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Biomedical subjects

S E Harris

Publications and source records attributed to S E Harris.

At least 19 recordsLinked to original sources
PubMed1992

Molecular cloning of a differentiation-related mRNA in the adipogenic cell line 1246.

The 1246 cell line is a C3H mouse teratoma-derived adipogenic cell line that can proliferate and differentiate in defined medium. We have constructed a recombinant phage library containing complementary DNAs (cDNAs) prepared from mRNA of differentiated 1246 cells. This library was screened using a differential hybridization technique. We have isolated five different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone corresponding to mRNA that is decreased during adipogenesis. Among the mRNAs expressed during adipose differentiation, some are not expressed in undifferentiated cells, whereas some are expressed at very low levels under these conditions. Moreover, the level of induction during differentiation and the temporal expression of the mRNAs corresponding to these cDNAs varied. Our results indicate that one of the cDNA clones isolated, called 154, which selects a 2.2-kilobase mRNA, was induced 100-fold at a very early time during the onset of the differentiation program in 1246 cells and also in adipocyte precursors in primary culture. Direct sequencing of 154 cDNA insert revealed no homology with sequences in GenBank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differentiation such as dexamethasone and isobutylmethylxanthine and inhibited by tumor necrosis factor alpha, transforming growth factor beta, and epidermal growth factor, which are known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipocytes was down-regulated by tumor necrosis factor alpha, but not by transforming growth factor beta or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Cloning of a mu-class glutathione S-transferase complementary DNA and characterization of its glucocorticoid inducibility in a smooth muscle tumor cell line.

A cDNA (designated hGSTYBX) encompassing the complete coding sequence of a hamster mu-class glutathione S-transferase (GST) subunit was cloned from a lambda ZAP library constructed with mRNA isolated from triamcinolone acetonide-treated smooth muscle tumor cells (DDT1 MF-2). Analysis of its nucleotide and deduced amino acid sequences demonstrated highest homology to the rat mu-class GST YB2 subunit. In proliferating subconfluent cells, in which constitutive expression of hGSTYBX mRNA was undetectable, glucocorticoid treatment induced hGSTYBX expression after a time lag of 3 h, and maximal induction occurred at 10 h. Nuclear run-on analysis showed that glucocorticoid induction resulted at least in part from an increased rate of transcription. Simultaneous treatment with glucocorticoid and cycloheximide prevented glucocorticoid induction, but had little effect on basal expression in confluent cells. In contrast, cycloheximide treatment 3 h after glucocorticoid treatment resulted in nearly full induction. These results taken together suggest that hGSTYBX induction may be a secondary glucocorticoid response.

Amino Acid Sequence↗
PubMed1991

Military policies regarding homosexual behavior: an international survey.

The military attachés of 110 non-Communist embassies in Washington, DC, were surveyed from 1982-1984 regarding their countries' military policies about homosexuality. Of the 57 that responded, 37% reported policies excluding persons who engage in homosexual behavior from military service, 14% reported policies that accept homosexual and bisexual persons, and 49% reported having no policies about homosexual behavior. Cultural factors appear to be related to the military policies.

Culture↗
PubMed1990

Structural characterization of the rat seminal vesicle secretion II protein and gene.

The gene encoding rat seminal vesicle secretion II (SVS II) protein has been cloned from a rat genomic DNA library using a cDNA probe generated from rat dorsal prostate androgen-dependent mRNA. The cloned 7.3-kilobase pair genomic fragment contains approximately 5000 base pairs (bp) of the 5'-flanking region and the entire coding region of the SVS II protein within two exons. A sequence of 4156 bp of the rat SVS II gene has been determined, including 2037 bp of the 5'-flanking region, exon 1 (95 bp), intron 1 (236 bp), exon 2 (1171 bp), and 614 bp of the 3'-flanking region. The 5'-flanking region contains three conserved elements found in other seminal vesicle secretion genes (SVS IV-VI proteins) within 250 bp of the transcription start site as well as a glucocorticoid response element at position -314 in the SVS II gene. The first exon encodes a 22-amino acid leader peptide plus the first 2 amino acids of the secreted protein. The second exon encodes the remaining amino acids in the SVS II protein sequence. The mature protein contains 392 residues and has an Mr of 43,116. Concomitant with the gene analysis, the rat SVS II protein was purified to homogeneity, and 333 residues (85%) of the amino acid sequence were determined by automated Edman degradation. The DNA-deduced sequence and that determined by direct analysis of the protein are in complete agreement. The blocked NH2-terminal amino acid was identified as pyroglutamic acid by mass spectrometry and aminopeptidase digestion. A 13-residue structure with the consensus sequence GSQLKSFGQVKSS is repeated 13 times within the SVS II protein and appears to be involved in the formation of the rat copulatory plug via a transglutaminase reaction cross-linking glutamine and lysine residues. Overall, the SVS II protein sequence exhibits little structural relatedness to any other known protein sequence; however, some similarity can be found between the 13-residue repeat and another repeating structure and apparent transglutaminase substrate in the guinea pig seminal vesicle clotting protein.

Amino Acid Sequence↗
PubMed1990

Characterization of the hamster DDT-1 cell aFGF/HGBF-I gene and cDNA and its modulation by steroids.

Syrian hamster DDT-1 cells are derived from smooth muscle of the ductus deferens. DDT-1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF-I and HBGF-II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF-I mRNA. The increase steady-state levels of aFGF/HBGF-I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF-I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3' noncoding sequences were on this clone. A 5' noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF-I's from hamster DDT-1 cells and several other species. The cDNA for hamster aFGF/HBGF-I was isolated from a DDT-1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF-I from four species shows a greater than 90% conservation of amino acid sequence.

Amino Acid Sequence↗
PubMed1990

Patient selection and outcomes for out-of-hospital births in one family practice.

This paper reports a study of the pregnancies followed to delivery in one family medicine group practice that offered a choice of childbirth settings. Those choosing out-of-hospital birth (OHB) were a self-selected group of highly motivated couples interested in natural childbirth or desiring to minimize the cost of pregnancy care. Of 790 singleton pregnancies followed to term, 71 (9.0%) planned home births, 510 (64.6%) planned clinic births, and 209 (26.5%) planned hospital births. Of those planning clinic or home birth, 73% gave birth outside the hospital as planned (44 at home and 379 in the clinic), 81 (14%) changed plans prenatally and gave birth in a local hospital, 46 (8%) were transferred to the local hospital intrapartum, and 29 (5%) were referred to tertiary care. Primiparas who initially chose OHB were more likely than multiparas to give birth in a hospital (46% vs 16%). Controlling retrospectively for obstetric risk and parity, there were few differences in outcome between local hospital and out-of-hospital births. The observed rates of serious complications for OHB were low, but overall, 27% of those initially considered candidates for birth outside the hospital required a change to a higher level of care. For primiparas initially planning clinic or home birth, discriminant analysis revealed five variables that together might have improved the prediction of the eventual decision for hospital delivery in 46%. Clinical pelvimetry was the most powerful variable, in keeping with the finding that most intrapartum transfers were for arrests of labor. The results also suggest that financial factors and other features of the physician-patient relationship influenced clinical decision making.

Adult↗
PubMed1989

Androgens and glucocorticoids modulate heparin-binding growth factor I mRNA accumulation in DDT1 cells as analyzed by in situ hybridization.

The ductus deferens smooth muscle tumor cell line (DDT1-MF-2) is very sensitive to steroids. Treatment with 10 nM testosterone accelerates the growth of DDT1 cells in the absence of serum. Glucocorticoids in the presence or absence of androgens inhibits growth. Stimulation of growth of DDT1 cells by testosterone can be replaced by the addition of heparin-binding growth factor I and II (HBGF). Addition of testosterone plus HBGF growth factors results in a further increase in cell number. By in situ hybridization, accumulation of HBGF-I mRNA is significantly increased by testosterone treatment of low density cultures. Testosterone treatment of high density cultures results in no stimulation of HBGF-I mRNA accumulation. Glucocorticoids alone, which block growth of DDT1 cells, have no effect of HBGF-I mRNA accumulation. However, the simultaneous addition of glucocorticoid and androgens to DDT1 cells results in a rapid accumulation of HBGF-I mRNA by 12 h, although growth is inhibited by the presence of both steroid analogs.

Animals↗
PubMed1988

Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures.

A 1173-base pair cDNA encoding bovine cellular retinaldehyde-binding protein (CRALBP) was cloned from a bovine retinal cDNA expression library using as probes both anti-CRALBP polyclonal and monoclonal antibodies. The amino acid sequence deduced from the cDNA corresponds exactly to that determined by direct analysis of NH2-terminally acetylated bovine CRALBP (Crabb, J. W., Johnson, C. M., Carr, S. A., Armes, L. G., and Saari, J. C. (1988) J. Biol. Chem. 263, 18678-18687). Nick-translated bovine CRALBP cDNA probes were then used to clone from a human retinal cDNA library a 1317-base pair cDNA encoding human CRALBP. Bovine and human CRALBP are 92% identical in amino acid sequence and not related to any other known protein sequence. Both the bovine and human proteins contain 316 residues and have calculated molecular weights of 36,378 and 36,347, respectively, exclusive of the NH2-terminal blocking groups. The CRALBP cDNA clones should prove valuable as tools for studying the physiological role of the protein in vision and visual disorders.

Animals↗
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↗