Lipid Peroxidation, Tissue Necrosis, and Metabolic and Mechanical Recovery of Isolated Reperfused Rat Heart as a Function of Increasing Ischemia

Isolated Langendorff-perfused rat hearts, after 30 min of preperfusion, were submitted to increasing times of global normothermic ischemia (1, 2, 5, 10, 20 and 30 min) or to the same times of ischemia followed by 30 min of reperfusion. Analysis of malondialdehyde, ascorbic acid, oxypurines, nucleosides, nicotinic coen-zymes and high-energy phosphates was carried out by HPLC on neutralized perchloric acid extracts of freeze-clamped tissues. In addition, maximum rate of intra-ventricular pressure development and cardiac output of malondialdehyde, lactate dehydrogenase, oxypurines and nucleosides were monitored during both preperfusion and reperfusion. Besides decreasing energy metabolites and nicotinic coenzyme pool, prolonged ischemia produced oxidation of significant amounts of hypoxanthine and xanthine to uric acid and generation of detectable levels of malondialdehyde (0.002 μmollg dry weight). After oxygen and substrate readmission, tissue and perfusate malondialdehyde increased only if previous ischemia was longer than 5 min, while lactate dehydrogenase was detected in perfusate of reperfused hearts following 10, 20, and 30 min of ischemia. Highest values of tissue malondialdehyde and total malondialdehyde output were recorded in reperfused hearts subjected to 30 min of ischemia (0.043 μmol/g dry weight and 0.069 μmol/ 30 min/g dry weight, respectively). Since tissue malondialdehyde was observed without detectable lactate dehydrogenase release in perfusate, it might be stated that malondialdehyde generation (i.e., lipid peroxidation) temporally preceded lactate dehydrogenase release (i.e., tissue necrosis). In reperfused hearts, evaluation of myocardial energy state and of mechanical recovery allowed us to determine times of ischemia beyond which reperfusion did not positively affect these metabolic and functional parameters. Main findings are that, under these experimental conditions, lipid peroxidation might be the cause and not the consequence of tissue necrosis and that duration of ischemia might be the factor deciding effectiveness of reperfusion.     

The relationship between codon boundaries and multiple reading-frame preferences: coding organization of bacterial insertion sequences

Theoretical considerations have shown that the five possible overlapping reading-frame configurations differ significantly in their coding flexibility and thus in their information content (Siegel and Fitch 1980; Smith and Waterman 1980). Contrary to expectation, the overlapping frame configuration allowing the greatest coding flexibility is rarely seen, whereas one of the most constraining is common. We point out here that this overlapping reading-frame paradox and an observed but unexplained preference in coding regions for a pyrimidine-purine at codon boundaries (Shepherd 1981; Jones and Kafatos 1982; Smith et al. 1983) are intimately linked. The codon boundary preference, which may be related to translation efficiency or accuracy, places constraints on the evolution of overlapping coding regions. These considerations may help identify actual coding regions in DNA sequences. We have analyzed five sequenced (enteric) bacterial insertion sequences for codon boundary incidences and reading-frame configurations and find that they are consistent with these proposed constraints.      

Factors influencing the release of proteins by cultured schwann cells

Cultured rat schwann cells grown in association with sensory neurons when labeled with [(3)H]leucinem, [(3)H]glucosamine, or [(35)S]methionine release labeled polypeptides into the culture medium. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the culture medium reveals a reproducible pattern of more than 20 polypeptides with molecular weights ranging from 15,000 to more than 250,000. Five major polypeptides (apparent molecular weights 225,000, 210,000, 90,000, 66,000, 50,000, and 40,000) account for approximately 40 percent of the leucine or methionine radioactivity in medium polypeptide. Schwann cells grown in a serum-free defined medium, in which schwann cells do not relate normally to axons, release approximately four times less labeled medium polypeptides tha cultures grown in medium supplemented with serum and chick embryo extract. In addition, there is a qualitative difference in the pattern of medium polypeptides resolved by SDS-PAGE, so that a single polypeptide (mol wt 40,000) accounts for nearly all of the label in medium polypeptides. Switching of cultures grown in defined medium to supplemented medium for 2 d results in a fourfold increase in the amount of labeled polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides as resolved by SDS-PAGE. This change in the pattern of polypeptides release by schwann cells is accompanied by changes in the association between schwann cells and axons. An early step in the establishment of normal axon-schwann cell relations appears to be an inward migration of schwann cells into axonal bundles and spreading of schwann cells along neurites. These changes are evident within 48 h after medium shift. Our results thus suggest that the release of proteins by schwann cells may be important for the development of normal axonal ensheathment.     

The relevance of malondialdehyde as a biochemical index of lipid peroxidation of postischemic tissues in the rat and human beings

By using a recently developed ion-pairing high-performance liquid chromatographic method for the direct determination of malondialdehyde (MDA) and several other acid-soluble low-mol-wt compounds (ascorbate, oxypurines, nucleosides, nicotinic coenzymes, high-energy phosphates), the variations of tissue and plasma MDA as a function of ischemia and reperfusion were determined in the rat (isolated Langendorff-perfused hearts and short-term incomplete cerebral ischemia) and in human beings (patients suffering from acute myocardial infarction subjected to fibrinolysis). In the rat, the data obtained indicate that, contrary to what had been previously reported in literature, MDA is not present either in control heart or in control brain. Oxygen deprivation induces the production of a low, but detectable amount of MDA in both heart and brain, whereas reperfusion causes a marked increase of MDA in both tissues. In human beings, plasma MDA was deeply affected only in patients suffering from acute myocardial infarction with successful thrombolysis, thus indicating the occurrence of oxygen radical-mediated tissue injury also in humans. On the whole, these results suggest that MDA is a valid biochemical marker of lipid peroxidation of postischemic tissues, which however needs a reliable analytical technique for its determination.     

Development and validation of the Axiom®Apple480K SNP genotyping array

Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome‐wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom^® genotyping array for apple and discuss its potential applications. The array has been built from the high‐depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non‐genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom^®Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom^®Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.     

A case of spontaneous coronary artery disease regression

Coronary angiographic trials have demonstrated that lowering cholesterol can slow the progression of atherosclerosis, limit the formation of new lesions and enhance atherosclerotic regression together with reducing the incidence of clinical events (Waters D, 1996). Spontaneous regression of coronary atherosclerotic lesions is rare. We report the case of a patient with a severe within-stent restenotic lesion whose coronary disease spontaneously regressed 12 months after initial diagnosis, allowing for medical treatment of symptoms rather than repeated intervention. (Int J Cardiovasc Interventions 1999; 2: 121-123)     

A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity

Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders.