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Biomedical subjects

R G Allen

Publications and source records attributed to R G Allen.

At least 55 records · Page 3Linked to original sources
PubMed1995

Expression and regulation of superoxide dismutase activity in human skin fibroblasts from donors of different ages.

We have determined the activities, protein, and mRNA abundances as well as the level of transcriptional activation of two intracellular forms of the free radical metabolizing enzyme superoxide dismutase in 29 human skin fibroblast lines established from donors of different ages. SOD-1 (a copper and zinc containing form of SOD) and SOD-2 (a manganese containing form of the enzyme) activities were both observed to be significantly lower in cell lines derived from fetal skin than in lines established from postnatal skin (ages 17-94 years). The percent of total activity contributed by SOD-1 decreased in an age-associated manner from approximately 50% in the fetal lines to less than 20% in lines established from old tissue donors. All of the cell lines were screened to exclude the possibility that they contained a polymorphism known to influence SOD-2 activity. Northern blot analysis revealed three SOD-1 mRNA transcripts that were 0.5, 0.7, and 1.9 kb in length. Although SOD-1 protein abundance was lower in fetal lines than in lines derived from postnatal donors, SOD-1 mRNA abundance did not differ between fetal cells and cell lines derived from young donors. SOD-2 protein abundance and mRNA abundance were both significantly lower in fetal lines than in postnatal lines. No postnatal age-dependent differences were observed in any of the SOD-2 parameters examined. Nuclear run-on analysis revealed that fetal cell lines exhibited a lower level of transcriptional initiation for SOD-1 than postnatal lines. The transcription of SOD-2 was readily detected in postnatal lines, but undetectable in fetal lines. These results are consistent with multiple levels of control for SOD-1 expression and with a strong transcriptional influence on SOD-2 expression.

Adolescent↗
PubMed1995

Identification of two classes of prolactin-releasing factors in intermediate lobe tumors from transgenic mice.

Targeted tumorigenesis, using the POMC gene promoter ligated to the simian virus 40 large T antigen, generated transgenic mice with massive tumors of the intermediate lobe (IL) of the pituitary. Inoculation of nude mice with the IL tumor cells resulted in very large secondary tumors. As the IL from several species produces a potent PRL-releasing factor (PRF), it was of interest to determine whether IL tumors from these mice also contain PRF. The objectives were to 1) measure serum PRL levels in mice with IL tumors, 2) determine whether these tumors contain PRF and examine its chromatographic properties, and 3) analyze whether this PRF is related to POMC, its derivatives, or other PRL secretagogues. Serum PRL levels were 5- to 6-fold higher in transgenic than in control mice. Primary and secondary IL tumors were acid extracted and successively fractionated using Sephadex G-100 gel filtration and reverse phase and gel permeation HPLC. PRF activity was determined using short term incubation of tissue extracts or column fractions with GH3 cells. Crude tumor extracts exhibited a strong and dose-dependent PRF activity. Upon chromatography, the PRF activity from either primary or secondary tumors resolved into two classes of compounds: a big PRF with an estimated mol wt of 70-80 kilodaltons and two small, very hydrophobic peptides. The elution profiles of the three PRFs differed from those of beta-endorphin, alpha MSH, beta MSH, ACTH, TRH, oxytocin, angiotensin II, vasoactive intestinal polypeptide, or corticotropin-like intermediate peptide. In summary, we have identified an animal model with IL tumors that has hyperprolactinemia and overproduces PRF. Two classes of PRFs, big and small, were resolved which differ from POMC derivatives and known regulators of PRL release. These data suggest that PRF is produced by melanotrophs, but is not a product of the POMC gene. The IL tumors should provide an excellent source for the purification and structural elucidation of PRFs.

Animals↗
PubMed1995

Targeted ablation of pituitary pre-proopiomelanocortin cells by herpes simplex virus-1 thymidine kinase differentially regulates mRNAs encoding the adrenocorticotropin receptor and aldosterone synthase in the mouse adrenal gland.

We have produced and characterized lines of transgenic mice expressing a fusion gene composed of the pituitary expression-specific promoter region of the POMC gene, driving the herpes simplex viral-1 thymidine kinase. Adult mice were treated with the antiherpes agent ganciclovir at 70 mg/kg body weight (ip, twice daily for 10-12 days). Approximately 98% of the pituitary intermediate lobe melanotropes and anterior lobe corticotropes were ablated as determined by immunocytochemistry and RIA specific for the POMC-derived peptides, ACTH, beta-endorophin, and alpha-MSH. The number of lactotropes, somatotropes, thyrotropes, and gonadotropes was not altered compared with controls, indicating that in the adult pituitary, POMC products are not required to maintain the distribution of cell types. As expected, plasma corticosterone levels were substantially decreased after POMC cell ablation. In situ hybridization studies showed that the mouse ACTH receptor was expressed uniformly throughout the adrenal cortex, and RNase protection assays revealed that the ACTH receptor mRNA decreased after pituitary POMC cell ablation. Additionally, RNase protection assays showed that pituitary POMC cell ablation resulted in the decrease of adrenal p450c11 beta transcripts while p450c11AS (aldosterone synthase) mRNA levels remained constant. These data demonstrate differential regulation of steroid pathway-specific enzymes by POMC products. Our results also suggest that the thymidine kinase cell obliteration technique may not be dependent on cell division as a prerequisite for cytotoxicity, thus supporting the idea that targeted molecular ablation using cell- and tissue-specific promoter sequences to drive viral thymidine kinase expression can be refined further to study other nonmitotic cells.

Adrenal Cortex↗
PubMed1994

Renal vascular effects of epinephrine infusion in the halothane-anesthetized piglet.

The objective of this study was to determine the dose-response effects of epinephrine, given by systemic intravenous infusion to the halothane-anesthetized newborn piglet, on renal blood flow, mean arterial blood pressure, and renal vascular resistance. Seven newborn piglets were acutely instrumented. A transit-time ultrasound flow probe was placed around the renal artery and a femoral arterial catheter was placed for blood pressure monitoring. Epinephrine was infused in doubling doses from 0.2 to 3.2 micrograms.kg-1.min-1. Mean arterial blood pressure increased from 54 mmHg (1 mmHg = 133.3 Pa) to an average of 96 mmHg at 3.2 micrograms.kg-1.min-1 of epinephrine. Renal blood flow increased from 165 mL.min-1 x 100 g-1 at baseline to 185 mL.min-1 x 100 g-1 at a dose of 0.2 microgram.kg-1.min-1 and increased further at 0.4 and 0.8 micrograms.kg-1.min-1 to reach 261 mL.min-1 x 100 g-1. Renal blood flow began to fall at a dose of 3.2 micrograms.kg-1.min-1, remaining however, significantly above baseline (211 mL.min-1 x 100 g-1). Consequently, calculated renal vascular resistance fell as the dose was increased from 0.2 to 0.8 micrograms.kg-1.min-1 and then rose again at 1.6 and 3.2 micrograms.kg-1.min-1, being significantly above baseline at 3.2 micrograms.kg-1.min-1. These results demonstrate that epinephrine when given by systemic infusion to the halothane-anesthetized newborn pig is a renal vasodilator at low doses and causes renal vasoconstriction at moderate to high doses. Renal blood flow remained above baseline at all doses tested, and thus, within the dosage range tested, epinephrine infusion should not cause renal ischemia.

Anesthesia↗
PubMed1994

Genetic alteration of cyclic adenosine 3',5'-monophosphate-dependent protein kinase subunit expression affects calcium currents and beta-endorphin release in AtT-20 clonal pituitary cells.

The role of the cAMP-dependent kinase (AK) in neurotransmission was investigated by genetic alteration of AK subunit expression in AtT-20 clonal pituitary cells. We characterized and compared wild-type [AK(wt)] cells and two clones with different AK activities. The first stably expresses a gene for a mutant AK regulatory subunit (RI) that does not bind cAMP [AK(-)]; the second stably expresses a gene for the catalytic subunit (C) of AK [AK(+)]. Western blot analysis of RI and C subunit expression showed increased expression of both subunits in AK(+) and AK(-) cells relative to AK(wt), with the transfection-induced expression of one subunit producing a compensatory increase in the expression of the other. The basal AK activities varied among the cell types, with AK(+) cells possessing 3-fold higher basal AK activity than AK(wt) cells, and AK(-) cells possessing half the AK activity of AK(wt) cells. Preincubation of cultures with 300 microM 8-(4-chlorophenylthio)-cAMP increased AK activity approximately 4-fold in AK(wt) and AK(+) cells, but was without effect in AK(-) cells. Subsequent addition of 1 microM cAMP in vitro increased AK activity an additional 2- to 3-fold in all cell types. The higher basal AK activity found in AK(wt) and AK(+) cells was associated with larger whole cell calcium currents (approximately 43% and approximately 75% larger than in AK(-) cells, respectively) and faster rates of current rundown. The currents from each cell line had similar voltage-dependent and pharmacological properties, however, and [3H]PN200-110 binding was similar among the cell types. Maximal currents were evoked at clamp potentials of 0-10 mV; currents were inactivated approximately 30% in the steady state at holding potentials of -40 mV compared to -80 mV, and currents were reduced approximately 45% in the presence of nifedipine at -40 mV, but were insensitive to omega-conotoxin GVIA. AK(wt) and AK(+) cells also had higher basal and cAMP-stimulated release of beta-endorphin; control rates were approximately 50% greater, but stimulated rates were approximately 400% greater compared to those in AK(-) cells. We conclude that a greater number of calcium channels were activated by depolarization in the phosphorylated state, that current rundown was largely due to dephosphorylation, and that activation of calcium channels was coupled to the release of beta-endorphin.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗
PubMed1993

Post-translational processing of proopiomelanocortin (POMC) in mouse pituitary melanotroph tumors induced by a POMC-simian virus 40 large T antigen transgene.

Mice harboring a transgene composed of proopiomelanocortin (POMC) gene promoter sequences (nucleotides -706 to +64) ligated to the simian virus (SV) 40 early gene encoding large T antigen developed large POMC-expressing pituitary tumors. Histologically the tumors arose from the intermediate lobe, contained nuclear SV40 T antigen and POMC peptides, but did not express other pituitary hormones. POMC processing in the pituitary tumors was indistinguishable from normal mouse intermediate lobe melanotrophs and was characterized by high proportions of acetylated and carboxyl-terminal shortened beta-endorphins, and amino-terminal acetylated alpha-melanocyte-stimulating hormone, and virtually no adrenocorticotropic hormone (ACTH)(1-39), beta-lipotropin, or POMC. The tumors contained abundant levels of mRNA for the prohormone convertase PC2 and undetectable levels of PC1. Normal mouse neurointermediate lobe also has a high ratio of PC2/PC1 expression that is distinct from the relative abundance of PC1 in anterior lobe and AtT-20 corticotroph cells. In contrast, extracts from tumors transplanted subcutaneously in nude mice contained predominantly nonacetylated forms of beta-endorphin(1-31) and -(1-27), very little ACTH(1-39), almost no corticotropin-like intermediate peptide or alpha-melanocyte-stimulating hormone, and higher proportions of intact POMC. Surprisingly, despite the less efficient proteolytic cleavage, a transplanted tumor expressed both PC1 and PC2. These studies are the first biochemical documentation of a melanotroph pituitary tumor in a rodent species and provide a new model for the investigation of pituitary oncogenesis and the molecular basis of tissue-specific prohormone post-translational processing.

Adrenocorticotropic Hormone↗
PubMed1992

Oxidants and antioxidants in development and differentiation.

Metabolic gradients are known to influence cellular differentiation at critical junctures of development. The existence of these gradients will stimulate variations in the rate of generation of active oxygen metabolites by metabolic pathways. Recent evidence suggests that cellular oxidation can induce changes in gene expression during normal development. Conversely, antioxidants such as ascorbate, alpha-tocopherol and beta-carotene are inhibitory to differentiation in many types of cells. One basis of oxidative influence on gene expression stems from changes in cellular calcium distribution.

Animals↗
PubMed1992

Comparison of arousal responses to tracheal and face mask occlusions in sleeping newborn piglets.

The arousal responses after occlusion of the airway at the mid-trachea were compared with the responses after occlusion of the airway in a face mask in chronically instrumented 3- to 5-day-old piglets. For each site of occlusion arousal latency was significantly longer from active sleep than from quiet sleep. There was a significant increase in the frequency of early arousals after face mask occlusions compared with tracheal occlusions in both sleep states. During quiet sleep the frequency of arousal by 1 s after occlusion was 0.55 with face mask occlusions compared with 0.28 with tracheal occlusion (P less than 0.01). During active sleep the frequency of arousal by 3 s after a face mask occlusion was 0.32 compared with 0.08 after tracheal occlusion (P less than 0.05). Arousal from quiet sleep occurred before changes in arterial oxygen saturation. During active sleep mean saturation at arousal was not different between face mask and tracheal occlusions. Exposure of the upper airway to the pressures generated during airway occlusions results in earlier arousal in both quiet and active sleep, indicating a potential role for upper airway mechanoreceptors in initiating arousal in the newborn piglet.

Airway Obstruction↗
PubMed1992

Differential regulation of proenkephalin expression in astrocytes by cytokines.

Astrocytes have previously been shown to respond to cytokines such as interleukin-1 beta, tumor necrosis factor-alpha, and gamma-interferon from multiple sources including microglia and astrocytes. Recently, astrocytes have also been shown to express the opioid precursor gene proenkephalin and proenkephalin-derived peptides. The objectives of the current study were to determine if immune cytokines regulate proenkephalin gene expression in primary cultures of neonatal rat cerebral astrocytes. Northern analysis of RNA from primary cultures of neonatal rat cerebral astrocytes indicated that proenkephalin transcript levels were decreased by approximately 50% with gamma-interferon treatment and increased approximately 100% by treatment with both tumor necrosis factor-alpha and interleukin-1 beta relative to untreated controls. Tumor necrosis factor-alpha treatment was unable to reverse the inhibitory effect of gamma-interferon pretreatment on proenkephalin messenger RNA levels in the astrocytes. In contrast, expression of the constitutively expressed glutamine synthetase gene was not altered by either tumor necrosis factor-alpha or gamma-interferon treatment. These cytokines also regulate the secretion of proenkephalin-derived peptides from astrocytes. The levels of immunoreactive Met-enkephalin-Arg6-Phe7 were increased by approximately 50% with tumor necrosis factor-alpha and decreased by approximately 40% with gamma-interferon relative to untreated controls. Tumor necrosis factor-alpha was again unable to reverse the inhibitory effect of gamma-interferon pretreatment on the secretion of proenkephalin-derived peptides. These results provide additional support for the hypothesis that rapidly proliferating astrocytes may serve an important and pivotal role in mediating the bi-directional neuroimmune interactions during central nervous system disease, infection, or trauma.

Animals↗
PubMed1991

Developmental patterns in the antioxidant defenses of the housefly, Musca domestica.

The activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S-transferases, GSSG reductase, thiol transferases, gamma glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione-oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.

Animals↗
PubMed1991

Leukemia and preleukemia in Fanconi anemia patients. A review of the literature and report of the International Fanconi Anemia Registry.

Fanconi anemia (FA) is an autosomal recessive disorder characterized clinically by a progressive pancytopenia, diverse congenital abnormalities, and increased predisposition to malignancy. Although a variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, the unique sensitivity of FA cells to the clastogenic effect of DNA cross-linking agents such as diepoxybutane (DEB) can be used to facilitate the diagnosis. We review all cases of FA reported to have leukemia, preleukemia, or a bone marrow (BM) clonal chromosomal abnormality and include for the first time an analysis of these conditions observed in patients in the International Fanconi Anemia Registry (IFAR). The incidence of acute myelogenous leukemia (AML) in FA patients is more than 15,000 times that observed in children in the general population. Cytogenetic studies of FA-associated leukemias disclose a high frequency of monosomy 7 and duplications involving 1q. There were no occurrences of t(8;21), t(15;17), or abnormalities of 11q, which are associated with M2, M3, and M5 leukemias, respectively, but not with preleukemia. Development of leukemia in FA patients was associated with an exceedingly poor prognosis, with a mean age of death of 15 years. We suggest that all FA patients may be considered preleukemic and that this disorder presents a model for study of the etiology of AML.

Acute Disease↗
PubMed1991

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q.

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.

Chromosome Mapping↗
PubMed1991

Antioxidant enzymes and steroid-induced proliferation of kidney tubular cells.

Diethylstilbestrol induces proliferation of Syrian hamster renal proximal tubular cells. By counting the number of cells in culture, we showed that liposomes containing superoxide dismutase or catalase suppressed diethylstilbestrol-induced proliferation, whereas empty liposomes or liposomes containing inactivated superoxide dismutase did not. Liposomes containing antioxidant enzymes did not suppress proliferation of cells in control media or of cells treated with ethinyl estradiol. In the absence of liposomes, exogenous superoxide dismutase did not suppress diethylstilbestrol-induced proliferation. The decrease in cell number when diethylstilbestrol-treated cells were treated with antioxidant enzyme-containing liposomes was not due to decreased cell viability. Results were confirmed by measuring a correlate of cell proliferation immunohistochemically, using an antibody to proliferating cell nuclear antigen. A larger proportion of diethylstilbestrol-treated cells than of control cells showed nuclear immunostaining with this antibody. The number of cells immunostained in diethylstilbestrol-treated cultures was sharply decreased by the addition of superoxide dismutase- or catalase-containing liposomes. Our studies suggest a role for active oxygen species in diethylstilbestrol-induced proliferation of cultured proximal tubular cells.

Animals↗
PubMed1991

Oxygen-reactive species and antioxidant responses during development: the metabolic paradox of cellular differentiation.

Metabolic gradients are established during early phases of development and their existence influences subsequent developmental events. Variations in oxygen supply and oxygen metabolism associated with the gradation of metabolic rate in embryos appear to form one basis for the influence of metabolic gradients on development. The rate of oxygen metabolism affects the rate of oxidant generation by various cellular biochemical pathways. Cells contain antioxidant defenses that respond to variations in cellular oxidant production. Large changes in the activity of the antioxidant enzyme superoxide dismutase and changes in cellular redox state occur during the differentiation of many types of cells. These changes correspond to an increased rate of oxidant production; the cellular environment becomes more prooxidizing during differentiation. Evidence is presented that implicates oxidants as a factor that can stimulate alterations in gene expression. Possible mechanisms by which oxidants influence gene expression are also discussed.

Animals↗
PubMed1990

The regulation of proenkephalin expression in a distinct population of glial cells.

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.

Animals↗