Interrelationship between oxidative damage and antioxidant enzyme activities: an easy and rapid experimental approach

This article describes a method for determining some antioxidant enzyme activities (catalase and/or glutathione peroxidase) and the oxidative status (protein oxidative damage and/or lipid peroxidation) of human blood. However, the main objective of the work is to illustrate the relationship between antioxidant defences and oxidative damage, showing to students their correlation and the general importance of the biochemical regulation in health and diseases.     

Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.     

TUBA8: A new tissue-specific isoform of alpha-tubulin that is highly conserved in human and mouse

We have cloned and sequenced a cDNA from a human adult skeletal muscle cDNA library, encoding for a novel isoform of alpha-tubulin (tuba8) that is preferentially expressed in heart, skeletal muscle, and testis. A genomic DNA sequence from the chromosomal region 22q11 allowed us to determine the complete structure of the TUBA8 gene that mirrors the canonical exon/intron organization of the vertebrate alpha-tubulin genes. We also cloned and sequenced the cDNA of its murine homologue (MMU-TUBA8). The latter encodes for a protein that differs from its human counterpart in only three amino acids, revealing an extreme rate of conservation that is even extended to both the 3' and 5' UTRs of the mRNAs. Sequence comparison of these novel isoforms with other known alpha tubulins shows that tuba8 is the most divergent member of the mammalian alpha-tubulin family. The sequence peculiarity of the human and murine tuba8 strongly suggests that they might have functional significance and, according to the multi-tubulin hypothesis, that they might play specific functional roles in the cell cytoskeleton.     

Elevated cortical extracellular fluid glutamate in transgenic mice expressing human mutant (G93A) Cu/Zn superoxide dismutase

Transgenic mice expressing a mutated (G93A) human Cu/Zn superoxide dismutase (SOD1) develop motor neuron pathology and clinical symptoms similar to those seen in patients with amyotrophic lateral sclerosis. Loss of motor neurons is most prominent in lumbar, followed by cervical cord and then brainstem. No significant cell death has been reported in motor cortex. The integrity of the cortical glutamate reuptake systems was evaluated using intracerebral microdialysis and western immunoblot assays for the glutamate transporters GLT-1, GLAST, and EAAC1. The basal extracellular fluid levels of aspartate, glutamate, glutamine, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid were evaluated by HPLC. The extraction fraction of L-3H]glutamate, corrected with [14C]mannitol, was also evaluated. GLT-1, EAAC1, and GLAST protein levels were determined by semiquantitative chemiluminescence immunoblot of proteins from membrane-enriched fractions. The relative optical density of film was translated into relative protein level by comparison with a standard control mouse. The SOD1 mutant mice demonstrated a significant (p < 0.05) increase in basal levels of extracellular aspartate and glutamate. In addition, when the glutamate extraction fraction was challenged with exogenous unlabeled glutamate (500 microM) by reversed microdialysis, the glutamate extraction fraction in the mutant SOD1 mice was decreased significantly from control levels. The SOD1 mutant mice demonstrated no difference in the cortical protein levels of the glutamate transporter subtypes. This study demonstrates that in areas of no visible pathology and no loss of glutamate transporter proteins, SOD1 mutant mice have elevated extracellular fluid aspartate and glutamate levels and a decreased capacity to clear glutamate from the extracellular space.     

Identification of PEX10, the gene defective in complementation group 7 of the peroxisome-biogenesis disorders.

The peroxisome-biogenesis disorders (PBDs) are a group of genetically heterogeneous, lethal diseases that are characterized by neuronal, hepatic, and renal abnormalities; severe mental retardation; and, in their most severe form, death within the 1st year of life. Cells from all PBD patients exhibit decreased import of one or more classes of peroxisome matrix proteins, a phenotype shared by yeast pex mutants. We identified the human orthologue of yeast PEX10 and observed that its expression rescues peroxisomal matrix-protein import in PBD patients'' fibroblasts from complementation group 7 (CG7). In addition, we detected mutations on both copies of PEX10 in two unrelated CG7 patients. A Zellweger syndrome patient, PBD100, was homozygous for a splice donor-site mutation that results in exon skipping and loss of 407 bp from the PEX10 open reading frame. A more mildly affected neonatal adrenoleukodystrophy patient was a compound heterozygote for a missense mutation in the PEX10 zinc-binding domain, H290Q, and for a nonsense mutation, R125ter. Although all three mutations attenuate PEX10 activity, the two alleles detected in the mildly affected patient, PBD052, encode partially functional PEX10 proteins. PEX10-deficient PBD100 cells contain many peroxisomes and import peroxisomal membrane proteins but do not import peroxisomal matrix proteins, indicating that loss of PEX10 has its most pronounced effect on peroxisomal matrix-protein import.     

Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes in Escherichia coli

Escherichia coli strains devoid of one or both of the two pyruvate kinase isoenzymes (PKA and PKF), were grown on minimal media in batch fermentations. The strain lacking both PKs showed a 28% decrease on its specific growth rate when compared to the wild type. However, protein and CO2 yields did not change. Using radioactive 1-C14 glucose and collecting the CO2 produced by the cultures, it was found that the mutant lacking both pyruvate kinases, metabolized glucose mainly through the pentose pathway (PP). The increased participation of the PP in glucose metabolism in this strain, was also reflected on the levels of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases.Copyright 1998 John Wiley & Sons, Inc.     

Free Amino Acid Composition of Phloem Sap and Growing Fruit of Lycopersicon esculentum

Pure phloem sap of tomato leaves was collected by stylectomy.Glutamine and glutamate were the predominant free amino acidstranslocated by the phloem stream. In developing fruits glutaminecontent increased significantly, reaching 35% of the total freeamino acids. Comparison in the amino acid composition betweenthe two tissues are discussed. (Received October 6, 1997; Accepted January 27, 1998)     

Transgenic tobacco plants expressing antisense ferredoxin-NADP(H) reductase transcripts display increased susceptibility to photo-oxidative damage

Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.     

Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.     

Calmodulin Inhibitors from Aspergillus stromatoides

An organic extract was prepared from the culture medium and mycelia of the marine fungus Aspergillus stromatoides Raper & Fennell . The extract was fractionated via column chromatography, and the resulting fractions were tested for their abilities to quench the fluorescence of the calmodulin (CaM) biosensor hCaM M124C‐mBBr. From the active fraction, emodin ( 1 ) and ω‐hydroxyemodin ( 2 ) were isolated as CaM inhibitors. Anthraquinones 1 and 2 quenched the fluorescence of the hCaM M124C‐mBBr biosensor in a concentration‐dependent manner with K_d values of 0.33 and 0.76 μM , respectively. The results were compared with those of chlorpromazine (CPZ), a classical inhibitor of CaM, with a K_d value of 1.25 μM . Docking analysis revealed that 1 and 2 bind to the same pocket of CPZ. The CaM inhibitor properties of 1 and 2 were correlated with some of their reported biological properties. Citrinin ( 3 ), methyl 8‐hydroxy‐6‐methyl‐9‐oxo‐9H‐xanthene‐1‐carboxylate ( 4 ), and coniochaetone A ( 5 ) were also isolated in the present study. The X‐ray structure of 5 is reported for the first time.