Identification of longevity-associated genes in long-lived Snell and Ames dwarf mice

Abstract Recent landmark molecular genetic studies have identified an evolutionarily conserved insulin/IGF-1 signal transduction pathway that regulates lifespan. In C. elegans, Drosophila, and rodents, attenuated insulin/IGF-1 signaling appears to regulate lifespan and enhance resistance to environmental stress. The Ames (Prop1 df/df ) and Snell (Pit1 dw/dw ) hypopituitary dwarf mice with growth hormone (GH), thyroidstimulating hormone (TSH), and prolactin deficiencies live 40-60% longer than control mice. Both mutants are resistant to multiple forms of environmental stress in vitro. Taken collectively, these genetic models indicate that diminished insulin/IGF-l signaling may play a central role in the determination of mammalian lifespan by conferring resistance to exogenous and endogenous stressors. These pleiotropic endocrine pathways control diverse programs of gene expression that appear to orchestrate the development of a biological phenotype that promotes longevity. With the ability to investigate thousands of genes simultaneously, several microarray surveys have identified potential longevity assurance genes and provided information on the mechanism(s) by which the dwarf genotypes (dw/dw) and (df/df), and caloric restriction may lead to longevity. We propose that a comparison of specific changes in gene expression shared between Snell and Ames dwarf mice may provide a deeper understanding of the transcriptional mechanisms of longevity determination. Furthermore, we propose that a comparison of the physiological consequences of the Pit1dw and Prop1df mutations may reveal transcriptional profiles similar to those reported for the C. elegans and Drosophila mutants. In this study we have identified classes of genes whose expression is similarly affected in both Snell and Ames dwarf mice. Our comparative microarray data suggest that specific detoxification enzymes of the P 450 (CYP) family as well as oxidative and steroid metabolism may play a key role in longevity assurance of the Snell and Ames dwarf mouse mutants. We propose that the altered expression of these genes defines a biochemical phenotype which may promote longevity in Snell and Ames dwarf mice

Ishemia-Reperfusion enhances GAPDH nitration in aging skeletal muscle

Aging and skeletal muscle ischemia/reperfusion (I/R) injury leads to decreased contractile force generation that increases severely with age. Our studies show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein expression is significantly decreased at 3 and 5 days reperfusion in the young mouse muscle and at 1, 3, 5, and 7 days in the aged muscle. Using PCR, we have shown that GAPDH mRNA levels in young and old muscle increase at 5 days reperfusion compared to control, suggesting that the protein deficit is not transcriptional. Furthermore, while total tyrosine nitration did not increase in the young muscle, GAPDH nitration increased significantly at 1 and 3 days reperfusion. In contrast, total tyrosine nitration in aged muscle increased significantly at 1, 3, and 5 days of reperfusion, with increases in GAPDH nitration at the same time points. We conclude that GAPDH protein levels decrease following I/R, that this is not transcriptionally mediated, that the aged muscle experiences greater oxidative stress, protein modification and GAPDH degradation, possibly contributing to decreased muscle function. We propose that tyrosine nitration enhances GAPDH degradation following I/R and that the persistent decrease of GAPDH in aged muscle is due to the prolonged increase in oxidative modification in this age group

Analysis of the Trypanosoma brucei Genome and Identification and Characterization of a Gene Family Encoding Putative EF-Hand Calcium-Binding Proteins

The flagellum of Trypanosoma brucei contains a family of antigenically related EF-hand calcium-binding proteins which are called the calflagins. Genomic Southern blots indicated that multiple copies of calflagin genes occur in T brucei. All of the copies were contained in a single 23 kb Xhol-Xhol fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. Two new members of the calflagin family were found from genomic clone sequences. The deduced amino acid sequences of the genomic clones showed the calflagin genes were arranged tandemly along the genomic fragments and were similar to previously described calflagins. The calflagin genes were related by two unrelated 3' flanking sequences. An open reading frame that was unrelated to any calflagin was found at the 5' end of the 2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. In general, variability among the T. brucei calflagins is greater than related proteins in T. lewisii and T. cruzi. This variability results from amino acid substitutions at the amino and carboxy termini, and duplication of internal segments

HMGB1 Expression in Fetal Membrane and Amniotic Fluid Cases and Controls A.

<p>Bar graph shows HMGB1 expression in fetal membranes as the mean and standard error of relative number of transcripts as determined by real time PCR. GAPDH was used as an internal control. Comparison among the clinical samples from women at term not in labor (Term Birth), with preterm premature rupture of membranes (pPROM), and with preterm birth with intact membranes (PTB). (ANOVA, *p<0.05). <b>B-E.</b> HMGB1 and acetylated lysine in human amniotic fluid (AF). <b>B.</b> pPROM AF without steroids has a greater amount of HMGB1 than term AF. <b>C</b>. Term AF has a greater amount of total protein acetylation than pPROM AF without steroids. <b>D</b>. pPROM AF with glucocorticoids has a greater amount of HMGB1 than term AF. <b>E</b>. Term AF has a greater amount of total protein acetylation than pPROM AF with steroids. However, HMGB1 retains acetylation in pPROM despite deacetylation of proteins by administered steroids (<i>D vs. E</i>) as seen by the preserved HMGB1 pattern. Interestingly, more proteins are acetylated in pPROM AF without steroid use than with steroid administration (<i>C vs. E</i>). In both term and pPROM, HMGB1 shows a consistent expression pattern (<i>B vs. D</i>).</p