Biomedical subjects

D G Gardner

Publications and source records attributed to D G Gardner.

At least 55 records · Page 3Linked to original sources

PubMed1996

Tissue-specific expression of the human brain natriuretic peptide gene in cardiac myocytes.

Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. To identify the cis-acting elements involved in regulation of the human BNP gene, we subcloned the full-length promoter (-1818 to +100) and deletions thereof upstream from a luciferase reporter gene and transiently transfected them into primary cultures of neonatal rat atrial and ventricular myocytes and myocardial fibroblasts. Luciferase activity of the full-length construct was higher in ventricular (39064 +/- 8488 relative light units, N=11) and atrial (11225 +/- 1907, N=17) myocytes than myocardial fibroblasts (329 +/- 113, n=5). Maximal promoter activity in ventricular and atrial myocytes was maintained by sequences positioned between -1818 and -1283 relative to the transcription start site. Deletion to -1175 resulted in a decrease, whereas further deletion to -500 effected an increase in reporter activity in both cell types. In ventricular and atrial myocytes, deletion from -500 to -40 reduced luciferase activity 20-fold and 2-fold, respectively, whereas in myocardial fibroblasts, deletion to -40 upregulated the BNP promoter 2-fold. Of note, deleting 16 bp between -127 and -111 reduced luciferase activity 7-fold and 4-fold in ventricular and atrial myocytes, respectively, but had essentially no effect on luciferase activity in fibroblasts. Placement of sequences lying between -127 and -40 upstream from a heterologous thymidine kinase promoter resulted in reporter expression that was 7.4-fold greater than the vector alone in ventricular myocytes, approximately 2-fold greater in atrial myocytes, and equivalent to the vector alone in fibroblasts. For study of activity of the human BNP promoter in adult myocytes, either 408 or 97 bp of 5' flanking sequence coupled to the luciferase reporter gene was injected into the apex of adult male Sprague-Dawley rat hearts. After 7 days, luciferase activity in the injected myocardium was 9.8-fold higher for the longer construct (70683 +/- 14744 versus 7223 +/- 3920, n=4, P < .01), consistent with our in vitro data. These data indicate that (1) the full-length human BNP promoter is more active in ventricular versus atrial myocytes and essentially inactive in fibroblasts, (2) the distal BNP promoter contains both positive and negative regulatory elements, (3) a region of the proximal BNP promoter located between -127 and -40 confers tissue specificity, and (4) the BNP promoter is active after injection into the adult rat heart.

Animals↗

PubMed1996

An E-box motif conveys inhibitory activity on the atrial natriuretic peptide gene.

Atrial natriuretic peptide (ANP) is a potent diuretic, natriuretic, and vasorelaxant hormone that is expressed early in ventricular hypertrophy. Expression of human ANP is controlled by a series of regulatory elements located in the 5' flanking sequence of its gene. We generated a series of 5' deletion mutations extending from -2600 to -1150 relative to the transcription start site and linked them to a chloramphenicol acetyltransferase reporter gene. Using transient transfection analysis, we have identified a negative regulatory element between -1206 and -1152 relative to the start site. Each of a series of 5' deletion mutants, when introduced into fibroblast cultures, expressed the reporter function at a level that was significantly less (< 20%) than that seen with the -1152 reporter construct, whereas comparably transfected atrial cardiocytes demonstrated no change in reporter activity, implying that the repressor function is specific to cell type. The critical region (from -1206 to -1152) associates with a soluble protein present in cardiac fibroblast extracts in a sequence-specific fashion. Deoxyribonuclease I footprint analysis demonstrated the presence of several protected regions, including one that overlies an E-box motif (CAACTG), an element that in other systems has been implicated in promoting differentiation in the myocyte lineage. Site-directed mutagenesis of the E-box motif suppressed both the protein-binding and inhibitory activities of the 54-bp fragment. In summary, we have found a region in the 5' flanking sequence of the human ANP gene that represses transcriptional activity in nonmyocardial cells. This element may play an important role in the restriction of ANP gene expression to cardiac myocytes.

Animals↗

PubMed1996

Selective regulation of the atrial natriuretic peptide gene by individual components of the activator protein-1 complex.

We used the human atrial natriuretic peptide (hANP) gene as a model to investigate the causal relationship between immediate early gene expression and the subsequent activation of the embryonic gene repertoire in cardiac hypertrophy. Using transient transfection analysis, we found that overexpression of individual Jun family members, alone or in combination, displayed unique activity that varied as a function of the promoter and the nature of the transfected myocyte populations under examination. In neonatal cardiac ventriculocytes, both c-jun and to a lesser extent, JunB stimulated hANP promoter activity (approximately 7- and 3- fold, respectively). When cotransfected together, a synergistic activation was observed (approximately 16-fold activation), a finding that stands in contrast to the behavior of JunB (i.e. neutral or inhibitory) with other 12-O- tetradeconoylphorbol 13-acetate response element-dependent promoters. In atriocytes, on the other hand, JunB did not itself activate the hANP promoter, and it antagonized c-jun- mediated transcription. JunD, a third member of this gene family, was devoid of activity in these transfected cultures. These findings suggest that the hANP gene promoter exhibits a broad range of responses to the individual products of the jun gene family. The response in any single situation is a function of the relative concentrations and subunit composition of the prevailing activator protein-1 complexes.

Animals↗

PubMed1996

Mechanisms contributing to renal resistance to atrial natriuretic peptide in rats with common bile-duct ligation.

Blunted volume expansion (VE) natriuresis and renal resistance to atrial natriuretic peptide (ANP) characterize states of pathological sodium retention. This study examined rats 1 to 3 wk after common bile-duct ligation (CBDL), at which time they had hyperbilirubinemia and hypoalbuminemia. Sham-operated normal rats (Sham) showed an increased sodium excretion rate (UNaV) from 1.0 +/- 0.1 to 16.3 +/- 3.9 muEq/min in response to acute VE (iv saline, 2 mL/100 g body wt over 5 min), whereas CBDL rats had a blunted response that was apparent after 1 wk and became maximal at 2 and 3 wk (0.3 +/- 0.1 to 3.2 +/- 0.4 muEq/min at 3 wk, P < 0.01 versus Sham response). The peak urinary cGMP excretion rate (UcGMPV) was also blunted (37.9 +/- 3.6 versus 87.5 +/- 8.3 pmol/min, P < 0.01) despite an even greater increase in plasma ANP concentration (Sham, 9.6 +/- 0.4 pg/mL in hydropenia to 22.8 +/- 2.6 pg/mL after VE; CBDL, 15.3 +/- 2.3 to 41.8 +/- 6.8 pg/mL). ANP-dependent cGMP accumulation by isolated inner medullary collecting duct (IMCD) cells from both Sham and CBDL rat kidneys was dose-dependent; however, at higher concentrations of ANP (> 10(-8) M), accumulation by cells from CBDL rats was significantly blunted, indicating resistance to ANP. Binding of 125I-ANP to IMCD cells was not different in CBDL rats compared with Sham control rats. Renal denervation improved but did not completely reverse the blunted natriuresis, and ANP resistance persisted in IMCD cells from denervated kidneys of CBDL rats. Incubation of IMCD cells with the phosphodiesterase inhibitors isomethylbutylxanthine or Zaprinast (each at 10(-3) M) restored ANP responsiveness in both innervated and denervated kidneys from CBDL rats, and intrarenal infusion of Zaprinast (10 micrograms/min) corrected the blunted increase in UNaV and UcGMPV after VE in rats with CBDL. These results suggest that ANP resistance in a model of abnormal sodium metabolism devoid of intrinsic renal disease may be related to increased activity of phosphodiesterase in renal target cells for ANP as well as to heightened renal nerve activity.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Atrial natriuretic peptide suppresses the transcription of its guanylyl cyclase-linked receptor.

Atrial natriuretic peptide (ANP) treatment of rat aortic smooth muscle cells suppressed both 125I-ANP binding and ANP-dependent cGMP accumulation, suggesting reductions in the type C (NPR-C) and type A (NPR-A) natriuretic peptide receptor populations, respectively. NPR-A, but not NPR-C, mRNA levels were reduced in a dose-dependent fashion by ANP. The latter effect appeared to be due, at least in part, to suppression of NPR-A gene promoter activity. ANP effected a dose- and time-dependent reduction in a transiently transfected NPR-A luciferase reporter (-1575LUC). Analysis of 5' deletion mutants of the NPR-A promoter demonstrated that the ANP-dependent sequence lies between -1575 and -1290 relative to the transcription start site. Inhibition of the ANP promoter was also effected by brain natriuretic peptide, type C natriuretic peptide, and 8-bromo-cGMP, but not by the NPR-C-selective ligand cANF. In the case of 8-bromo-cGMP, the responsive element(s) was localized to the same 285-base pair region linked to the ANP effect above. These findings indicate that ANP autoregulates its own receptors in these cells and, at least in the case of NPR-A, it does so through suppression of receptor gene expression and receptor synthesis. This suppression may operate through a cGMP-dependent element located more than a kilobase upstream from the transcription start site.

Animals↗

PubMed1995

Multiple calcifying hyperplastic dental follicles.

This article describes two additional examples of this unusual condition; two others were reported previously. The microscopic features are those of the hyperplastic dental follicles that occur in regional odontodysplasia. However, the teeth are not defective, as in regional odontodysplasia. This rare condition is sufficiently distinctive to be considered a pathologic entity.

Adult↗

PubMed1995

Canine acanthomatous epulis. The only common spontaneous ameloblastoma in animals.

This article describes the acanthomatous epulis in dogs, a quite common lesion that appears to be the counterpart of human ameloblastoma. Histologically, it resembles closely the acanthomatous pattern of human ameloblastoma and, like ameloblastomas in human beings, it infiltrates cancellous bone; moreover, it has no tendency to metastasize. Veterinarians have sometimes treated the acanthomatous epulis with therapeutic irradiation, providing one example when this tumor served as an animal model for human ameloblastoma. Although this treatment has cured some animals, others have developed postirradiation malignancies.

Ameloblastoma↗

PubMed1995

Feline inductive odontogenic tumor (inductive fibroameloblastoma)--a tumor unique to cats.

The inductive fibroameloblastoma is a rare odontogenic tumor that occurs in young cats of either sex, predominantly in the anterior maxilla. This article critically reviews the previously published examples with emphasis on microscopic features, relationship to ameloblastic fibroma, and biologic behavior; an additional example is described. This tumor differs microscopically from human ameloblastic fibromas in that it is not well-circumscribed but rather originates multifocally within the supporting connective tissue as characteristic, spherical condensations of fibroblastic connective tissue (ectomesenchyme) associated with islands of odontogenic epithelium. Its biologic behavior requires further study but the tumor apparently is unique to cats and is distinct from human ameloblastic fibroma. Consequently, feline inductive odontogenic tumor is suggested as being a more appropriate designation than inductive fibroameloblastoma.

Ameloblastoma↗

PubMed1995

1,25(OH)2D3 suppresses expression and secretion of atrial natriuretic peptide from cardiac myocytes.

We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the rat atrial natriuretic peptide (ANP) gene and the secretion of the encoded protein product in neonatal rat cardiac myocyte cultures. 1,25(OH)2D3 effected a dose- and time-dependent inhibition of agonist-stimulated ANP secretion, which was accompanied by a reduction in the levels of the ANP mRNA transcript. The latter effect appeared to derive, at least in part, from suppression of ANP gene transcription. Of interest, both the reduction in mRNA levels and the inhibition of transcriptional activity were amplified by simultaneous treatment with retinoic acid, suggesting that heterodimerization of liganded 1,25(OH)2D3 receptor and retinoic acid receptor (likely retinoid X receptor) may underlie the inhibitory mechanism in the cardiac myocyte. Neither the secretory effect nor the effect on transcription proved to be calcium dependent. 22-Oxacalcitriol, a nonhypercalcemic analogue of 1,25(OH)2D3, was equally effective in suppressing ANP mRNA levels and transcription of the gene. These findings add to a growing body of data that imply an important role for 1,25(OH)2D3 in the regulation of cardiovascular function.

Animals↗

PubMed1995

Natriuretic peptides inhibit DNA synthesis in cardiac fibroblasts.

We have examined the effects of the natriuretic peptides on DNA synthesis in primary cultures of neonatal rat cardiac fibroblasts. Binding analysis using 125I-labeled atrial natriuretic peptide identified a single class of high-affinity binding sites (Kd = 0.03 +/- 0.01 nmol/L) in these cells. Of these sites, 80% appear to be of the natriuretic peptide C receptor subtype, with the remainder being A and B receptor subtypes. Northern blot analysis confirmed the presence of all three natriuretic peptide receptors in these cells. Atrial natriuretic peptide (10(-7) mol/L) effected a modest but consistent reduction in both agonist- and stretch-stimulated [3H]thymidine incorporation (17% to 41%). Moreover, brain natriuretic peptide (10(-7) mol/L), C-type natriuretic peptide (10(-7) mol/L), and des-[Gln18,Ser19,Gly20,Leu21,Gly22]-ANF 4-23-NH2 (10(-7) to 10(-6) mol/L) all proved capable of antagonizing growth factor-dependent [3H]thymidine incorporation (the inhibition ranged from 14% to 28%) and cell proliferation, suggesting that all three natriuretic peptide receptor subtypes are involved in the regulation of mitogenesis in these cultures. The inhibition by atrial natriuretic peptide was amplified by cotreatment with phosphodiesterase inhibitors. Similar reduction in [3H]thymidine incorporation was seen after treatment with 8-bromo-cGMP (10(-4) to 10(-3) mol/L) or nitroprusside (10(-4) to 10(-3) mol/L). These results suggest an important paracrine role for the natriuretic peptides in regulating fibroblast growth during cardiac hypertrophy.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Fra-1, a Fos gene family member that activates atrial natriuretic peptide gene transcription.

Previous studies suggested that individual components of the activator protein 1 (AP-1) complex behave in a highly idiosyncratic fashion at the level of the human atrial natriuretic peptide (ANP) gene promoter. ANP gene transcription is activated by c-jun and is generally suppressed by c-fos. In the present study, fra-1, a close relative of the c-fos gene product in terms of its structure and functional activity, behaved like fos in cardiac atriocytes, effecting an approximately 50% reduction in c-jun-activatable expression of a human ANP chloramphenicol acetyltransferase (CAT) reporter. In cardiac ventriculocytes, however, fra-1 effected a synergistic amplification of the c-jun response (a 2.5-fold increase over c-jun alone). In atrial cells, fos-like proteins were not uniformly inhibitory in that a carboxy terminal deletion mutant of c-fos activated a human ANP-CAT reporter in the atriocyte cultures. Finally, using a series of domain-swap mutations in the fos/fra structural sequences, we showed that sequences at both the amino and the carboxy termini are required to realize the full fra-1-dependent stimulatory effect as well as the c-fos-dependent inhibition of ANP gene transcription. These findings suggest considerable heterogeneity in the response of the ANP promoter to different components of the AP-1 complex. Such heterogeneity may serve to broaden the range of biological responses available to this promoter as the cardiac cell attempts to adapt to perturbations in the extracellular environment.

Animals↗

PubMed1995

Familial gingival fibromatosis with unusual histologic findings.

This article describes the occurrence of numerous calcifications, amyloid deposits, and islands of odontogenic epithelium in the gingiva of 3 siblings with familial gingival fibromatosis. These microscopic features have not been reported previously in patients with this condition.

Adolescent↗

PubMed1995

Modulation of ANP-dependent effects of endothelin by inhibitors of neutral endopeptidase and cGMP phosphodiesterase.

Endothelin 3 (ET3) infusion increases the concentration of atrial natriuretic peptide (ANP) in plasma, which in turn raises hematocrit (Hct) through a transcapillary shift of plasma fluid and proteins into the interstitium, thereby reducing plasma volume (PV). The level of ANP bioactivity in peripheral target tissues is a function of ANP secretory rate and the turnover rate of ANP and its intracellular effector mechanisms. Neutral endopeptidase (NEP) is a widely distributed enzyme that participates in ANP catabolism, whereas cGMP-phosphodiesterase (PDE) degrades the intracellular second messenger of ANP. Therefore, we examined the consequences of inhibition of NEP and PDE on the ANP-dependent activity described above using the NEP inhibitor SQ 28,603 and the cGMP-PDE inhibitor zaprinast (M&B 22,948). In anesthetized, bilaterally nephrectomized rats, infusion of SQ 28,603 alone reduced mean arterial pressure (MAP) by 2.5 +/- 0.5% and increased Hct by 4.6 +/- 0.3% (P < .01 for both), leading to a calculated decrease in PV of 7.5 +/- 0.6%. These changes were prevented by pretreatment with rabbit anti-rat ANP antiserum. Simultaneous infusion of ET3 (25 ng/kg/min) and SQ 28,603 caused MAP to increase by 12.8 +/- 2.2%, an effect identical with that observed after ET3 alone (12.7 +/- 2.3%), whereas the increase in Hct of 9.4 +/- 0.4% was greater (P < .05) than the 7.5 +/- 0.4% increase seen with ET3 alone. ET3 increased plasma ANP concentration (599 +/- 135 vs. 108 +/- 13 pg/ml in vehicle-infused rats; P < .0001); ET3 and SQ 28,603 infused simultaneously increased plasma ANP even further (810 +/- 166 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1994

Negative regulation of the human atrial natriuretic peptide gene by 1,25-dihydroxyvitamin D3.

We have examined the effect of 1,25-dihydroxyvitamin D3 on the promoter activity of the atrial natriuretic peptide (ANP) gene in cultured neonatal rat atrial myocytes. In acute transfection studies 1,25-dihydroxyvitamin D3 inhibited the expression of a human ANP (hANP) promoter-driven chloramphenicol acetyltransferase reporter (-1150 hANP CAT) in a dose-dependent fashion (10(-10)-10(-8) M). When an expression vector for the vitamin D receptor (VDR) (pSVL-VDR) was introduced together with the reporter plasmid, there was a significant ligand-dependent amplification of the vitamin D-dependent inhibition. Deletion analysis of the 5'-flanking sequence localized the suppressible promoter sequence to within 104 base pairs of the transcription start site of the hANP gene. Thyroid hormone, glucocorticoid, estrogen, and retinoic acid receptor were incapable of mimicking the VDR-dependent inhibition. Retinoid X receptor, on the other hand, effected a significant reduction in hANP promoter activity which was at least additive with that produced by the liganded VDR. The VDR-dependent inhibition displayed promoter selectivity. Both the SV40 promoter and a conventional vitamin D response element linked to a truncated SV40 promoter were activated by the liganded vitamin D receptor, whereas the Rous sarcoma virus promoter was unaffected. On the other hand, the cardiac-specific troponin T promoter was suppressed in a fashion similar to ANP. These findings imply a potentially important role for vitamin D3 in the regulation of gene transcription in myocardial cells.

Animals↗

PubMed1994

Injury to the oral mucous membranes caused by the common houseplant, dieffenbachia. A review.

The common houseplant, dieffenbachia, causes painful edematous swelling of the oral mucous membranes when chewed. This property, which is well known to the staffs of poison control centers, can be dangerous to the unwary or to victims of practical jokes. The microscopic features of the injury are those of acute inflammation, but the nature of the plant's toxicity remains controversial. The numerous needles (raphides) of calcium oxalate, which are contained in specialized cells (idioblasts) in the plant, and proteases have both been implicated. When stimulated, the idioblasts fire the raphides with some force for a distance of two to three cell lengths. One possibility is that the local toxicity of dieffenbachia may be caused by a combination of this mechanical release of the raphides, which results in traumatic injury, and the effects of a chemical toxin carried on their surfaces and in their grooves.

Animals↗

PubMed1994

The so-called calcifying epithelial odontogenic tumour in dogs and cats (amyloid-producing odontogenic tumour).

This paper describes the microscopical features of a rare odontogenic tumour that occurs in dogs and cats and which has been referred to as the calcifying epithelial odontogenic tumour (CEOT), although it is not the counterpart of the human tumour of that name. We have proposed amyloid-producing odontogenic tumour (APOT) as an appropriate alternative term. The tumour is composed of irregularly shaped strands of squamous epithelium, which in some areas exhibit palisading of the basal cells, similar to that seen in ameloblastomas. Stellate reticulum occurs focally in some examples. The second prominent feature is the presence of amyloid which tends to calcify. Finally, in some examples, a collagenous matrix, which is apparently a form of dentine, is present focally, adjacent to the epithelium. These histological features are compared with those of the canine keratinizing ameloblastoma and the human CEOT. To date, to few examples have been reported to determine accurately the biological behaviour of APOTs, but some have recurred after excision; none has metastasized.

Ameloblastoma↗

PubMed1994

Ameloblastomas in the horse: a critical review and report of an additional example.

Previously published cases of ameloblastoma in the horse are reviewed in detail for their acceptability as examples of that tumor; an additional one is described. So far, this rare equine lesion has been shown to have two histologic patterns. The first consists of islands and sheets of epithelium that exhibit the basal cell characteristics of ameloblastoma; the central cells comprise stellate reticulum. The second exhibits these basal cell features less markedly and the central cells are spindle-shaped and closely packed. The biologic behavior of the equine ameloblastoma is thought to be the same as in human beings, a slowly growing, invasive lesion that does not metastasize. However, this conclusion requires confirmation.

Ameloblastoma↗

PubMed1994

Differential regulation of natriuretic peptide receptor activity in vascular cells.

We studied the regulation of the individual natriuretic peptide receptor subtypes by 12-O-tetradecanoylphorbol 13-acetate (TPA) and forskolin in cultured bovine aortic endothelial and smooth muscle cells. In cultured endothelial cells, 10(-8) mol/L TPA caused a reduction in atrial natriuretic peptide (ANP) receptor binding activity that was seen as early as 2 hours after the treatment and reached a maximum (88 +/- 3% of control) after 24 hours, whereas the inhibition of ANP-stimulated cyclic GMP (cGMP) accumulation peaked at 2 hours (62 +/- 13% of control) and returned to control levels after 12 hours. After 24 hours of forskolin (10(-4) mol/L) treatment, ANP binding activity fell to 47 +/- 6%, and cGMP accumulation was 52 +/- 11% of control. Northern blot analysis revealed that 10(-8) mol/L TPA suppressed natriuretic peptide C receptor transcript levels, and forskolin increased levels modestly after 24 hours of treatment. Natriuretic peptide A receptor transcript levels remained unchanged by either treatment. In cultured smooth muscle cells, 10(-8) mol/L TPA suppressed ANP binding activity and ANP-stimulated cGMP formation in a fashion similar to that seen in endothelial cells. TPA treatment also resulted in an inhibition of C-type natriuretic peptide-stimulated cGMP production (59 +/- 7% of control); however, this response persisted for as long as 24 hours after addition of the agonist. Treatment with 10(-4) mol/L forskolin produced a time-dependent inhibition of ANP binding activity and did not inhibit cGMP production stimulated by either ANP or C-type natriuretic peptide. In contrast to the effects seen with endothelial cells, TPA caused a dose-dependent stimulation of natriuretic peptide C receptor mRNA, whereas forskolin was inhibitory in smooth muscle cells. These results indicate that the effects of the kinase activators are a function of the individual receptor subtype as well as the cell in which it is expressed and imply a considerable degree of flexibility in the response to regulatory stimuli.

Animals↗