Extraction,isolation and analysis of chondroitin sulfate from broiler chicken biomass

This study was undertaken to investigate whether chondroitin sulfate (CS) can be efficiently isolated from broiler chicken by-product from mechanical deboning (BMD), which is a mixture of crushed bone, cartilage, skin, adipose tissue and muscle. With BMD as a starting material, it may be possible to maximize CS production from chicken cartilage. CS was liberated from BMD by proteolysis using proteolytic activities from papain, pancreatin, kiwi fruits and flavourzyme. The final product isolated by anion-exchange chromatography and differential precipitation with varying concentrations of ethanol contained CS-peptides with glucuronosyl-N-acetylgalactosamine-4-sulfate as a predominant disaccharide. All the enzyme sources used had sufficient activities to produce CS-peptides with undetectable amount of glucosamine. However, the size of peptide attaching to CS chain and the antigenicity to anti-keratan sulfate (KS) monoclonal antibody varied among the CS-peptides prepared with different proteinases. Papain digestion resulted in the smallest size of peptide with no detectable antigenicity to the anti-KS antibody. In contrast, pancreatin digestion provided CS-peptide having a very weak but positive antigenicity to the anti-KS antibody, which was much lower than that seen with CS-peptide prepared with kiwi fruit proteinase or flavourzyme. Pancreatin is suggested as an economical source of proteinase to liberate CS-peptides from BMD.     

Reactive oxygen species produced by the mitochondrial respiratory chain are involved in Cd-induced injury of rat ascites hepatoma AS-30D cells

Using AS-30D rat ascites hepatoma cells, we studied the modulating action of various antioxidants, inhibitors of mitochondrial permeability transition pore and inhibitors of the respiratory chain on Cd²⁺-produced cytotoxicity. It was found that Cd²⁺ induced both necrosis and apoptosis in a time- and dose-dependent way. This cell injury involved dissipation of the mitochondrial transmembrane potential, respiratory dysfunction and initial increase of the generation of reactive oxygen species (ROS), followed by its decrease after prolonged incubation. Inhibitors of the mitochondrial permeability transition pore, cyclosporin A and bongkrekic acid, and inhibitors of respiratory complex III, stigmatellin and antimycin A, but not inhibitor of complex I, rotenone, partly prevented necrosis evoked by exposure of the cells to Cd²⁺. Apoptosis of the cells was partly prevented by free radical scavengers and by preincubation with N-acetylcysteine. Stigmatellin, antimycin A and cyclosporin A also abolished Cd²⁺-induced increase in ROS generation. It is concluded that Cd²⁺ toxicity in AS-30D rat ascites hepatoma, manifested by cell necrosis and/or apoptosis, involves ROS generation, most likely at the level of respiratory complex III, and is related to opening of the mitochondrial permeability transition pore.     

Effect of long-chain fatty acids and acyl-CoA on mitochondrial permeability,transport, and energy-coupling processes

The following effects of fatty acids and acyl-CoA thioesters on energy metabolism of mitochondria can now be assumed: (1) Inhibition of adenine nucleotide translocation. This effect may increase the energy state of mitochondria respiring under state 3 conditions and decrease phosphorylation potential in the surrounding medium (the cytoplasm). (2) Increased permeability to monovalent cations. This may lead to a partial energy dissipation due to a futile recycling of K+ (or another cation), namely and energy-dependent uptake and a passive outflow. (3) True uncoupling due to increased permeability to protons. This effect probably occurs at high concentrations of fatty acids only. (4) Substrate effect. Fatty acids in the form of acyl-CoA are excellent respiratory substrates for mitochondria of most tissues. Their oxidation is coupled to the generation of high energy state of the mitochondrial membrane and, consequently, to ATP synthesis.     

Effect of monovalent cations on the inhibition by NAD+ of NADH oxidation in submitochondrial particles.

Oxidation of NADH in submitochondrial particles, with O₂ or ferricyanide as electron acceptor, was inhibited by micromolar concentrations of NAD⁺ when measured in 240 mM sucrose or, in a lesser extent, in 120 mM NaCl or LiCl. In 120 mM solutions of either KCl, RbCl, CsCl or NH₄Cl the inhibition by up to 100 μM concentrations of NAD⁺ did not occur. The inhibition observed in the sucrose medium disappeared after solubilization of the particles with detergents and re-appeared when the membranes were reconstituted. The inhibitory effect was potentiated by palmitoyl-CoA. The possibility is discussed that the inhibition of NADH oxidation by low concentrations of NAD⁺ and its release by K⁺, Rb⁺, Cs⁺ and NH₄⁺ depend on the interaction between NAD⁺ and the negatively charged mitochondrial membrane.