Search PubMed⌕ Search

Biomedical subjects

D G Beer

Publications and source records attributed to D G Beer.

63 records · Page 4Linked to original sources
PubMed1984

Functional changes in the regulatory subunit of the type II cyclic adenosine 3':5'-monophosphate-dependent protein kinase isozyme during normal and neoplastic lung development.

The abilities of cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic 8-azidoadenosine 3':5'-[32P]monophosphate (8-N3-[32P]cAMP) to bind to the regulatory subunit (RII) of the type II cAMP-dependent protein kinase isozyme and to cause subsequent dissociation of the holoenzyme were compared in extracts from adult and neonatal mouse lung and lung adenoma. RII in extracts from adult lung exhibits equal numbers of high- (Kd 15 nM) and low- (Kd 230 nM) affinity 8-N3-[32P]cAMP binding sites. In the neonate, the proportion of high-affinity sites is reduced to 20% while, in lung adenoma, only low-affinity RII binding is observed. Low-affinity RII binding is correlated with an inability of cAMP to dissociate the type II holoenzyme completely. Sucrose gradient sedimentation of adult lung cytosol in the presence of cAMP shows complete dissociation of the type I isozyme, while only some of the type II holoenzyme is dissociated. This is in contrast to the case with lung tumor cytosol, in which only low-affinity binding is observed and no apparent dissociation of the type II isozyme occurs. cAMP does promote RII dephosphorylation within the holoenzyme, however, suggesting that cAMP can bind to RII without dissociating the tetramer. Consistent with this interpretation, photoincorporation of 8-N3-[32P]cAMP prior to sucrose gradient sedimentation results in the formation of a photolabeled RII complex which sediments at the same rate as does the holoenzyme. Two-dimensional gel electrophoresis of RII photolabeled at low and high concentrations of 8-N3-[32P]cAMP suggests that these altered binding and dissociation characteristics of the type II isozyme are not due to the presence of a structurally altered RII molecule. After DEAE-cellulose chromatography of lung cytosol, only high-affinity RII binding is observed, and all of the RII can now be dissociated with cAMP. Low-affinity binding may thus reflect either an altered conformational state of RII or the interaction of the type II kinase with other cytosolic molecules which can affect RII binding and dissociation without altering the functional properties of the type I isozyme.

Adenoma↗
PubMed1984

Localization of specific [3H]dexamethasone binding in urethan-induced mouse lung tumors.

Pulmonary tumors induced in A/J mice 14 months after a single i.p. injection of urethan vary greatly in size. Since glucocorticoids may play a major role in regulating lung cell proliferation, glucocorticoid binding was examined in these tumors to determine whether tumor size was related to any specific pattern of [3H]dexamethasone [( 3H]DEX) binding. Tumor samples were incubated in vitro with 17 nM [3H]DEX for 90 min at 37 degrees, washed extensively to reduce nonspecific binding, and either fractionated by differential centrifugation to quantify nuclear and cytosolic binding or processed for autoradiography. Quantitative binding studies demonstrate a reduction in specific nuclear [3H]DEX binding and an increase in nonspecific cytosolic binding in all of the tumors examined as compared to normal adult lung. Autoradiographic studies reveal pulmonary tumors of different morphology which vary in their [3H]DEX binding characteristics. Small tumors were of two histological patterns, alveolar adenomas which are probably derived from alveolar type II cells and papillary adenomas which are probably derived from bronchiolar Clara cells. The alveolar adenomas contain little nuclear [3H]DEX binding, whereas the papillary adenomas show extensive nuclear localization of [3H]DEX. These results indicate that nuclear localization of [3H]DEX can provide a biochemical criterion for distinguishing alveolar from papillary adenomas. Most of the larger tumors were either papillary or anaplastic in morphology and localized [3H]DEX in their nuclei. This suggests that these larger and possibly more malignant tumors are derived from papillary adenomas.

Animals↗
PubMed1983

Time course of the carbon tetrachloride-induced decrease in mitochondrial aldehyde dehydrogenase activity.

Hepatic microsomal enzymes like cytochrome P-450 and glucose 6-phosphatase are inhibited after exposure to CCl4 in vivo. Since comparatively less is known about the effects of CCl4 on nonmicrosomal enzymes, we investigated the rapidity by which CCl4 inhibits the low Km mitochondrial aldehyde dehydrogenase (ALDH) isozyme, an enzyme known to be inhibited 24 hr after CCl4 treatment. The activity of this ALDH isozyme was significantly lowered 6 and 12 hr after a single 1 ml/kg intragastric dose of CCl4. The mitochondrial low Km ALDH specific activities exhibited a similar pattern of destruction/inhibition to the documented target enzyme microsomal cytochrome P-450 in that lowest values were observed 6 hr after CCl4. These values were 44 and 37% of control for cytochrome P-450 content and the low Km ALDH activity, respectively. Alcohol dehydrogenase activity, expressed as activity per gram liver, was depressed 12 hr after CCl4 dosing. Finally, the activity of the low Km cytosolic ALDH, the isozyme that metabolizes malondialdehyde at low concentrations, was not affected by CCl4 treatment. The CCl4-induced decline in the activity of the matrix ALDH isozyme occurs earlier than previously reported mitochondrial damage. The study of sensitive enzymes like the low Km ALDH may provide valuable information by which it may be possible to determine the relationship of the truly rapid biochemical effects of CCl4 such as microsomal lipid peroxidation with later effects on nonmicrosomal components.

Aldehyde Dehydrogenase↗
PubMed1983

Methods in laboratory investigation. Autoradiographic demonstration of the specific binding and nuclear localization of 3H-dexamethasone in adult mouse lung.

This report describes the first autoradiographic demonstration of specific nuclear localization of 3H-dexamethasone in different cell types of the lung. Adult mouse lung tissue was incubated in vitro for 90 minutes with 17 nM 3H-dexamethasone in the presence or absence of various nonradioactive steroids. After extensive washing to remove any nonspecifically bound ligand, the specimens were processed for autoradiography using the thaw-mount method. In the absence of competing steroids, silver grains were localized in the nuclei of alveolar type II cells, bronchiolar and arteriolar smooth muscle cells, fibroblasts, and endothelial cells of the pulmonary vasculature. No significant nuclear concentration of label was observed in the bronchiolar epithelium, however. The specificity of 3H-dexamethasone labeling was demonstrated by incubating 17 nM 3H-dexamethasone with a 600-fold excess of either unlabeled dexamethasone, estrogen, dihydrotestosterone, or progesterone. These autoradiographic binding and steroid competition studies were confirmed by quantifying with liquid scintillation counting the specific 3H-dexamethasone binding in nuclear and cytosolic fractions prepared from lung tissues that had undergone identical incubation and washing procedures as those for autoradiography. These results demonstrate that many cell types in adult lung are targets for glucocorticoids and may respond to physiologic concentrations of this hormone.

Animals↗
PubMed1981

Inhibition of rat liver aldehyde dehydrogenase by carbon tetrachloride.

Current data suggests that aldehydic products of lipid peroxidation possess substantial cytotoxic properties. Carbon tetrachloride (CCl4), a potent stimulator of hepatic lipid peroxidation, was tested for possible effects on hepatocellular aldehyde metabolism. CCl4 (1 ml/kg) produced an elevation in serum alanine aminotransferase activity, hepatic fatty infiltration, centrilobular necrosis and significant decreases in the content of hepatic microsomal cytochrome P-450. Concurrently, the aldehyde dehydrogenase (E.C. 1.2.1.3) activity of mitochondrial and cytosolic fractions was significantly depressed. The lower Km aldehyde dehydrogenase located in the mitochondria showed the largest degree of inhibition (46%). An in vitro system which contained the low Km mitochondrial aldehyde dehydrogenase was employed to determine the role of microsomal lipid peroxidation in the inhibition of the enzyme. Aldehyde dehydrogenase was shown to be extremely sensitive to inhibition under conditions of NADPH or NADPH and CCl4-stimulated lipid peroxidation. Reduced glutathione (6 mM) provided complete protection of aldehyde dehydrogenase activity under conditions of NADPH-stimulated lipid peroxidation but could not protect activity loss during CCl4-stimulated microsomal lipid peroxidation. The degree of enzyme activity loss related well with the amount of thiobarbituric reacting substances present in the incubation mixture. These findings show that CCl4 decreases the activity of the aldehyde oxidizing enzyme, aldehyde dehydrogenase. This effect may accentuate cytotoxic effects of reactive aldehydic products generated during lipid peroxidation.

Aldehyde Dehydrogenase↗
PubMedDate not supplied

Indomethacin-induced apoptosis in esophageal adenocarcinoma cells involves upregulation of Bax and translocation of mitochondrial cytochrome C independent of COX-2 expression.

The prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to exert a chemopreventive effect in esophageal and other gastrointestinal tumors. The precise mechanism by which this occurs, however, is unknown. While the inhibition of COX-2 as a potential explanation for this chemopreventive effect has gained a great deal of support, there also exists evidence supporting the presence of cyclooxygenase-independent pathways through which NSAIDs may exert their effects. In this study, immunohistochemical analysis of 29 Barrett's epithelial samples and 60 esophageal adenocarcinomas demonstrated abundant expression of the COX-2 protein in Barrett's epithelium, but marked heterogeneity of expression in esophageal adenocarcinomas. The three esophageal adenocarcinoma cell lines, Flo-1, Bic-1, and Seg-1, also demonstrated varying expression patterns for COX-1 and COX-2. Indomethacin induced apoptosis in all three cell lines, however, in both a time- and dose-dependent manner. In Flo-1 cells, which expressed almost undetectable levels of COX-1 and COX-2, and in Seg-1, which expressed significant levels of COX-1 and COX-2, indomethacin caused upregulation of the pro-apoptotic protein Bax. The upregulation of Bax was accompanied by the translocation of mitochondrial cytochrome c to the cytoplasm, and activation of caspase 9. Pre-treatment of both cell lines with the specific caspase 9 inhibitor, z-LEHD-FMK, as well as the broad-spectrum caspase inhibitor, z-VAD-FMK, blocked the effect of indomethacin-induced apoptosis. These data demonstrate that induction of apoptosis by indomethacin in esophageal adenocarcinoma cells is associated with the upregulation of Bax expression and mitochondrial cytochrome c translocation, and does not correlate with the expression of COX-2. This may have important implications for identifying new therapeutic targets in this deadly disease.

Adenocarcinoma↗
PubMedDate not supplied

Expression of the glucocorticoid receptor and K-ras genes in urethan-induced mouse lung tumors and transformed cell lines.

Glucocorticoids influence cell proliferation and differentiation in the lung. We examined the expression of the glucocorticoid receptor (GR) gene in urethan-induced mouse lung tumors and transformed lung cell lines to determine whether any altered responsiveness to these steroids is involved in the neoplastic development of some lung tumors. We find that a GR mRNA of similar size and amount is expressed in both normal lung and urethan-induced lung tumors. The K-ras gene is activated in urethan-induced lung adenomas and transformed lung cell lines. Both alveolar and papillary lung adenomas express slightly elevated levels of K-ras mRNA and similar levels of H-ras mRNA, but variable levels of c-myc mRNA, GR and K-ras mRNAs are concurrently expressed in a cyclic manner during the proliferation of nontransformed C10 and transformed A5 lung cell lines. Treatment of the C10 cells with dexamethasone (Dex) results in the inhibition of cell proliferation and the down-regulation of both the GR and K-ras mRNA. Dex treatment also down-regulated GR mRNA levels in A5 and LM2 cells, but no inhibitory effect was observed on K-ras mRNA levels or cell proliferation. These results suggest that glucocorticoids can inhibit K-ras expression in nontransformed lung cells. Although transformed lung cells respond to the steroid by down-regulation of the GR, the presence of an activated K-ras allele may override the inhibitory effects of these hormones on cell proliferation.

Animals↗