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J D Baxter

Publications and source records attributed to J D Baxter.

At least 109 records · Page 6Linked to original sources
PubMed1990

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin.

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.

Animals↗
PubMed1989

The rat growth hormone gene contains multiple thyroid response elements.

The thyroid hormone receptor exerts transcriptional control over a variety of genes. This report describes four sites that bind this receptor with high affinity within the 5'-flanking DNA of the rat growth hormone gene, approximately centered at -180, -160, -60 and -20 nucleotides from the transcription start site. These sites were defined by gel retardation of short synthetic oligonucleotides using native receptor purified several hundred-fold from rat liver. Binding sites were also defined by methylation interference and methidium-propyl-EDTA footprinting. Alignment of the four binding sites suggests that each contains two purine-rich regions, the more downstream of which, GGGATCGC, is highly conserved. Mutations made within each of the two upstream sites reduce receptor binding affinity. For one mutation, a partial loss of receptor binding strength correlated with a change in electrophoretic mobility, indicating that receptor binding may alter DNA conformation. Mutations at each of the four sites also reduce thyroid hormone responsiveness of the -237/+11 promoter linked to the chloramphenicol acetyltransferase gene coding sequences and transfected into cultured pituitary (GC) cells. These results suggest that several different receptor-binding elements interact to control thyroid hormone responsiveness of the rat growth hormone gene and reveal common sequences that may be important for receptor-DNA recognition.

Animals↗
PubMed1989

Structure of recombinant human renin, a target for cardiovascular-active drugs, at 2.5 A resolution.

The x-ray crystal structure of recombinant human renin has been determined. Molecular dynamics techniques that included crystallographic data as a restraint were used to improve an initial model based on porcine pepsinogen. The present agreement factor for data from 8.0 to 2.5 angstroms (A) is 0.236. Some of the surface loops are poorly determined, and these disordered regions border a 30 A wide solvent channel. Comparison of renin with other aspartyl proteinases shows that, although the structural cores and active sites are highly conserved, surface residues, some of which are critical for specificity, vary greatly (up to 10A). Knowledge of the actual structure, as opposed to the use of models based on related enzymes, should facilitate the design of renin inhibitors.

Aspartic Acid Endopeptidases↗
PubMed1989

Will the increased military low-level flying activity in Labrador be detrimental to the hearing of humans in the region?

The Government of Canada has directed the Department of National Defence to encourage our NATO allies to increase use of their facilities at Goose Bay, Labrador. This has already resulted in a substantial increase in the amount of military flying in the area, and more is projected. Much of the flying is done at very low altitude. The aboriginal people in the region (the Innu and, to a lesser extent, the Inuit) are demanding a halt to low-level military flying, and their representatives claim that the noise from the low-flying jet aircraft can cause hearing loss and ear disease. A survey on the ground in the area measured noise levels up to 127.7 dBA with very brief exposure levels. A task force commission to look at the problem concluded that occasionally low-level subsonic overflights might produce noise levels that were potentially damaging to hearing, but that the probability of this happening is at present very low because of the low frequency of flights. This might change if the frequency of flights increases.

Aircraft↗
PubMed1988

Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer.

Although steroid hormone receptors are known to activate gene expression by binding to specific hormone-dependent enhancers, the mechanisms by which steroids inhibit the transcription of specific genes are unknown. It is shown here by gene transfer studies that the same glucocorticoid receptor that activates gene expression can negatively regulate expression of the human glycoprotein hormone alpha-subunit gene. Glucocorticoid inhibition was conferred by a 52-nucleotide region that also contains elements crucial both for adenosine 3',5'-monophosphate (cAMP) responsiveness and for placental-specific expression of this gene and was observed only under conditions in which these elements were functioning as enhancers. Purified glucocorticoid receptor was found to bind to DNA that overlap the cAMP responsive elements sites in this region. It is hypothesized that steroid receptors negatively regulate gene expression by interfering with the activity or binding of other important transcription factors.

Cell Line↗
PubMed1988

The thyroid hormone receptor binds to multiple domains of the rat growth hormone 5'-flanking sequence.

Gel shift assays have been employed to examine the association of the thyroid hormone receptor with specific DNA sequences in the 5'-flanking DNA of the rat growth hormone (rGH) gene. This DNA is known to have structure(s) that mediate thyroid hormone effects on the rGH promoter. The receptors used were obtained from preparations purified 300-500-fold from rat liver nuclear extracts and contained about 1% pure receptors. Thyroid hormone receptor binding to DNA was assessed by monitoring protein-bound 32P-labeled restriction endonuclease fragments in parallel with L-tri[125I]iodothyronine-labeled protein-DNA complexes. The receptors were found to bind specifically to four different regions of the rGH 5'-flanking DNA (nucleotides -1730 to -1230, -530 to -230, -181 to -149, and -149 to +12) numbered with respect to the transcriptional start site. The specificity of the binding was documented by the finding that the receptor did not bind to other rGH 5'-flanking DNA sequences or to several other DNAs and by the fact that only the DNAs exhibiting specific binding could block the binding of radiolabeled DNA. The binding was also detected in NaCl concentrations up to 140 mM, reduced by Mg2+ concentrations up to 5 mM, and inhibited by 1 mM zinc. The DNA sequence-specific binding of the receptor was found to require occupancy of the receptor by the hormone (L-triiodothyronine) and could also be observed when the receptor was occupied by the thyroid hormone antagonist amiodarone. These results indicate that thyroid hormone receptors interact specifically with several sites on the 5'-flanking DNA of the rGH gene and that hormone occupancy is not required for the binding. Thus, thyroid hormone may act by stimulating a transcriptional activation function of the receptor rather than by stimulating DNA binding per se.

Animals↗
PubMed1988

Large scale purification of the nuclear thyroid hormone receptor from rat liver and sequence-specific binding of the receptor to DNA.

Methodology is reported for extracting thyroid hormone receptors from rat liver nuclei and for purifying these such that certain receptor properties can be examined. The extraction technique resulted in 1700 pmol of receptor/2 kg of liver and bypasses centrifugation in dense sucrose. The receptor was then purified by sequential heparin-Sepharose, DEAE-Sepharose, and phospho-Ultrogel chromatography and size exclusion and hydrophobic interaction high performance liquid chromatography. These steps yielded 23-35 micrograms of receptor at 0.7-1.5% purity from two 2-kg liver preparations. The cross-linkers disuccinimidyl suberate and N-succinimidyl-6-(4-azido-2-nitrophenylamino)hexanoate were employed to covalently attach 125I-labeled 3,5,3'-triiodo-L-thyronine (T3) to the purified receptor. Autoradiography after denaturing polyacrylamide gel electrophoresis revealed major 49,000 Mr and minor 58,000 Mr specific T3-binding proteins. The purified receptors exhibited high affinity (Kd = 100 pM) single site T3-binding activity. Because of the high affinity and specificity of [125I]T3 for the receptor, it was possible to uniquely identify the receptor containing DNA-protein complexes in a gel retardation assay and thus directly demonstrate for the first time that the receptor can specifically recognize sequences in the 5'-flanking DNA of the rat growth hormone gene. [125I]T3-labeled receptor migrated at the same position as the major gel-retarded 32P-labeled DNA band. Specific DNA competed for the binding much more strongly than nonspecific DNA. Thus, the purification procedure results in relatively large quantities of receptor at a purity sufficient for detecting and studying a number of its properties including specific DNA binding activity.

Animals↗
PubMed1988

Cell-specific expression of transfected brain identifier repetitive DNAs.

To define the neural-specific expression of rat repetitive identifier (ID) DNA, we co-transfected an intron B subclone of the rat growth hormone (rGH) gene, containing a tandem array of two type 2 repeats and a single ID monomer, and a plasmid conferring neomycin resistance into human SK-N-MC neuroblastoma, HeLa epidermal carcinoma, 293 kidney and 251 MG glioblastoma cells. Transcript analysis from both individual and pools of G418-resistant cells revealed that rGH intron B repeats were expressed only in SK-N-MC neuroblastoma cells as small, cytoplasmic RNAs of 85, 110, 155 and 180 bases. Primer-extension studies show these repetitive RNAs to contain a common 5' end that maps precisely to the beginning of the ID element and that type 2 transcripts are not stably expressed. However, ID DNA expression from two other transfected plasmids, each containing only the ID core sequence, was not restricted to the SK-N-MC cell line. These data show that the transfected rGH ID sequence is selectively expressed in a neural-specific manner resulting in BC-like RNAs, and furthermore, suggest that flanking DNA may play a role in cell-specific expression of certain repetitive DNA elements.

Animals↗
PubMed1988

The human growth hormone gene contains both positive and negative control elements.

A subset of DNA sequences in the 5'-flanking DNA of the human growth hormone (hGH) gene was examined by protein-DNA binding and gene transfer-expression experiments. Two adjacent cis-acting elements (I and II) were identified between nucleotides -308/-235 of the hGH gene that modulated the expression of a linked reporter gene in transfected HeLa cells. Elements I and II repressed gene expression whereas element II alone activated it. HeLa whole cell extracts contain two factors that bind hGH DNA carrying elements I and II. Factor I binds to single-stranded DNA, and its binding is correlated with repression of gene expression. Factor II binds between nucleotides -275/-257 of the hGH gene. This region is homologous to the binding site for the adenovirus major late transcription factor, and factor II binding to hGH DNA is competed by adenovirus major late promoter DNA, indicating that the hGH and major late adenovirus promoters share a transcription regulatory element.

Animals↗
PubMed1988

Processing of the primary transcript for the rat growth hormone gene in vivo.

Processing of the rat growth hormone (rGH) gene primary transcript and the effects of thyroid and glucocorticoid hormones on rGH pre-mRNA levels have been studied using subcloned radiolabeled DNA fragments from each of the four introns of this gene as probes. Blot-hybridization analysis of poly(A)+RNA from GC cells, GH3 cells, and normal pituitary gland indicates that processing of intron sequences from the precursor transcript takes place in a qualitatively similar fashion in each of these cell types. The data indicate that, in general, those introns closest to the termini of the primary transcript are removed first followed by removal of the internal introns. The suggested order of removal is IA, ID, IC, and IB. This process is unaffected qualitatively by thyroid or glucocorticoid hormones, both of which increase the rate of transcription of the gene. In addition to the primary transcript and the partially processed intermediate transcripts, GC and GH3 cells were found to contain a heterogenous group of intron-containing polyadenylated rGH gene transcripts which cannot be accounted for by any combination of intron deletions. These transcripts could arise either from internal start sites in the gene, premature termination of transcription, or inefficient processing of rGH mRNA precursors in the transformed cells. Thyroid hormone rapidly increases the levels of intron C-containing transcripts with kinetics that parallel the binding of thyroid hormone receptor to nuclei, but does not alter the ratio of primary to partially processed transcripts. These data suggest that most of the stimulatory activity of this hormone is due to effects on rGH gene transcription and not on pre-mRNA processing.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Characterization of recombinant human prorenin and renin.

A cell line that secretes substantial quantities of recombinant human prorenin was prepared by transfecting Chinese hamster ovary cells with a gene encoding preprorenin. The prorenin was purified to homogeneity and was found to have a single amino terminus, reflecting cleavage after a typical 23 amino acid signal sequence. The purified inactive prorenin was not a substrate for active renin and was not capable of self-activation. Prorenin could be converted to renin by addition of exogenous protease, and deglycosylation of the prorenin did not alter the sensitivity to protease activation. The enzymatic activity of deglycosylated renin was kinetically identical to that of the native protein. Multimilligram quantities of recombinant human renin and prorenin were purified, providing suitable material for studies directed toward greater understanding of the function of these proteins and for structural studies such as x-ray diffraction for use in design of renin inhibitors.

Animals↗
PubMed1988

Progesterone induction of metallothionein-IIA gene expression.

Expression of the metallothionein gene is known to be induced by glucocorticoids in a variety of cells. Here we show that in human cell lines containing functional progesterone receptors, the endogenous metallothionein-IIA (hMTIIA) gene is inducible by the synthetic progestins R5020 and medroxy-progesterone acetate. That this effect reflects a direct interaction with the metallothionein gene is supported by our finding that the partially purified progesterone receptor binds to the promoter region of the gene in vitro. The limits of the DNase I footprint and the guanine residues protected in methylation studies with the progesterone receptor are similar to those previously described for the glucocorticoid receptor. Thus, the hormone regulatory element of the human metallothionein-IIA gene can mediate regulation by both glucocorticoids and progestins, as does the hormone regulatory element of mouse mammary tumor virus.

Animals↗
PubMed1988

[Clinical trial of BOBCAT: 1st report on the reliability and validity of computerized pure-tone audiometry].

Computerized diagnostic audiometry is quickly emerging as a viable productivity tool in the audiology clinic. To date, there has been little reported on its reliability and validity with the hearing-impaired. The 'Battery of Basic Computerized Audiometric Tests' (BOBCAT) is a computer program which puts a wide variety of clinical hearing tests under computer control. The purpose of the present study was to ascertain the reliability and validity of BOBCAT in the measurement of hearing sensitivity. A field study was conducted in a group of 92 workers exposed to noise to measure air- and bone-conduction thresholds. Coefficients of reliability of 0.85 and higher were obtained between air and bone thresholds for both methods of testing; that is, manual and computer-controlled audiometry. The same measurement between manual and computerized air-conduction pure-tone thresholds gave values ranging from 0.93 to 0.98. A principal-components analysis documented content validity of computerized audiometry. These results are interpreted as clear evidence of both reliability and validity of the BOBCAT procedure, with one exception: 6.7% of all observations are showing air-conduction computerized thresholds of -10 dB at one or more frequencies, with no confirmation by manual audiometry. This is attributed to insufficient randomization of intervals between stimuli, a problem which should be taken care of by new versions of the software.

Adult↗
PubMed1987

Human renin is correctly processed and targeted to the regulated secretory pathway in mouse pituitary AtT-20 cells.

Renin is formed by intracellular processing of prorenin and catalyzes the conversion of angiotensinogen to angiotensin I, the precursor to angiotensin II. Several tissues synthesize prorenin. However, in man, the kidney is the only known source of circulating renin, raising the possibility that the processing enzyme is unique to that tissue. We have transfected a gene that directs prorenin synthesis in pituitary AtT-20 cells, which are capable of processing other prohormones. The results demonstrate that transfected AtT-20 cells can secrete inactive prorenin, accurately process prorenin to active renin, and be stimulated to release active renin in response to a secretagogue. These data imply that cellular elements capable of directing the processing of prorenin to renin and its correct subcellular compartmentalization may be present in nonrenal cell types and that critical elements of the regulated release of renin that occur in the kidney can be reconstituted in cells in culture.

Amino Acid Sequence↗