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Biomedical subjects

T A Cooper

Publications and source records attributed to T A Cooper.

At least 37 records · Page 2Linked to original sources
PubMed1997

Effects of parathyroidectomy on induced renal failure in dogs.

OBJECTIVE: To determine the effects of parathyroid hormone (PTH) depletion on dogs with induced chronic renal failure. ANIMALS: 2 groups of 26 mixed-breed dogs of both sexes (13 were parathyroidectomized [PTX] and 13 had sham surgery). PROCEDURE: After surgical reduction of renal mass and PTX, dogs were selected for a 24-month period of study and monitored for clinical, hematologic, blood biochemical, and organ function status. On development of uremia or after 24 months, dogs were euthanatized, and tissues were examined. RESULTS: Higher survival rate and smaller decrement in renal function (glomerular filtration rate) were observed in PTX dogs, compared with those that had sham surgery, but values did not reach statistical significance. The PTX dogs remained hypocalcemic during the study and had lower serum Ca2+ X P product values. Regardless of parathyroid state, survivors and fatalities could be separated on the basis of serum Ca2+ X P product values. Parathyroidectomy did not prevent renal deposition of calcium, and renal lesions were poorly correlated with renal cortical calcium concentration. Abnormalities reported in dogs with renal failure, which were attributed to PTH (glucose intolerance, pulmonary hypertension), were not observed in PTX dogs or those that had sham surgery. CONCLUSIONS AND CLINICAL RELEVANCE: PTX had beneficial effects, but these were mediated via changes in mineral homeostasis rather than via direct effects of PTH. Results attributable to PTX were similar to those previously obtained by dietary restriction of phosphate intake.

Acidosis↗
PubMed1997

Reliability of using random urine samples for "spot" determination of fractional excretion of electrolytes in cats.

OBJECTIVE: To determine whether the "spot" method of determining fractional excretion (FE) of electrolytes in cats is accurate. ANIMALS: 5 clinically normal young adult female cats. PROCEDURE: Cats were acclimated to metabolism cages, and 2 consecutive 72-hour collections of urine were made to determine FE of total calcium, potassium, total magnesium, sodium, and phosphorus by conventional methods, using endogenous creatinine clearance as an estimate of glomerular filtration rate. During collections, small samples of urine were obtained by cystocentesis at 8 AM, 3 PM, and 9 PM for determination of FE of the electrolytes by use of the "spot" method. RESULTS: Values from "spot" determinations were highly variable, compared with 72-hour values, with a high percentage falling outside the range of mean +/- 2 SD for 72-hour FE values. CONCLUSIONS AND CLINICAL RELEVANCE: The "spot" method for determining FE is not precise, and if used, caution and judgement should be exercised in interpretation of the results.

Animals↗
PubMed1996

Muscle-specific splicing enhancers regulate inclusion of the cardiac troponin T alternative exon in embryonic skeletal muscle.

The alternative exon 5 of the striated muscle-specific cardiac troponin T (cTNT) gene is included in mRNA from embryonic skeletal and cardiac muscle and excluded in mRNA from the adult. The embryonic splicing pattern is reproduced in primary skeletal muscle cultures for both the endogenous gene and transiently transfected minigenes, whereas in nonmuscle cell lines, minigenes express a default exon skipping pattern. Using this experimental system, we previously showed that a purine-rich splicing enhancer in the alternative exon functions as a constitutive splicing element but not as a target for factors regulating cell-specific splicing. In this study, we identify four intron elements, one located upstream,and three located downstream of the alternative exon, which act in a positive manner to mediate the embryonic splicing pattern of exon inclusion. Synergistic interactions between at least three of the four elements are necessary and sufficient to regulate splicing of a heterologous alternative exon and heterologous splice sites. Mutations in these elements prevent activation of exon inclusion in muscle cells but do not affect the default level of exon inclusion in nonmuscle cells. Therefore, these elements function as muscle-specific splicing enhancers (MSEs) and are the first muscle-specific positive-acting splicing elements to be described. One MSE located downstream from the alternative exon is conserved in the rat and chicken cTNT genes. A related sequence is found in a third muscle-specific gene, that encoding skeletal troponin T, downstream from an alternative exon with a developmental pattern of alternative splicing similar to that of rat and chicken cTNT. Therefore, the MSEs identified in the cTNT gene may play a role in developmentally regulated alternative splicing in a number of different genes.

Alternative Splicing↗
PubMed1995

A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon.

Alternative Splicing↗
PubMed1995

A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer.

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion.

Alternative Splicing↗
PubMed1995

The sense of effort and two models of single-joint motor control.

Two sets of experiments were carried out. In the first set, human subjects were asked to make the same effort with the elbow flexors at different joint angles under isometric conditions. In some experiments, the subjects were standing with the arm in a vertical (parasagittal) plane; in others, they were seated with the arm in a horizontal (transverse) plane. When muscular torque at a given effort level (ordinate) was plotted as a function of elbow joint angle (abscissa), the resulting isoeffort torque-angle profiles tended to be flat or negatively sloping over a range from 45 degrees to 135 degrees, and they were often nonmonotonic. Increases in effort up to near-maximal levels caused the isoeffort torque-angle profiles to shift upward with little alteration in shape. In the second set of experiments, seated subjects with the arm horizontal resisted baseline torques produced by a motor that acted to extend the elbow joint. Unexpected increases and decreases in torque were superimposed on the baseline torque. The subjects either were instructed to intervene and return the elbow to the initial (90 degree) position, or were told, "Do not intervene voluntarily; let the motor move your arm." Effort was reported both under baseline conditions and after the changes in torque. It was found that changes in effort were a function of the changes in torque opposed by the elbow flexors, and were similar whether the subject had repositioned the arm or allowed it to be moved by the motor. In the latter case, the arm came to rest after displacements that were a function of the size and direction of the torque change. For individual subjects, the largest angular displacements ranged from +/- 10 degrees to +/- 20 degrees for changes in torque of +/- 10 N.m. There was no evidence for any angular dependence of the effort judgements at a given torque over this angular range. Depending on whether effort is primarily an efferent perception proportional to voluntary motor activity or also has a significant afferent (involuntary) component, different models of motor control are supported by these data.

Adult↗
PubMed1993

The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element.

We have characterized a novel positive-acting splicing element within the developmentally regulated alternative exon (exon 5) of the cardiac troponin T (cTNT) gene. The exon splicing element (ESE) is internal to the exon portions of the splice sites and is required for splicing to the 3' splice site but not the 5' splice site flanking the exon. Sequence comparisons between cTNT exon 5 and other exons that contain regions required for splicing reveal a common purine-rich motif. Sequence within cTNT exon 5 or a synthetic purine-rich motif facilitates splicing of heterologous alternative and constitutive splice sites in vivo. Interestingly, the ESE is not required for the preferential inclusion of cTNT exon 5 observed in primary skeletal muscle cultures. Our results strongly suggest that the purine-rich ESE serves as a general splicing element that is recognized by the constitutive splicing machinery.

Alternative Splicing↗
PubMed1992

In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron.

A single cardiac troponin T (cTNT) gene generates two mRNAs by including or excluding the 30-nucleotide exon 5 during pre-mRNA processing. Transfection analysis of cTNT minigenes has previously demonstrated that both mRNAs are expressed from unmodified minigenes, and mutations within exon 5 can lead to complete skipping of the exon. These results suggested a role for exon sequence in splice site recognition. To investigate this potential role, an in vitro splicing system using cTNT precursors has been established. Two-exon precursors containing the alternative exon and either the upstream exon or downstream exon were spliced accurately and efficiently in vitro. The mutations within the alternative exon that resulted in exon skipping in vivo specifically blocked splicing of the upstream intron in vitro and had no effect on removal of the downstream intron. In addition, the splicing intermediates of these two precursors have been characterized, and the branch sites utilized on the introns flanking the alternative exon have been determined. Potential roles of exon sequence in splice site selection are discussed. These results establish a system that will be useful for the biochemical characterization of the role of exon sequence in splice site selection.

Animals↗
PubMed1989

Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing.

The cardiac troponin T (cTNT) pre-mRNA contains a single alternative exon (exon 5) which is either included or excluded from the processed mRNA. Using transient transfection of cTNT minigenes, we have previously localized pre-mRNA cis elements required for exon 5 alternative splicing to three small regions of the pre-mRNA which include exons 4, 5, and 6. In the present study, nucleotide substitutions were introduced into the region containing exon 5 to begin to define specific nucleotides required for exon 5 alternative splicing. A mutation within the 5' splice site flanking the cTNT alternative exon that increases its homology to the consensus sequence improves splicing efficiency and leads to increased levels of mRNAs that include the alternative exon. Surprisingly, substitution of as few as four nucleotides within the alternative exon disrupts cTNT pre-mRNA alternative splicing and prevents recognition of exon 5 as a bona fide exon. These results establish that the cTNT alternative exon contains information in cis that is required for its recognition by the splicing machinery.

Animals↗
PubMed1988

Cis requirements for alternative splicing of the cardiac troponin T pre-mRNA.

The cardiac troponin T (cTNT) pre-mRNA splices 17 exons contiguously but alternatively splices (includes or excludes) the fifth exon. Because both alternative splice products are processed from the same pre-mRNA species, the cTNT pre-mRNA must contain cis-acting sequences which specify exon 5 as an alternative exon. A cTNT minigene (SM-1) transfected into cultured cells produces mRNAs both including and excluding exon 5. The junctions of exons 4-5-6 and 4-6 in the cTNT minigene mRNAs are identical to those of endogenous cTNT mRNAs and no other exons are alternatively spliced. Thus, the SM-1 pre-mRNA is correctly alternatively spliced in transfected cells. To circumscribe the pre-mRNA regions which are required for the alternative nature of exon 5, we have constructed a systematic series of deletion mutants of SM-1. Transfection of this series demonstrates that a 1200 nt pre-mRNA region containing exons 4, 5, and 6 is sufficient to direct alternative splicing of exon 5. Within this region are two relatively large inverted repeats which potentially sequester the alternative exon via intramolecular base-pairing. Such sequestration of an alternative exon is consistent with models which propose pre-mRNA conformation as being determinative for alternative splicing of some pre-mRNAs. However, deletion mutants which remove the majority of each of the inverted repeats retain the ability to alternatively splice exon 5 demonstrating that neither is required for cTNT alternative splice site selection. Taken together, deletion analysis has limited cis elements required for alternative splicing to three small regions of the pre-mRNA containing exons 4, 5, and 6. In addition, the cTNT minigene pre-mRNA expresses both alternative splice products in a wide variety of cultured non-muscle cells as well as in cultured striated muscle cells, although expression of the cTNT pre-mRNA is normally restricted to striated muscle. This indicates that cis elements involved in defining the cTNT exon 5 as an alternative exon do not require muscle-specific factors in trans to function.

Animals↗
PubMed1988

Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells.

The chicken gene encoding cardiac troponin T (cTNT) is expressed in both cardiac and skeletal muscle during early embryonic development, but is specifically repressed in skeletal muscle during fetal development. To determine if the cis-acting sequences governing transcription of a single gene in these two related cell types are the same, we have transfected promoter/upstream segments of the cTNT gene coupled to the bacterial chloramphenicol acetyltransferase gene into primary cultures of early embryonic cardiac and skeletal muscle cells. Using this assay system, chloramphenicol acetyltransferase activity directed by the cTNT promoter/upstream region was between two and three orders of magnitude higher in cardiac or skeletal muscle cells than in fibroblast cells, indicating that cis elements responsible for cell-specific expression reside in this region of the cTNT gene. Deletion experiments showed that a 67-nucleotide DNA segment residing between 268 and 201 nucleotides upstream of the cTNT transcription initiation site is required for cTNT promoter activity in embryonic cardiac cells. This region is not required in embryonic skeletal muscle cells because a cTNT promoter construction containing only 129 upstream nucleotides is transcriptionally active in these cells. These results demonstrate that different cis-acting sequences are required for cTNT expression in early embryonic cardiac and skeletal muscle cells. Nonessential regions residing farther upstream, on the other hand, affected the level of expression of these minimum regions in a similar manner in both cell types. The data from these experiments indicate, therefore, that transcription of the cTNT promoter in early embryonic cardiac and skeletal muscle cells is governed both by common and divergent regulatory elements in cis and in trans.

Acetyltransferases↗
PubMed1987

Interrelationships of opioidergic and adrenergic mechanisms controlling the secretion of corticotrophin releasing factor in the rat.

The effects of morphine and naloxone on hypothalamo-pituitary-adrenocortical (HPA) activity and blood pressure were studied in rats in which adrenergic transmission had been impaired pharmacologically. The alpha 1-adrenoceptor antagonist, prazosin and the tyrosine hydroxylase inhibitor, alpha-methyl-para-tyrosine (alpha-MPT), increased the hypothalamic corticotrophin releasing factor (CRF) content and the plasma corticotrophin (ACTH) concentration and exaggerated the HPA response to stress. In addition, the spontaneous secretion of CRF by hypothalami in vitro was increased by alpha-MPT-treatment. Morphine enhanced the basal and stress-induced activity of the HPA system in vivo. It also stimulated the secretion of CRF by hypothalami in vitro. Naloxone did not affect resting HPA activity but reduced markedly the stress-induced release of corticotrophin. The effects of morphine and naloxone on the HPA axis, in vivo, were reduced by pretreatment with alpha-MPT and prazosin, respectively. The HPA responses could not be correlated with the changes in blood pressure which the drugs caused. The results suggest that opioid substances stimulate HPA activity by depressing the activity of the adrenergic pathways which inhibit the secretion of CRF.

Adrenocorticotropic Hormone↗
PubMed1986

Effects of naloxone on hypothalamo-pituitary-adrenocortical activity in the rat.

The influences of morphine and naloxone on hypothalamo-pituitary-adrenocortical (HPA) function were studied in the rat to investigate further the role of opioidergic mechanisms in the control of the secretion of corticotrophin and its hypothalamic releasing factor (CRF). Morphine not only caused rises in hypothalamic CRF content and plasma ACTH concentration but also potentiated the HPA response to stress. Its effects were antagonized by naloxone which, when given alone, did not influence basal plasma concentrations of ACTH and corticosterone but which inhibited, in a dose-dependent manner, the release of both of these hormones which normally occurs in response to stress. Naloxone also attenuated the exaggeration in stress-induced HPA activity but did not affect the increases in plasma ACTH concentration which followed adrenalectomy. The findings suggest that opioidergic mechanisms may be involved in the regulation of the HPA response to stress.

Adrenalectomy↗
PubMed1986

Pharmacological characterization of opioid receptors influencing the secretion of corticotrophin releasing factor in the rat.

The effects of selective agonists and antagonists of opioid receptors on the secretion of corticotrophin releasing factor (CRF) by isolated rat hypothalami in vitro were studied. Morphine (10(-8)-10(-6) M) and the mu-opioid receptor agonists, FK33-824CH (10(-8)-10(-6) M) and Tyr-D-Ala-Gly-MePhe-NH(CH2)2OH (10(-8)-10(-6) M), caused dose-related increases in the release of CRF from isolated hypothalami. The kappa-opioid receptor agonist, U50,488 (10(-8)-10(-6) M), was also weakly active in this respect but the delta-opioid receptor agonist, (D-Pen2,D-Pen5)-enkephalin (2 X 10(-10)-2 X 10(-7) M), was not. The stimulatory actions of morphine and Tyr-D-Ala-Gly-MePhe-NH(CH2)2OH on CRF release were antagonized by naloxone (10(-8) M) and by the mu/delta-opioid receptor antagonist, beta-funaltrexamine (beta-FNA, 10(-9) M), but not by the delta-opioid receptor antagonist, ICI-154129 (5 X 10(-6) M). The effects of U50,488 on CRF release were unaffected by either beta-FNA or ICI-154129 but were antagonized by high doses of naloxone (10(-6) M). The results suggest that both mu- and kappa-opioid receptors are involved in the stimulation of CRF secretion but that delta-opioid receptors are not important in this respect.

Animals↗
PubMed1985

A single cardiac troponin T gene generates embryonic and adult isoforms via developmentally regulated alternate splicing.

A single cardiac troponin T gene generates two mRNA products by developmentally regulated alternate splicing. Nucleotide sequence of the entire 18 exon gene and both representative cDNAs demonstrate that the two mRNAs differ by the presence or absence of a single internal coding exon. Both mRNA products appear to be generated from a single primary transcript; however, one mRNA splice product predominates in early embryonic cardiac muscle while the other vastly predominates in adult cardiac muscle. The corresponding embryonic and adult cardiac troponin T proteins differ by the inclusion or exclusion, respectively, of an internal, highly acidic 10 amino acids near the amino terminus. Unusual features of the variable peptide region and its restriction to embryonic stages suggest that it might play a specialized role during sarcomere assembly in embryonic striated muscle. In addition to developmental regulation of RNA processing, the cardiac troponin T gene also demonstrates complex tissue-specific expression. We have previously shown that this single cardiac troponin gene is regulated according to two different and tissue-specific regulatory programs. Here we demonstrate that a single promoter is utilized for both expression patterns. The cardiac troponin T gene also has several interesting structural features including a pseudoexon and an exon only six nucleotides in length. The size of this exon establishes a new lower limit for the number of nucleotides that can be recognized as an exonic sequence.

Amino Acid Sequence↗
PubMed1984

Complete cDNA-derived amino acid sequence of chick muscle creatine kinase.

cDNA clones of chick muscle creatine kinase mRNA were prepared by conventional procedures and then extended by primer extension to include the entire coding region of the mRNA. The nucleotide sequence of the cDNA clones permits, for the first time, determination of the complete sequence of the 381 amino acids comprising the muscle creatine kinase subunit of a higher vertebrate. In addition, available information regarding amino acid residues, intergral or proximal to the active site, allows tentative positioning of part of the active site within the primary sequence. Comparison of the chick muscle creatine kinase amino acid sequence with partial amino acid sequence for rat and rabbit muscle creatine kinase indicates that the primary structure of this enzyme is strongly conserved in evolution.

Amino Acid Sequence↗
PubMed1984

A single troponin T gene regulated by different programs in cardiac and skeletal muscle development.

A cloned complementary DNA derived from a messenger RNA transiently present at low abundance levels in early chick embryonic skeletal muscle hybridizes to a messenger RNA present at high abundance levels in cardiac muscle. Genomic DNA hybridization and nucleotide sequence identity of complementary DNA's from both heart and skeletal muscle demonstrate that the messenger RNA's from both sources are encoded by the same gene. The encoded polypeptide is a troponin T sequence which is probably a cardiac isoform. This single copy troponin T isogene is governed by different regulatory programs in heart and skeletal muscle differentiation.

Animals↗