[Canadian Consensus Conference on Congenital Heart Defects in the Adult 1996].
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Adult↗
Biomedical subjects
R McKay
Publications and source records attributed to R McKay.
The JUN kinase/stress-activated protein kinase pathway is required for epidermal growth factor stimulation of growth of human A549 lung carcinoma cells.
Epidermal growth factor (EGF) plays a major role in non-small cell lung cancer cell autocrine growth and has been reported to activate the JUN kinase/stress-activated protein kinase (JNK/SAPK) pathway in model cells. Activation of JNK/SAPK leads to the phosphorylation of c-JUN protooncogene on serines 63 and 73. This mechanism is required for and cooperates in the transformation of rat embryo fibroblasts by Ha-RAS. However, the function of JNK/SAPK in human tumor growth is unknown. We have tested several lung carcinoma cell lines. All exhibited UV-C-inducible JNK/SAPK activity; two exhibited constitutive activity in low serum, and two (M103 and A549) exhibited EGF-inducible JNK/SAPK activity. In A549 cells, EGF induced a rapid and prolonged (up to 24 h) activation of the JNK/SAPK pathway that correlated with a 150-190% growth stimulation. Stably transfected clones of A549 cells expressing c-JUN(S63A,S73A), a transdominant inhibitor of c-JUN, completely blocked the EGF-stimulated proliferation effect but did not alter the basal proliferation rate. Consistent with these results JNK antisense oligonucleotides targeted to JNK1 and JNK2 entirely eliminated the EGF-stimulated JNK/SAPK activity and blocked EGF-stimulated growth but not basal growth. In contrast, specific inhibition of the RAF/ERK pathway by PD98059 (MEK1 inhibitor) completely blocked ERK activation by EGF and basal cell growth but not EGF-stimulated growth, thereby dissociating the growth-promoting roles of each pathway. Our observations indicate, for the first time, that JNK/SAPK may be a preferential effector pathway for the growth properties of EGF in A549 cells.
Animals↗
Stem cells in the central nervous system.
In the vertebrate central nervous system, multipotential cells have been identified in vitro and in vivo. Defined mitogens cause the proliferation of multipotential cells in vitro, the magnitude of which is sufficient to account for the number of cells in the brain. Factors that control the differentiation of fetal stem cells to neurons and glia have been defined in vitro, and multipotential cells with similar signaling logic can be cultured from the adult central nervous system. Transplanting cells to new sites emphasizes that neuroepithelial cells have the potential to integrate into many brain regions. These results focus attention on how information in external stimuli is translated into the number and types of differentiated cells in the brain. The development of therapies for the reconstruction of the diseased or injured brain will be guided by our understanding of the origin and stability of cell type in the central nervous system.
Animals↗
Definition by specific antisense oligonucleotides of a role for protein kinase C alpha in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes.
Epidermal keratinocyte differentiation is a tightly regulated, stepwise process that requires protein kinase C (PKC) activation. Studies using cultured mouse keratinocytes induced to differentiate with Ca2+ have indirectly implicated the alpha isoform of PKC in upregulation of "late" (granular cell) epidermal differentiation markers. Activation of this isoform is also implicated in the suppression of "early" differentiation markers keratin (K) 1 and 10 that characterizes the neoplastic phenotype produced by the v-Ha-ras oncogene. We used antisense oligonucleotides (AS) to directly address the role of PKC alpha in regulating expression of these markers in normal and v-Ha-ras-transduced primary keratinocytes and a keratinocyte cell line (SP-1) containing an activating mutation of the c-Ha-ras gene. Transfection of PKC alpha AS reduced the PKC alpha protein level in a dose-dependent manner, with a maximum effect at doses of 100 nM or higher. Immunoblot analysis with antibodies against PKC alpha, PKC delta, PKC epsilon, and PKC eta confirmed that PKC alpha AS selectively reduced the level of PKC alpha but not the other isoforms. In vitro kinase assays also revealed suppression of Ca(2+)-dependent PKC activity, which is the PKC alpha activity in this cell type, after transfection of PKC alpha AS. When PKC alpha AS-treated normal keratinocytes were stimulated to terminally differentiate with Ca2+, induction of the late differentiation markers loricrin, filaggrin, and SPR-1, as well as transglutaminase K mRNA, was suppressed when compared with their induction in scrambled AS-treated controls. In neoplastic v-Ha-ras-transduced keratinocytes and SP-1 cells, transfection of PKC alpha AS, but not the scrambled AS control, selectively downregulated PKC alpha and restored differentiation-specific expression of K1. These findings directly confirm that PKC alpha is an important component of the signaling pathway regulating terminal differentiation of normal keratinocytes and that activation of PKC alpha contributes to the altered differentiation program of neoplastic murine keratinocytes.
Animals↗
Antisense oligonucleotide inhibitors of isozymes of protein kinase C: in vitro and in vivo activity, and clinical development as anti-cancer therapeutics.
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Animals↗
Functional and structural alterations with 24-hour myocardial hibernation and recovery after reperfusion. A pig model of myocardial hibernation.
BACKGROUND: Short-term myocardial hibernation of 3 hours resulting from a moderate resting coronary flow reduction has been reproduced in pigs. This study was designed to determine whether any structural changes accompany short-term hibernation caused by a moderate flow reduction maintained for 24 hours and whether any such structural alterations are reversible after reperfusion. METHODS AND RESULTS: A severe left anterior descending coronary artery (LAD) stenosis was created with a reduction of resting flow to approximately 60% of baseline and maintained for 24 hours. Regional coronary flow was measured by a flowmeter; wall thickening was determined by echocardiography, and local metabolic changes were measured. Of 17 pigs, 11 completed the study protocol of 24 hours. The LAD flow was reduced from 0.91 +/- 0.11 to 0.52 +/- 0.13 mL.min-1.g-1, a 43% mean decrease, at 15 minutes after the LAD stenosis and was maintained at 0.56 +/- 0.11 mL.min-1.g-1 at 24 hours. The reduction of regional coronary flow initially produced acute myocardial ischemia, as evidenced by reduced regional wall thickening (from 37.2 +/- 6.9% at baseline to 11.5 +/- 6.8%), regional lactate production (-0.34 +/- 0.28 mumol.g-1.min-1), and a decrease in regional coronary venous pH (from 7.41 +/- 0.035 at baseline to 7.30 +/- 0.030). At 24 hours, the reductions in coronary flow and wall thickening were maintained relatively constant and the rate-pressure product was relatively unchanged, but lactate production ceased and regional H+ concentration normalized, with a tendency toward a further reduction in regional oxygen consumption, from 3.10 +/- 0.90 mL.min-1.100 g-1 at 15 minutes after stenosis to 2.52 +/- 0.95 mL.min-1.100 g-1 at 24 hours (P = .06), indicating metabolic adaptation of the hypoperfused regions. Of 11 pigs, 6 were free of myocardial infarction; 3 had patchy necrosis involving 4%, 5%, and 6% of the area at risk; and 2 other pigs had a few scattered myocytes with necrosis, detected only by light and electron microscopy. Ultrastructural changes consisted of a partial loss of myofibrils and an increase in mitochondria and glycogen deposition. Regional wall thickening recovered 1 week after reperfusion in most pigs, and the ultrastructural changes reverted to normal. CONCLUSIONS: In this pig model, moderately ischemic myocardium undergoes metabolic and structural adaptations but preserves the capacity to recover both functionally and ultrastructurally after reperfusion.
Actin Cytoskeleton↗
Inhibition of growth of human tumor cell lines in nude mice by an antisense of oligonucleotide inhibitor of protein kinase C-alpha expression.
A 20-mer phosphorothioate oligodeoxynucleotide (ISIS 3521) designed to hybridize sequences in the 3'-untranslated region of human protein kinase C-alpha (PKC-alpha) mRNA has been shown to inhibit the expression of PKC-alpha in multiple human cell lines. In human bladder carcinoma (T-24) cells, inhibition of PKC-alpha was both concentration dependent and oligonucleotide sequence specific. ISIS 3521 had a IC50 of 50-100 nM for PKC-alpha mRNA reduction and was without effect on the expression of other members of the PKC family of genes (PKC-eta and zeta). Toxicity studies in mice revealed that the oligodeoxynucleotide was well tolerated at repeat doses of 100 mg/kg i.v. for up to 14 days, with no acute toxicity apparent. The oligodeoxynucleotide was found to also inhibit the growth of three different human tumor cell lines, the T-24 bladder, human lung carcinoma (A549), and Colo 205 colon carcinoma grown in nude mice. The inhibition was dose dependent with ID50 values for the growth inhibition between 0.06 and 0.6 mg/kg daily when given i.v., depending on the cell line examined. Three control phosphorothioate oligodeoxynucleotides not targeting human PKC-alpha were without effect on the growth of the tumors at doses as high as 6 mg/kg. Recovery of ISIS 3521 from tumor tissue and resolution by capillary gel electrophoresis revealed that 24 It after the final dose of oligodeoxynucleotide, intact, full-length 20-mer material was present as well as some apparent exonuclease degradation products (e.g., n-1 and n-2 mers). These studies demonstrate the in vivo antitumor effects of an antisense oligodeoxynucleotide targeting PKC-alpha and suggest that this compound may be of value as a chemotherapeutic agent in the treatment of human cancers.
Animals↗
The coronary arteries in hearts with discordant atrioventricular connections.
Gross morphologic study of 14 hearts with congenitally corrected transposition or discordant atrioventricular connections and double-outlet right ventricle or pulmonary atresia disclosed consistently the origins of the coronary arteries from two aortic sinuses. With usual atrial arrangement, the artery arising in sinus 1 (right-hand facing), as seen from the noncoronary aortic sinus, supplied the morphologically right ventricle. Coronary blood supply to the morphologically left ventricle usually came from sinus 2 (left-hand facing), although in two hearts the circumflex branch was a continuation of the artery from sinus 1. The sinus nodal artery arose from the circumflex coronary artery, and histologic studies of two hearts demonstrated that blood supply to the anterior atrioventricular node also came from this vessel. Early branching and entrapment in fat or right ventricular muscle, as well as malalignment of aortic and pulmonary sinuses, occurred frequently. These findings may have implications for coronary arterial transfer in the double switch operation in hearts with discordant atrioventricular connections.
Adolescent↗
Antisense oligonucleotides as inhibitors of signal transduction: development from research tools to therapeutic agents.
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Animals↗
Nerve growth factor amplifies cyclic AMP production in the HT4 neuronal cell line.
There has been considerable interest and controversy in the relationship between nerve growth factor (NGF) and the cyclic AMP (cAMP) second messenger system. We have used a novel, neuronal cell line (HT4) to investigate the effect of NGF on the adenylyl cyclase signaling system. Treatment of cells with NGF (100 ng/ml, 15 min) amplified cAMP accumulation (approximately 75%) in response to activation of adenosine A2 receptors (5 min) with 5'-N-ethylcarboxamidoadenosine or activation of adenylyl cyclase directly with forskolin. Basal cAMP accumulation was not altered by NGF. This amplification appears to be mediated by activation of protein kinase C (PKC) because (1) it was mimicked by activators (phorbol esters and a diacylglycerol analogue) of PKC, (2) the effects of NGF and phorbol ester on cAMP accumulation were not additive, (3) NGF amplification of cAMP accumulation was abolished by down-regulation of PKC, (4) NGF increased cytosolic PKC activity, and (5) inhibitors of PKC blocked the NGF-induced amplification of cAMP accumulation. Although NGF-induced amplification of cAMP accumulation was dependent upon PKC, mechanisms other than the classic activation pathway (i.e., hydrolysis of inositol phospholipids or the production of diacylglycerol) appeared to mediate PKC activation by NGF. The tyrosine kinase inhibitor, lavendustin A, blocked NGF-mediated amplification of cAMP accumulation, suggesting a novel interaction between a tyrosine kinase and protein kinase C.
Adenosine↗
Identification of target genes regulated by homeotic proteins in Drosophila melanogaster through genetic selection of Ultrabithorax protein-binding sites in yeast.
A method based on the transcriptional activation of a selectable reporter in yeast cells was used to identify genes regulated by the Ultrabithorax homeoproteins in Drosophila melanogaster. Fifty-three DNA fragments that can mediate activation by UBX isoform Ia in this test were recovered after screening 15% of the Drosophila genome. Half of these fragments represent single-copy sequences in the genome. Six single-copy fragments were investigated in detail, and each was found to reside near a transcription unit whose expression in the embryo is segmentally modulated as expected for targets of homoeotic genes. Four of these putative target genes are expressed in patterns that suggest roles in the development of regional specializations within mesoderm derivatives; in three cases these expression patterns depend on Ultrabithorax function. Extrapolation from this pilot study indicates that 85-170 candidate target genes can be identified by screening the entire Drosophila genome with UBX isoform Ia. With appropriate modifications, this approach should be applicable to other transcriptional regulators in diverse organisms.
Animals↗
Dopaminergic microtransplants into the substantia nigra of neonatal rats with bilateral 6-OHDA lesions. II. Transplant-induced behavioral recovery.
Transplants of fetal ventral mesencephalic (VM) dopamine neurons implanted into the substantia nigra in 6-hydroxy-dopamine (6-OHDA)-lesioned neonatal pups establish axonal connections with the denervated caudate putamen (Nikkhah et al., 1995). In the present study, we have explored the functional capabilities of these animals after they reached adulthood on a battery of spontaneous and drug-induced behavioral tasks. The results demonstrate that unilateral intranigral VM grafts in bilaterally lesioned neonates induce a marked bias in spontaneous- and stress-induced rotation contralateral to the implant not present in the lesion-only controls. Amphetamine and apomorphine induced vigorous contra- and ipsilateral rotation, respectively. Moreover, grafted animals achieved 75% of the performance level in contralateral skilled forelimb use when compared to normal controls, which was significantly above lesion-only animals (50% of normal). Spontaneous nocturnal locomotor activity was elevated 2.2-fold in the grafted animals. Sensorimotor orientation and disengage behavior was spared by the neonatal dopamine lesion and unaffected by the grafts. The level of functional restoration seen in the present study was more extensive than reported previously in neonatally 6-OHDA-lesioned rats where the VM grafts were implanted ectopically into the striatum. However, functional recovery remained incomplete also after intranigral graft placement compared to normal intact animals. The present approach should provide a new promising avenue for the continued exploration of the mechanisms involved in functional recovery and structural repair in the damaged nigrostriatal system.
Amphetamine↗
Inhibition of protein kinase C-alpha expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides.
A 20-mer phosphorothioate oligodeoxynucleotide designed to hybridize to the AUG translation initiation codon of mRNA encoding murine protein kinase C-alpha (PKC-alpha) inhibits the expression of PKC-alpha both in vitro and in vivo. In mouse C127 mammary epithelial cells, the reduction in PKC-alpha mRNA expression was both dose and time dependent. The oligodeoxynucleotide exhibited an IC50 value of 100-200 nM and reduced PKC-alpha mRNA expression for up to 48 hr. This reduction was specific for PKC-alpha versus other PKC isozymes (delta, epsilon, and zeta) and completely dependent upon oligodeoxynucleotide sequence. When administered intraperitoneally in mice, the same oligodeoxynucleotide caused a dose-dependent, oligodeoxynucleotide sequence-dependent reduction of PKC-alpha mRNA in liver, with an IC50 value of 30-50 mg/kg of body weight. Inhibition of expression was 64 +/- 11% after a single 50-mg/kg dose. The expression of PKC-delta, epsilon, and zeta mRNA was unaffected by this treatment. The oligodeoxynucleotide activity in vivo did not require the presence of cationic liposomes or any other delivery systems, although in vitro, the oligodeoxynucleotide required cationic liposomes for inhibition of PKC-alpha expression. This study demonstrates the utility of phosphorothioate oligodeoxynucleotides as specific inhibitors of gene expression in vivo after systemic administration.
3T3 Cells↗
Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters.
We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.
Base Sequence↗
Transplantation strategies for the analysis of brain development and repair.
Fetal tissue provides the best functional restoration in transplantation therapies for neurodegenerative diseases. Lineage analysis using in vivo cell marking methods shows that a multipotential cell gives rise to different neuronal types in the mammalian central nervous system. Culture methods that allow this cell to be expanded in vitro would provide important additional tools in the development of brain transplant therapies. The stem cells of the mammalian central nervous system express nestin, an intermediate filament protein that is abundant in neuronal and glial precursors but not present in the normal adult brain. Primary and immortal cultures of nestin-positive cells have been established and the differentiation of these cells analyzed both in vitro and by transplantation into the developing central nervous system. We have expanded neuronal precursor cells in vitro as both primary and immortal cells. Following implantation into the hippocampus, neurons derived from cultured stem cells are integrated into the host tissue. These findings encourage re-examination of the use of cultured cells in therapeutic transplants to the adult brain. In addition to increasing cell number, the period of culture may also permit genetic manipulation to define mechanisms of neuronal function and to enhance the therapeutic utility of transplanted cells.
Animals↗
Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors.
Changes in intermediate filament gene expression occur at key steps in the differentiation of cell types in the mammalian CNS. Neuroepithelial stem cells express the intermediate filament protein nestin and down-regulate it sharply at the transition from proliferating stem cell to postmitotic neuron. Nestin is also expressed in muscle precursors but not in mature muscle cells. We show here that in transgenic mice, independent cell type-specific elements in the first and second introns of the nestin gene consistently direct reporter gene expression to developing muscle and neural precursors, respectively. The second intron contains an enhancer that functions in CNS stem cells, suggesting that there may be a single transcriptional mechanism regulating the CNS stem cell state. This enhancer is much less active in the PNS. The identification of these elements facilitates analysis of mechanisms controlling the switch in gene expression that occurs when muscle and brain precursors terminally differentiate.
Animals↗
Cavopulmonary connection in repair of atrioventricular septal defect with small right ventricle.
Between February 1991 and May 1992, 9 patients with a nonrestrictive inlet ventricular septal defect or complete atrioventricular septal defect and hypoplasia of the right ventricle underwent successful two-ventricle correction incorporating a bidirectional cavopulmonary anastomosis. Despite a satisfactory early postoperative course, large serous pleural effusions developed in 5 of them between 2 and 6 weeks after operation. Serial echocardiography showed an evolution of flow patterns in the superior vena cava from marked systolic reversal immediately after operation to nearly continuous forward flow into both pulmonary arteries about 6 weeks later. This pattern was consistent with the transition from cardiac-dependent to respiratory-dependent caval flow. The development of pleural effusions appeared to coincide with the loss of systolic caval flow reversal and resolved after establishment of a predominantly respiratory-dependent flow pattern.
Adolescent↗
Aerosol penetration and leakage characteristics of masks used in the health care industry.
BACKGROUND: Historically, surgical masks have been worn to protect patients from being infected by large, pathogen-containing aerosol droplets emitted by health care personnel. Today, emphasis has shifted from solely protecting the patient to protecting the health care worker as well. As a result of new procedures used in operating rooms and clinical areas, aerosolized hazardous agents in the submicrometer size range are being produced, posing a potential threat to health care workers. METHODS: Eight surgical masks were tested for aerosol particle penetration through their filter media and through induced face-seal leaks. RESULTS: The percentage of filter penetration ranged from 20% to nearly 100% for submicrometer-sized particles. In comparison, a dust-mist-fume respirator used in industrial settings had significantly less penetration through its filter medium. When the surgical masks had artificially induced face-seal leaks, the concentration of submicrometer-sized particles inside the mask increased slightly; in contrast, the more protective dust-mist-fume respirator showed a fourfold increase in aerosol penetration into the mask with an artificial leak 4 mm in diameter. CONCLUSION: We conclude that the protection provided by surgical masks may be insufficient in environments containing potentially hazardous submicrometer-sized aerosols.
Aerosols↗