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A F Stewart

Publications and source records attributed to A F Stewart.

At least 127 records · Page 7Linked to original sources
PubMed1990

In vivo monitoring of a cAMP-stimulated DNA-binding activity.

The transcriptional activity of the tyrosine aminotransferase (TAT) gene is influenced by two major signal transduction pathways, by glucocorticoids and by glucagon acting via cAMP. We analyzed the effect of cAMP on protein-DNA interactions in vivo and on the transcription rate of the TAT gene. We demonstrate that a cAMP-responsive element (CRE) is located in a tissue-specific DNase I-hypersensitive region, 3.6 kb upstream of the start site of transcription. By using the genomic footprinting technique, we show that this sequence is occupied by protein in uninduced cells and that the in vivo footprint is transiently increased upon cAMP induction. Protein binding at the TAT-CRE correlates with the rate of transcription of the TAT gene. Cycloheximide treatment reveals that the genomic footprint is subject to rapid turnover; however, subsequent cAMP induction in the continued presence of cycloheximide restores the footprint partially. We conclude that as a part of the signal transduction pathway, a cAMP-dependent, post-translational modification increases the DNA-binding activity of a protein to the TAT-CRE and thereby stimulates the transcription rate of the TAT gene.

Base Sequence↗
PubMed1990

Chromatin structures of the rat tyrosine aminotransferase gene relate to the function of its cis-acting elements.

The relationship between DNase I-hypersensitive sites (HSs) and transcriptional enhancers of the rat tyrosine aminotransferase (TAT) gene was examined by comparing HSs in and around the TAT gene with the activity of the corresponding DNA sequences in transient transfection assays. In this manner, we identified two HSs as liver-specific enhancers. Of three hepatoma cell lines examined, only one sustained TAT mRNA levels comparable to those of liver. In this cell line, both enhancers were strongly active, and strong hypersensitivity in chromatin over the enhancers was evident. The other two hepatoma cell lines had reduced levels of TAT mRNA and no or altered hypersensitivity over either the enhancers or the promoter. One of these lines carried a negative regulator of the TAT gene, the tissue specific extinguisher Tse-1. This cell line exhibited all HSs characteristic of the strongly active gene except at the promoter; however, one enhancer was inactive even though hypersensitive in chromatin. In a TAT-nonexpressing cell line, inactivity of both enhancers correlated with absence of the respective HSs. We conclude that although hypersensitivity in chromatin necessarily accompanies cell-type-specific enhancer activity, the occurrence of cell-type-specific HSs does not imply that the underlying sequences harbor enhancers active in transient transfection assays.

Animals↗
PubMed1990

Effect of parathyroid hormone-like peptides on 25-hydroxyvitamin D-1 alpha-hydroxylase activity in rodents.

The role of vitamin D metabolism in the humoral hypercalcemia of malignancy syndrome (HHM) is unclear. We studied in vivo and in vitro effects of synthetic parathyroid hormone-like peptides (PTH-LPs) on rodent renal 25-OHD-1 alpha-hydroxylase activity. Infusion of mice with PTH-LP-(1-36) at 10 pmol/h for 12 and 24 h showed significant (429 +/- 139% and 937 +/- 413%, respectively) stimulation of control enzyme activity. Infusion for 36 h demonstrated diminution of activity to levels nearer to the unstimulated state (228 +/- 36% of control). In that maximal activity was observed after 24 h of infusion, we examined 1 alpha-hydroxylase activity after variable dosages of PTH-LP-(1-36) at this time point. Animals infused with PTH-LP-(1-36) at dosages of 2.5, 10, and 30 pmol/h for 24 h demonstrated 1 alpha-hydroxylase activities of 0.71 +/- 0.12, 4.74 +/- 2.09, and 9.91 +/- 1.01 ng.mg protein-1.20 min-1 (means +/- SD), respectively, all significantly greater than control activity (0.51 +/- 0.20 ng.mg protein-1.20 min-1). PTH-LP-(1-36) and PTH-LP-(1-74) were comparable in potency to bovine (b)PTH-(1-34) in stimulating 1 alpha-hydroxylase. Direct in vitro incubation of PTH-LP-(1-36) with renal slices resulted in stimulation of 1 alpha-hydroxylase activity up to 200% of control levels, comparable to that seen with equimolar concentrations of bPTH-(1-34).(ABSTRACT TRUNCATED AT 250 WORDS)

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1990

Clinical review 16: Parathyroid hormone-related proteins: coming of age in the 1990s.

The last 3 yr have yielded a fertile harvest of new information on the HHM clinical syndrome and on the novel peptide hormone family responsible for the syndrome. Whereas the clinical riddle enshrouding the HHM syndrome first posed in the early 1940s appears to have been largely solved, a whole new field, concerning the physiological role(s) of PTHRP has opened. The field has evolved rapidly and provides an example of fruitful clinical investigation: the original problem was a clinical one (the HHM syndrome); understanding the clinical disorder led to pursuit of the problem in the laboratory (adenylate cyclase assays, protein purification, molecular cloning, PTHRP synthesis); and observations made in the laboratory have rapidly yielded clinical fruits (PTHRP immunoassays) and opened a new window on normal physiology.

Gene Expression↗
PubMed1989

Characterization of canine renal receptors for the parathyroid hormone-like protein associated with humoral hypercalcemia of malignancy.

Parathyroid hormone-like proteins (PTHLP) display actions in the kidney which are similar to those of parathyroid hormone (PTH). We compared the binding properties of PTHLP and PTH in canine renal cortical membranes to determine if they interacted with the same or different receptors. Radioiodination to high specific activity (greater than 400 microCi/micrograms) of [Nle8,18,Tyr34]human PTH-(1-34)amide and [Tyr36]PTHLP-(1-36)amide was performed using the lactoperoxidase method. Complete enzymatic digestion of both radioligands demonstrated that the peptides were monoiodinated. Both radioligands retained full biological activity in the renal adenylate cyclase assay, and neither was significantly degraded during incubation with highly purified canine renal membranes under binding assays conditions. Specific binding reached equilibrium by 20 min at 20 degrees C. Competition binding studies using unlabeled [Nle8,18,Tyr34]human PTH-(1-34)amide, [Tyr36] PTHLP-(1-36)amide, and bovine PTH-(1-34) with either radioligand revealed similar binding affinities for all three peptides. Biologically inactive PTHLP fragments did not show significant displacement. In contrast to its similar binding affinity, [Tyr36]PTHLP-(1-36)amide was 6-15-fold less potent than bovine PTH-(1-34) in the renal adenylate cyclase assay, suggesting less efficient receptor-effector coupling. Photoaffinity cross-linking using either radioligand in canine renal membrane labeled indistinguishable 70,000-dalton proteins. In the presence of multiple protease inhibitors, binding to an 85-kDa component was observed. Labeling of both receptor forms was specifically abolished by an excess of either cold peptide and dose-response curves using affinity cross-linked membranes corroborated the apparent binding affinities determined by conventional radioligand binding assays. We conclude that PTHLP-(1-36) and amino-terminal PTH analogues bind to indistinguishable receptors in canine renal cortical membranes, but display differential coupling to post-receptor events.

Affinity Labels↗
PubMed1989

A myosin isoform repressed in hypertrophied ALD muscle of the chicken reappears during regeneration following cold injury.

A library of monoclonal antibodies specific for myosin heavy chain (HC) was used to study myosin expression in regenerating fibers. The response to cold injury of slow skeletal ALD muscle previously induced to eliminate SM1 myosin by weight overload was compared to that of its contralateral control. Native gel electrophoresis combined with immunoblotting demonstrated that slow SM1 myosin HC eliminated from hypertrophic muscle reappeared both at the site of active regeneration and unexpectedly, also distal to the site of injury. The regeneration response of hypertrophied muscles was similar to that of the controls. In addition to SM1 myosin HC, ventricular-like and embryonic/fast isoforms were also expressed in both muscles during the early stages of regeneration and disappeared as the muscle fibers matured. These observations demonstrate that regenerating slow muscle fibers reexpress myosins' characteristic of developing muscle irrespective of the myosin phenotype prior to injury. The reappearance of repressed myosin HC in the hypertrophied ALD muscle is consistent with the presence of newly differentiated myonuclei.

Animals↗
PubMed1989

Native and a synthetic analogue of the malignancy-associated parathyroid hormone-like protein have in vitro transforming growth factor-like properties.

A human parathyroid-like protein (PLP) has recently been isolated and cloned from human tumors associated with the paraneoplastic syndrome, humoral hypercalcemia of malignancy. PLP shares NH2-terminal amino acid sequence similarity with PTH but has a unique primary structure thereafter. Studies reported to date have indicated that both native and synthetic amino-terminal PLP polypeptides display actions in vivo and in vitro that are similar to those of PTH. We report here that purified native PLP and synthetic 36Tyr(1-36)amide human PLP induce epidermal growth factor-dependent transformation of NRK 49F cells in soft agar. Further, the synthetic peptide induces a significant increase in the biosynthesis of fibronectin by human dermal fibroblasts. (1-34)PTH does not display either of these biological activities. These data indicate that there are qualitative differences between PTH and the recently identified PLP. The latter hormone appears to possess transforming growth factor-like properties that may be relevant to its physiological actions.

Cell Transformation, Neoplastic↗
PubMed1989

Synthetic parathyroid hormone-like protein-(1-74): biochemical and physiological characterization.

PTH-like protein-(1-74) [PTHLP-(1-74)] was synthesized and purified. On the basis of chromatographic criteria and amino acid composition of the full-length peptide, direct amino acid sequencing of the N-terminus, and amino acid composition and internal sequence of proteolytic fragments of PTHLP-(1-74), the synthetic peptide appears to be of high quality and purity. Physiological comparison of PTHLP-(1-74) to [Tyr36]-PTHLP-(1-36) amide and bovine (b) PTH-(1-34) indicates that all three peptides are of equivalent potency in the fetal rat long bone and rat osteosarcoma 17/2.8 adenylate cyclase assays. However, as in earlier studies with native and N-terminal PTHLPs, PTHLP-(1-74) is considerably less potent (2%) in stimulating the canine renal cortical adenylate cyclase assay than is bPTH-(1-34). Further, PTHLP-(1-74) displayed only 12% of the activity of bPTH-(1-34) in inducing hypercalcemia when infused into rats in vivo. These studies support the possibility that subclasses of PTH receptors or varying PTH- and PTHLP-signalling transduction mechanisms may exist. In addition, they emphasize the need to precisely define the naturally occurring secretory and circulating species of this novel class of peptide hormones.

Adenylyl Cyclases↗
PubMed1989

Parathyroid hormone-related protein modulates the effect of transforming growth factor-beta on deoxyribonucleic acid and collagen synthesis in fetal rat bone cells.

Proteins with biochemical function and sequence similarity to PTH are produced by many tumors associated with hypercalcemia and may have a role in pathological bone remodeling. Synthetic polypeptides comprising the amino-terminus of human PTH-related protein (PTH-rp) were examined for effects in intact fetal rat calvariae, and in osteoblast-enriched (ob) cultures isolated from fetal rat parietal bone. In cultured calvariae, 0.5-5 nM PTH-rp stimulated [3H]thymidine incorporation into DNA by 25-70% after 24 h of treatment and decreased relative [3H]proline incorporation into collagen by 50%; the inhibitory effect on collagen production was not altered by hydroxyurea, which decreased DNA synthesis by 85%. PTH-rp also increased [3H]hydroxyproline levels by 100% in culture medium from bones prelabeled with [3H]proline, indicating accelerated matrix turnover. In contrast to results in intact calvariae, PTH-rp had little effect on basal DNA and collagen synthesis in serum-deprived ob cultures. However, when ob cultures were treated with transforming growth factor type beta at concentrations similar to those found in calvarial culture medium, 0.02-2 nM PTH-rp enhanced DNA synthesis and decreased collagen production. Furthermore, equimolar PTH-rp and PTH concentrations similarly displaced 125I-PTH-rp binding and enhanced cAMP synthesis in ob cultures. These studies suggest that PTH-rp regulates osteoblastic cell activity primarily through PTH-related pathways and may act in part by modulating the effects of locally produced transforming growth factor-beta in bone.

Animals↗
PubMed1988

Isolation and characterization of the bovine kappa-casein gene.

1. The bovine kappa-casein gene has been isolated as a series of overlapping lambda clones and shown to consist of five exons distributed over a total length of approximately 13 kb. Most of the mature protein-coding sequence is contained in a single large exon. 2. Approximately 65% of the gene has been sequenced together with portions of the 5'- and 3'-flanking sequences. The immediate 5'-flanking sequence contains several motifs which are characteristic of upstream regions including a TATA box, a CAAT box, a sequence similar to that recognized by transcription factor AP-1 and a purine-rich sequence resembling that found upstream in all other lactoprotein genes. Other possible regulatory sequences are found upstream of exon 4. 3. The organization of the kappa-casein gene, together with its upstream sequence, confirms previous conclusions that it is unrelated to the calcium-sensitive-casein gene family to which it is linked. Evidence is presented which supports a previous suggestion that kappa-casein and the fibrinogens are evolutionarily related. 4. Intron sequences contain several examples of the A family of the artiodactyl Alu-like repeated sequences, together with a single example of a C-family sequence. The remainders of the introns of the kappa-casein gene, compared with the repeat elements and exons, are A + T-rich. 5. Among the lambda clones isolated, representatives were found of the A and B genetic variants which can be distinguished by restriction-enzyme analysis. Several other examples of polymorphisms in the non-coding region were found.

Alleles↗
PubMed1988

Complete nucleotide sequence of the bovine beta-casein gene.

The beta-casein gene is a member of a small gene family encoding the calcium-sensitive caseins, which are specifically synthesized and secreted by the mammary gland during lactation in response to both peptide and steroid hormones. The caseins are involved in the transport of calcium phosphate in milk, which is important for bone development in the infant mammal. We report here the organization and complete DNA sequence of the 8.5 kb long bovine beta-casein gene. Comparison with the rat beta-casein gene reveals that the exons of both genes correspond exactly. The 5' flanking sequences of all Ca-sensitive casein genes are conserved within the proximal 200 bp and contain several elements that probably function as cis-acting regulatory elements, including an octamer-like motif, and SV40-type core enhancer and a sequence that appears to be common to all lactoprotein genes. The latter sequence is flanked on either side by 12 bp direct repeats. These direct repeats are themselves each part of sequences that display two-fold symmetry. The first 30 nucleotides of the 3' flanking regions in the bovine and rat beta-caseins are well conserved, indicating that they are likely to be involved in the mechanism of 3' end processing of the primary transcript.

Amino Acid Sequence↗
PubMed1988

Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy.

Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome that appears to be mediated in many instances by a parathyroid hormone-like peptide. Poly(A)+ RNA from a human renal carcinoma associated with this syndrome was enriched by preparative electrophoresis and used to construct an enriched cDNA library in phage lambda gt10. The library was screened with a codon-preference oligonucleotide synthesized on the basis of a partial N-terminal amino acid sequence from a human tumor-derived peptide, and a 2.0-kilobase cDNA was identified. The cDNA encodes a 177 amino acid protein consisting of a 36 amino acid leader sequence and a 141 amino acid mature peptide. The first 13 amino acids of the deduced sequence of the mature peptide display strong homology to human PTH, with complete divergence thereafter. RNA blot-hybridization analysis revealed multiple transcripts in mRNA from tumors associated with the humoral syndrome and also in mRNA from normal human keratinocytes. Southern blot analysis of genomic DNA from humans and rodents revealed a simple pattern compatible with a single-copy gene. The gene has been mapped to chromosome 12.

Amino Acid Sequence↗
PubMed1988

Synthetic human parathyroid hormone-like protein stimulates bone resorption and causes hypercalcemia in rats.

Parathyroid hormone-like adenylate cyclase-stimulating proteins (hACSPs) have been implicated as one of the calcemic, bone-resorbing agents in patients with humoral hypercalcemia of malignancy. We report the synthesis of an amino-terminal hACSP fragment, Tyr36 hACSP (1-36) amide. The synthetic hACSP is a potent agonist of renal membrane adenylate cyclase (Km, 1.7 X 10(-10)) and of bone cell adenylate cyclase (Km 1 X 10(-9)M). It is a potent bone-resorbing agent in vitro, stimulating 45Ca release from fetal rat long bones at a concentration of 10(-9) M. When infused via osmotic minipumps into rats, it is also a potent calcemic factor in vivo, inducing a rise in serum calcium from (mean +/- SD) 10.6 +/- 0.6 to 19.7 +/- 3.2 mg/dl when infused at 1.4 micrograms/h and from 9.9 +/- 0.7 to 11.4 +/- 1.2 mg/dl when infused at 0.14 micrograms/h. These findings indicate that biologically active hACSP fragments can be synthesized. One such synthetic peptide possesses the in vitro and in vivo bioactivities demonstrated in native, tumor-derived hACSPs. It is also a potent calcemic, bone-resorbing agent.

Adenylyl Cyclases↗