Cellular oxidative stress and peroxisomal enzyme activities in pediatric liver transplant patients

In this study, we examined the activities of key peroxisomal enzymes in peripheral blood lymphocytes [PBLs] of pediatric liver transplant patients. Venous blood was drawn from 14 patients aged 5-16 years on FK-506 treatment and 18 healthy subjects for isolation of lymphocytes. Beta -Oxidation of very long chain fatty acids [VLCFAs] and activities of superoxide dismutase [SOD], glutathione peroxidase [GPx], NADPH oxidase [NOX], catalase and peroxisomal enzyme acyl CoA oxidase [ACO] were measured in cellular homogenates. Levels of malondialdehyde [MDA] were measured as an index of lipid peroxidation. Protein content and mRNA levels of catalase, peroxisomal membrane protein-70 [PMP-70] and ACO were measured using Western blotting and PCR techniques. PBLs isolated from liver transplant patients showed significantly [p < 0.01] increased levels [226.9 +/- 24.5 micro mol/mg protein] of MDA as compared to the levels in controls [162.8 +/- 19.6 micro mol/mg protein], whereas enzyme activities of SOD and NOX remained unaltered in patients' cells. Enzyme activities of catalase and GPx were markedly [p < 0.01] decreased in cells isolated from liver transplant patients. ACO activity and beta -oxidation of VLCFAs in PBLs from liver transplant patients were however found to be significantly increased by 38 and 52% respectively when compared with controls. Gene expression of PMP-70 and ACO was also significantly increased [p < 0.01] in PBLs of patients. Our results clearly showed that peroxisomal metabolic activities are markedly altered in lymphocytes of liver transplant patients and might contribute to the development of cellular oxidative stress

Molecular mechanisms of organelle biogenesis and related metabolic diseases

Organelle biogenesis is regulated by transcriptional networks that modulate expression of specific genes encoding organellar proteins. Structural and functional specificity of organelles requires not only the transcription of specific genes and translation of resulting mRNAs, but also the transfer of encoded polypeptides to their site of function through signaling peptides. A defect in targeting of proteins to their subcellular site of function may not necessarily prevent biogenesis of the organelle, but would definitely lead to formation of a defective organelle with respect to that specific function. Several metabolic diseases are associated with dysfunction or defects in organelle biogenesis; among these, peroxisome biogenesis disorders, mitochondrial biogenesis defects and lysosomal storage disorders are an extensively studied group of genetic diseases where biogenesis of the organelle is compromised either due to a defect in assembly of the organelle itself or impaired import of matrix proteins into the organelle. Recent advances in biochemical and molecular aspects of biogenesis of subcellular organelles have not only unraveled the mechanisms for organization of cellular networks, but have also provided new insights into the development of metabolic diseases that are caused by disruption of organelle biogenesis. This article reviews the molecular mechanisms of biogenesis of mitochondria, lysosomes and peroxisomes in relation to the metabolic diseases of genetic or nongenetic origin

Very-long-chain fatty acids activate lysosomal hydrolases in neonatal human skin tissue

The aim of this study was to examine the in vitro effect of peroxisomal dysfunction on lysosomal enzymes, the autophagic machinery in the cell, in order to understand the mechanisms of pathogenesis of peroxisomal disorders. Foreskin samples were obtained immediately after circumcision of 1- to 2-day-old infants at the Maternity Hospital, Kuwait. Skin tissues were cleaned, cut into slices of 1-2 mm[2] in size and treated with lignoceric acid [1-20 micro g/ml], a very-long-chain fatty acid [VLCFA], in the presence or absence of 1-5 mM aminotriazole [ATZ]. A battery of lysosomal enzymes were assayed following treatment of dermal tissue with VLCFA or ATZ. Treatment of skin slices with lignoceric acid significantly increased [p < 0.001] the enzymic activities of acid lipase, acid phosphatase, alpha -glucosidase, alpha -galactosidase, N-acetyl- alpha -D-glucosaminidase [NAGA] and N-acetyl- alpha -D-galactosaminidase [NAGTA]. ATZ [1-5 mM], an inhibitor of key peroxi somal enzyme catalase, also markedly increased the enzymic activities of acid phosphatase, alpha -glucosidase [23%] and alpha -galactosidase [18%] without any significant effect on NAGA or NAGTA. Western blot analysis further revealed that both VLCFA and ATZ significantly increased the protein expression of lysosomal enzymes, beta -galactosidase and beta -glucuronidase. Experimen tal dysfunction of peroxisomes mimicked by elevated VLCFA or ATZ-mediated catalase inhibition significantly increased the activities of lysosomal hydrolases in human dermal tissue, suggesting that activation of the lysosomal system could be one of the factors responsible for cellular damage during pathogenesis of peroxisomal diseases

Interleukin-1 and nitric oxide increase NADPH oxidase activity in human coronary artery smooth muscle cells

Cytokines, nitric oxide [NO] and reactive oxygen species [ROS] are well known for their pathogenic effects in development of cardiovascular diseases. Interleukin-1 [IL-1] is known to induce NO generation, however it is not well established if IL-1beta or NO regulate production of ROS, such as superoxide anion. Therefore, the main objective of this study was to evaluate the effect of IL-1beta or NO on enzyme activity of NADPH oxidase [NOX], a superoxide-generating system recently documented to participate in a variety of vascular functions. Human coronary artery smooth muscle cells [SMC] obtained from Clonetics were treated with IL-1 beta and NO donor, sodium nitroprusside [SNP], in culture. Nitrites accumulated in supernatants of SMC cultures were measured as an index of NO released following treatment with IL-1beta. NOX enzyme activity was assayed using cytochrome c as the electron acceptor. Treatment with IL-1beta resulted in a 3-fold increase in the production of NO by SMC. Both IL-1beta and SNP enhanced NOX activity, by 67 and 45%, respectively, following 24 h of treatment. This study suggests that NO or NO- generating cytokines might regulate the production of ROS in the cardiovascular system through modulation of superoxide-generating systems such as NOX