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R Wade

Publications and source records attributed to R Wade.

At least 37 records · Page 2Linked to original sources
PubMed1995

Delineation of a slow-twitch-myofiber-specific transcriptional element by using in vivo somatic gene transfer.

Contractile proteins are encoded by multigene families, most of whose members are differentially expressed in fast- versus slow-twitch myofibers. This fiber-type-specific gene regulation occurs by unknown mechanisms and does not occur within cultured myocytes. We have developed a transient, whole-animal assay using somatic gene transfer to study this phenomenon and have identified a fiber-type-specific regulatory element within the promoter region of a slow myofiber-specific gene. A plasmid-borne luciferase reporter gene fused to various muscle-specific contractile gene promoters was differentially expressed when injected into slow- versus fast-twitch rat muscle: the luciferase gene was preferentially expressed in slow muscle when fused to a slow troponin I promoter, and conversely, was preferentially expressed in fast muscle when fused to a fast troponin C promoter. In contrast, the luciferase gene was equally well expressed by both muscle types when fused to a nonfiber-type-specific skeletal actin promoter. Deletion analysis of the troponin I promoter region revealed that a 157-bp enhancer conferred slow-muscle-preferential activity upon a minimal thymidine kinase promoter. Transgenic analysis confirmed the role of this enhancer in restricting gene expression to slow-twitch myofibers. Hence, somatic gene transfer may be used to rapidly define elements that direct myofiber-type-specific gene expression prior to the generation of transgenic mice.

Animals↗
PubMed1995

Developmental regulation of troponin I isoform genes in striated muscles of transgenic mice.

The differentiation and diversification of striated muscle is a complex process involving numerous temporal and spatial alterations in the pattern of contractile protein isoform gene expression. In order to gain insight into the regulation of contractile protein isoform changes during skeletal and cardiac muscle formation, the expression of a transgene comprising a chloramphenicol acetyltransferase (CAT) reporter gene linked with sequences from -4200 to +12 of the human slow skeletal troponin I (TnIs) gene, and all three endogenous mouse troponin I (TnI) isoform genes, was investigated in embryonic, neonatal, and postnatal mice. The -4200 TnIsCAT transgene was properly activated in the limb and trunk skeletal muscle primordia and the early embryonic atrium and ventricle of the heart. Along with the endogenous mouse TnIs gene, expression of the CAT transgene began to segregate into the presumptive slow-twitch myofibers at late fetal stages and expression declined in the neonatal and postnatal heart except for the conductive tissues, in which expression persisted into adulthood. However, expression of the CAT transgene during development did not completely follow the endogenous mouse TnIs gene. The expression of the CAT transgene was aberrantly low in the embryonic cardiac outflow tract and the ventricles of the fetal heart. In addition to its expression in striated muscles, the transgene was expressed aberrantly in the primordial axial skeleton. We conclude that the upstream sequences from the human TnIs gene contain sufficient regulatory information to confer appropriate transgene expression during the early differentiation of skeletal muscles and during the establishment of fiber type upon the maturation of myofibers. However, additional regulatory elements are likely to be required for correct temporal and spatial regulation in the heart and somitic mesoderm during development. In vitro DNA transfection of cultured skeletal and cardiac muscle cells identified a cell type-specific enhancer element within the first intron of the TnIs gene whose absence in the transgene may account for the aberrant expression observed in vivo. In addition, we provide the first evidence that the fast-twitch skeletal muscle isoform of troponin I, TnIf, is transiently expressed during early cardiac muscle development.

Animals↗
PubMed1995

Use of thapsigargin to study Ca2+ homeostasis in cardiac cells.

Several reports have documented that thapsigargin is a potent inhibitor of the SR Ca2+ ATPase isolated from cardiac or skeletal muscle. We have characterized the specificity of this agent in intact rat cardiac myocytes using cells maintained in the whole cell voltage clamp configuration. We have shown that thapsigargin decreases the magnitude of the Ca2+ transient and the twitch by about 80% while it slows the decay rate for these responses. These changes were not accompanied by any alterations in sarcolemmal currents or in the trigger Ca2+ generated by the inward calcium current. Taken together these results reveal that the action of thapsigargin is restricted to the SR Ca2+ ATPase in intact cardiac myocytes. Furthermore, it is demonstrated unambiguously that SR intracellular Ca2+ stores are an absolute requirement for the development of contractile tension in rat heart myocytes. It is shown that thapsigargin is a valuable probe to examine the importance of SR pools of Ca2+ and the role of the Ca2+ ATPase in intact myocytes as well as in genetically altered heart cells.

Animals↗
PubMed1994

Human cardiac troponin T: identification of fetal isoforms and assignment of the TNNT2 locus to chromosome 1q.

The troponin complex is located on the thin filament of striated muscle and is composed of three component polypeptides: troponin T, troponin I, and troponin C. Three troponin T genes have been described on the basis of molecular cloning in humans and other vertebrates. These are expressed in a tissue-specific manner and encode the troponin T isoforms expressed in cardiac muscle, slow skeletal muscle, and fast skeletal muscle, respectively. Each of these genes is subject to alternative splicing, resulting in the production of multiple tissue-specific isoforms. We have cloned cDNAs encoding human cardiac troponin T from adult heart and have used these to demonstrate that multiple cardiac troponin T mRNAs are present in the human fetal heart, resulting from alternative splicing in the 5' coding region of the gene. Hybridization of the cloned cDNAs to genomic DNA identifies a single-copy gene, and using somatic cell hybrid analysis, we have mapped the corresponding gene locus (designated TNNT2) to the long arm of chromosome 1 (1cen-qter).

Amino Acid Sequence↗
PubMed1994

Structure and expression of the human slow twitch skeletal muscle troponin I gene.

The contractile protein troponin I is encoded by a multigene family whose members are expressed differentially in various classes of muscle fibers. In vertebrates, the "slow" isoform of troponin I is expressed during early heart and skeletal muscle development but is restricted to slow twitch skeletal muscle in the adult. This diverse expression pattern offers an opportunity to study the regulation of a single gene within different developmental contexts. To initiate such studies, we have cloned the gene encoding the human slow twitch skeletal muscle isoform of troponin I and have identified 5'-flanking sequences required for its expression in skeletal muscle cells. The slow troponin I gene spans 12.5 kilobases and is divided into nine exons. In contrast to many muscle-specific genes, the troponin I promoter does not contain consensus CCAAT or TATA elements. Moreover, the sequence from -9 to +11 resembles an "initiator element" previously shown to direct transcription of some tissue-specific genes lacking TATA boxes (Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113; Brand, N. J., Petkovich, M., and Chambon, P. (1990) Nucleic Acids Res. 18, 6799-6806; Weis, L., and Reinberg, D. (1992) FASEB J. 6, 3300-3309). A transcriptional fusion construct, comprising 4.2 kilobases of troponin I 5'-flanking DNA linked to the bacterial chloramphenicol acetyl-transferase gene, exhibited cell type-specific and developmentally regulated expression. A muscle-specific enhancer regulated slow troponin I promoter activity.

Adult↗
PubMed1994

Sequencing of a cDNA encoding the human fast-twitch skeletal muscle isoform of troponin I.

A cDNA encoding the human fast-twitch skeletal muscle isoform of troponin I (TnIfast) has been sequenced. This cDNA is 701 base pairs in length, and encodes a protein of 182 amino acids. TnIfast is the last of the three known human TnI isoforms to be sequenced. Comparison of the deduced human TnIfast protein sequence with a variety of troponin I isoforms from other species has revealed a high degree of cross-species sequence conservation between TnIfast proteins.

Amino Acid Sequence↗
PubMed1993

Troponin I gene expression during human cardiac development and in end-stage heart failure.

Recent reports have demonstrated the presence of two isoforms of troponin I in the human fetal heart, namely, cardiac troponin I and slow skeletal muscle troponin I. Structural and physiological considerations indicate that these isoforms would confer differing contractile properties on the myocardium, particularly on the phosphorylation-mediated regulation of contractility by adrenergic agonists. We have investigated the developmental expression of these isoforms in the human heart from 9 weeks of gestation to 9 months of postnatal life, using Western blots revealed with troponin I antibodies to detect troponin protein isoforms and Northern blots to detect the corresponding mRNAs. The results show the following: 1) Slow skeletal muscle troponin I is the predominant isoform throughout fetal life. 2) After birth, the slow skeletal isoform is lost, with cardiac troponin I being the only isoform detectable by 9 months of postnatal development. 3) The protein isoforms and their corresponding mRNAs follow the same pattern of accumulation, suggesting that the transition in troponin expression is regulated at the level of gene transcription. The developmental transition in troponin I isoform content has implications for contractility of the fetal and postnatal myocardium. We further analyzed right and left ventricular muscle samples from 17 hearts in end-stage heart failure resulting from pulmonary hypertension, ischemic heart disease, or dilated cardiomyopathy. Cardiac troponin I mRNA remained abundant in each case, and slow skeletal muscle troponin I mRNA was not detectable in any of sample. We conclude that alterations in troponin I isoform content do not therefore contribute to the altered contractile characteristics of the adult failing ventricle.

Adolescent↗
PubMed1992

Intrafollicular insemination for male factor infertility.

Intrafollicular insemination is a promising new development that may have a major impact on ART, notably in the treatment of male factor infertility. Reported is the first successful intrafollicular insemination in the United States and the first successful intrafollicular insemination for male factor infertility. Although the results are encouraging, further investigations are currently underway to critically evaluate intrafollicular insemination.

Adult↗
PubMed1991

Pain efficacy in home hospice patients. A longitudinal study.

A descriptive, retrospective study was conducted to determine pain intensity in 30 home hospice patients identified with pain problems. Charts were reviewed, from admission to death, for numerical pain intensity ratings, changes in the route of administration, and/or changes in medication/scheduling. Patients were grouped into three categories according to their pattern of pain as follows: (a) Group I, 20 patients who had a pattern of increasing pain, a significant lowering of the pain intensity means, and frequent changes in pain management; (b) Group II, seven patients who showed little or no increase in pain; and (c) Group III, three patients who showed a decreased intake of pain medication. In contrast to Group I, both Groups II and III demonstrated few route changes, received few pain intensity ratings, and were maintained on oral medications through death. Based upon the nurses' pain intensity ratings and frequent interventions, a t test of admission and final means demonstrated a significant lowering of pain intensity, over time, for 15 patients in Group I who received consistent pain ratings.

Aged↗
PubMed1990

Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism.

We have isolated tropomyosin cDNAs from human skeletal muscle and nonmuscle cDNA libraries and constructed gene-specific DNA probes for each of the four functional tropomyosin genes. These DNA probes were used to define the regulation of the corresponding mRNAs during the process of myogenesis. Tropomyosin regulation was compared with that of beta- and gamma-actin. No two striated muscle-specific tropomyosin mRNAs are coordinately accumulated during myogenesis nor in adult striated muscles. Similarly, no two nonmuscle tropomyosins are coordinately repressed during myogenesis. However, mRNAs encoding the 248 amino acid nonmuscle tropomyosins and beta- and gamma-actin are more persistent in adult skeletal muscle than those encoding the 284 amino acid nonmuscle tropomyosins. In particular, the nonmuscle tropomyosin Tm4 is expressed at similar levels in adult rat nonmuscle and striated muscle tissues. We conclude that each tropomyosin mRNA has its own unique determinants of accumulation and that the 248 amino acid nonmuscle tropomyosins may have a role in the architecture of the adult myofiber. The variable regulation of nonmuscle isoforms during myogenesis suggests that the different isoforms compete for inclusion into cellular structures and that compensating autoregulation of mRNA levels bring gene expression into alignment with the competitiveness of each individual gene product. Such an isoform competition-autoregulatory compensation mechanism would readily explain the unique regulation of each gene.

Animals↗
PubMed1990

Regulation of contractile protein gene family mRNA pool sizes during myogenesis.

During myogenesis, muscle contractile protein gene expression is induced and the products are used to assemble the contractile apparatus characteristic of striated muscle. The different muscle proteins are accumulated in a fixed stoichiometric ratio related to their organization in the contractile apparatus. We have examined the relationship between contractile protein gene expression and the maintenance of stoichiometry at different stages of human myogenesis. Essentially all of the known components of adult human skeletal muscle thick and thin filaments have been cloned in the form of cDNAs and used to generate isoform-specific DNA probes. The expression of fast, slow, and cardiac isoforms was measured in human myogenic primary culture and in fetal and adult human skeletal muscle. We observed that neither fast nor slow nor cardiac isoforms are coordinately regulated at the level of comparative transcript accumulation throughout myogenesis. Thus, the stoichiometry of contractile protein levels cannot be explained by coordination of expression in each of these isoform classes. However, we find that the stoichiometry of mRNA accumulation of each gene family is very similar among three developmental stages: myotubes, fetal skeletal muscle, and adult skeletal muscle. This is consistent with the possibility that the maintenance of stoichiometry between the contractile proteins could be largely regulated by the total accumulation of mRNA from each of these gene families.

Actins↗