Biomedical subjects

R G Allen

Publications and source records attributed to R G Allen.

At least 37 records · Page 2Linked to original sources

PubMed1999

Effects of donor age on the expression of a marker of replicative senescence (EPC-1) in human dermal fibroblasts.

EPC-1 (early population doubling level cDNA-1) is a quiescence-specific gene expressed at high levels by early passage WI-38 fibroblasts under conditions of either density-dependent growth arrest or serum deprivation. Late passage WI-38 cells lose the ability to express EPC-1 under all conditions tested. The decline in EPC-1 mRNA is gradual during the replicative life span and correlates inversely with the population doubling level (PDL) of the cells. The objective of this study was to determine whether the decline in EPC-7 mRNA abundance observed during proliferative senescence also occurs in cultures derived from donors of different ages. To address this question, we examined the abundance of EPC-1 mRNA in 28 skin fibroblast lines established from healthy donors of different ages ranging from 12 fetal weeks to 94 years. EPC-1 expression was measured, under conditions of growth arrest, prior to the end of the replicative life span of the cultures. Despite some variability in steady-state transcript levels among the cell lines, EPC-1 expression was significantly lower in cells derived from the fetal donor group (12-20 gestational weeks) than in cells derived from adult donors. An in vitro age-dependent decline in EPC-1 expression was observed in all the skin lines examined, independent of donor age; however, no significant difference was observed between the young adult donor group (17-33 years) and the old adult donor group (78-94 years). Thus, expression of EPC-1 is linked to the replicative age of the cells and whether the cells are derived from fetal skin or adult skin. In adults, EPC-1 expression is independent of donor age.

Adolescent↗

PubMed1999

Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38).

The activities and mRNA abundances of enzymes that regulate the rate of electron flow through the electron transport chain (ETC), including NADH dehydrogenase, succinate dehydrogenase, and cytochrome c oxidase, were examined in young and senescent fetal lung fibroblasts (WI-38). We also determined the activities and mRNA abundances of antioxidant defenses including superoxide dismutase, catalase, and glutathione peroxidase. We confirmed our previous report of a senescence-related increase in the abundance of ND4, a mitochondrially encoded subunit of NADH dehydrogenase. The activities of cytochrome c oxidase and NADH dehydrogenase were also elevated in senescent cultures. No differences were observed in the mRNA abundances of COX-1, a mitochondrially encoded subunit of cytochrome c oxidase or of nuclearly encoded subunits of various electron transport components (SD, COX-4, and ND 51). Lucigenin-detected chemiluminescence and H2O2 generation were both elevated in senescent cells. Catalase activity was also elevated in senescent fibroblasts. However, no differences in catalase mRNA abundance were observed. A small decrease in GSH peroxidase (GPx) mRNA abundance was observed in senescent cells. No other changes in the activities or mRNA abundances of any of the antioxidant defenses were observed in early and late passage cultures. The relationships between oxidant generation, mitochondrial enzyme activities, and antioxidant defense observed during proliferative senescence are dissimilar to those detected between fetal and postnatal fibroblasts as well as those found between fibroblast lines obtained from young and old individuals. The relevance of the differences between these models is discussed.

Acridines↗

PubMed1999

Monitoring glycosaminoglycan replenishment in cartilage explants with gadolinium-enhanced magnetic resonance imaging.

We previously devised a magnetic resonance imaging method that allows for the nondestructive and quantitative determination of glycosaminoglycan concentration in excised cartilage. The technique measures the concentration of the charged contrast agent Gd-DTPA2- (gadolinium diethylenetriamine-pentaacetic acid) equilibrated within cartilage, from which the tissue distribution of glycosaminoglycan can be calculated. The goals of our study were to determine the practicality of nondestructively monitoring glycosaminoglycan concentration in cartilage explants over a long-term culture period and to determine if glycosaminoglycan could be restored to glycosaminoglycan-depleted cartilage explants maintained in long-term culture. To meet our objectives, we harvested bovine cartilage explants, treated them initially with trypsin to reduce the glycosaminoglycan concentration, and cultured them for as long as 8 weeks. Images depicting glycosaminoglycan concentration were calculated from magnetic resonance images acquired at selected intervals during the trypsinization process and the subsequent culture period. The results indicate that gadolinium-enhanced magnetic resonance imaging can follow the reduction of glycosaminoglycan concentration over the course of enzymatic digestion and the replenishment of glycosaminoglycan over several weeks of culture and that cultured cartilage explants are capable of restoring glycosaminoglycan to 85% of its initial concentration. Of particular interest, samples cultured for 5 weeks indicated a depth dependence of glycosaminoglycan regeneration to values similar to the initial physiologic distribution. Thus, this magnetic resonance imaging method may be a very powerful means for exploring the spatial and temporal evolution of glycosaminoglycan in cartilage.

Animals↗

PubMed1999

Nociceptin, Phe(1)psi-nociceptin(1 - 13), nocistatin and prepronociceptin(154 - 181) effects on calcium channel currents and a potassium current in rat locus coeruleus in vitro.

1. The actions of the neuropeptide nociceptin, the putative nociceptin receptor antagonist [Phe1psi(CH(2)-NH)Gly(2)]-nociceptin-(1 - 13)NH(2) (Phe(1)psi-nociceptin(1 - 13)) and the putative nociceptin precursor products nocistatin (rat prepronociceptin(125 - 132)) and rat prepronociceptin(154 - 181) were examined on membrane properties of rat locus coeruleus (LC) neurons using whole cell patch clamp techniques. 2. Nociceptin inhibited I(Ba) in all LC neurons, (pD(2) of 8.9, maximum inhibition 50%). The inhibition of I(Ba) by nociceptin was associated with slowing of the activation of I(Ba) and could be significantly reversed by a strong depolarizing prepulse. Phe(1)psi-nociceptin(1 - 13) also inhibited I(Ba) in LC neurons (notional pD(2) of 7.6, maximum inhibition 18%). Application of Phe(1)psi-nociceptin(1 - 13) (1 microM) significantly occluded the subsequent effects of a co-application of nociceptin (3 nM) on I(Ba). 3. As previously reported for nociceptin, Phe(1)psi-nociceptin(1 - 13) caused an outward current in LC neurons voltage clamped at -60 mV (pD(2) of 7.1, maximum current 50% of that of methionine enkephalin, 10 microM). The Phe(1)psi-nociceptin(1 - 13) induced current reversed polarity at -112 mV and exhibited pronounced inward rectification. Phe(1)psi-nociceptin(1 - 13) (1 microM) reversibly inhibited the current caused by nociceptin (300 nM) by 30%. 4. Neither nocistatin nor rat prepronociceptin(154 - 181) inhibited I(Ba) in LC neurons, or prevented the subsequent inhibition by nociceptin. Neither nocistatin or prepronociceptin(154 - 181) affected the membrane properties of LC neurons. 5. This study demonstrates that nociceptin modulates somatic I(Ba) in rat LC neurons. The putative ORL1 antagonist Phe(1)psi-nociceptin(1 - 13) exhibited partial agonist activity at inhibiting I(Ba) and opening K(+) channels in LC. Other putative nociceptin precursor products were without effect on LC cells.

Animals↗

PubMed1999

Cyclic AMP analogs induce synthesis, processing, and secretion of prepro nociceptin/orphanin FQ-derived peptides by NS20Y neuroblastoma cells.

Recent studies have shown that cAMP analogs can induce expression of prepro (pp) orphanin FA (OFQ)/nociceptin-related gene products in NS20Y mouse neuroblastoma cells (Saito et al. [1996]. J Biol Chem 271, 15615-15622). Additionally, exposure of NS20Y cells to cAMP analogs promoted neurite outgrowth and large dense-core vesicle formation. Even though an OFQ-like precursor (called 27K) was identified in NS20Y cell extracts, no secretion of OFQ-related peptides was detected. We have used reversed-phase high-performance liquid chromatography combined with a specific radioimmunoassay for OFQ(1-17) to determine if NS20Y cells secrete ppOFQ-derived peptides when stimulated by the cAMP analog ctp-cAMP. We found that NS20Y cells secreted abundant amounts of OFQ-derived products when stimulated by cAMP analogs. We also have determined that secretion of OFQ peptides was both time and concentration dependent and reversible on removal of cAMP analogs from the culture medium. In addition, the opioid agonist D-Pen2-D-Pen5-enkephalin inhibited forskolin-stimulated OFQ peptide secretion. Further, the synthetic glucocorticoid dexamethasone virtually abolished ctp-cAMP-stimulated OFQ peptide secretion. These results suggest that the biosynthesis, processing, and secretion of the OFQ neuropeptide transmitter system can be modulated through intracellular cAMP levels and that these functions are regulated by opioids and molecules involved in mediating the stress response. The NS20Y cell system will be extremely valuable for studying the regulation of OFQ-derived peptides by a variety of intra-cellular and extracellular signaling pathways.

Animals↗

PubMed1999

Effects of ibuprofen and indomethacin on the regional circulation in newborn piglets.

We investigated the effects of clinically comparable doses of ibuprofen and indomethacin on renal, gastrointestinal and cerebral perfusion in newborn piglets, and hypothesized that ibuprofen would have less effect on regional circulation. Animals were randomly assigned to receive ibuprofen (20 mg/kg, n = 8), indomethacin (0.3 mg/kg, n = 7) or vehicle (n = 6). Fluorescent microspheres were injected prior to and at 20, 40, 60, 90 and 120 min after drug administration. Regional blood flow was measured and vascular resistances were calculated. Cardiovascular and respiratory variables were not significantly affected by either study drug or vehicle. Ibuprofen increased renal cortical and medullary resistance by 44 and 52% (p < 0.05). However, ibuprofen had no significant effects on gastrointestinal or cerebral resistance. Indomethacin raised renal cortical and medullary resistance by 66 and 71% at 60 min postinjection, respectively (p < 0.05). Indomethacin increased duodenojejunal, ileal and colon resistance by 97, 102 and 75% at 60 min, respectively (p < 0.05). Indomethacin increased cerebral cortical and cerebellar resistance by 92 and 86% at 90 min (p < 0. 05). While indomethacin and, to a lesser extent, ibuprofen both increase renal vascular resistance, indomethacin vasoconstricts the gastrointestinal and cerebral circulations which are unaffected by ibuprofen.

Animals↗

PubMed1998

Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation.

Normal human diploid fibroblasts have a finite replicative lifespan in vitro, which has been postulated to be a cellular manifestation of aging in vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared "healthy," according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.

Adolescent↗

PubMed1998

Antioxidants stimulate transcriptional activation of the c-fos gene by multiple pathways in human fetal lung fibroblasts (WI-38).

We have examined the effects of three structurally distinct antioxidants (N-acetylcysteine [NAC], Trolox C [a water-soluble vitamin E derivative], and nordihydroguaiaretic acid [NGA]) on the expression of the c-fos gene over a 2-hour period. Determination of cellular glutathione concentration (the primary determinant of the cellular redox state) over the same time-course verifies that all the compounds studied cause an increase in cellular reduction potential. The level of c-fos messenger RNA increased rapidly in response to micromolar concentrations of these compounds, reaching a peak in 30-60 minutes. Induction of c-fos expression by these antioxidants is at least partly due to an increase in transcription, as determined by nuclear run-on assay. Down regulation of protein kinase C (PKC) by pretreatment for 24 hours with 500 nm PMA prevents induction by subsequent stimulation with either PMA or NGA. NAC induction of c-fos is unaffected by PMA pretreatment, while Trolox C superinduced c-fos following PMA pretreatment. None of these treatments stimulated translocation of PKC-alpha from the cytosol to the membrane. These results suggest that increasing the intracellular reducing potential induces c-fos expression through multiple pathways.

Acetylcysteine↗

PubMed1998

Orphanin FQ is the major OFQ1-17-containing peptide produced in the rodent and monkey hypothalamus.

In order to investigate the processing of OFQ containing peptides in the hypothalamus we have developed a sensitive and quantitative radioimmunoassay for OFQ. We fractionated rodent and monkey hypothalamic extracts by reversed-phase high performance liquid chromatography and found that the extracts contained multiple peaks of OFQ immunoreactivity with the major peak co-eluting with synthetic OFQ1-17. Mouse hypothalamic extracts were also fractionated by SDS-PAGE to determine the apparent molecular weights of molecules containing the OFQ peptide. Multiple peaks of OFQ immunoreactivity, ranging in size from approximately 1 to 30 kilodaltons, were detected by this method. These results suggest that OFQ1-17 is processed to smaller peptides in mouse and monkey hypothalamic neurons.

Amino Acid Sequence↗

PubMed1998

Scintigraphic measurement of regional gastrointestinal transit in the dog.

Scintigraphic techniques can measure sequentially gastric emptying, small bowel transit, and colonic transit in humans, and comparable methods for experimental studies in animals would be useful. We developed such a method in dogs and examined the effects of prokinetic drugs on regional transit. Two isotopes were given to fasting dogs. Polystyrene pellets labeled with 99mTc were mixed in a can of dog food and 111In- labeled pellets were given in a gelatin capsule coated with a pH-sensitive polymer, designed to dissolve in the distal bowel. Gamma camera images were obtained for up to 24 h. Prokinetic drugs were given by intravenous injection. Duplicate baseline studies showed good agreement in seven dogs. In a second group (n = 4), intra- and interanimal variabilities were established. Two novel prokinetic drugs (AU-116 and AU-130) accelerated small bowel and colonic transit. A simple noninvasive method for measuring whole gut transit in dogs was developed and validated. Two new prokinetics accelerated small bowel and colonic transit.

Administration, Oral↗

PubMed1997

Development and age-associated differences in electron transport potential and consequences for oxidant generation.

We determined the activities of NADH dehydrogenase (ND), succinate dehydrogenase, and cytochrome c oxidase (COX) in 29 skin fibroblast lines established from donors ranging in age from 12 gestational weeks to 94 years. The results of this study demonstrate that all three of the enzyme activities examined are greater in adult-derived fibroblasts than in the fetal cell lines. The ratio of enzyme activities that control electron entry into and exit from the electron transport chain varied directly with lucigenin-detected chemiluminescence (an indicator of .O2- generation) and inversely with H2O2 generation. These results indicate a clear difference in the predominant oxidant species generated during fetal and adult stages of life. We also examined the mRNA abundances of different components of the electron transport chain complexes. We observed higher abundances of mitochondrial encoded mRNAs (COX 1 and ND 4) in cell lines established from adults than in fetal cells. No differences in the mRNA abundances of the nuclear encoded sequences (COX 4 and ND 51) were observed in fetal and postnatal-derived lines. Succinate dehydrogenase mRNA abundance was greater in cell lines established from postnatal donors than in fetal cell lines. No significant differences between cell lines established from young and old adults were detected in any of the parameters examined.

Adult↗

PubMed1997

Abundance of alpha 1(I) and alpha 1(III) procollagen and p21 mRNAs in fibroblasts cultured from fetal and postnatal dermis.

The steady-state abundance of alpha 1(I) and alpha 1(III) procollagen mRNAs, p21Sdi1 mRNA, and beta-actin mRNA was determined in 29 skin fibroblast lines established from fetal, young and old donors. Donor ages ranged from 12 gestational weeks to nonagenarian. Adult donors were members of the Baltimore Longitudinal Study of Aging. The abundance of alpha 1(I) procollagen mRNA was decreased in cell lines from both young and old donors compared with fetal lines. Additionally, one alpha 1(I) transcript observed in the fetal lines was not detected in postnatal lines. The abundance of alpha 1(III) procollagen mRNA was decreased in postnatal lines from old donors compared with fetal lines. The abundance of beta-actin mRNA was lower in postnatal lines from both young and old donors compared to fetal lines. These results suggest that cultures of fetal skin fibroblasts exhibit a greater capacity for synthesis of procollagens and beta-actin than postnatal lines. In contrast, the abundance of p21Sdi1 mRNA was elevated in lines established from postnatal donors. These results are consistent with developmental changes in amounts of procollagen, beta-actin and p21.

Actins↗

PubMed1997

Pituitary lactotroph hyperplasia and chronic hyperprolactinemia in dopamine D2 receptor-deficient mice.

Dopamine secreted from hypophysial hypothalamic neurons is a principal inhibitory regulator of pituitary hormone secretion. Mice with a disrupted D2 dopamine receptor gene had chronic hyperprolactinemia and developed anterior lobe lactotroph hyperplasia without evidence of adenomatous transformation. Unexpectedly, the mutant mice had no hyperplasia of the intermediate lobe melanotrophs. Aged female D2 receptor -/- mice developed uterine adenomyosis in response to prolonged prolactin exposure. These data reveal a critical role of hypothalamic dopamine in controlling pituitary growth and support a multistep mechanism for the induction and perpetuation of lactotroph hyperplasia, involving the lack of dopamine signaling, a low androgen/estrogen ratio, and a final autocrine or paracrine "feed-forward" stimulation of mitogenesis, probably by prolactin itself.

Animals↗

PubMed1996

Analysis of the role of calmodulin binding and sequestration in neuromodulin (GAP-43) function.

We demonstrated previously that forced expression of the neuronal phosphoprotein neuromodulin (also known as GAP-43, F1, B-50, and p57) in mouse anterior pituitary AtT-20 cells enhances depolarization-mediated secretion and alters cellular morphology. Here we analyze the role of calmodulin binding by neuromodulin in these responses. In cells expressing wild-type neuromodulin, a complex with calmodulin that is sensitive to intracellular calcium and phosphorylation is localized to the plasma membrane. Transfection of several mutant forms of neuromodulin shows that the effects of this protein on secretion are dependent on both calmodulin binding and association with the plasma membrane. In contrast, the morphological changes depend only on membrane association. Thus, the multitude of effects of neuromodulin noted in previous studies may result from divergent properties of this protein.

Animals↗

PubMed1996

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Amino Acid Sequence↗

PubMed1996

Growth-associated protein-43 (GAP-43) facilitates peptide hormone secretion in mouse anterior pituitary AtT-20 cells.

The neuronal growth-associated protein (GAP)-43 (neuromodulin, B-50, F1), which is concentrated in the growth cones of elongating axons during neuronal development and in nerve terminals in restricted regions of the adult nervous system, has been implicated in the release of neurotransmitter. To study the role of GAP-43 in evoked secretion, we transfected mouse anterior pituitary AtT-20 cells with the rat GAP-43 cDNA and derived stably transfected cell lines. Depolarization-mediated beta-endorphin secretion was greatly enhanced in the GAP-43-expressing AtT-20 cells without a significant change in Ca2+ influx; in contrast, expression of GAP-43 did not alter corticotropin-releasing factor-evoked hormone secretion. The transfected cells also displayed a flattened morphology and extended processes when plated on laminin-coated substrates. These results suggest that AtT-20 cells are a useful model system for further investigations on the precise biological function(s) of GAP-43.

Animals↗

PubMed1996

Effects of cellular aging on the induction of c-fos by antioxidant treatments.

The proto-oncogene c-fos (the cellular homolog of v-fos, Finkel-Biskis-Jenkins (FBJ) murine osteogenic sarcoma virus) encodes a major component of the activator protein-1 (AP-1) transcription factor. Serum stimulation as well as oxidizing treatments induce transitory increases in c-fos mRNA abundance. The induction of c-fos by serum stimulation is also known to decline during proliferative senesence. In this study, we examined the effects of two classes of antioxidants on the induction of c-fos in early and late passage human fetal lung fibroblasts (WI-38). N-acetyl cysteine (NAC) induces c-fos transcription in both early and late passage cells, while nordihydroguaiaretic acid (NGA) induced c-fos transcription in early passage cells but fails to stimulate it in late passage cells. Since we had previously observed an age-related decline in protein kinase C (PKC) translocation from the cytosol to the membrane, following its activation, and because PKC activation appears to be involved in the NGA induction of c-fos we examined the relative protein abundances of several PKC isoforms in early and late passage cells. Additionally, we examined the protein abundance of several members of the MAP kinase pathway which could play a role in c-fos induction by the PKC-dependent pathway. We were unable to detect PKC-beta or theta in early or late passage cells. Late passage cells contained a slightly greater abundance of PKC alpha, gamma and epsilon than cells at an early passage. No other differences in PKC isoforms or in members of the MAP kinase family were observed in early or late passage cells. These results clearly demonstrate that at least some pathways leading to c-fos induction remain intact in late passage cells. While we were unable to detect any decreases in PKC isoforms or MAP kinase proteins we cannot exclude the possibility that functional decrements accumulate in these proteins during senesence.

Aging↗

PubMed1996

Expression of hydrogen peroxide and glutathione metabolizing enzymes in human skin fibroblasts derived from donors of different ages.

We have examined the activities and mRNA abundance of two hydrogen peroxide metabolizing enzymes (glutathione peroxidase and catalase), glutathione concentration, and the activities of several enzymes that influence glutathione concentration, including glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PD), and gamma-glutamylcysteine synthetase (gamma-GCS), in 29 skin fibroblast lines derived from donors ranging in age from 14 gestational weeks to 94 years of age. H2O2 metabolizing enzyme activities and mRNA abundances were greater in skin fibroblast cultures established from postnatal donors than in fetally derived cultures. There were no significant differences in either of these parameters in cell lines established from postnatal donors of different ages. Total glutathione concentration decreased with age, but GR activity appeared to be unaffected by age. In order to estimate the ability of the cultures to produce NADPH (an important component of cellular redox status and a cofactor for GR), we determined glucose-6-phosphate dehydrogenase activity and mRNA abundance. We were unable to directly measure gamma-GCS activity or mRNA abundance in any of the skin lines or in fetal lung fibroblast; however, we were able to indirectly demonstrate the presence of this enzyme by stimulating fetal lung fibroblasts with H2O2 following treatment with L-buthionine-S,R-sulfoximine (BSO), an inhibitor of gamma-GCS activity. These results show that some, but not all, age-associated differences in antioxidant defense levels are maintained in a culture environment and are consistent with the hypothesis that developmental stages of life are associated with lower antioxidant defense levels than are present in postnatal phases of life.

Adolescent↗