PubMed Health⌕ Search

Biomedical subjects

J W Hamilton

Publications and source records attributed to J W Hamilton.

At least 19 recordsLinked to original sources
PubMed1995

Preferential effects of the chemotherapeutic DNA crosslinking agent mitomycin C on inducible gene expression in vivo.

The immediate effects of a single dose of the chemotherapeutic DNA crosslinking agent, mitomycin C (MMC), on the expression of several constitutive and drug-inducible genes were examined in a simple in vivo system, the 14 day chick embryo. We observed no effect of MMC on the steady-state mRNA expression of the constitutively expressed beta-actin, transferrin, or albumin genes. In contrast, MMC treatment significantly altered both the basal and drug-inducible mRNA expression of two glutethimide-inducible genes, 5-aminolevulinic acid (ALA) synthase and cytochrome P450 CYP2H1. The basal expression of these genes was transiently but significantly increased over a 24 hr period following a single dose of MMC. Conversely, MMC significantly suppressed the glutethimide-inducible expression of these genes when administered 1 to 24 hr prior to the inducing drug. The effects of MMC on both basal and drug-inducible ALA synthase and CYP2H1 mRNA expression were principally a result of changes in the transcription rates of these genes. In contrast, MMC treatment had little or no effect on glutethimide-induced expression of ALA synthase or CYP2H1 when administered 1 hr after the inducing drug, suggesting that a very early event in the induction process represents the target for these MMC effects. Covalent binding studies demonstrated that the effects of MMC on gene expression were closely correlated temporally with formation of [3H]-porfiromycin-DNA adducts. These results support the hypothesis that genotoxic chemicals specifically target their effects to inducible genes in vivo.

5-Aminolevulinate Synthetase↗
PubMed1995

The accuracy of acoustic rhinometry using a pulse train signal.

The signal used in the original acoustic rhinometers was an impulse of short duration and wide energy spectrum. A rhinometer utilizing a new signal of a similarly broad energy but different time course has been developed. We investigated the accuracy of area reconstructions computed using this signal by comparing them with the original areas of simple models. This study demonstrates that, at present, acoustic rhinometry using a pulse train signal is subject to significant systematic errors. Some of these are intrinsic to the rhinometer. Others are caused by the geometry of the models. Areas reconstructions beyond narrow constrictions are particularly inaccurate. The findings are significant because the nasal valve can act as such a constriction. The errors can cause the parameters used in the clinical application of acoustic rhinometry to deviate substantially from the true values.

Acoustic Stimulation↗
PubMed1994

Development regulation of basal and hormone-inducible phosphoenolpyruvate carboxykinase gene expression in chick embryo liver in vivo.

We have examined cytosolic phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) gene transcription and steady-state mRNA expression in chick embryo liver in vivo both constitutively and in response to dexamethasone, cAMP, retinoic acid, and the protein synthesis inhibitors, cycloheximide and pactamycin. We report here that PEPCK mRNA is constitutively expressed between 12 and 16 days of development. PEPCK expression was also transiently but highly inducible on Day 14 by dexamethasone, cAMP, cycloheximide, and pactamycin, but not by retinoic acid. Cycloheximide pretreatment had an additive or synergistic effect on the induction by dexamethasone and/or cAMP. Dexamethasone- and cycloheximide-induced increases in mRNA expression were principally due to increases in the rate of PEPCK gene transcription. Following an initial induction by dexamethasone, PEPCK expression was no longer responsive to a second administration of dexamethasone but was still responsive to cycloheximide and cAMP. PEPCK induction by dexamethasone or cycloheximide progressively diminished between 12 and 15 days of development. By Day 16, PEPCK expression was no longer responsive to dexamethasone, but was still inducible by cAMP and this induction was increased by cycloheximide. These results indicate that PEPCK is transiently inducible by glucocorticoids in chick embryo liver and that there are two developmental switches to the adult phenotype between Days 14 and 16 of development and between Day 16 and 4 days posthatching. Our results also suggest the presence of a developmentally regulated repressor of PEPCK gene expression in chick embryo liver.

Animals↗
PubMed1994

Effects of the genotoxic carcinogen chromium(VI) on basal and hormone-inducible phosphoenolpyruvate carboxykinase gene expression in vivo: correlation with glucocorticoid- and developmentally regulated expression.

Previous studies have shown that a number of different genotoxic carcinogens that induce different types of DNA damage preferentially alter the expression of inducible genes in vivo. To investigate further the mechanistic basis for these effects, we examined the effects of the human lung carcinogen chromium(VI) on expression of the hormone-inducible cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene in chick embryo liver. Chromium(VI) pretreatment had significant effects on both basal and glucocorticoid-inducible PEPCK expression in 14-d-old embryo liver. These effects were principally a result of changes in PEPCK transcription. In contrast, treatment with chromium(VI) 1 h after treatment with glucocorticoid had no effect on PEPCK induction, suggesting that an early event in the induction process is the target for carcinogen effects. In 16-d-old liver, in which PEPCK expression is no longer responsive to glucocorticoid induction, both basal and inducible PEPCK expression were also refractory to chromium(VI) effects, indicating that carcinogen responsiveness is a phenotypic rather than an inherent property of inducible genes and is related to their competence for induction. Chromium(VI) had no effect on cAMP induction of PEPCK expression, demonstrating that carcinogens target their effects to specific regulatory pathways. Comparison of the effects of chromium(VI) with those of cycloheximide suggests that chromium(VI) targets its effects to a labile, constitutively expressed repressor involved in PEPCK gene regulation.

Animals↗
PubMed1994

Processing of chromogranin A by bovine parathyroid secretory granules: production and secretion of N-terminal fragments.

Chromogranin A (CgA) is a glycoprotein located in the secretory granules of multiple neuroendocrine tissues, including the parathyroid gland. Although the function of CgA is not known, a role has been proposed for CgA as a prohormone for biologically active peptides. Using 3H-CgA as a substrate for bovine parathyroid secretory granule extracts, we demonstrate a precursor product relationship between intact CgA and multiple N-terminal fragments of CgA. N-terminal CgA fragments of mol wt 24, 26, and 33 k are generated in a time dependent manner in the presence of bovine parathyroid secretory granule enzymes. The generation of the 33 kilodalton (kDa) N-terminal CgA fragment is calcium dependent. In the presence of EDTA, intermediate CgA fragments of mol wt 36 and 45 are generated. The effect of EDTA is reversible with added calcium. Based on immunodetection on Western blots, the 26 kDa N-terminal fragment of CgA is secreted by bovine parathyroid cells in a time and calcium-dependent manner in parallel with PTH and intact CgA. The secretion of the 26 kDa N-terminal fragment of CgA increases in response to low calcium incubation conditions and is suppressed by high calcium incubation conditions. We conclude that bovine parathyroid secretory granules contain enzymatic activity capable of processing CgA to multiple N-terminal fragments. The secretion of at least one N-terminal fragment (26 kDa) is calcium responsive. The physiological significance of CgA processing in parathyroid secretory granules is as yet unknown.

Amino Acids↗
PubMed1993

Preferential alteration of inducible gene expression in vivo by carcinogens that induce bulky DNA lesions.

Our laboratory is interested in whether chemical carcinogen-induced DNA damage is nonrandomly distributed in the genome, i.e., "targeted," at the level of individual genes. To examine this, we have been investigating whether carcinogen treatment in vivo differentially alters the expression of specific genes. In this study, we examined the effects of four model carcinogens that induce bulky lesions in DNA--benzo[a]pyrene (B[a]P), aflatoxin B1 (AFB1), 7,12-dimethylbenz[a]anthracene (DMBA), and 2-acetylaminofluorene (AAF)--on the steady-state mRNA expression of several constitutive and drug-inducible genes in vivo. We specifically tested the hypothesis that carcinogen-induced DNA damage is preferentially targeted to inducible genes relative to constitutively expressed genes using the chick embryo as a simple in vivo test system. In summary, the four carcinogens had no effect on the steady-state mRNA expression of constitutively expressed beta-actin, transferrin, or albumin genes over a 24-h period after a single dose of each carcinogen. In contrast, each of these same treatments significantly altered the mRNA expression of two glutethimide-inducible genes, ALA synthase and CYP2H1. Both the basal expression of these genes and their drug-inducible expression was altered. B[a]P and AFB1 had similar effects on expression of the two inducible genes and caused similar levels of covalent adducts in total DNA, even though the administered doses differed by 30-fold. B[a]P binding to DNA, and the basal expression of CYP2H1 were similar in liver and lung. However, B[a]P significantly altered basal CYP2H1 mRNA expression in liver, a tissue in which this gene is highly inducible by glutethimide, and had no effect on basal CYP2H1 mRNA expression in lung, a tissue in which this gene is not drug-inducible. These data support the hypothesis that inducible gene expression is a target for carcinogen-induced DNA damage in vivo.

5-Aminolevulinate Synthetase↗
PubMed1992

Regulated, but not constitutive, secretory proteins bind porcine chymotrypsinogen.

Endocrine and exocrine cells exhibit both a constitutive and a regulated secretory pathway. In the latter pathway, secretory proteins are stored at a high concentration in secretory granules and are released by exocytosis in response to appropriate external stimuli. Sorting between the two secretory pathways is believed to take place in the trans-Golgi tubular network. To account for experimental data, it has been proposed that sorting receptors exist which bind a variety of regulated secretory proteins, including foreign secretory proteins introduced into the cells by transfection. In support of the sorting receptor hypothesis Chung et al. (Chung, K.-N., Walter, P., Aponte, G. W., and Moore, H.-P.H. (1989) Science 243, 192-197) isolated a group of 25-kDa canine pancreatic "hormone-binding proteins" that bound regulated but not constitutive secretory proteins. To determine if similar proteins are present in other species and tissues, we have screened porcine pancreas, parathyroid, adrenal medulla, and pituitary glands. A 31-kDa protein, similar to that identified by Chung et al. (1989), which binds to regulated but not to constitutive secretory proteins was identified in porcine pancreas. This protein was not detected in the parathyroid, adrenal medulla, or pituitary glands, however, which argues against it serving as a general sorting receptor. NH2-terminal sequencing, immunoreactivity, and proteolytic activity data indicate that the porcine 31-kDa protein is similar if not identical to porcine chymotrypsinogen A or B.

Adrenal Medulla↗
PubMed1992

Inhibition of protein synthesis increases the transcription of the phenobarbital-inducible CYP2H1 and CYP2H2 genes in chick embryo hepatocytes.

The mechanism by which phenobarbital and similar compounds regulate gene expression has remained elusive for many years despite intense investigation. We had previously reported that the mRNA expression for the phenobarbital-inducible CYP2H1 gene was increased by cycloheximide treatment as rapidly and to a similar extent as by the phenobarbital-type drugs glutethimide and 2-propyl-2-isopropylacetamide (PIA), in primary cultures of chick embryo hepatocytes or in chick embryo liver in vivo (J. W. Hamilton, W. J. Bement, P. R. Sinclair, J. F. Sinclair, and K. E. Wetterhahn, 1988, Biochem. J. 255, 267-275). To examine the mechanism of this induction further, we determined the effects of various structurally related and unrelated inhibitors of protein synthesis on CYP2H1 expression in cultured chick embryo hepatocytes. Cycloheximide increased the transcription rate of the CYP2H1/2 genes to a similar extent as did PIA, and had little or no effect on CYP2H1 mRNA half-life. A number of other protein synthesis inhibitors, including streptovitacin, acetoxycycloheximide, pactamycin, and ricin, all increased CYP2H1 mRNA expression to a similar extent. The dose responses for induction of CYP2H1 mRNA and inhibition of protein synthesis by these agents were closely correlated. There was no relationship between the effectiveness of these agents to induce CYP2H1 mRNA expression and their structures or lipophilicity. Cycloheximide acetate required deesterification to cycloheximide for both inhibition of protein synthesis and induction of CYP2H1 mRNA. These results suggest that a labile negative regulatory protein is involved in CYP2H1/2 gene expression. It is also possible that this factor is involved in regulating the phenobarbital response of CYP2H1/2.

Animals↗
PubMed1992

Oropharyngeal swallowing in normal adults of different ages.

In an effort to evaluate the effect of normal aging on oropharyngeal events of swallowing, 80 normal volunteers, stratified by gender into four age groups, were studied. Liquid and semisolid swallows were performed and recorded simultaneously using videofluoroscopy and manometry. Several parameters, including total duration of oropharyngeal swallowing, were significantly longer in the oldest age group than in any other age group. A delay in initiation of maximal hyolaryngeal excursion primarily accounted for the longer durations with increased age. Significant durational changes also were found as a function of bolus consistency and presence or absence of the manometry tube. Females had a longer duration of upper esophageal sphincter (UES) opening. The amplitude of pharyngeal pressures, duration of peak pharyngeal pressures, and rate of propagation of the contractions were not significantly different for age, gender, or consistency of bolus. No significant differences were found between age groups or between genders in UES pressure. Normal aging affects some parameters of swallowing, while others are preserved.

Adult↗
PubMed1992

Pancreastatin and bovine parathyroid cell secretion.

Chromogranin A (CgA) is an acidic glycoprotein found in secretory granules of multiple peptidergic tissues and cosecreted with the resident peptide hormones. Pancreastatin is an amidated, biologically active peptide whose sequence is contained within CgA. We investigated the effect of the C-terminal fragment of bovine pancreastatin (bP32-47) on bovine parathyroid cell secretion. bP32-47 amide inhibited low-calcium-stimulated PTH secretion by 44% and chromogranin A (CgA) secretion by 33%. We were able to identify a pancreastatin-like peptide as a very minor component of the endogenous breakdown peptides from CgA. However, using several approaches, we were unable to detect pancreastatin in secretory granule extracts or in incubation media. We conclude that although exogenous bovine pancreastatin has inhibitory effects on secretion, detectable pancreastatin is not secreted under normal incubation conditions. Based on our current data, we would question the physiologic importance of pancreastatin in bovine parathyroid glands.

Animals↗
PubMed1991

Elevation of delta-aminolevulinic acid synthase and cytochrome PB1 P450 messenger RNA levels by dihydropyridines, dihydroquinolines, sydnones, and N-ethylprotoporphyrin IX.

A series of compounds that increase the activity of delta-aminolevulinic acid synthase (ALAS) in chick embryo hepatocyte cultures were studied for their effects on steady-state levels of mRNA for ALAS and phenobarbital-inducible cytochrome PB1 P450. N-Ethylprotoporphyrin IX (N-EtPP), which is believed to lower heme levels by inhibition of ferrochelatase (FC), had little effect on steady-state ALAS mRNA levels. 3,5-Diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4- isobutylpyridine (4-isobutyl DDC), which is believed to lower heme levels by repetitive destruction of the heme moiety of cytochrome P450, increased steady-state levels of ALAS mRNA levels approximately 2-fold. 3,5-Diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DCC) which inhibits FC activity and destroys the heme moiety of cytochrome P450, increased ALAS mRNA levels approximately 4-fold. A combination of N-EtPP and 4-isobutyl DDC produced a synergistic increase in ALAS mRNA levels to approximately 6-fold over control levels. The synergistic increase in ALAS activity observed previously with this combination can be explained, at least in part, by a synergistic increase in ALAS mRNA levels. Other porphyrinogenic agents, which function as mechanism-based inactivators of cytochrome P450 and elevate ALAS activity, were found to elevate ALAS mRNA. These compounds included 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), 2,4-diethyl-2-methyl-1,2-dihydroquinoline (DMDQ), and 2,2,4-trimethyl-1,2,dihydroquinoline (TMDQ). The elevation of ALAS mRNA by these porphyrinogenic agents is probably due to their lowering of cellular heme levels by a combination of ferrochelatase inhibition and repetitive destruction of the heme moiety of cytochrome P450. The lowering of heme levels should result in an enhancement of ALAS mRNA half-life as it has been demonstrated by others that heme shortens the half-life of ALAS mRNA. It was of interest that some of these drug treatments also caused an elevation in steady-state levels of cytochrome PB1 P450 mRNA; the exception was TTMS, which along with its analogue 3-(2-phenylethyl)-4-methylsydnone (PEMS), did not alter cytochrome PB1 P450 mRNA levels. Increases in steady-state levels of cytochrome PB1 P450 mRNA subsequent to increases in steady-state levels of ALAS mRNA were observed with 4-ethyl DDC, 4-isobutyl DDC, DMDQ, and TMDQ. The data obtained with N-EtPP and a combination of N-EtPP and 4-isobutyl DDC on cytochrome PB1 P450 mRNA levels do not support the contention that heme functions as a positive regulator of cytochrome P450 gene expression.

5-Aminolevulinate Synthetase↗
PubMed1991

Sulfated secreted forms of bovine and porcine parathyroid chromogranin A (secretory protein-I).

Chromogranin A (secretory protein-I) is an acidic sulfated glycoprotein found in secretory granules of most endocrine and neuroendocrine cells. In the parathyroid it is co-stored and secreted with parathormone in response to hypocalcemia. Differences in post-translational modifications have been reported between chromogranin A from the bovine adrenal and porcine parathyroid glands. The former has been reported to be sulfated mainly on oligosaccharide residues and apparently includes a proteoglycan form, whereas the latter was previously reported to be tyrosine sulfated with little of the proteoglycan form present. Here we have directly compared 35SO4-labeled parathyroid chromogranin A from the pig and the cow to determine if these reported differences were tissue or species specific. We find that the chromogranin A secreted by the bovine gland contains a proteoglycan form, whereas that from the porcine gland does not. Moreover, chromogranin A of both species is primarily sulfated on oligosaccharide residues with little if any tyrosine sulfate detected. Differences were detected in the structure of sulfated O-linked oligosaccharides in bovine and porcine parathyroid chromogranin A.

Animals↗
PubMed1991

Inhibition of parathyroid hormone secretion correlates with increased incorporation of 32P into phosphatidylinositol and lysophosphatidylinositol.

We have studied the incorporation of radioactive P (32P) into lipids of bovine parathyroid tissue under conditions of stimulated and inhibited hormone secretion. Utilizing low (0.5 mM) and high (3.0 mM) concentrations of calcium to regulate parathyroid hormone secretion, we initially found that the labeling of the cellular phospholipids with 32P was greater in those tissues incubated in high-calcium medium. Thin-layer chromatography of lipid extracts prepared from tissue incubated in either low- or high-calcium media revealed that the increased incorporation of 32P (high or low) was localized primarily to two phospholipids. To determine whether the increases were due directly to the different calcium concentrations, the experiments were performed in media containing normal calcium concentrations (1.25 mM) and low (0.5) or high (3.0) magnesium concentrations to modulate hormone secretion. The results were identical to those obtained using low and high calcium, indicating that the increased 32P incorporation was not an effect of high calcium but rather correlated with the inhibition of hormone secretion. The use of other secretagogues confirmed this correlation. The identity of the two phospholipids was established, by two-dimensional thin-layer chromatography, to be phosphatidylinositol (PI) and lysophosphatidylinositol (LPI). The correlation of increased 32P incorporation with inhibition of secretion led us next to examine isolated secretory granules from tissues exposed to either high-or low-calcium conditions. Thin-layer chromatography of granule lipid extracts yielded chromatograms containing PI and LPI, and the radioactivity of each was greater in the high-calcium sample than in the low-calcium sample.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Heme regulates hepatic 5-aminolevulinate synthase mRNA expression by decreasing mRNA half-life and not by altering its rate of transcription.

Hepatic 5-aminolevulinate (ALA) synthase, the first and rate-limiting enzyme in the heme biosynthetic pathway, is known to be feedback repressed by the end product of the pathway, heme. We investigated whether heme regulates ALA synthase mRNA expression transcriptionally or post-transcriptionally in primary cultures of chick embryo hepatocytes. 2-Propyl-2-isopropylacetamide increased the rate of transcription of the ALA synthase gene, whereas heme or an inhibitor of heme biosynthesis, desferrioximine, had no effect on the drug-induced transcription rate. Heme decreased the half-life of ALA synthase mRNA from approximately 3.5 h to 1.2 as recently reported by Drew and Ades (1989, Biochem. Biophys. Res. Commun. 162, 102-107). We also found that the heme-mediated decrease in mRNA stability was prevented by cycloheximide treatment, suggesting that the heme effect was mediated by a labile protein. These results support a model for hepatic ALA synthase regulation in which inducing drugs directly stimulate ALA synthase gene transcription, whereas heme regulates ALA synthase expression post-transcriptionally by modulating mRNA stability as well as by blocking translocation of ALA synthase enzyme into the mitochondrion.

5-Aminolevulinate Synthetase↗
PubMed1991

Bovine parathyroid glands secrete a 26-kDa N-terminal fragment of chromogranin-A which inhibits parathyroid cell secretion.

Chromogranin-A (CgA) is a ubiquitous protein which colocalizes in secretory granules of multiple endocrine tissues and cosecretes with peptide hormones from these tissues. Although the function of CgA has remained unknown, there has been recent interest in its potential role as a prohormone for smaller, biologically active peptides. We isolated and characterized a 26-kDa N-terminal fragment of CgA which is a natural breakdown product of bovine parathyroid CgA in storage. A similar, if not identical, fragment of CgA is secreted by bovine parathyroid glands. The secreted fragment elutes on HPLC and migrates on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gels in the same position as the 26-kDa N-terminal fragment. When added to the incubation medium of dispersed bovine parathyroid cells, the 26-kDa N-terminal fragment of CgA inhibits the low calcium-stimulated secretion of both PTH and CgA. This N-terminal fragment is homologous to betagranin, which is a 21-kDa N-terminal fragment of CgA that is generated from CgA in rat insulin granules. Thus, a naturally occurring betagranin-like N-terminal fragment of bovine parathyroid CgA is not only secreted itself, but can inhibit the secretion of PTH and intact CgA by bovine parathyroid cells. The processing of intact CgA to fragments such as the N-terminal fragment that we describe may be important in the autocrine or paracrine regulation of secretion.

Amino Acid Sequence↗
PubMed1990

Uptake of L-carnitine by rat jejunal brush border microvillous membrane vesicles. Evidence of passive diffusion.

We have previously described apparent active transport of carnitine into rat intestinal mucosa with intracellular accumulation against a concentration gradient in a process dependent upon the presence of sodium ions, oxygen, and energy. In the work described here, we sought to define the interaction between carnitine and the brush border membrane, which we presumed contained the transport mechanism. Using isolated rat jejunal brush border microvillous membrane vesicles, we found evidence of passive diffusion alone. We found no evidence of carrier-mediated transport--in particular no saturation over a concentration range, inhibition by structural analogs, transstimulation phenomenon, and no influence of sodium ions, potential difference or proton gradients. We conclude that a carnitine transporter does not exist in the brush border membrane of enterocytes and that other cellular mechanisms are responsible for the apparent active transport observed.

Animals↗