Role of free radicals in senescence.
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Aging↗
Biomedical subjects
R G Allen
Publications and source records attributed to R G Allen.
Oxidative stress as a causal factor in differentiation and aging: a unifying hypothesis.
In this article, the authors have pointed out flaws in the current version of the free radical hypothesis of aging and have advanced a new hypothesis that reconciles and encapsulates existing information. The main premise of this hypothesis is that aging is a continuation of development and is thus influenced by genetically programmed phenomena. Completion of various genetic programs and the duration of life are linked to a metabolic potential which is itself a genetically determined sum of energy expenditure. Nevertheless, the rate at which metabolic potential is reached is linked to the rate of metabolism and the level of oxidative stress both of which are influenced by epigenetic stimuli. The current version of the free radical hypothesis postulates that partially reduced oxygen species are produced in aerobic cells in an uncontrolled fashion and do not play any useful physiological function. The principle tenet of the free radical hypothesis is that molecular damage is the underlying cause of aging and that O2- radicals and derivatives induce most of the damage sustained by cells during aging. The authors regard this hypothesis as flawed because it fails to explain either low randomly occurring damage can lead to age-associated changes that are species-specific, or the sequential nature of the changes that occur in aging organisms. In contrast to the free radical hypothesis, our hypothesis can explain the specific and sequential nature of aging-related changes because they are postulated to be neither dependent upon uncontrolled damage nor the cellular capacity to prevent it. Instead, the authors suggest that the damage accumulated during aging is a secondary effect rather than a direct cause of senescence. The authors have shown that cells exert control not only on their level of antioxidant defense but also on their rate of oxidant production. The authors postulate that aging is the terminal stage of development, and as such is influenced genetically. The authors also postulate that a definite sum of energy is required to complete the genetic programs associated with aging. Thus, the rate of aging is linked to the level of oxidative stress; the rate of energy utilization is postulated to determine the level of oxidative stress. Oxidative stress is one of the factors which appears to govern changes in gene expression during differentiation and we suggest that it causes alterations in gene expression during aging. In the authors revised hypothesis, free radicals promote aging by affecting specific genetic programs and the incidental damage they inflict in cells is only a by-product of this process.(ABSTRACT TRUNCATED AT 400 WORDS)
Aging↗
Prevalence of antibodies to the core protein P17, a serological marker during HIV-1 infection.
Studies on monitoring the immune response to viral structural proteins during human immunodeficiency virus (HIV-1) infection have established the significance of antibodies to the core protein p24 during the progression of the disease. We have studied the prevalence of antibodies to the core protein p17 in order to study their diagnostic and prognostic significance in the pathogenesis of HIV-1. Full-length HIV-1 p17, molecularly cloned and expressed in Escherichia coli was purified by immunoaffinity chromatography using an HIV-1 p17-specific monoclonal antibody. A highly sensitive enzyme-linked immunoassay was developed using the purified recombinant p17 as the serological target to detect antibodies to p17. The results indicated that antibodies to p17 decline during progression of disease, with the decline being more dramatic as patients moved from asymptomatic to AIDS-related complex (ARC). Patient specimens deficient in p24 antibody, but having detectable levels of antibody to p17 were almost always positive for p24 antigen. Under these conditions, p17 antibody is an important serological marker because it provides a more consistent marker for core antigens during HIV-1 infection.
Acquired Immunodeficiency Syndrome↗
Discrimination of HIV-2 infection from HIV-1 infection by western blot and radioimmunoprecipitation analysis.
Serum and plasma samples were collected from blood donors who were confirmed positive for antibodies to HIV-1 in the United States, and from blood donors and individuals in West Africa and Portugal who were positive for antibodies to HIV-1, HIV-2, or both. Western blots and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) radioimmunoprecipitation assays (RIPA) utilizing native HIV-1 and HIV-2 proteins were performed on these specimens to determine the ability of these procedures to discriminate between HIV-1 and HIV-2 infections. Extensive serologic cross reactivity between HIV-1 and HIV-2 p24 was found in both populations. Antibody reactivity to the envelope protein gp120 was able to discriminate 20 of 20 (100%) U.S. specimens as HIV-1 infections. In specimens from West Africa and Portugal, Western blot and RIPA were in complete agreement on 33 of 42 samples (78.6%). Among these 33 specimens, 10 were found to be reactive for antibodies to HIV-1 only, 10 were reactive to HIV-2 only, and 13 were considered to be dually reactive, having antibodies reactive with both HIV-1 gp120 and HIV-2 gp120. Nine of the 42 specimens were discordant by Western blot and RIPA classification, being dually reactive by one procedure and reactive with only one viral gp120 by the other technique. Because of the serological cross reactivities between HIV-1 and HIV-2, in certain populations it is difficult to ascertain whether an individual is infected with HIV-1, HIV-2, a new viral type, or whether the individual is infected simultaneously with multiple viruses. More specific tests such as viral isolation or molecular probes may be necessary to distinguish between infections with these viruses in certain populations.
Acquired Immunodeficiency Syndrome↗
Corticotropin-releasing factor differentially regulates anterior and intermediate pituitary lobe proopiomelanocortin gene transcription, nuclear precursor RNA and mature mRNA in vivo.
The proopiomelanocortin (POMC) gene and its peptide products are under complex regulation in the pituitary by multiple hormonal, neurohormonal and neurotransmitter factors. Corticotropin-releasing factor (CRF) stimulates the release of POMC-derived peptides in both anterior and intermediate lobes of the pituitary, while having differential long-term effects on levels of POMC mRNA in the two pituitary lobes in vivo. In the present study, we have analyzed the release of POMC-derived peptides, as well as changes in POMC gene transcription, primary transcript, nuclear mRNA and cytoplasmic mRNA levels following acute and chronic in vivo CRF administration in an attempt to elucidate the mechanism whereby this tissue-specific differential regulation occurs. Subcutaneous injection of CRF led to a substantial increase in plasma adrenocorticotropin, but only a minor sustained rise in plasma alpha-melanocyte-stimulating hormone. CRF was shown to induce rapid, time-dependent increases in POMC gene transcription in both anterior and intermediate pituitary lobes, which were reflected by increases in the level of POMC primary transcript in the nucleus. POMC primary transcript remained 2-fold elevated in the anterior lobe for at least 4 h after a single injection of CRF; in contrast, CRF stimulation of POMC primary transcript in the intermediate lobe was short-lived, and had returned to control values by 60 min after injection. After 7 days of repetitive CRF administration, POMC primary transcript, mature nuclear mRNA and cytoplasmic mRNA were 2.5 to 3.0-fold elevated in the anterior pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenocorticotropic Hormone↗
Association of proenkephalin transcripts with polyribosomes in the heart.
Previous results have shown that the relative abundance of proenkephalin mRNA in the rat heart is comparable to the levels found in the brain; however, the extractable enkephalin-containing peptide levels are much lower in the heart. This lack of correspondence between the levels of transcript and peptide could arise from either the inefficient translation of proenkephalin transcripts or the translation of proenkephalin transcripts into peptides that are rapidly secreted or degraded. To distinguish between these possibilities, the translational status of proenkephalin mRNA in the rat heart was established by Northern blot analysis of sucrose density gradient-sedimented polysomal fractions and compared to the striatum, which is known to efficiently translate proenkephalin transcripts. In both tissues, we detected 1.5-kilobase transcripts, but an additional larger transcript of approximately 3.6 kilobases was detected in the heart. Both transcripts were associated primarily with polyribosomes, suggesting active translation of proenkephalin mRNA in the rat heart. RIA of the culture media and extracts from primary cultures of neonatal rat cardiomyocytes indicated the presence of immunoreactive Met-enkephalin-Arg6-Phe7, which was stimulated by 8-(4-chlorophenylthio)cAMP. These results suggest that proenkephalin transcripts are translated in the heart and that detectable levels of immunoreactive Met-enkephalin-Arg6-Phe7 are present in the media and cell extracts of primary cultures of neonatal rat cardiomyocytes.
Animals↗
Superoxide dismutase induces differentiation of Friend erythroleukemia cells.
Friend erythroleukemia cells (FELC) served as a model system for cell differentiation because these cells can be triggered to differentiate by a variety of chemical agents. Treatment with the classical inducer of differentiation, hexamethylene bisacetamide (HMBA), stimulated superoxide dismutase (SOD) activity, which increased in parallel with HMBA-induced differentiation. Furthermore, FELC were shown to differentiate in response to the addition of liposomes containing SOD. Oxidative treatment with liposomes containing D-amino acid oxidase or xanthine oxidase, cumene peroxide, or potassium superoxide also induced differentiation, whereas antioxidants such as alpha-tocopherol, butylated hydroxytoluene, or beta-carotene did not induce differentiation. Also, HMBA induction of differentiation was suppressed by treatment with antioxidants.
Acetamides↗
Biological oxidation and the mobilization of mitochondrial calcium during the differentiation of Physarum polycephalum.
We have previously reported that calcium is required for the starvation-induced differentiation of the slime mold, Physarum polycephalum. With the exception of calcium, each component of the complex starvation medium may be withheld and the organism will still differentiate into spherules. The results of the present study reveal that spherulation will proceed normally when the microplasmoidal cells are transferred from nutrient medium to a citrate buffer containing only 8 mM CaCl2. Electron microscopy and X-ray microprobe analysis reveal that there is an initial increase in the population of calcium-containing mitochondrial granules when the microplasmodia are induced to differentiate. However, as differentiation proceeds, these granules decrease in number and are virtually absent from the mitochondria of mature spherules. The accumulation and depletion of calcium-containing granules is not observed in a nondifferentiating strain of Physarum cultured under standard conditions, but is observed when this strain is first treated with a calcium-enriched nutrient medium that conditions it for spherulation. Changes in the cellular concentrations of NADH and lipid peroxides, and in the activity of superoxide dismutase, correspond temporally to the pattern of increase and depletion of the calcium-containing inclusions. The oxidative stress associated with starvation-induced spherulation may be a consequence of the active accumulation of calcium; the mobilization of this calcium may then be the event that initiates differentiation.
Animals↗
Oxidative influence on development and differentiation: an overview of a free radical theory of development.
Metabolic gradients exist in developing organisms and are believed to influence development. It has been postulated that the effects of these gradients on development result from differential oxygen supplies to tissues. Oxygen has been found to influence the course of development. Cells and tissues in various stages of differentiation exhibit discrete changes in their antioxidant defenses and in parameters of oxidation. Metabolically generated oxidants have been implicated as one factor that directs the initiation of certain developmental events. Also implicated as factors that modulate developmental processes are the cellular distribution of ions and the cytoskeleton both of which can be influenced by oxidants. The interaction of oxidants with ion balance and cytoskeleton is discussed.
Animals↗
In situ hybridization detection of marked differences in pre-proopiomelanocortin messenger ribonucleic acid content of individual corticotropes and melanotropes.
Recently, heterogeneity of POMC mRNA content between intermediate lobe melanotropes of the rat pituitary gland was demonstrated by in situ hybridization of tissue sections. In the present study the heterogeneity of POMC mRNA content in dispersed rat pituitary cells has been investigated. Acutely dispersed cells from adult male rat anterior or neurointermediate lobe tissues were adhered to poly-L-lysine-coated coverslips. The cells were fixed and then hybridized with 35S-labeled POMC or 1B15 (cyclophilin) cRNA. Parallel studies measuring constitutively expressed cellular 1B15 mRNA content were undertaken to ensure that the apparent single cell differences in POMC mRNA were not inherent to the in situ hybridization procedure. When classified by image analysis, extensive differences in silver grain densities were seen over POMC mRNA-containing cells from both lobes. To determine if mRNA in polysomal configurations was less accessable for hybridization with probes than naked mRNA, cells were preincubated with pactamycin, a potent inhibitor of ribosomal initiation of protein synthesis. Pactamycin had no effect on these results. Thus, there appears to be large differences in POMC mRNA content between individual pituitary cells expressing the same gene product.
Adrenocorticotropic Hormone↗
Molecular bases of biologic aging.
The authors outline the progression of thought on the mechanism of the aging process, giving emphasis to environmental factors that influence genetic events. Discussion is limited to those theories that explain fundamental causes of aging and have a firm thermodynamic basis. The authors propose that the cumulative result of continual oxidative stress and other thermodynamic processes (such as aminoacid racemization and nonenzymatic glycosylation), resulting in altered function and increasing the net entropy of living systems, governs the rate of the aging process.
Aging↗
Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells.
Mammalian cell lines (BSC-40, NG108-15, and GH4C1) that cannot process the murine neuroendocrine peptide precursor prepro-opiomelanocortin (mPOMC) when its synthesis is directed by a vaccinia virus vector were coinfected with a second recombinant vaccinia virus carrying the yeast KEX2 gene, which encodes an endopeptidase that cleaves at pairs of basic amino acid residues. mPOMC was cleaved intracellularly to a set of product peptides normally found in vivo, including mature gamma-lipotropin and beta-endorphin1-31. In GH4C1 cells (a rat pituitary line), product peptides were incorporated into stored secretory granules. These results suggest that the inability of any particular cell line to process a prohormone precursor is due to the absence of a suitable endogenous processing enzyme.
Cell Line↗
Superoxide dismutase induces differentiation in microplasmodia of the slime mold Physarum polycephalum.
Evidence is presented that supports a role for the enzyme superoxide dismutase (SOD) in the differentiation of the slime mold, Physarum polycephalum. SOD activity increases 46-fold during differentiation. A strain of Physarum that does not differentiate exhibits no change in SOD activity. Addition of SOD, via liposomes, to the nondifferentiating strain induces differentiation; this effect is enhanced by an inhibitor of glutathione synthesis. Other antioxidants selected for study failed to induce differentiation. Conversely, oxidative treatments including introduction of D-amino acid oxidase, via liposomes, induced differentiation. Cellular oxidation is the probable cause of the SOD effect.
Isoenzymes↗
Human fibroblast antioxidant defense response to alteration in oxygen tension.
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Antioxidants↗
Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. I. Adrenocorticotropin (ACTH) and alpha-melanotropins (alpha-MSHs).
We have studied the post-translational processing of POMC-derived peptides during fetal monkey development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Pituitary tissues obtained from fetal monkeys ranging from Gestational Day 50 to 155 were fractionated and analyzed for ACTH- and alpha-MSH-related peptides and compared to adult forms. Extracts of whole pituitary from Fetal Days 50 and 55 contained ACTH(1-39) and very small amounts of CLIP (corticotropin-like intermediate-lobe peptide; ACTH(18-39))-like immunoactivity. Acetylated alpha-MSHs were not detectable at Day 50. alpha-MSHs were barely detectable at Day 55. By Day 65, when pituitary lobes were separable, small amounts of des-, mono-, and diacetyl alpha-MSH were detectable in NIL extracts, but not in anterior lobe extracts. ACTH(1-39) levels were negligible when compared to increasing alpha-MSHs through Fetal Day 80 to 155 in the intermediate lobe. The CLIP immunoactivity was negligible in Day 80 and adult anterior lobe extracts. Thus, lobe-specific proteolytic processing of ACTH-related peptides was well established by midterm gestation. Marked increases of alpha-N- and alpha-N,O-acetylated forms of alpha-MSHs were detected during middle and late stage fetal development. Diacetyl alpha-MSH was the predominant form of alpha-MSH in adult NIL extracts. No acetylated alpha-MSHs were found in anterior lobe tissues, thus adult anterior lobe extracts contained almost exclusively ACTH(1-39). However adult NIL extracts contained two distinct forms of CLIP-related immunoactivity. Therefore changes in post-translational processing patterns of ACTH-related and alpha-MSH-related peptides continued to some extent, postnatally. These data indicate that marked changes in post-translational processing of POMC-derived ACTH-related products occur during the first half of monkey gestation.
Adrenocorticotropic Hormone↗
Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. II. beta-Lipotropin (beta-LPH)-related peptides.
We have studied the post-translational processing of POMC-derived peptides during fetal monkey pituitary development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Whole pituitary glands obtained from Day 50 and 55 fetal monkeys and separated lobes From Day 65 to 155 were extracted, fractionated, and analyzed for beta-melanotropin (beta-MSH), midportion beta-endorphin (beta-EP), and acetylated beta-EP immunoactivity. Separated adult pituitary lobes were analyzed for comparison. At Day 50, POMC-containing cells were located in both the anterior and intermediate pituitary lobes by immunofluorescence staining, the majority of these cells were localized in the anterior lobe. The Day 50 and 55 whole pituitaries contained predominantly beta-lipotropin (beta-LPH), gamma-lipotropin (gamma-LPH), beta-EP(1-31), and 2.2-kda beta-MSH. No acetylated products were found in Day 50 whole pituitary extracts. By Day 55, carboxy-shortened and acetylated beta-EPs were barely detectable in whole pituitary extracts. These forms were more apparent in the Day 65 separated neurointermediate lobe (NIL) extracts, and were similar to adult proportions by Day 80. The adult anterior lobe contained predominantly beta-LPH, beta-EP, and gamma-LPH. Adult NILs contained almost exclusively 2.2-kda beta-MSH, alpha-N-acetyl beta-EP(1-31) and alpha-N-acetyl beta-EP(1-27). The production of 2.2-kda beta-LPH in the monkey NIL indicates that monkey beta-LPH is different from rat beta-LPH in that it must contain the paired-basic cleavage site required for the formation of 2.2-kda beta-MSH that is known to be lacking in rat beta-LPH. Another finding was that monkey beta-EP contains a Tyr residue at position 27 as found in human beta-EP but appears to have the rat Gln substitution at position 31. The post-translational processing patterns characteristic of each lobe were well established by midterm fetal development (Day 80).
Animals↗
Oxidative stress and cellular differentiation.
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Calcium↗
Developmental changes in the superoxide dismutase activity of human skin fibroblasts are maintained in vitro and are not caused by oxygen.
Confluent cultures of human skin fibroblast lines established from fetal and postnatal donors were exposed to a broad range of oxygen tensions (10-600 mmHg) for 1 wk; superoxide dismutase (SOD) activity was subsequently determined. Hyperoxia increased SOD activity slightly in postnatal lines but not in fetal lines. The magnitude of the increase in postnatal lines was not significant. Fetal lines exhibit only about one-fifth the SOD activity observed in postnatal lines. The results indicate that, while development-associated changes in SOD do occur in human cells, these alterations do not result from variations in ambient oxygen tension.
Adolescent↗