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Biomedical subjects

M S Miller

Publications and source records attributed to M S Miller.

At least 91 records · Page 5Linked to original sources
PubMed1989

Differential induction of fetal mouse liver and lung cytochromes P-450 by beta-naphthoflavone and 3-methylcholanthrene.

Previous studies have shown that the incidences of liver and lung tumors in mice exposed transplacentally to 3-methyl-cholanthrene (MC) were significantly influenced by the sensitivity of both mothers and fetuses to induction of cytochrome(s) P-450 by polycyclic aromatic hydrocarbons. In order to delineate further the biochemical and molecular processes underlying the observed biological effects, the inductive effect of MC and beta-naphthoflavone (beta NF) on cytochrome P-450 was determined at the biochemical and molecular levels. C57BL/6 females were mated with DBA/2 males and treated i.p. on day 17 of gestation with olive oil alone, 150 mg/kg of beta NF or different doses of MC. At various times after injection the mothers were sacrificed and the fetuses removed for biochemical and molecular studies. MC caused maximal induction of aryl hydrocarbon hydroxylase (AHH) activity by 8 h in both the liver and lung. beta NF caused nearly maximal induction of AHH activity by 8 h in the lung but had little effect on liver AHH activity at this time. Maximal induction with beta NF occurred by 24 h in both organs. Addition of monoclonal antibody 1-7-1, specific for the MC-inducible forms of cytochrome P-450 (P-450IA1 and A2), to the incubation mixtures resulted in a 55-70% inhibition of AHH activity in both lung and liver assays, regardless of the inducing agent used, while having no effect on AHH activity from oil-treated mice. RNA blot analysis carried out in parallel with enzyme assays demonstrated that the levels of enzyme activity correlated very well with the levels of steady-state RNAs. MC caused maximal induction of P-450IA1 RNA levels 4 h after injection in both organs and a biphasic secondary increase was observed in the lung. Maximal levels of P-450IA1 RNA were seen at 12-16 h following injection of beta NF. However, the ratio of P-450IA1 RNAs present at 16 versus 2 h in the beta NF-treated liver appeared greater than that in the lung. P-450IA2 was also induced in fetal liver and lung, but at low levels relative to P-450IA1. The results indicate that the increase in functional AHH activity was primarily due to induction of cytochrome P-450IA1. The differences in induction kinetics observed for cytochromes P-450IA1 and A2 suggest that these enzymes exhibit both tissue- and inducer-dependent specificity.

Animals↗
PubMed1989

Conformational change in the floor of the human rhinovirus canyon blocks adsorption to HeLa cell receptors.

A series of eight antiviral compounds complexed with human rhinovirus 14 (HRV-14) were previously shown to displace segments of polypeptide chains in the floor of the "canyon" by as much as 0.45 nm in C-alpha positions from the native conformation (J. Badger, I. Minor, M. J. Kremer, M. A. Oliveira, T. J. Smith, J. P. Griffith, D. M. A. Guerin, S. Krishnaswamy, M. Luo, M. G. Rossman, M. A. McKinlay, G. D. Diana, F. J. Dutko, M. Fancher, R. R. Rueckert, and B. A. Heinz, Proc. Natl. Acad. Sci. USA 85:3304-3308, 1988). Because the canyon is thought to serve as the viral receptor-binding site (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H. J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London] 317:145-153, 1985; M. G. Rossmann and R. R. Rueckert, Microbiol. Sci. 4:206-214, 1987), these compounds were assessed for their ability to block adsorption of HRV-14 to HeLa cell membrane receptors. In parallel experiments, the compounds were assessed directly for antiviral activity in an in vitro plaque reduction assay in intact HeLa cells. All eight compounds blocked the adsorption of 50% of HRV-14 at approximately the same concentration required to reduce the number of visible plaques by 50% (MIC). A structurally related compound which was inactive in the plaque reduction assay had no effect on HRV-14 binding. A drug-resistant mutant of HRV-14 (Leu-1188), which was less sensitive to the eight compounds in plaque reduction assays was similarly less sensitive in the adsorption assay. We propose that the conformational changes in the floor of the HRV-14 canyon induced by these compounds substantially decrease adsorption of the virion to its receptor. These results provide further evidence for the role of the HRV canyon in receptor binding.

Adsorption↗
PubMed1989

Metabolism of transplacental carcinogens.

Literature on fetal metabolism of carcinogens since 1980 has been systematically listed and selectively discussed. The published data continues to support the conclusion that animal and human fetal tissues have the capacity to metabolize carcinogens, but at a low rate compared to the adult. Metabolism of low-molecular-weight chemicals, including nitrosamines, appears only near term in the rodent and is poorly inducible transplacentally; these agents are correspondingly relatively ineffective as fetal carcinogens. Metabolism of aromatic carcinogens, by contrast, appears early in gestation and is highly inducible transplacentally in rodents by chemicals such as polycyclic aromatic hydrocarbons (PAH) and tetrachlorodibenzo-p-dioxin, resulting in dramatic percent increases in enzyme activity. Transplacental induction has not been unequivocally demonstrated for the human fetus. Phase II enzymes, metabolizing aromatic compounds to water-soluble forms, generally have higher constitutive activity but lower degree of inducibility in the fetus, compared with phase I (activating) enzymes, and appear to show quantitatively different patterns in the human compared with the rodent. Specific and sensitive new technologies, including 32P-postlabelling, immunodetection of specific proteins, and use of cDNA probes, are beginning to be applied to fetal systems and are providing a more definitive and detailed understanding of the ontogeny and modulation of fetal carcinogen metabolizing enzymes. Fetal and maternal metabolism of PAH, especially methylcholanthrene (MC), have been found to be important determinants in susceptibility of the fetus to tumorigenesis. In particular, we have utilized a mouse model system wherein a single dominant gene, Ah, confers responsiveness to induction of PAH metabolism by cytochrome Pl450 (IA1); the recessive allele, Ah, is associated with nonresponsiveness. In appropriate backcrosses between C57BL/6 (AhAh) and DBA/2 (AhAh) mice, responsive and nonresponsive fetuses were carried together in mothers who were, themselves, either responsive or nonresponsive. In both cases, responsive fetuses later developed more lung and liver tumours after transplacental MC, compared with nonresponsive littermates. Fetuses of responsive mothers, however, experienced a lower cancer risk than did those of nonresponsive mothers at a comparable MC dose. Pretreatment of the pregnant mice with the noncarcinogenic inducer, beta-naphthoflavone (BNF) had a uniform protective effect for all of the fetuses, especially the responsive ones, if the mother was responsive. For nonresponsive mothers, by contrast, BNF pretreatment led to an enhancement of tumorigenesis, in the responsive fetuses only, under certain conditions of dose and fetal sex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Increased metallothionein gene expression in 5-aza-2'-deoxycytidine-induced resistance to cadmium cytotoxicity.

The pyrimidine analog, 5-azacytidine (AZA-CR), has been shown to increase the expression of the metallothionein (MT) gene and to induce tolerance to cadmium toxicity. Since incorporation into DNA of AZA-CR appears to be required for this effect, the deoxynucleoside of AZA-CR should also be effective. Therefore, this study was undertaken to assess the effect of 5-aza-2'-deoxycytidine (AZA-CdR) pretreatment on cadmium-induced cytotoxicity and MT expression in cultured cells. TRL 1215 cells in log phase of growth were exposed to AZA-CdR (0.4, 0.8, 4.0, 8.0 microM) followed 48 h later by the addition of cadmium (10 microM). MT concentrations were measured 24 h after the addition of cadmium. AZA-CdR alone caused modest, dose-related increases in MT levels (2.3-fold maximum), while cadmium alone resulted in a 9.5-fold increase. Pretreatment with AZA-CdR in combination with cadmium caused a 19--24-fold increase in cellular MT at all doses of AZA-CdR. Addition of the DNA synthesis inhibitor, hydroxyurea (HU), to the incubation medium during AZA-CdR exposure prevented the enhancing effect of the analog on cadmium induction of MT accumulation. Time course studies revealed that AZA-CdR pretreatment reduced the time required for cadmium to induce MT levels from 4--8 h to 0--2 h. AZA-CdR pretreated cells placed in suspension with cadmium (125 microM) showed a marked reduction in cadmium-induced cytotoxicity as reflected by reduced glutamic-oxaloacetic transaminase (GOT) loss. Uptake studies showed that AZA-CdR pretreatment had no effect on cadmium transport during the initial phases of exposure, indicating that an alteration in the toxicokinetics of the metal did not account for the reduction in toxicity. AZA-CdR did, however, cause hypomethylation of the MT-I gene. These results suggest that AZA-CdR pretreatment induces tolerance to cadmium toxicity by increasing the genetic expression of MT possibly through hypomethylation of the MT gene.

Animals↗
PubMed1987

Early changes in degenerating mouse sciatic nerve are associated with endothelial cells.

This study determines the earliest reproducible biochemical change in nerves undergoing Wallerian degeneration by assessing the timing, magnitude, and specificity of changes in markers of cellular function in distal stumps of transected mouse sciatic nerves. Analysis of temporal changes in synthesis of DNA, RNA, protein, and in activity of ornithine decarboxylase in transected and sham-operated nerves 0-5 days postoperatively indicated that incorporation of [3H]thymidine, a marker of premitotic activity, was the earliest and only specific marker of early Wallerian degeneration. Although the 3-4 day peak in [3H]thymidine incorporation in distal stumps at the onset of the [3H]thymidine response (1 day post-transection) revealed preferential labeling of endothelial cells lining intrafascicular capillaries.

Animals↗
PubMed1987

Sign iconicity: single-sign receptive vocabulary skills of nonsigning hearing preschoolers.

This study examined hearing preschoolers' ability to use iconic cues to comprehend signs. Twelve nonsigning 3-year-old hearing children served as subjects. Scores obtained from oral presentations of the Peabody Picture Vocabulary Test--Revised (PPVT-R) were compared with the scores obtained from the manual (signed) presentations of the PPVT-R. Results suggest that even in a limited choice context, the resemblance of signs to their referents does not enable hearing children to decipher the meaning of most of the presented signs. Discussion focuses on the relevance of these findings to the selection and use of adapted assessment instruments with young hearing-impaired children.

Association Learning↗
PubMed1987

Temporal analysis of dithiobiuret neurotoxicity in rats and assessment of potential nonneural causes.

To evaluate the hypothesis that depressed neuromuscular transmission causes dithiobiuret (DTB)-induced muscle weakness in rats, the temporal development of impaired treadmill performance and deficits in the nerve-elicited muscle contractions were compared during daily treatment with the toxicant (DTB, 1 mg/kg/day X 6 days). Diminished treadmill test performance after 4 days of treatment marked the initial detection of impaired motor function. At this time fading (loss of tension during tetanus) of gastrocnemius contractions elicited in response to 100-Hz sciatic nerve stimulation occurred in DTB-treated rats but not in controls. After 5 and 6 days of treatment, treadmill failure became complete, tetanic fade worsened dramatically, and peak contractile tension measured during trains of nerve stimulation (10-250 Hz) decreased progressively. Appearing by Day 6 were marked body weight loss, dehydration, hypothermia, and a depression in serum concentrations of thyroid hormones. Total oxygen content of the blood was not reduced at any time during treatment, and serum concentrations of glucose, sodium, potassium, calcium, chloride, and phosphorus in DTB-treated rats on Day 6 were similar to those of control animals. Therefore, hypoxia, hypoglycemia, or a serum electrolyte imbalance do not initiate or modulate the neuromuscular toxicity. Light microscopic evaluation of liver, kidney, lung, thyroid, and other organs in intoxicated rats was unremarkable and in skeletal muscles and selected sites of brain, spinal cord, and sciatic nerve no morphologically significant lesions were observed. Even when DTB-intoxicated rats were maintained in a state of flaccid muscle weakness for 5 continuous days, peripheral nerve lesions proximal to the intramuscular nerves were not detected. Thus, depressed neuromuscular transmission appears to be the primary cause of the flaccid muscle weakness and no evidence was obtained that nonneural effects of DTB initiate or modulate this effect.

Animals↗
PubMed1987

N-methyl-N'-nitro-N-nitrosoguanidine and benzo[a]pyrene-7,8-diol-9,10-epoxide inhibit glucocorticoid-inducible polyoma virus middle-T gene expression in rat mT-1 cells by a post-transcriptional mechanism.

Treatment of rat mT-1 cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) inhibited the steroid-induced expression of the polyoma virus middle-T (mT) gene. Rat mT-1 cells were derived from normal F111 fibroblasts transfected with a plasmid (pSVM-mT) containing a mT cDNA sequence linked to the mouse mammary tumor virus long terminal repeat, thus rendering the mT gene responsive to glucocorticoid hormones. Following a 1-h treatment period with either 100 ng/ml of MNNG or 100 ng/ml of BPDE, administration of 1 microM dexamethasone (DEX) for 24 h resulted in, respectively, 51 and 33% less induction of phosphorylated mT protein in carcinogen-treated than in solvent-treated cells. MNNG appeared to increase the basal level of phosphorylated mT antigen expression by 44%, whereas BPDE had little or no effect on the level of phosphorylated mT protein in uninduced cells. MNNG and BPDE had little or no effect on the level of total steroid-induced mT RNA following addition of DEX for 4 or 20 h. The concentrations of MNNG and BPDE used in these experiments caused, respectively, an 18 and 30% decline in cell number 24 h following carcinogen treatment and had no effect on cell viability as determined by trypan blue exclusion. Previous studies demonstrating the inhibitory effects of chemical carcinogens on the steroid-mediated induction of endogenous genes suggested that carcinogens mediate their effects via a pretranslational mechanism. The present study, using a recombinant DNA construct inserted into the cell genome, provides evidence that carcinogens can inhibit hormone-induced gene expression by a post-transcriptional mechanism as well.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1987

Rapid anterograde spread of premitotic activity along degenerating cat sciatic nerve.

Peripheral nerve transection triggers a series of phenotypic alterations in Schwann cells distal to the site of injury. Mitosis is one of the earliest and best characterized of these responses, although the mechanism by which axonal damage triggers this critical event is unknown. This study examines the appearance and spatio-temporal spread of premitotic activity in distal stumps of transected cat tibial nerves. Premitotic activity was determined by measuring incorporation of [3H]thymidine (a marker of DNA synthesis during the S-phase of the cell cycle) into consecutive segments of desheathed tibial nerve. Incorporation of [3H]thymidine spread proximo-distally within distal nerve stumps between 3 and 4 days posttransection with an apparent velocity of at least 199 +/- 67 mm/day. This suggests that anterograde fast axonal transport may directly or indirectly be associated with the Schwann cell mitotic response to axon transection.

Animals↗
PubMed1986

Inhibition of steroid-inducible tyrosine aminotransferase gene expression by N-methyl-N'-nitro-N-nitrosoguanidine in a rat hepatoma cell line.

Treatment of rat hepatoma cells (the Fao clone of Reuber H35 cells) with a 500 ng/ml concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a marked inhibition of steroid-inducible tyrosine aminotransferase (TAT) gene expression. Following a 1-h treatment period with MNNG, addition of 1 microM dexamethasone (DEX) for 6 and 24 h resulted in, respectively, 78 and 51% less induction of TAT enzyme activity in MNNG-treated than in solvent-treated cells. MNNG had little effect on the basal level of TAT activity 6 h after treatment with the carcinogen, but the uninduced level of TAT activity declined to 36% of the control value 24 h following MNNG treatment. MNNG-treated cells concurrently exhibited 75% less total steroid-induced TAT RNA following addition of 1 microM of DEX for 4 h, but showed little or no change in the basal level of total TAT RNA or in the level of total alpha-tubulin RNA. In addition, MNNG treatment resulted in 45% less induction of TAT enzyme activity following a 4-h treatment period with 0.5 mM N6,O2'-dibutyryl-adenosine 3',5'-cyclic monophosphate. The concentration of MNNG used in these experiments caused only a 14% decline in cell number 24 h following carcinogen treatment and had no effect on cell viability as determined by trypan blue exclusion. The data indicate that the inhibitory effects of MNNG appear to be mediated at a pre-translational step. MNNG inhibits the accumulation of total DEX-inducible TAT RNA either by preventing the steroid-mediated increase of the rate of TAT RNA transcription or decreasing total TAT RNA stability, or both.

Animals↗
PubMed1985

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry.

The capability of fast atom bombardment mass spectrometry for characterization of phosphorylation sites in a tryptic peptide from chicken egg yolk riboflavin-binding protein has been evaluated. The quality of information about molecular weight, amino acid sequence, phosphorylation sites, and microheterogeneity is evaluated as a function of the sign of the ions analyzed, the nature of the counter ions associated with the phosphate substituents, sample matrix, and various instrumental parameters. The intact octaphosphorylated 23-residue peptide was found to be susceptible to mass spectral analysis. Information from the negative ion spectrum was used in conjunction with complete sequence information and experiments which showed that all phosphates were attached to serine residues. Phosphorylated and unphosphorylated serine residues were identified and the sample was shown to be homogeneously octaphosphorylated.

Amino Acid Sequence↗
PubMed1985

The separation and identification of enolase isozymes of brain and sciatic nerve by high-pressure liquid anion-exchange chromatography.

A rapid technique for separating and quantitating the three enolase isozymes present in rodent brain and sciatic nerve was developed using high-pressure liquid anion-exchange chromatography. At pH 7.9, one cationic and two anionic enzyme forms were separated with baseline resolution in an imidazole buffer containing ethylenediaminetetraacetic acid (EDTA) and magnesium. The recovery of enolase activity was 90% or greater for brain and 85% for sciatic nerve. Chromatography of liver and axon-free (degenerated) sciatic nerve allowed the identification of non-neuronal, hybrid, and neuron-specific enolase isozymes. These enzyme forms, respectively, constituted 40%, 29% and 19% of total activity in brain, and 63%, 13% and 4% of total activity in normal sciatic nerve.

Animals↗