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S E Harris

Publications and source records attributed to S E Harris.

At least 91 records · Page 5Linked to original sources
PubMed1988

Structure of the androgen dependent SVS VI protein as derived from cDNA.

The 11S poly(A+)RNA from rat seminal vesicle was cloned. By hybrid selection of clones reacting to the low molecular weight region of the 11S peak, a clone containing a new seminal vesicle cDNA sequence called SVS VI was isolated. The DNA sequence of two overlapping cDNAs (pSV24 and pSV33) is presented. The sequence of SVS VI was compared to the previously isolated SVS IV and SVS V cDNAS. Dot hybridization showed that SVS VI is androgen responsive after giving testosterone to castrated rats. The hydrophilicity was analyzed using standard Bionet procedures. All three proteins are extremely variable, rich in alpha-helix and very water soluble. The computer predicted hydrophilicity is compared for SVS VI, V and IV. A small region in the 3'-non-coding area of SVS VI has high similarity to a region in SVS IV mRNA.

Amino Acid Sequence↗
PubMed1988

Endotoxin-induced suppression of pulmonary antibacterial defenses against Staphylococcus aureus.

In order to evaluate the effect of endotoxin on lung host defenses, Sprague-Dawley rats were intravenously injected with either placebo or 5 mg/kg of Escherichia coli lipopolysaccharide B. Two hours after treatment, animals were challenged with Staphylococcus aureus by either low dose aerosol inhalation or high dose intratracheal instillation of the bacteria into the lungs. Quantitative lung bacteriologic examination and bronchoalveolar lavage (BAL) for total and differential cell counts were performed immediately (zero hour) and at 4 h after bacterial challenge. Lung phagocytic defenses against aerosolized S. aureus challenges are provided solely by the alveolar macrophage (AM) in the absence of inflammation. In aerosol-challenged control rats, 20.6 +/- 2.0% of the initial deposited bacterial challenge remained viable in the lung at 4 h. Animals pretreated with endotoxin, however, showed a significant decrease in pulmonary bactericidal activity (31.3 +/- 3.4% bacteria remaining at 4 h), indicating a defect in alveolar macrophage (AM) function. Further assessment of the bactericidal oxidative metabolism of endotoxin-treated AM by luminol-enhanced chemiluminescence indicated an increased production of free radical oxygen species when compared with control nontreated cells in both the unstimulated (66 +/- 4 versus 38 +/- 7 x 10(3) cpm in control) and stimulated (250.5 +/- 17.1 versus 147.1 +/- 6.2 x 10(3) cpm in control) states. Total and differential cell counts in both control and endotoxin-treated aerosol-challenged rats were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosols↗
PubMed1987

Toxicological interactions between carbon monoxide and carbon dioxide.

Fischer 344 male rats were subjected to 30-min individual or combined exposures of carbon monoxide (CO) and carbon dioxide (CO2). All deaths from CO occurred during the exposures, and the LC50 values were 4600 and 5000 ppm, depending on experimental conditions. Animals exposed to CO2 concentrations ranging from 1.3 to 14.7% for 30 min were neither incapacitated nor fatally injured. The addition of nonlethal concentrations of CO2 (1.7 to 17.3%) to sublethal concentrations of CO (2500 to 4000 ppm) caused deaths of the exposed rats both during and following (up to 24 h) the 30-min exposures. The most toxic combination of these two gases (2500 ppm CO plus 5% CO2) increased the rate of carboxyhemoglobin (COHb) formation 1.5 times that found in rats exposed to 2500 ppm of CO alone. The COHb equilibrium levels were the same. Exposure to both CO and CO2 produced a greater degree of acidosis and a longer recovery time than that observed with either single gas. The results fit a mathematical model indicating a synergistic interaction. Combustion of 11 materials at their LC50 values indicated that CO was probably the primary toxicant in one case and that the combined CO plus CO2 was the cause of the deaths in three other cases. Additional fire gases need to be studied to explain deaths from the other materials.

Animals↗
PubMed1987

Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells.

Oxidative damage to DNA in cultured bovine adrenocortical cells was investigated by exposing cells to a sublethal concentration (10 microM) of cumene hydroperoxide under conditions previously shown to be deficient in the biological antioxidants selenium and alpha-tocopherol (vitamin E). DNA prepared from cells incubated for 4 h with 10 microM cumene hydroperoxide had a greater fraction showing resistance to S1 nuclease after denaturation and reassociation to a log C0t of -3. Cross-linking by cumene hydroperoxide was abolished in cells that had been grown in the presence of 20 nM selenite or 1 microM alpha-tocopherol for 96 h prior to peroxide addition, whereas such cells remained susceptible to cross-linking by nitrogen mustard. Extensive strand breaks in DNA from peroxide-treated cells as assessed by alkaline sucrose gradient centrifugation were greatly reduced in cells grown in selenite or alpha-tocopherol. Despite the evidence of damage to DNA, cumene hydroperoxide was not detectably mutagenic, in contrast to 5 microM methylnitronitrosoguanidine (MNNG), when assessed as the incidence of resistance to 25 microM ouabain. We confirmed that cumene hydroperoxide at greater than 10 microM lowers cloning efficiency and that this is largely prevented by selenite or alpha-tocopherol. Additionally, selenite or alpha-tocopherol produced increased clonogenicity in cells not incubated with peroxide. To examine effects of the biological antioxidants on replicative lifespan, cells were grown continuously in fetal bovine serum (FBS), fibroblast growth factor (FGF), and selenite or alpha-tocopherol. Selenium increased replicative lifespan by 10-20% and alpha-tocopherol by 22-30%. Levels of DNA cross-links and strand breaks did not differ under any circumstances between early (second) passage and late (30th) passage cells. The experiments on replicative potential were all performed in the presence of FGF. When FGF was omitted from the culture medium, replicative lifespan was reduced by 85%. We conclude that types of damage to DNA resulting from peroxide exposure are not present in cells under standard culture conditions at early or late stages of the lifespan. Other work has noted a relationship between clonogenicity and replicative lifespan; thus, the increase in cloning efficiency seen with selenium and alpha-tocopherol may cause the observed slight increase in replicative lifespan. Oxidative damage does not appear to be a major determinant of cellular senescence in adrenocortical cells.

Adrenal Cortex↗
PubMed1987

Effects of exposure to single or multiple combinations of the predominant toxic gases and low oxygen atmospheres produced in fires.

The toxicity of single and multiple fire gases is studied to determine whether the toxic effects of the combustion products from materials can be explained by the toxicological interactions (as indicated by lethality) of the primary fire gases or if minor, more obscure gases need to be considered. LC50 values for Fischer-344 rats have been calculated for the individual gases, carbon monoxide (CO), hydrogen cyanide (HCN), or decreased oxygen (O2), for 30-min exposures plus relevant postexposure periods using the NBS Toxicity Test Method. Combination experiments with CO and HCN indicate that they act in an additive manner. Synergistic effects have been found when the animals are exposed to certain combinations of CO and carbon dioxide (CO2). Five percent CO2 raised the threshold for deaths due to hypoxia and decreased the LC50 of HCN. Decreasing the O2 concentration in the presence of various mixtures of the other major fire gases increased the toxicity even further. A comparison of the concentrations of the major combustion products generated from a number of polymeric materials at their LC50 (30-min exposure plus 14-day postexposure) values with the combined pure gas results indicates that, in most cases, the observed toxicity may be explained by the toxicological interactions of the examined primary toxic fire gases. These results provide necessary information for the computer model currently being developed at the Center for Fire Research to predict the toxic hazard that people will experience under various fire scenarios.

Air↗
PubMed1986

Methylcholanthrene: a possible pseudosubstrate for adrenocortical 17 alpha-hydroxylase and aryl hydrocarbon hydroxylase.

In cultured bovine adrenocortical cells, loss of 17 alpha-hydroxylase activity was observed after incubation with 3-methylcholanthrene (3-MC). The suppression of 17 alpha-hydroxylase by 3-MC was rapid (50% loss of activity in 10 hr at 1 microM 3-MC), did not exhibit a lag period, and was not affected by cycloheximide. Direct effects of 3-MC on 17 alpha-hydroxylase were observed only at high concentrations, but the concentration for 50% loss of activity was 0.3 microM when 3-MC was added for 24 hr prior to assay of 17 alpha-hydroxylase. High concentrations (to 40 microM) of substrate (progesterone), did not affect the loss of activity due to 3-MC. Loss of 17 alpha-hydroxylase activity was specific; 11 beta-hydroxylase was unaffected and cell growth was unaltered. However, 22-amino-23,24-bisnorchol-5-en-3 beta-ol, an inhibitor of 17 alpha-hydroxylase, partially prevented the loss of 17 alpha-hydroxylase at 1-30 nM. 3-MC is thought to induce cytochrome P-450s via a receptor with high affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD was without effect on 17 alpha-hydroxylase over the range of 10 nM to 10 microM. Benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, chrysene, and methylphenanthrenes suppressed 17 alpha-hydroxylase at high concentrations (10-50 microM for 50% loss of activity). Some steroids that lack a substituent at position 17 also caused loss of 17 alpha-hydroxylase. Like 17 alpha-hydroxylase, bovine adreno-cortical cell AHH was found to be suppressed by exposure to 3-MC. Compounds that caused loss of 17 alpha-hydroxylase caused loss of AHH, with a similar order of potency and at similar concentrations. Suppression of AHH by 3-MC did not require protein synthesis and was prevented by an inhibitor of enzymatic activity, alpha-naphthoflavone. This implies a degree of similarity of the cytochrome P-450s for 17 alpha-hydroxylase and adrenal AHH, but the activities were shown to be likely due to different enzymes. The suppression of 17 alpha-hydroxylase and AHH by 3-MC appears not to occur by a receptor-mediated mechanism but to be similar to the suppression of 11 beta-hydroxylase and 21-hydroxylase by steroid pseudosubstrates previously observed.

Adrenal Cortex↗
PubMed1986

Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence.

During cellular senescence, non-clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin- or prostaglandin E1-stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH-stimulated cAMP production rates were measured in clones immediately after isolation, during long-term growth following isolation, and after subcloning. ACTH-stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/10(6) cells/min, whereas mass cultures at this PDL showed approximately 50 pmol/10(6) cells/min. Forskolin-stimulated cAMP production rates in clones varied only over the range of 59-119 pmol/10(6) cells/min and showed no correlation with the ACTH-stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP-inducible 17 alpha-hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub-subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eight-fold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.

Adrenal Cortex↗
PubMed1985

Hormonal regulation of benzo[a]pyrene metabolism in human adrenocortical cell cultures.

In cultured fetal human adrenocortical cells, metabolism of the carcinogen benzo[a]pyrene was found to be unresponsive to the xenobiotic inducers 3-methylcholanthrene, benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, exposure of cultures to the hormone adrenocorticotropin (ACTH) for 48 hours stimulated benzo[a]pyrene metabolism 3-fold. The major metabolite was the 7,8-diol. Other compounds which stimulate the production of adrenocortical cell cyclic AMP (forskolin and cholera toxin) as well as monobutyryl cyclic AMP also increased benzo[a]pyrene metabolism. Human adrenocortical cells thus provide an unusual example of hormonal regulation of the metabolism of a carcinogen.

Adrenal Cortex↗
PubMed1985

Mode of action of butylated hydroxyanisole (BHA) and other phenols in preventing loss of 11 beta-hydroxylase activity in cultured bovine adrenocortical cells.

When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11 beta-hydroxylase (cytochrome P-45011 beta), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11 beta-hydroxylase against loss of enzymatic activity in the presence of pseudosubstrates:BHA (butylated hydroxyanisole,2(3)-tert-butyl-4-methoxyphenol), dimethyl sulfoxide (DMSO), and metyrapone. The present experiments examine the protective effects of several phenolic analogs of BHA in this system, and compare their activities to that of DMSO and metyrapone. When a variety of analogs of BHA were tested for their abilities to prevent loss of 11 beta-hydroxylase activity in cultured adrenocortical cells incubated with 50 microM cortisol for 24 hr, phenol itself was found to be about equipotent with BHA. Addition of methyl, methoxy and benzyl groups to phenol did not diminish protective activity of the compound, but addition of one and particularly two tert-butyl groups greatly diminished activity. Thus, BHT(2,6-di-t-butyl-4-methylphenol) was inactive, in contrast to BHA. The hydroxy group of phenol was essential since benzene and fluorobenzene were inactive. Compounds with multiple hydroxyl groups were not as active as phenol itself, with the exception of catechol. No products of phenol formed during incubations of cells with cortisol were detected by high performance liquid chromatography. Estimated EC50 values for protection of 11 beta-hydroxylase by phenols were about 100 microM, whereas the EC50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarithmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11 beta-hydroxylase, phenols and DMSO were tested for prevention of loss of 11 beta-hydroxylase activity at three different oxygen concentrations (2, 5, and 19% O2). Lowering the oxygen concentration itself resulted in a small diminution of the loss of 11 beta-hydroxylase. Phenols and dimethyl sulfoxide were more effective at low oxygen and less effective in air. Because the cytochrome P-450 inhibitor metyrapone was found previously to be very effective in protecting 11 beta-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11 beta-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11 beta-hydroxylase, but at concentrations that ranged from 4- to greater than 100-fold higher than those required for protection.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex↗
PubMed1985

Selenium deficiency in cultured adrenocortical cells: restoration of glutathione peroxidase and resistance to hydroperoxides on addition of selenium.

Cultured bovine adrenocortical cells were previously shown to be functionally deficient in selenium and vitamin E when grown in medium supplemented with fetal bovine serum. In the present experiments, the lack of significant bioavailable amounts of selenium in the medium was demonstrated by the finding of only low levels of glutathione peroxidase in the cultured cells (0.008 U/mg protein compared with 0.045 U/mg protein in fresh adrenocortical tissue). When 20 nM selenium as selenite was added to the cultured adrenocortical cells, glutathione peroxidase activity increased continuously over 72 h, with a total increase of about eightfold over this period. Over the same time-course, the highest concentration of cumene hydroperoxide tolerated by the cells without cell death increased progressively from 10 microM to 50 microM. Addition of 1 microM alpha-tocopherol also increased the amount of cumene hydroperoxide tolerated to 50 microM. Cell death was measured by cloning efficiency after removal of cumene hydroperoxide. Addition of either selenium or alpha-tocopherol had little effect on the growth rate of the cells over six passages, even when residual vitamin E was removed from the serum by extraction with ether and residual low molecular weight selenium compounds were removed by dialysis. It is concluded that combined deficiency of selenium and vitamin E, at least in the presence of other components of fetal bovine serum, has little effect on the ability of the cells to survive under normal conditions, as evidenced by continued long-term proliferation. However, the low levels of glutathione peroxidase resulting from selenium deficiency cause an increase susceptibility to peroxide-mediated toxicity. The combined deficiency of selenium and vitamin E impairs the ability of cells to survive under adverse conditions, as well as altering mitochondrial functions, as previously demonstrated.

Adrenal Cortex↗
PubMed1985

Growth of seminal vesicle epithelial cells in serum-free collagen gel culture.

Epithelial cells from mouse seminal vesicles were enzymatically dissociated, enriched by gradient centrifugation, and maintained in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morphologically, immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 microgram/ml). The cells required EGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin, and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free media. A time-course study revealed that a lag period preceded rapid growth. The collagen gel, serum-free culture provides a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells.

Animals↗
PubMed1985

The role of ascorbic acid in the function of the adrenal cortex: studies in adrenocortical cells in culture.

To investigate the role of ascorbic acid in the function of the adrenal cortex, we studied the effects of ascorbate on the regulation of 11 beta-hydroxylase in culture. When primary bovine adrenocortical cells were cultured in a serum-free defined medium in the absence of ACTH, 11 beta-hydroxylase activity declined with a half-time of about 40 h. When 50 microM cortisol, which acts as a pseudosubstrate for 11 beta-hydroxylase, was added to such cultures, 11 beta-hydroxylase activity declined with a half-time of about 6 h. Ascorbate (5 mM) markedly reduced the rate of loss of 11 beta-hydroxylase activity in the presence of cortisol. Previous studies showed that phenolic and sulfoxide antioxidants, which also prevent loss of 11 beta-hydroxylase activity, inhibited the enzyme at concentrations somewhat higher than those required for protective activity. However, ascorbate at concentrations from 10 microM to 5 mM did not inhibit 11 beta-hydroxylase. The same range of ascorbate concentrations added to cells during a 24-h preincubation with cortisol showed increasing prevention of loss of 11 beta-hydroxylase activity. Ascorbate and a lowered concentration of oxygen were synergistic in their protective action. At 2% oxygen, 5 mM ascorbate almost completely prevented loss of 11 beta-hydroxylase activity in the presence of 50 microM cortisol. 11 beta-Hydroxylase activity was reinduced over a period of 5 days in third passage cultures by addition of 1 microM ACTH in defined lipoprotein-free medium. Addition of ascorbate enhanced the reinduction about 2-fold. The action of ascorbate in prevention of pseudosubstrate-mediated loss of activity and in enhancing reinduction of 11 beta-hydroxylase is specific; neither alpha-tocopherol nor selenium prevented loss of 11 beta-hydroxylase in the presence of cortisol or enhanced reinduction of 11 beta-hydroxylase in the presence of ACTH. As an additional test of specificity, it was shown that reinduction of 17-hydroxylase activity was completely unaffected by ascorbate, selenium, or alpha-tocopherol, and addition of cortisol to cultures with high 17-hydroxylase did not result in any loss of enzyme activity. Thus, a major function of ascorbate in the adrenal cortex is as a protective compound for cytochrome.

Adrenal Cortex↗
PubMed1984

Mode of action of sulfoxides in preventing loss of activity of 11 beta-hydroxylase in cultured bovine adrenocortical cells.

Previously, loss of 11 beta-hydroxylase activity when adrenocortical cells are incubated with the pseudosubstrate cortisol was found to be reduced when the concentration of oxygen was lowered, or when butylated hydroxyanisole (BHA) or dimethyl sulfoxide (Me2SO) were included in the medium. In the present experiments, we tested the hypothesis that Me2SO protects 11 beta-hydroxylase by scavenging OH. radicals. Substances known to react with OH. at high rates and non-toxic enough to be used at concentrations of 10-100 mM, including several alcohols, benzoate and radioprotectant thiols, did not prevent loss of activity of 11 beta-hydroxylase in the presence of 50 microM cortisol. Two of the alcohols, ethanol and glycerol, as well as Me2SO, were radioprotective in cultured bovine adrenocortical cells. Therefore free OH. radicals do not appear to be involved in loss of 11 beta-hydroxylase activity. When sulfoxides other than dimethyl sulfoxide were tested for their ability to protect 11 beta-hydroxylase in the presence of cortisol, several aryl sulfoxides, particularly dibenzyl sulfoxide, as well as dipropyl sulfoxide, were active at concentrations to 1/200 of that required for Me2SO. Previously, we have demonstrated that 11 beta-hydroxylase inhibitors, particularly metyrapone, effectively protect against loss of 11 beta-hydroxylase activity in the presence of pseudosubstrates and therefore we examined whether sulfoxides may act by directly inhibiting 11 beta-hydroxylase. Me2SO showed an ED50 for inhibition of 11 beta-hydroxylase activity of greater than 1 M, in contrast to its ED50 for protection of 34 mM. For metyrapone, however, the ED50 for inhibition of the enzyme (250 nM) was close to that for protection of activity (270 nM). The other sulfoxides showed ED50-values for inhibition of 11 beta-hydroxylase that were substantially higher than the ED50-values for protection. Sulfoxides may have a mixed mode of action in protection of 11 beta-hydroxylase activity, as previously shown for phenols; they may protect by radical scavenging, but may also need to bind close to the active site of the enzyme where destructive radicals may be formed.

Adrenal Cortex↗
PubMed1984

Molecular differentiation of the mouse genital tract: altered protein synthesis following prenatal exposure to diethylstilbestrol.

Exposure in utero to the synthetic estrogen diethylstilbestrol (DES) has been associated with the subsequent development of reproductive tract lesions in both women and experimental animals. Using the techniques of organ culture and two-dimensional (2-D) gel electrophoresis, the effects of DES on protein synthetic patterns were studied during fetal and neonatal development of the CD-1 mouse. The protein patterns, analyzed by comparing 2-D fluorograms after [35S] methionine incorporation at different developmental stages, were correlated with the histology at the same age. Several qualitative and quantitative changes in protein synthesis were observed after prenatal DES exposure. A protein, apparent by Day 14 of gestation, with molecular weight approximately 70,000 and pI of 5.8, was observed to be greatly diminished in all reproductive tract tissues exposed to DES during prenatal development. This alteration, induced in utero, persists through the early postnatal differentiation of the genital tract (17 days old). This protein may provide an early marker for alterations in normal reproductive tract function.

Animals↗