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Biomedical subjects

J D Baxter

Publications and source records attributed to J D Baxter.

At least 145 records · Page 8Linked to original sources
PubMed1986

Extra-atrial expression of the gene for atrial natriuretic factor.

Atrial natriuretic factor (ANF) is a group of peptides, originally isolated from the cardiac atria, that have a number of important effects on blood pressure, renal function, and salt balance. In the current study, expression of the ANF gene in certain extra-atrial tissues of the rat has been examined by radioimmunoassay of extracted ANF protein and by blot-hybridization, nuclease S1 analysis, and primer-extension analysis of the ANF mRNA. ANF peptides and mRNA were detected in cardiac ventricles, lung, and pituitary gland at levels generally less than or equal to 1% those of cardiac atria. The ANF transcripts in extra-atrial tissue appear to be very similar to those synthesized in the atria. They are polyadenylylated, are equivalent in overall length (950-1050 nucleotides), and have identical 5' termini. A secondary transcription start site mapping approximately 80 base pairs upstream from the primary start site is employed in atria and to a lesser extent in other tissues. The ANF transcript is present throughout the cardiac ventricles from apex to base and in the septum as well as the ventricular free walls. The transcript is more prevalent in the left ventricle and interventricular septum than in the right ventricle. Immunocytochemistry using various anti-rat ANF antibodies localized ANF immunoreactivity to the atrial myocytes; the ventricular myocytes, particularly along the endothelial surface of the ventricular chamber; perialveolar cells in the lung; and the gonadotropin-producing cells of the pituitary. The data indicate that the capacity for ANF gene expression extends beyond atrial tissue, albeit at much reduced levels, and may suggest alternative, perhaps paraendocrine, functions for the peptide in these tissues.

Animals↗
PubMed1986

Analysis by immunocytochemistry and in situ hybridization of renin and its mRNA in kidney, testis, adrenal, and pituitary of the rat.

Renin gene expression in cells and tissues of the rat was examined by in situ hybridization histochemistry and immunocytochemistry. By using a mouse cDNA probe, hybridization histochemistry revealed renin mRNA in the renal juxtaglomerular cells, testicular Leydig cells, adrenal zona glomerulosa cells, the intermediate lobe of the pituitary, and scattered cells of the anterior lobe of the pituitary. With four separate antisera to mouse submaxillary renin, there was immunoreactivity in the renal juxtaglomerular cells. However, only one of the antisera stained the Leydig cells, a second stained the adrenal zona glomerulosa, a third stained the intermediate lobe of the pituitary, and a fourth stained scattered cells of the anterior lobe of the pituitary that were identified as gonadotrophs. The variations with the different antisera in detecting extrarenal renin are unexplained but could imply that posttranslational proteolysis or glycosylation of preprorenin varies in different tissues with consequent variations in immunoreactivity. The finding of renin mRNA and renin-like immunoreactivity in these tissues supports the notion that these tissues are sites for production of renin.

Adrenal Glands↗
PubMed1986

The 5'-flanking sequences of the human growth hormone gene contain a cell-specific control element.

The 5'-flanking sequences of the human growth hormone (hGH) gene contain a cell-specific control element. Hybrid genes containing truncated 5'-flanking DNA fragments from the hGH gene fused to the chloramphenicol acetyl transferase (cat) gene were examined using transient transfection of rat anterior pituitary (GC) and nonpituitary cell lines (HeLa, Rat 2, and KB); preferential expression of these gene hybrids was only observed in GC cells. Deletions through the 5'-flanking sequences of the hGH gene revealed that the region containing nucleotides -230 to -180 is required for efficient cat gene expression in GC cells. This region of DNA is highly homologous to a region of the rGH gene that contains a tissue-specific control element. A hybrid gene containing the tissue-specific control element, but lacking the proximal promoter elements, of the hGH gene upstream from the promoter of the thymidine kinase gene (TKp) from herpes simplex virus ligated to the cat gene produced more CAT activity than the TKp.cat gene in GC cells but not in HeLa cells. These data suggest that the tissue-specific control element located in the 5'-region of the hGH gene can act in the presence of a heterologous promoter and is specific for expression in pituitary cells.

Acetyltransferases↗
PubMed1986

Biochemical similarity of expressed human prorenin and native inactive renin.

Prorenin is secreted by mammalian cells transfected with a human preprorenin expression construct. The purpose of this investigation was to compare the physicochemical properties of expressed prorenin in culture medium with the known characteristics of human inactive renin, which accounts for nearly half the renin in plasma and kidney. We found that expressed human prorenin strongly resembles human renal and plasma inactive renin. The expressed prorenin was inactive and could be equally activated by acid (dialysis to pH 3.3) or trypsin. Acid activation was completely reversible; reexposure to acid could reactivate the expressed inactive renin. Exposure to cold (-5 degrees C for 3 days) could also activate expressed renin. The Michaelis-Menten constant of acid-activated expressed renin with sheep substrate was 0.29 microM, and the pH optimum was 7.8. Expressed inactive renin bound to a cibacron-blue affinity column and could be eluted with 0.5M NaCl. All the above characteristics resemble those of human renal and plasma inactive renin. In addition, the molecular weight of expressed prorenin and human chorionic renin was 47,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 46,000, as measured by high-performance liquid chromatography. These data, taken together with the published observation that native human inactive renin cross-reacts with antibodies generated against amino acid sequences in the prosegment of renin, provide strong support for the hypothesis that human inactive renin is prorenin.

Cold Temperature↗
PubMed1986

Observations on the prevalence of ear disease in the Inuit and Cree Indian school population of Kuujjuaraapik.

In the last twenty years it has been recognized that hearing loss as the result of middle ear infection and/or noise exposure is a major problem among Canadian Inuit. In the past ten years in the Eastern Canadian Arctic attempts have been made to alleviate the problem and physicians, audiologists and educators have been involved in treatment, training programs and research with varying degrees of success. In the last few years the Quebec Inuit have become more aware of these problems and have asked for assistance. Whatever evolves, Inuit co-operation and advice is essential; their cultural identity must be respected if any project is to be successful. In February, 1984, a program outline working paper entitled "Program for Combatting Hearing Disorders in the Inuit Population of Nouveau Quebec" was circulated by Project Nord-Laval University. The goal of the program was "to ensure the integrity of hearing for the Inuit by preventing hearing loss, identifying hearing loss and minimizing the effects of hearing loss." In October, 1984 a Pilot Project involving the school population at Kuujjuaraapik was carried out involving personnel from the Project Nord-Laval University, the Department of Otolaryngology and the School of Human Communication Disorders-McGill University.

Adolescent↗
PubMed1985

Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene.

In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.

Animals↗
PubMed1985

Interaction of triiodothyronine-receptor complexes with simian virus 40 minichromosomes in monkey kidney CV-1 cells.

Thyroid hormone-responsive tissues contain chromatin-localized receptors that bind to DNA and may associate preferentially with actively transcribed chromatin. To study such receptor-chromatin localization, we have used cultured CV-1 cells permissive for simian virus 40 (SV40), in which viral minichromosomes can be separated from the cellular chromatin. CV-1 cells were found to contain intranuclear thyroid hormone-binding sites with an affinity for T3 and T4 and a site concentration similar to those in other thyroid hormone-responsive tissues. When these cells were infected with SV40 or an SV40-human GH gene recombinant, T3 did not affect SV40 replication, early or late gene transcription, or human GH gene expression. However, in both cases, these infections resulted in the association of about 7.5% of the total specific T3-binding activity with the SV40 minichromosome, representing about 1 receptor molecule/65 minichromosomes and a 10-fold enrichment over the cellular chromatin-associated activity (4.3 fmol/micrograms SV40 minichromosomal DNA vs. 0.43 fmol/micrograms chromosomal DNA); 30% of this could be covalently cross-linked to the minichromosome with dissuccinimidyl suberate. The minichromosomes were also found to be transcriptionally active. Thus, thyroid hormone receptors interact preferentially with the SV40 minichromosome, possibly owing to their tendency to associate with transcriptionally active chromatin. This system provides an alternate approach to study the association of thyroid hormone receptors with defined chromosomal segments.

Animals↗
PubMed1985

Acute effects of dexamethasone on intraocular pressure in glaucoma.

The effects of 3 mg orally administered dexamethasone on the intraocular pressure (IOP) were examined in four patients with primary open-angle glaucoma hospitalized for this study. Plasma-free glucocorticoid activity was measured by a radioreceptor assay. Diurnal rhythms of IOP and plasma-free glucocorticoid activity were detected prior to administration of dexamethasone. The plasma-free glucocorticoid activity rose two- to threefold in the 30-min period following steroid administration and then declined throughout the rest of the day. IOP was approximately 2 mmHg higher in the 0-4-hr period and approximately 5.5 mmHg higher in the 4-8-hr period following the pharmacologic doses of dexamethasone compared with similar periods on control days. The increase in the IOP was highly significant (P less than 0.006) in the latter time period. These findings suggest that the glucocorticoids may have a greater role in regulating IOP than generally has been appreciated.

Adult↗
PubMed1985

Intranasal presentation of a pituitary adenoma.

Unilateral intranasal polypoidal lesions should always arouse suspicion of unusual pathology. A case of an intranasal extension of a pituitary adenoma is presented. Fourteen other cases that have been reported in the literature are summarized and modalities of treatment of this entity are briefly discussed.

Adenoma↗
PubMed1985

Management of hearing impairment in the Canadian Inuit--an update.

In recent years, the teachers in the north and the Inuit have become increasingly aware of the educational effects of hearing los frequently associated with chronic otitis media. In the Baffin Zone, N.W.T. and in Nouveau Quebec a surge of enthusiasm has surfaced to do something positive and on-going about the problem. A description of events and changes in attitude occurring among the Inuit, educators, and medical services is presented.

Canada↗
PubMed1984

Analysis of a major human chorionic somatomammotropin gene. Evidence for two functional promoter elements.

The sequence of one of the two major expressed human chorionic somatomammotropin genes (hCS-1) was determined. The hCS-1 gene and the human growth hormone gene (hGH-1) share 92% nucleotide sequence homology in their 5'- and 3'-flanking regions, introns and exons. This finding, in addition to the existence of multiple closely linked hGH and hCS genes, suggests that these genes are evolving by concerted mechanisms. S1 nuclease, hybridization, and primer extension analysis of placental poly(A+) RNA demonstrated the presence of two functional initiation sites within the hCS-1 and/or the hCS-2 gene(s). The majority (about 95%) of the transcripts initiate 30 nucleotides downstream from the TATAAA sequence, and about 5% of the transcripts initiate 30 nucleotides downstream from a CATAAA sequence located 55 nucleotides 5' to the TATAAA sequence. The presence of two functional promoter elements as well as direct repeated sequences flanking the TATAAA sequence and exon I of the hCS genes are consistent with the hypothesis (Cooke, N. E., and Baxter, J. D. (1982) Nature (Lond.) 297, 603-606) that an important regulatory element may have been inserted into the gene in a separate evolutionary event. An analysis of other direct repeats, internal homology, and homology between other growth hormone and chorionic somatomammotropin genes offers a more extensive conceptualization of how this gene family evolved.

Amino Acid Sequence↗
PubMed1984

Transcription of two classes of rat growth hormone gene-associated repetitive DNA: differences in activity and effects of tandem repeat structure.

The rat growth hormone (rGH) gene contains two classes of repetitive DNA arranged as clusters within intron B and the 3' flanking region. The major family is equivalent to the CHO type 2 DNA. The second ("truncated repeat", TR) is a truncated version of the first and occurs in certain neural-specific transcripts and genes ("identifier" elements, ID). Here we report, using the HeLa cell-free transcription assay, that RNA polymerase III (Pol III) efficiently initiates at internal promoters within a tandem array of rGH gene repetitive DNA monomers and results in a novel organization of overlapping Class III transcription units. Transcription competition studies revealed that the rat type 2 structures share Pol III transcription factors with a tRNA gene, a human Alu repeat, and a mutant VA1 gene. Also, the rGH type 2 but not the TR DNA efficiently promotes Pol III initiation, yet other TR members, which differ only in flanking DNA, are transcribed. Thus, the rGH gene is strikingly enriched with 10 repetitive DNA monomers; multimeric type 2 elements are actively transcribed; rGH-TR sequences are expressed only as part of larger transcripts promoted by type 2 DNA; and, type 2 DNA uses tRNA gene transcription factors. These studies show that flanking sequences, promoter organization and factor competition may all affect rat repetitive DNA expression.

Animals↗
PubMed1984

Interactions of the nuclear thyroid hormone receptor with core histones.

These studies concern the interactions of the rat liver thyroid hormone nuclear receptor with histones and factors influencing the receptor's assay and stability. Heating certain crude receptor preparations at 50 degrees C produces a selective loss of triiodothyronine (T3) but not thyroxine (T4) binding activity, whereas, with more purified preparations, such heating decreases both T3 and T4 binding. The selective T3-binding loss in crude preparations was found to be due to the simultaneous denaturation of the receptor's high-affinity hormone-binding activity for both T3 and T4 and generation of new low-affinity T4-binding sites. The fraction in which T4 binding can be activated could be separated from the receptors by Sephadex G-100 chromatography. Core histones stimulated both T3- and T4-binding activity of 6-fold-purified receptor preparations, and data from several different experimental approaches suggest that this stimulation is due to the capability of the core histones to prevent the receptor from binding to or being denatured by Sephadex G-25 assay columns. The core histones were also found to stabilize 500-fold-purified but not 6-fold-purified or crude receptor preparations. A number of other acidic or basic proteins had little or none of these stimulatory effects, whereas a few proteins (such as the insulin B chain and histone H1) did have activity, although it was less than that of the core histones. There were no significant differences between the purified core histone subfractions (H2A, H2B, H3, and H4). That core histones can interact with the thyroid hormone receptors was demonstrated more directly by the finding that the receptors bind to histone-Sepharose but not Sepharose or insulin- or ovalbumin-Sepharose columns and that this binding was blocked by core histones at concentrations suggestive of an affinity for the receptor-core histone interaction of around 3 microM at 0.15 M salt concentration. The results demonstrate the utility of the histones in the assay and stabilization of purified thyroid hormone receptors, but they fail to support our previous hypothesis of a receptor subunit where T3- but not T4-binding activity is regulated selectively by histones. However, the results indicate that histones may interact with the receptors with some degree of specificity, and they raise the possibility that the histones participate in the nuclear localization of the receptors.

Animals↗
PubMed1984

Mechanisms of glucocorticoid hormone action.

This work summarizes some of our studies of the mechanisms of glucocorticoid action, including aspects of steroid binding to receptors, the activation of glucocorticoid-receptor complexes and the regulation of expression of endogenous and transferred glucocorticoid-responsive genes. Studies of the receptor-steroid interaction support the notion that steroid entry is passive. A comparative analysis of binding in isolated cytosol and intact cells suggests that the initial receptor-steroid binding reaction and not subsequent steps such as activation and nuclear binding, is predominantly responsible for the high-affinity state that is generated. The binding is driven by entropy and enthalpy changes at low temperature; at higher temperatures it is driven by entropy changes, with enthalpy working against it. Studies of the activation of the receptor-glucocorticoid complex with the use of highly purified receptors suggest that this step is associated with a change in charge of the receptor-glucocorticoid complex (such as would occur with a dephosphorylation reaction), whereas the data do not support the notion that dissociation of a bound RNA or of receptor oligomers is responsible for generating the nuclear- and DNA-binding activity of the complex. Studies of the regulation by glucocorticoids of expression of the endogenous rat growth hormone (rGH) gene in cultured rat pituitary tumor (GC, GH3D6) cells suggest that glucocorticoids increase the expression of this gene by multiple mechanisms. First, there is a modest direct stimulation of transcription by a mechanism(s) that does not depend on protein synthesis; however, if the cells have been exposed to thyroid hormone for several hours, the steroid exerts a much greater increase in rGH pre-mRNA levels. Secondly, the steroid appears to stimulate some relatively stable function or functions that increase the ability of thyroid hormone to increase rGH levels. Thirdly, the steroid probably increases rGH mRNA stability, since the fold-increases in rGH mRNA exceed those of transcription. Finally, the steroid may, by unknown mechanisms, affect rGH mRNA polyadenylation. The gene transfer experiments utilized the rat and human (h) GH genes and hybrid genes containing either rGH and Herpes Simplex virus thymidine kinase (TK) gene sequences or the human metallothionein-IIA (hMT-IIA) and TK gene sequences. The steroid was found to regulate hMT-IIA gene expression in all glucocorticoid-responsive cell types tested by actions on its 5'-flanking DNA. By contrast, the glucocorticoid regulated GH gene expression in some but not all glucocorticoid-responsive cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Interaction of sodium molybdate with highly purified glucocorticoid receptor.

Sodium molybdate can affect the properties of the glucocorticoid receptor in relatively crude preparations. To obtain more information as to whether these effects are due to direct interactions of the ion with the receptor or with other components present in the receptor-containing mixtures, the effects were examined of sodium molybdate on glucocorticoid receptors purified 3000-5000-fold to about 10% homogeneity from rat liver cytosol. The ion was found to: (1) increase the stability of the purified receptor at either 0 or 20 degrees C, although the effect was more pronounced at 20 degrees C (2) induce an apparent dimerization of the receptors as judged by sephadex G-150 gel filtration and sucrose density gradient sedimentation and (3) decrease the ionic strength required for elution of the purified receptor from DEAE-cellulose columns. Although, it is conceivable that each of these observed effects is due to indirect actions of the ion on contaminants in the preparations, it is more likely that the ion exerts its effects through direct interactions with the receptor.

Animals↗
PubMed1984

Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA.

Human metallothionein-IIA (hMT-IIA) gene expression is regulated by heavy metals and glucocorticoids. When the cloned hMT-IIA gene or its 5'-flanking DNA structure fused to herpes simplex virus thymidine kinase (HSV-TK) structural gene sequences were transferred into TK- Rat 2 fibroblasts, both genes were inducible by Cd++ and/or dexamethasone. Placement of the hMT-IIA gene 5'-flanking region, either intact of deleted in its TATA box and cap site, upstream of the HSV-TK gene promoter rendered the latter both glucocorticoid- and heavy metal-inducible. Thus the structure that mediates both Cd++ and glucocorticoid responsiveness is present in the hMT-IIA gene 5'-flanking DNA, does not require its TATA box or cap site, and can activate a heterologous promoter.

Animals↗