Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of agedependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H͞HeJ ؋ DW͞J)F1 background. Mutant dw J ͞dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhr lit mutation, which like the Pit1 dw mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1 dw mutant, and the closely related, long-lived Prop-1 df (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.T he analysis of single-gene mutations in flies (1) and nematode worms (2-4) has begun to yield important clues to the molecular basis of aging and genetic control of longevity in invertebrates. At present there are four examples of single gene mutations that extend longevity in mammals (5-8). The best documented of these is the Ames dwarf mutation, now known as Prop-1 df , which in homozygous form has been shown to extend longevity by Ͼ50% in both males and females (5). Homozygous df͞df mice show defects in embryonic development of the anterior pituitary that lead to an absence of cells responsible for the production of growth hormone (GH), thyroid-stimulating hormone, and prolactin (PRL). The small body size of these mice is apparent within the first 3 weeks of age, and young adults are approximately one-third of the size of ϩ͞df or ϩ͞ϩ littermates, which are themselves phenotypically indistinguishable from one another. Extended longevity (about 20%) and small body size also are seen in transgenic mice that express high brain levels of urokinase-type plasminogen activator (6); in this case the phenotypes are thought to reflect a loss of appetite and diminished food intake similar to that seen in genetically normal mice and rats subjected to involuntary food restriction (9). In a third instance, targeted deletion of the p66 shc signal transduction protein has been shown to lead to increased lifespan presumably mediated by increased cellular resistance to apoptosis (7).The fourth example, the Snell dwarf mutation Pit1 dw and the coallelic mutation Pit1 dwJ are the topic of...

Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH-/- mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.

We have proposed that the age-associated increase of reactive oxygen species (ROS) by electron transport chain (ETC) dysfunction may cause the elevated basal level of p38 MAPK stress response pathway activity. However, the mechanism by which ROS activates this pathway is not clear. Here we propose that activation of the p38 MAPK pathway by complex I (CI) generated ROS, in response to rotenone (ROT) treatment, is based on the ability of reduced Trx to bind to and inhibit ASK 1 and its release from the complex upon oxidation. This balance of free vs. bound ASK1 regulates the level of p38 MAPK pathway activity. To support this mechanism we demonstrate that the production of ROS by ROT treated AML12 hepatocyte cells dissociates the Trx-ASK1 complex, thereby increasing p38 MAPK pathway activity. This mechanism is supported by the ability of N-acetyl cysteine (NAC) to prevent dissociation of Trx-ASK1 and activation of the p38 MAPK pathway. We also demonstrated that the ratio of ASK1/Trx-ASK1 increases in aged mouse livers and that this correlates with the increased basal activity of the p38 MAPK pathway. The longevity of Snell dwarf mice has been attributed to their resistance to oxidative stress. A comparison of the levels of Trx-ASK1 in young and aged dwarfs showed a higher abundance of the complex than in their age-matched controls. These results, which are indicative of a decreased level of oxidative stress, suggest that increased ROS production in aged liver may alter the ratio of ASK1 and Trx-ASK1, thereby increasing the age-associated basal level of p38 MAPK pathway activity.-Hsieh, C.-C., Papaconstantinou, J. Thioredoxin-ASK1 complex levels regulate ROS-mediated p38 MAPK pathway activity in livers of aged and long-lived Snell dwarf mice. Keywordsrotenone; MKK3 kinase; MKP-1 levels; MAPK signaling THE FREE RADICAL THEORY OF AGING proposes that endogenously produced oxygen radicals (ROS) are a basic cause of the progressive age-associated declines in tissue function, and that oxidative stress generated by extrinsic environmental factors accelerate this decline (1-6). Some of the biochemical characteristics of aged tissues are the consequences of an increase in their prooxidant state, and it has been hypothesized that this affects the activity and function of key proteins of signal transduction pathways that regulate stress response (7,8). Specifically, our past studies have shown that the regulation of translation of C/EBPα and C/EBPβ transcription factors and the increased basal levels of activities of p38 MAPK and SAPK/JNK stress response signaling pathways suggest the development of a state of chronic stress in aged tissues (4, NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript 7-13). However, although these studies suggest an interaction between the increased levels of intrinsic oxidative stress and of stress response signal transduction pathway activities, little is known about the mechanism that links these physiological characteristics of aged tissues.It has been shown that...

The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBP␣ and C/EBP␤) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse ␣ 1 -acid glycoprotein promoter, we detected multiple forms of C/EBP␣ and C/EBP␤ proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBP␣s with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42-and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBP␤ isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBP␣ forms specific complexes with the ␣ 1 -acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP␤ forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBP␣ and C/EBP␤ isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBP␣ and C/EBP␤ isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42-kDa C/EBP␣ and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBP␣ and the third AUG codon of the 20-kDa C/EBP␤. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors.

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ObjectiveOxidative stress is a postulated etiology of spontaneous preterm birth (PTB) and preterm prelabor rupture of the membranes (pPROM); however, the precise mechanistic role of reactive oxygen species (ROS) in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE), a predicted cause of pregnancy complications, on human amnion epithelial cells.MethodsAmnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2′7′-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE) assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1), phospho-p38 (P-p38 MAPK), and p19arf. Expression of senescence-associated β-galectosidase (SAβ-gal) was used to confirm cell ageing in situ.ResultsROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min) and significantly (p<0.01) at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19Arf correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC) prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19Arf were not significantly affected by NAC.ConclusionsThe findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.