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Biomedical subjects

J W Hamilton

Publications and source records attributed to J W Hamilton.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1990

Isolated cytomegalovirus ileitis detected by colonoscopy.

Cytomegalovirus infections in immunocompromised patients mimic graft-versus-host disease by causing abdominal pain, watery diarrhea, and protein-losing enteropathy. The cases of three bone marrow transplant patients with diarrheal illness and biopsy-proven graft-vs.-host disease are reported. Isolated cytomegalovirus enteritis was subsequently identified by endoscopic examination and biopsy of the terminal ileum. All three improved with the eventual institution of 9-(1,3-dihydroxy-2-propoxymethyl) guanine. Ileoscopy is important in addition to colonoscopy in bone marrow transplant patients with diarrhea if cytomegalovirus enteritis is to be identified and appropriately treated.

Adult↗
PubMed1989

Molecular basis of hexavalent chromium carcinogenicity: effect on gene expression.

The carcinogenicity of chromium(VI) compounds is explained in terms of the "uptake-reduction" model. According to this model, chromium (VI) enters cells as the anionic tetrahedral species chromate, CrO4(2-), using normal cellular anion transport systems, such as the sulfate transport system. Redox-active enzymes and small molecules reduce chromium (VI) intracellularly and produce "reactive intermediates" capable of damaging cellular constituents. DNA is presumed to be a critical target within the cell; however, chromium (VI) itself is unreactive toward DNA under physiological conditions in vitro. Thus, the ability of chromium(VI) to damage DNA depends on the presence of cellular components capable of producing putative DNA-damaging agents, such as chromium(VI) thioester, chromium(V), chromium(IV), chromium(III), thiyl radical and hydroxyl radical, upon reaction with the chromium(VI). The exact nature of the DNA damage will be strongly dependent on the reactive intermediates produced by cellular components and systems active in chromium(VI) metabolism. The chromium(VI)-induced DNA damage disrupts the normal functioning of DNA in critical cellular processes, including transcription and replication. The effect of chromium(VI)-induced DNA damage on the function of DNA as a template for transcription has been examined in chick embryo liver in vivo. Chromium(VI) induced DNA-protein and DNA interstrand cross-links and suppressed the induction of 5-aminolevulinic acid synthase and cytochrome P-450 mRNA by porphyrinogenic drugs. In contrast, chromium(VI) increased the basal steady-state mRNA levels of these two inducible genes, but had no effect on the steady-state mRNA levels of the constitutive genes, beta-actin, conalbumin and albumin. Nuclear "run-off" transcription experiments showed that the effect of chromium(VI) on the steady-state levels of basal and drug-inducible 5-aminolevulinic acid synthase mRNA was principally the result of changes in the transcription rate of the 5-aminolevulinic acid synthase gene. Chromium(VI)-induced changes in gene expression correlated with the presence of DNA interstrand and DNA-protein cross-links, suggesting that chromium(VI)-induced DNA lesions lead to changes in expression of the targeted genes.

Animals↗
PubMed1989

Toxic effects of methyl methanesulfonate (MMS) on activated macrophages from chickens.

Adherent peritoneal exudate cells rich in macrophages were harvested from Cornell K-strain chickens 42 hr after i.p. stimulation with Sephadex G-50. Glass-adherent monolayers were obtained on coverslips and subjected to in vitro exposure to methyl methanesulfonate (MMS) at various doses for 1 hr. Solvent (0.17% ethanol final concentration) and sham (RPMI 1640 growth media) exposures were also performed. At selected times after exposure, the macrophages were analyzed for cell viability, adherence, DNA damage, and functional activity. Although MMS doses of 5 x 10(-3) M and 1 x 10(-3) M concentrations resulted in significant cytoxicity, 2 x 10(-4) M had no significant cytotoxic effect. However, this exposure resulted in DNA damage as measured by alkaline elution. Concomitant with the DNA damage was a significant decrease in the phagocytic activity of macrophages. Repair of MMS-induced DNA lesions in macrophages was indicated by a normal DNA alkaline elution profile 10 hr postrecovery. Functional activity of cells also returned to normal levels. In contrast, the incidence of Fc receptor-positive cells detected by rosetting increased immediately after MMS exposure, and phagocytosis of opsonized SRBCs was not affected by 2 x 10(-4) M MMS treatment. Similarly, MMS treatment did not alter the acid phosphatase activity of macrophages. However, bactericidal ability of MMS-treated macrophages for unopsonized Escherichia coli was significantly depressed. These results suggest that the avian macrophage is a useful target cell for examining possible relationships between genotoxic and immunotoxic effects of environmental mutagens.

Animals↗
PubMed1989

Differential effects of chromium(VI) on constitutive and inducible gene expression in chick embryo liver in vivo and correlation with chromium(VI)-induced DNA damage.

The effect of DNA damage induced by the carcinogen chromium(VI) on the function of DNA as a template for transcription of constitutive and inducible genes was examined in chick embryo liver in vivo. Changes in gene expression, determined using solution hybridization and northern blot analyses to measure steady-state mRNA levels and a nuclear run-off assay to measure gene transcription rates, were compared to chromium-DNA binding and to chromium(VI)-induced DNA damage as previously measured by DNA alkaline elution. Chromium(VI) treatment had little or no effect on either the steady-state mRNA levels or the transcription rates of the constitutively expressed genes for albumin, conalbumin (avian transferrin), or beta-actin. In contrast, chromium(VI) treatment had significant but opposite effects on the basal and drug-inducible expression of 5-aminolevulinate synthase and cytochrome PB1 P450. The changes in steady-state expression of these two inducible genes were similar to the changes in transcription rate, indicating that the effects of chromium were principally transcriptional. Chromium(VI) treatment increased the basal expression of both inducible genes four- to fivefold at maximum, and the time course of this effect was similar to the time course for chromium(VI)-induced DNA damage and repair. In contrast, chromium(VI) pretreatment suppressed by 60-70% at maximum the subsequent induction of these genes by glutethimide, a phenobarbital analog, and the time course of this effect also corresponded to that of chromium(VI)-induced DNA damage and repair. The time courses of the changes in expression of these genes were bimodal, with the second peak corresponding closely to that of chromium(VI)-induced DNA cross-links. However, the first peak occurred during a period when no DNA cross-links or strand breaks were detectable by alkaline elution, although significant levels of chromium were bound to DNA. This suggests that chromium(VI), like cisplatin, may initially produce a DNA monoadduct that subsequently leads to DNA cross-link formation and that both types of chromium(VI)-induced lesions have a significant effect on the expression of targeted genes.

5-Aminolevulinate Synthetase↗
PubMed1989

Development of a three-channel, 24-h ambulatory esophageal pressure monitor.

We have developed a three-channel ambulatory esophageal pressure monitor and tested it with a series of 24-h studies. The monitor is a battery-operated, microprocessor-based device that measures pressures from three transducers positioned in the esophagus, stores the data in its memory, and transfers the data to an IBM PC computer system at the end of the recording period. Programs on the PC then analyze the data and identify contractile events, categorizing them according to specific parameters. Other programs display the pressure waveforms on the PC and allow visual inspection of the entire recordings or, alternatively, of particular events of interest. The system detects contractile abnormalities in patients with intermittent, noncardiac chest pains. We tested the system on ten normal subjects and found a relatively high incidence of what are usually considered "abnormal" contractions.

Esophagus↗
PubMed1989

Bovine parathyroid tissue: a model to compare the biosynthesis and secretion of parathyroid hormone and parathyroid hormone-related peptide.

Bovine parathyroid tissue was evaluated as a model to compare parathyroid hormone-related peptide (PTH-rP) and parathyroid hormone (PTH) secretion. Tissue was incubated in variable calcium levels (n = 5). A parathyroid cell digest was prepared from collagenase-treated glands. PTH-rP and PTH levels were determined by radioimmunoassay. PTH-rP bioactivity was determined by 3H-cAMP production in a UMR 106 cell bioassay. PTH-rP levels in the incubation medium were 2.0 ng/mg protein (0.25 mmol Ca++), 2.2 ng/mg protein (1.25 mmol Ca++), and 1.9 ng/mg protein (2.5 mmol/L Ca++). PTH levels were 321 ng/mg protein (0.25 mmol/L Ca++) and 200 ng/mg protein (2.5 mmol Ca++). Therefore, calcium significantly inhibited PTH but not PTH-rP secretion (p = 0.03). Addition of incubation medium to the bioassay resulted in 3H-cAMP levels that were 8 to 10 times greater than basal levels. Greater than 50% of the activity persisted after addition of PTH antibody, demonstrating that a significant amount of the activity was caused by PTH-rP. Tissue PTH-rP was 5.1 ng/mg protein, compared with 2080 ng/mg protein for PTH. We conclude that (1) bovine parathyroid tissue contains bioactive PTH-rP and is a useful model to compare the biosynthesis and secretion of PTH-rP and PTH in normal tissue and (2) unlike PTH, PTH-rP secretion is not regulated by calcium.

Animals↗
PubMed1988

Hormone-containing peptides from normal and goiter human thyroglobulins.

A series of low iodine human thyroglobulin samples derived from colloid-rich goiter tissue was examined by HPLC mapping of tryptic digests and compared to normal human thyroglobulin. These samples ranged in iodine content from 2 to 8 gram-atoms of iodine (g.a. I) per mole and were not further iodinated in vitro. Peptides containing the principal hormonogenic sequence were detected using the long wavelength absorbance of the iodotyrosine derivatives at 325 nm. Two such peptides were isolated and sequenced. Their thyroxine content was confirmed by radioimmunoassay. The number of 325-nm-absorbing peaks was significantly lower in the normally iodinated human thyroglobulin than that observed the thyroglobulins of cattle and dog. This suggests a more restricted iodination in the human protein. Sodium dodecyl sulfate gel patterns of the reduced and alkylated proteins showed significant molecular size heterogeneity in all of the samples. Polypeptide fragments ranged in molecular size from approximately 330 to 45 kDa in the goiter derived material and from approximately 330 to 15 kDa in the normal human material. This difference between the proteins is consistent with earlier observations that peptides less than 45 kDa appear concomitantly with hormone formation. These data confirm that the human thyroglobulin molecule is capable of forming at least limited amounts of thyroid hormone at iodine levels as low as 4 g.a. I per mole. The hormone detected in this study was located at residue 5 near the amino terminus of the thyroglobulin molecule.

Chromatography, High Pressure Liquid↗
PubMed1988

Expression of 5-aminolaevulinate synthase and cytochrome P-450 mRNAs in chicken embryo hepatocytes in vivo and in culture. Effect of porphyrinogenic drugs and haem.

To examine current models for the co-ordinate regulation of 5-aminolaevulinate (ALA) synthase and cytochrome P-450 we have determined the effect of drugs, inhibitors of haem biosynthesis, haem and cycloheximide on the steady-state expression of mRNAs for ALA synthase and a phenobarbital-inducible cytochrome P-450 (PB1 P-450), in chick embryo hepatocytes in vivo and in primary culture. We found that the mRNAs for ALA synthase and PB1 P-450 were rapidly and simultaneously induced by the porphyrinogenic drugs glutethimide and 2-propyl-2-isopropylacetamide. Inhibitors of haem biosynthesis when administered alone had a small effect on ALA synthase mRNA induction, but in combination with the drugs synergistically increased induction of both ALA synthase mRNA and enzyme activity. However, there were concentrations of inhibitors that increased induction of enzyme activity without increasing mRNA induction. Haem suppressed ALA synthase mRNA induction by drugs by only 50%, whereas induction of ALA synthase enzyme activity was completely suppressed. This suppression of ALA synthase mRNA by haem was blocked by cycloheximide treatment which did not block the induction of ALA synthase mRNA by drugs. In fact, cycloheximide synergistically increased the drug induction of ALA synthase mRNA, suggesting the presence of a labile protein factor which may interact with a haem-responsive element of the ALA synthase gene. Cycloheximide treatment alone did not significantly affect ALA synthase mRNA expression, but induced PB1 P-450 mRNA to a similar extent to that caused by porphyrinogenic drugs, suggesting the presence of a labile repressor which modulates PB1 P-450 gene expression. Basal and drug-inducible PB1 P-450 mRNA levels were unaffected by haem or by inhibitors of haem biosynthesis, indicating that the PB1 P-450 gene is not regulated by haem in chick embryo hepatocytes. Our results indicate that drugs simultaneously induce ALA synthase and PB1 P-450 mRNA expression, and that ALA synthase activity is regulated by haem principally at a post-transcriptional site rather than at the transcriptional level.

5-Aminolevulinate Synthetase↗
PubMed1988

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides.

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.

Amino Acid Sequence↗
PubMed1988

A comparison of 30-kDa and 10-kDa hormone-containing fragments of bovine thyroglobulin.

Studies have been carried out on reduced and alkylated 19 S bovine thyroglobulin to characterize naturally occurring, iodine-rich fragments. In this report, the purification and properties of a 30-kDa, hormone-enriched polypeptide (TgE) are described and compared to that of a previously reported 10-kDa fragment (TgF). The amino acid sequence of TgF was found to overlap with that of TgE. In spite of its larger size, TgE contains only a single hormone bearing site. Both the 10- and 30-kDa fragments are derived from the NH2-terminal end of the bovine thyroglobulin. These fragments contain the principal hormone-forming site at residue 5 of the thyroglobulin sequence and appear to be formed by cleavage of the parent polypeptide chain. The mechanism which generates these cleavages is not clear since the sequences surrounding the cleavage points which give rise to these peptides are quite different. These two fragments may be precursor and product in such a process. The amino acid sequence contained within TgE includes two putative sites for N-linked glycosylation. Since no glucosamine was observed and only small amounts of neutral sugar were detected, it appears that this part of the molecule is not extensively glycosylated.

Amino Acid Sequence↗
PubMed1988

Clinical evaluation of methylcellulose as a bulk laxative.

We studied a bulk laxative containing methylcellulose in a group of normal subjects as well as in a group of chronically constipated individuals. The initial study in normal subjects was performed to show that the compound could increase fecal weight without significant side effects. Fifty healthy subjects were studied. Methylcellulose in daily doses of 4 g demonstrated a statistically significant increase in fecal frequency, fecal water, and fecal solids. In the second phase, we studied a group of 59 chronically constipated individuals treated with daily doses of the laxative containing either 1, 2, or 4 g of methylcellulose or 3.4 g psyllium. All of these doses resulted in statistically significant increases in stool frequency, water content, and fecal solids. There was no increase in individual stool weight from any of the laxative doses. Methylcellulose, in a daily dose as low as 1 g, is an effective laxative.

Adolescent↗