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Biomedical subjects

K Walsh

Publications and source records attributed to K Walsh.

At least 19 recordsLinked to original sources
PubMed1995

Cloning, chromosomal localization and expression analysis of the mouse Akt2 oncogene.

We isolated mouse cDNA clones containing the entire coding region of the putative oncogene Akt2. Sequence analysis revealed that, like its human homolog, Akt2 encodes a protein-serine/threonine kinase containing a pleckstrin homology domain at its amino terminus. Fluorescence in situ hybridization of the mouse cDNA to rodent metaphase spreads demonstrated that the Akt2 gene maps to mouse chromosome band 7B1 and rat chromosome 1q22. Expression levels of mouse Akt2 mRNA and Akt2 protein varied among tissues, with the highest levels in skeletal muscle. Akt2 expression was low in a multipotent fibroblast cell line, but it was upregulated when these cells were transformed with Myod and induced to differentiate into myocytes. These data demonstrate that Akt2 expression is activated during cellular differentiation and suggest that it functions in the signaling pathways of some adult tissues.

Amino Acid Sequence↗
PubMed1995

Expression of gax, a growth arrest homeobox gene, is rapidly down-regulated in the rat carotid artery during the proliferative response to balloon injury.

gax is a recently described homeobox gene whose expression in the adult is largely confined to cardiovascular tissues, gax has been shown to be rapidly down-regulated in cultured vascular smooth muscle cells (VSMC) upon stimulation by serum or platelet-derived growth factor. The temporal profile of gax expression in vitro matches that of two families of growth arrest genes: the gas genes and the gadd genes. All of these genes are expressed at their highest levels in quiescent cells and are down-regulated following mitogen activation. Here we report that gax is also down-regulated in vivo in the vascular wall in response to endothelial denudation by balloon angioplasty. The reduction in steady state levels of gax mRNA is transient and occurs with a similar time course to that seen in vitro. The down-regulation of gax in response to balloon injury mirrors the up-regulation seen in a number of early response genes such as c-myc and c-fos. This report is the first to document the in vivo expression of a growth arrest gene which regulates proliferation of vascular smooth muscle cells. In addition, in contrast with previous reports which have demonstrated up-regulation of several genes following balloon injury and/or angioplasty, the present report demonstrates the down-regulation of a regulatory gene within hours of balloon injury. The characteristics of gax suggest it may be required to maintain the gene expression of proteins in VSMC that are associated with the nonproliferative or contractile phenotype in smooth muscle cells.

Angioplasty, Balloon↗
PubMed1995

Parameters that influence the extent of site occupancy by a candidate telomere end-binding protein.

The MF3 protein specifically recognizes telomeric and non-telomeric DNA probes that can form G.G base-paired structures (Gualberto, A., Patrick, R. M., and Walsh, K. (1992) Genes & Dev. 6, 815-824). Here we further characterize the nucleic acid recognition properties of MF3 and present a mathematical analysis that evaluates the potential extent of telomere site occupancy by this factor. The substitution of dI at dG positions in telomeric DNA probes revealed that a single dG at any position within the internal repeat was sufficient for high affinity binding to MF3. The RNA analogs of high affinity DNA sites were not bound specifically by MF3, but the substitution of dU for dT in a DNA probe had little or no effect on binding. These data demonstrate that ribose ring structure is a critical feature of nucleoprotein complex formation, and this ribose specificity may enable MF3 to occupy sites of unusual DNA structure while minimizing interactions with cellular RNAs. Collectively, the nucleic acid binding properties of MF3 suggest that it may occupy a significant fraction of sites at telomere ends or other G-rich regions of altered DNA structure in vivo.

Amines↗
PubMed1995

Serum induction of MEF2/RSRF expression in vascular myocytes is mediated at the level of translation.

Vascular smooth muscle cells (VSMCs) reversibly coordinate the expression of VSMC-specific genes and the genes required for cell cycle progression. Here we demonstrate that isoforms of the MEF2/RSRF transcription factor are expressed in VSMCs and in vascular tissue. The MEF2A DNA-binding activity was upregulated when quiescent VSMCs were stimulated to proliferate with serum mitogens. The serum-induction of MEF2A DNA-binding activity occurred approximately 4 h following serum activation, and this correlated with an increase in the level of MEF2A protein without changes in the level of MEF2A mRNA or protein stability. These results indicate that MEF2A induction by serum is regulated at the level of translation.

Animals↗
PubMed1995

MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation.

The terminal differentiation of C2C12 skeletal muscle cells involves the activation of unique sets of genes and an irreversible withdrawal from the cell cycle. This process is associated with a decrease in cdk2 activity in cell extracts. The decrease in cdk2 activity correlates with diminished levels of cdk2 and cyclin A and with a marked induction of the p21 cyclin-dependent kinase (cdk) inhibitor. The upregulation of p21 occurred at the levels of mRNA and protein, and p21 formed a complex with the cyclin kinases in myotubes. Further, the immunodepletion of p21 from myotube extracts neutralized the heat-stable cdk2 inhibitory activity that was induced upon myogenic differentiation. The levels of p21 mRNA, protein, and activity remained constant in myotubes when they were reexposed to mitogen-rich growth medium, indicating that permanent changes in the cell's genetic program contribute to its sustained expression following terminal differentiation. Indeed, 10T1/2 fibroblasts transformed with the myogenic factor MyoD, but not the parental multipotent cells, upregulated p21 transcript levels when induced to differentiate by serum withdrawal, demonstrating that the upregulation is an integral feature of myogenic commitment and differentiation. The functional consequences of this upregulation were indicated by ectopically expressing p21 in myoblasts; this was sufficient for cell cycle arrest in mitogen-rich growth medium. The induction and sustained expression of p21 appears to be a contributory mechanism by which myocytes irreversibly exit the cell cycle upon terminal differentiation.

Base Sequence↗
PubMed1995

Regulation of Gax homeobox gene transcription by a combination of positive factors including myocyte-specific enhancer factor 2.

Homeobox-containing genes play an essential role in basic processes during embryogenesis and development, but little is known about the regulation of their expression. To elucidate regulatory networks that govern homeobox gene expression, we defined the core promoter of the mouse Gax homeobox gene and characterized its interactions with cellular proteins. Transient transfection experiments revealed Gax promoter activity in several cell types. Deletion analysis defined a 138-bp minimal promoter fragment between positions -125 and +13 relative to the transcription initiation site. Mutagenesis and protein-DNA binding assays suggested that at least three positive factors interact with this fragment and are required for transcriptional activity. One of these factors, HRF-1, recognizes a cis element consisting of an inverted palindromic motif. A second factor is Sp1, that binds to a G/C-rich element. The third is the MADS box factor referred to as MEF2 or RSRF. Mutations in the MEF2/RSRF site had the greatest effect on transcription in cell types that expressed the highest levels of endogenous MEF2 activity. Conversely, overexpression of MEF2A transactivated the Gax promoter more efficiently in cells lacking endogenous MEF2. These data provide evidence for a direct transcriptional link between members of the MADS and homeobox families of transcription factors.

Amino Acid Sequence↗
PubMed1995

Seizure activity causing loss of cardiac output after a Fontan operation.

After a Fontan operation pulmonary blood flow is heavily dependent on respiration. Two patients with a Fontan and normal sinus rhythm developed episodes of collapse with loss of cardiac output secondary to the tonic phase of an epileptic fit. The forced expiratory effort during the tonic phase severely compromised systemic venous return and led to collapse due to loss of cardiac output.

Cardiac Output, Low↗
PubMed1995

Two proximal CArG elements regulate SM alpha-actin promoter, a genetic marker of activated phenotype of mesangial cells.

Mesangial cells express smooth muscle alpha-actin (SM alpha-actin) in response to glomerular injury in vivo, and SM alpha-actin gene expression serves as a genetic marker characterizing the activated phenotype of mesangial cells. We used a molecular genetic approach to analyze the SM alpha-actin promoter and evaluate transcriptional mechanisms that might direct the genetic switch of mesangial cells to the activated phenotype. The sequence spanning -894 to +1 of the SM alpha-actin promoter directed high levels of transcription that were attenuated in serum-restricted cells and upregulated upon treatment with serum or endothelin-1. Deletional analysis revealed a core promoter fragment, from positions -122 to +1, that was necessary and sufficient for transcription. This core activity was modulated by upstream sequences between -670 and -122. The 122-bp core promoter contains two highly conserved CArG box motifs (designated CB1 and CB2), and introduction of deletion mutations of either CB1 or CB2 reduced transcription in mesangial cells to near basal levels. Further analysis revealed that CB1 and CB2 acted synergistically when subcloned upstream of a heterologous, minimal thymidine kinase promoter. CB2 alone was sufficient to confer serum inducibility to a heterologous promoter, but both CB2 and CB1 were required for maximal levels of serum-induced transcription. Collectively, these results demonstrate that CB1 and CB2 cooperate to mediate serum-induced activation of the SM alpha-actin promoter in mesangial cells.

Actins↗
PubMed1995

Rapid characterization of growth-arrest genes in transient transfection assays.

We developed a rapid assay for identifying growth-arrest genes to facilitate studies of cell cycle regulation. A7r5 vascular smooth muscle cells were transiently transfected with two plasmids: (i) a pMSV beta Gal reporter construct expressing beta-galactosidase (beta-gal) under transcriptional control of the murine sarcoma virus long terminal repeat; and (ii) a eukaryotic expression vector driving transcription of a potential growth inhibitory c-DNA under control of the cytomegalovirus promoter/enhancer. Twenty-four hours after transfection, cellular DNA was labeled for an additional 24 h with 5-bromo-2-deoxyuridine (BrdU) to label cellular DNA. After fixation, transfected cells were identified by histochemical staining with a beta-gal substrate, 6-chloro-3-indolyl-beta-D-galactopyranoside (i.e., Red-Gal). Transfected cells (beta-gal-positive) that traversed S phase (i.e., DNA synthesis) were quantified by indirect immunocytochemical staining for BrdU. Since autoradiography was not required to score for DNA synthesis, the length of experiments was much shorter than previously described growth-arrest assays performed with transiently transfected cells. Experiments with two growth-arrest genes, p53 and the p21 cyclin-dependent kinase inhibitor, demonstrated the utility of this assay.

Base Sequence↗
PubMed1994

The gene encoding rat phosphoglycerate mutase subunit M: cloning and promoter analysis in skeletal muscle cells.

The expression of the gene encoding the muscle-specific (M)-subunit of phosphoglycerate mutase (PGAM-M) is restricted to adult skeletal and cardiac muscle. In order to study its expression in muscle, the rat PGAM-M gene has been isolated and sequenced. Rat PGAM-M spans about 2.2 kb and is composed of three exons: 442, 181 and 186-bp long, and two introns of 97 bp and 1.3 bp. The analysis of the 5'-flanking region reveals a promoter which contains multiple DNA regulatory elements and constitutes an ideal model to study muscle gene transcriptional regulation. Thus, the elements responsible for rat PGAM-M muscle-specific expression have been identified by transient transfection in chicken embryo primary cultures, using chimeric constructs of the rat promoter linked to a cat reporter gene. Here, we report that in spite of the abundance of E-box motifs in the rat PGAM-M promoter known for their involvement in muscle gene expression, two DNA elements regulate the muscle-specific transcription of rat PGAM-M: an A/T motif, the putative MEF-2-binding site (myocyte-specific enhancer-binding factor 2), and a proximal 27-bp element which is conserved between the rat and human genes. These two elements define a small promoter (170 bp) sufficient to support potent and skeletal-muscle-specific expression. The conserved 27-bp region contains a transcriptional regulatory element able to confer muscle-specific expression when located upstream from a heterologous TATA box.

Amino Acid Sequence↗
PubMed1994

The mouse creatine kinase paired E-box element confers muscle-specific expression to a heterologous promoter.

E-box elements, with the CANNTG sequence motif, occur in numerous promoters and enhancers. We evaluated the tissue-specific expression properties of the paired murine E-box element from the mouse muscle creatine kinase (MCK) enhancer in a minimal heterologous promoter construct. A 46-bp fragment containing the paired E-box element in its wild-type (wt) configuration conferred high levels of muscle-specific expression in transfected embryonic chicken cell cultures. The expression from this paired E-box element was similar to that of the simian virus 40 (SV40) promoter/enhancer, but a 21-bp fragment containing a single E-box was inactive. We conclude that the paired E-box element from the MCK enhancer is sufficient for high levels of muscle-specific expression when placed upstream from a non-muscle TATA element.

Actins↗
PubMed1994

Molecular cloning and localization of the human GAX gene to 7p21.

The GAX homeobox gene is expressed in the cardiovascular tissues of the adult rat, including heart, lung, kidney, and blood vessels. In the vasculature it is specifically expressed in quiescent smooth muscle cells, but its expression is rapidly down-regulated when these cells are stimulated to proliferate with mitogens. Since vascular smooth muscle cell proliferation is important in the pathology of blood vessel disorders, the human GAX gene was isolated and characterized. The human GAX cDNA was obtained by an anchored-PCR approach using cDNA templates from cardiovascular tissues and amplification primers designed from sequence information of the rat GAX cDNA and the homeodomain-containing exon of the human GAX gene. The human and rat GAX gene coding sequences are 98% conserved at the amino acid level and 83% conserved at the nucleotide level. Similar to rat, the human homolog contains a CAX trinucleotide repeat N-terminal to the homeodomain that encodes for a stretch of 17 consecutive histidine or glutamine residues. The human GAX locus was mapped by fluorescence in situ hybridization to the short arm of chromosome 7 at band p21. The human cDNA sequence will be useful for analyses of GAX gene expression in cardiovascular diseases.

Amino Acid Sequence↗
PubMed1994

Guidelines for the prevention and control of tuberculosis in the elderly.

Tuberculosis (TB) in the elderly is on the rise although the disease is both preventable and curable. The primary practitioner plays a pivotal role in the diagnosis and prevention of TB. The article provides guidelines designed to bring the practitioner up-to-date on the latest recommendations for the prevention and control of TB in the elderly. An overview of TB, current epidemiology, and information on the uniqueness of this disease as it presents in the elderly client is addressed. An in-depth guideline and explanation for all aspects of care are included for both the institutionalized and community-based elder. Current Centers for Disease Control and Prevention recommendations and research are presented. The role of the primary practitioner in the prevention and control of this disease is included with a quick reference tool. The guidelines also address the current diagnostic testing and recommended treatment for TB.

Aged↗
PubMed1994

Incidence and prognosis of obstruction of the left ventricular outflow tract in Liverpool (1960-91): a study of 313 patients.

OBJECTIVE: To determine the incidence of the various types of obstruction of the left ventricular outflow tract in patients born in the five health districts of Liverpool and to compare their prognosis into early adult life. DESIGN: Notes of all patients with obstruction of the left ventricular outflow tract born in the study area between 1960 and 1991 were reviewed. Patients with hypoplastic left ventricle, mitral valve atresia, and those with discordant atrioventricular or ventriculoarterial connections were excluded. Survivors were traced and assessed clinically; eight were lost to follow up. RESULTS: Obstruction of the left ventricular outflow tract occurred in 313 patients (67% male), giving an incidence of 6.1/10,000 live births. The median (range) age at presentation was 13.9 months (0-20 yr). Aortic valve stenosis occurred in 71.2%: subvalve in 13.7%, supravalve in 7.7%, and multilevel in 7.4%. The median (range) duration of follow up was 10.0 (1-29) yr. Aortic regurgitation at presentation occurred more often (p < 0.001) in patients with subvalve stenosis than in those with other types of obstruction, but there was an increased incidence (p < 0.001) at follow up in patients with valve stenosis. Ninety eight patients (31.3%) underwent operation. The reoperation rate was 27% for valve stenosis and 9% for subvalve obstruction. No patients with supravalve stenosis underwent reoperation. The median duration from first operation to aortic valve replacement (17 patients) was 12.3 years. Hazard analysis confirmed that the risk of death was higher in patients presenting at a younger age, with more severe stenosis, and those with subaortic, multilevel obstruction or a syndrome. Hazard analysis also showed that the risk of a clinical event (surgery, balloon dilatation, or endocarditis) was greater in patients who presented at a younger age, with more severe stenosis or aortic regurgitation, and in those with subvalve or multilevel obstruction. CONCLUSIONS: Aortic valve stenosis was the most common type of obstruction. Hazard analysis indicates that the age and severity of obstruction at presentation have a significant effect on survival and freedom from a clinical event. The risk of premature death in patients presenting with moderately severe valve stenosis is reasonably small, but increases considerably in those with subvalve, supravalve, and multilevel obstruction. Patients who present with mild valve stenosis have a good prognosis. The risk of sudden death is less than previous predictions. Patients with subvalve and multilevel obstruction, even when mild at presentation, are more likely to undergo intervention or develop endocarditis than those with valve or supravalve stenosis. Follow up into adult life is essential.

Adolescent↗