induced by pneumoperitoneum is a well-studied entity, which increases oxidative stress during laparoscopic operations. The reported anti-inflammatory action of aprotinin was measured in a pneumoperitoneum model in rats for the first time in this study. MATERIALS AND METHODS: A total of 60 male Albino Wistar rats were used in our protocol. Prolonged pneumoperitoneum (4 h) was applied, causing splanchnic ischemia and a period of reperfusion with a duration of 60 or 180 min followed. Several cytokines and markers of oxidative stress were measured in liver, small intestine, and lungs to compare the aprotinin group with the control group. Tissue inflammation was also evaluated and compared between groups using a five-scaled histopathologic score. RESULTS: In aprotinin group values of biochemical markers (tumor necrosis factor α, interleukin 6, endothelin 1, C reactive protein, pro-oxidant-antioxidant balance, and carbonyl proteins) were lower in all tissues studied. Statistical significance was greater in liver and lungs (P < 0.05). Histopathologic examination revealed significant difference between control and aprotinin groups in all tissues examined. Aprotinin groups showed mild to moderate lesions, while in control groups severe to very severe inflammation was present. Aprotinin subgroup with prolonged reperfusion period (180 min) showed milder lesions in all tissues than the rest of the groups. CONCLUSIONS: Aprotinin reduced inflammatory response and oxidative stress induced by pneumoperitoneum in liver, small intestine, and lungs.

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Oxidative folding that occurs in a crowded cellular milieu is characterized by multifaceted interactions that occur among nascent polypeptides and resident components of the endoplasmic reticulum (ER) lumen. Macromolecular crowding has been considered an essential factor in the folding of polypeptides, but the excluded volume effect has not been evaluated for small, disulfide-rich peptides. In the research presented, we examined how macromolecular crowding agents, such as albumin, ovalbumin, and polysaccharides, influenced the kinetics and thermodynamics of forming disulfide bonds in four model peptides of varying molecular size from 13 residues (1.4 kDa) to 58-residues (6.5 kDa): conotoxins: GI, PVIIA, r11a, and bovine pancreatic trypsin inhibitor. Our results indicate that the excluded volume effect does not significantly alter the folding rates nor equilibria for these peptides. In stark contrast, folding reactions were dramatically accelerated, when protein-based crowding agents were present at concentrations lower than those predicted to provide the excluded volume effect. Submillimolar albumin alone was as effective as glutathione in promoting the oxidative folding of GI conotoxin at concentrations typically found in the ER. To the best of our knowledge, this is the first report and quantitative characterization of oxidative folding of peptides mediated by other than thioredoxin-based protein disulfide bonds. Our work raises a possibility that concurrent secretory and ER-resident proteins may influence the oxidative folding of small, cysteine-rich peptides not as crowding agents, but as redox-active factors.

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In the presence of denaturant and thiol initiator, the native bovine pancreatic trypsin inhibitor (BPTI) denatures by shuffling its native disulfide bonds and converts to a mixture of scrambled isomers. The extent of denaturation is evaluated by the relative yields of the scrambled and native species of BPTI. BPTI is an exceedingly stable molecule and can be effectively denatured only by guanidine thiocyanate (GdmSCN) at concentrations higher than 3-4 M. The denatured BPTI consists of at least eight fractions of scrambled isomers. Their composition varies under increasing concentrations of GdmSCN. In the presence of 6 M GdmSCN, the most predominant fraction of scrambled BPTI accounts for 56% of the total structure of denatured BPTI. Structural analysis reveals that this predominant fraction contains the bead-form isomer of scrambled BPTI, bridged by three pairs of neighboring cysteines, Cys5-Cys14, Cys30-Cys38 and Cys51-Cys55. The extreme conformational stability of BPTI has important implications in its distinctive folding pathway.

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Peptides and unfolded proteins with the sequence Xaa-Pro-Tyr or Xaa-Pro-Phe have a relatively strong local interaction, present about 64% of the time at 10 degrees C, of the aromatic ring with the side-chain of Pro and the C alpha H of residue Xaa, but only when the Xaa-Pro peptide bond is cis. With the sequence Tyr-Yaa-Gly (Yaa not equal to Pro), there is a somewhat present about 26% of the time, of the aromatic ring with the Gly residue. When present together, in the sequence Xaa-Pro-Tyr-Yaa-Gly, the two interactions of the Tyr side-chain compete and have the expected strengths. Both interactions have an enthalpic basis, with enthalpies of about -3 and -2.8 kcal/mol, respectively. The two interactions responded differently to the denaturants urea and guanidinium chloride; urea had little effect on either, but the second was weakened by guanidinium chloride, whereas the first interaction was strengthened slightly. This explains why such local interactions can be observed in unfolded proteins under strongly denaturing conditions. Interactions such as these are stronger than anticipated in an otherwise disordered peptide; they are probably important for determining the conformational tendencies of unfolded polypeptide chains and may play a role in protein folding. Similar interactions probably occur in protein-ligand interactions.

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DsbA is a protein found in the periplasm of Escherichia coli that is required for the formation of disulfide bonds in secreted proteins. It contains only two cysteine residues, which can form reversibly a very unstable disulfide bond that has been proposed to be the oxidant that introduces disulfide bonds into secreted proteins. The present study investigates the effect of DsbA on the well-characterized disulfide-coupled refolding processes of BPTI and of alpha-lactalbumin. Disulfide-bonded DsbA in stoichiometric amounts proved to be a very potent donor of disulfide bonds to reduced BPTI but showed little catalytic activity at neutral pH in the presence of a glutathione redox buffer. In contrast to the related eukaryotic enzyme protein disulfide isomerase, DsbA did not substantially catalyze the usual intramolecular disulfide bond rearrangements of quasi-native folding intermediates of BPTI. Neither did DsbA catalyze the intramolecular rearrangements observed in the three disulfide-bonded "molten globule" form of alpha-lactalbumin at neutral pH. Thiol-disulfide exchange is normally very slow at acidic pH but occurs rapidly with DsbA; consequently, DsbA catalyzed the disulfide folding of BPTI under acidic conditions. It was then possible to detect some increase in the rates of disulfide rearrangements, but only with stoichiometric amounts of DsbA and on the hour time scale. These results suggest that the primary role of DsbA in the bacterial periplasm is to introduce disulfide bonds into newly secreted proteins.

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The increased use of arterial conduits in coronary artery bypass grafting is reflected in numerous publications addressing indications, choice of conduits, and possible side-effects. Besides the internal thoracic artery, the right gastroepiploic artery is becoming established, and the inferior epigastric artery is being subjected to clinical trials. The latter conduit provides good patency and can be combined with the internal thoracic arteries, but harvesting must be done carefully to prevent local complications. Arterial conduits can lead to hypoperfusion, and additional saphenous vein grafting may become necessary; careful vasodilatation of the conduit before implantation is necessary. Xenografts and allogenic implants demonstrate poor late patency and should be used only as a last resort. Aprotinin reduces blood loss during surgery and seems to be particularly useful in reoperations; but it prolongs the activated clotting time and underheparinization can occur. Retrograde cardioplegia seems to be particularly indicated in reoperations, whereas topical cooling can be omitted. Coronary revascularization can be safely combined with carotid endarterectomy; the exact indication for this simultaneous procedure is still being explored.

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The kinetics of disulphide bond formation and breakage have been measured in five analogues of the single-disulphide intermediates that occur in folding of bovine pancreatic trypsin inhibitor (BPTI), in which the cysteine residues not involved in disulphide bonds have been replaced by serine residues. Only a single disulphide bond can be made in each analogue, allowing the rates and equilibrium constants of the corresponding microscopic steps to be determined. These steps cannot be characterized in normal BPTI with six cysteine residues, as only the total overall rate of forming all 15 possible disulphide bonds can be measured. The intramolecular rate of forming each disulphide bond was found in the dithiol forms of reduced BPTI to be approximately proportional inversely to the size of the disulphide loop formed. On this basis, the 30-51 disulphide bond is not formed preferentially in the fully reduced protein, even though it predominates amongst the one-disulphide intermediates as a result of rapid intramolecular disulphide rearrangements. It is found to be more stable than the other one-disulphide intermediates because its disulphide is more stable to reduction than the others. This is undoubtedly due to the stable partially folded structure of (30-51), as the other intermediates are much less structured under folding conditions. Urea (8 M) uniformly decreased by a factor of 5 the effective concentrations of cysteine thiol groups in the analogues and in reduced BPTI, suggesting that reduced BPTI under normal refolding conditions is not an ideal random coil; any non-random conformations do not, however, favour formation of any specific disulphide bonds. Findings from these studies have led to further refinement and development of various approaches to measuring and interpreting the intramolecular rates of disulphide bond formation and breakage in proteins.

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