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Biomedical subjects

R Strong

Publications and source records attributed to R Strong.

At least 19 recordsLinked to original sources
PubMed2002

Transcriptional and post-transcriptional regulation of tyrosine hydroxylase messenger RNA in PC12 cells during persistent stimulation by VIP and PACAP38: differential regulation by protein kinase A and protein kinase C-dependent pathways.

VIP and PACAP38 are closely related peptides that are released in the adrenal gland and sympathetic ganglia and regulate catecholamine synthesis and release. We used PC12 cells as a model system to examine receptor and second messenger pathways by which each peptide stimulates transcriptional and post-transcriptional mechanisms that regulate the level of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzymatic step in catecholamine synthesis. Concentration-response studies revealed that PACAP38 had both greater efficacy and potency than VIP. The specific PAC1 receptor antagonist PACAP[6-38] blocked the effects of each peptide on TH mRNA content while the PACAP/VIP type II receptor antagonist (N-AC-Tyr(1)-D-Phe(2))-GRF-(1-29)-NH(2) was without effect. At equipotent concentrations, each peptide stimulated a transient increase in TH gene transcription lasting less than 3h. Continuous VIP treatment stimulated a transient increase in TH mRNA lasting less than 24h. In contrast, continuous exposure to PACAP38 stimulated a stable increase in TH mRNA that persisted for 2 days in the absence of elevated transcription, pointing to different post-transcriptional effects of the two peptides. PACAP38 alone had no effect on the magnitude of TH gene transcription or TH mRNA in A126-1B2 PKA-deficient PC12 cells. However, when combined with dexamethasone, PACAP38 produced a synergistic increase in TH mRNA in the absence of PACAP38-stimulated TH gene transcription. In contrast, VIP had no effect on either TH mRNA content or TH gene transcription in this model. PACAP38, but not VIP, stimulated PKC activity. Calphostin C antagonized the effect of PACAP38 on the persistent post-transcriptional elevation in TH mRNA. Thus, the results support the conclusion that VIP and PACAP38 each stimulate PAC1 receptors to increase TH gene transcription through a PKA-controlled pathway, but their divergent post-transcriptional effects result at least partly from differing abilities to stimulate PKC.

Animals↗
PubMed2001

A sensitive, single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis.

We describe a fast and robust new assay format to measure poly(A) polymerase (PAP) activity in a microtiter plate format. The new assay principle uses only natural nucleotide triphosphates and avoids a labour-intensive filtration step. A coupled enzymatic system combining PAP and reverse transcriptase forms the basis of the assay. The PAP generates a poly(A) tail on a RNA substrate and the reverse transcriptase is used to quantify the polyadenylated RNA by extension of a biotinylated oligo-dT primer. We demonstrate the principle of the assay using influenza virus RNA polymerase and yeast PAP as examples. A specific increase in the K(m) value for ATP and the observation of burst kinetics in the polyadenylation dependent, but not in the polyadenylation independent, assay suggest that a rate limiting step of influenza polymerase activity occurs after transcription elongation. Yeast PAP was used to validate the assay as an example of a template independent PAP. The new yeast PAP assay was approximately 100-fold more sensitive than the conventional TCA precipitation assay for yeast PAP, but the kinetic analysis of the PAP reaction gave similar results in both assays. The two enzymes show important differences with respect to inhibition by 3'-deoxy-ATP. Whereas the K(i) value for 3'-deoxy-ATP (105-117 microM) is similar to the K(m) value for ATP (186 microM) in the case of influenza RNA polymerase, the K(i) value for 3'-deoxy-ATP (0.4-0.6 microM) is approximately 100-fold lower than the K(m) value for ATP (50 microM) in the case of yeast PAP.

Adenosine Triphosphate↗
PubMed2001

A tetracycline-repressible transactivator approach suggests a shorter half-life for tyrosine hydroxylase mRNA.

Long-term increases in catecholamine release result in elevated levels of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of these compounds. This increase is due, in part, to increased transcription. However, recent evidence suggests that increased stability of TH mRNA may also play a role. One of the problems in studying the stability of the TH message is the limitation of current methods for assessing transcript half-life. In this study the regulation of the expression of the rat TH gene was placed under the control of a tetracycline (Tet)-repressible transactivator (tTA). In the absence of doxycycline (Dox), an analogue of Tet, TH mRNA was synthesized. However, when Dox was present, transcription of TH message was essentially totally suppressed, and the resulting degradation of the TH mRNA provided an index of the half-life of this message. With this approach the computed half-life of TH mRNA was significantly shorter than that determined following actinomycin D administration. This effect was not due to some unique feature of the chimeric gene used to synthesize TH mRNA or to an untoward effect of the Tet analogue used to suppress TH transcription.

Anti-Bacterial Agents↗
PubMed2001

Identification of a glucocorticoid-responsive element in the promoter region of the mouse tyrosine hydroxylase gene.

It has been known for nearly 30 years that glucocorticoid receptor stimulation induces increased tyrosine hydroxylase (TH) gene expression. However, the mechanism mediating this effect has remained elusive. Sequences with homology to known glucocorticoid-responsive elements (GRE) have been identified in the 5' flanking region of the TH gene of several vertebrate species, but none has been shown to be functional. To identify the GRE element(s) in the TH promoter, we generated chimeric constructs in which different lengths of the 5' flanking sequences of the mouse TH gene (3.6, 1.1 and 0.8 kb) were ligated to a luciferase reporter gene. Dexamethasone treatment increased luciferase expression only in cells transiently transfected with the construct containing 3.6 kb of the TH 5' flanking DNA. Co-administration of mifepristone (RU486), a glucocorticoid receptor antagonist, blocked this effect. We identified a TH-GRE sequence (5'-GGCACAGTGTGGTCT) in the mouse 5' flanking DNA between -2435 and -2421 from the transcription start. Responsiveness to dexamethasone was lost following deletion of this sequence. To determine the ability of this element to function in a heterologous promoter, we prepared a chimeric construct in which the TH-GRE sequence was cloned just upstream of a minimal thymidine kinase (TK) promoter. Promoter activity was increased 2-fold in dexamethasone-treated PC12 cells transfected with the TH-GRE-TK construct. These results provide strong evidence that the 15 base-pair sequence in the 5' flanking DNA of the mouse TH gene functions as a glucocorticoid response element. This is the first report identifying a functional glucocorticoid response element in the promoter region of the TH gene of any species.

Animals↗
PubMed2001

Interaction of a glucocorticoid-responsive element with regulatory sequences in the promoter region of the mouse tyrosine hydroxylase gene.

The purpose of the work reported here was to determine whether the tyrosine hydroxylase glucocorticoid-responsive element (TH-GRE) interacts with the cyclic AMP pathway and the CRE in regulating mouse TH promoter activity, and whether an additional, previously identified downstream GRE-like element also participates in the function of the TH-GRE and CRE. To determine the role of the cAMP pathway on TH-GRE function, we compared the effects of forskolin and dexamethasone on TH mRNA, TH gene transcription and TH promoter activity in a mutant PC12 cell line (A126-1B2) deficient in cAMP-dependent protein kinase A (PKA) with their effects in the wild-type parental strain. Forskolin treatment increased TH mRNA content, transcriptional activity and the activity of a chimeric gene with 3.6 kb of the TH promoter in wild-type cells, but not in PKA-deficient cells. In contrast, dexamethasone treatment stimulated equivalent increases in TH mRNA, TH gene transcription and TH promoter activity in each cell type. Mutation of the CRE in chimeric constructs containing 3.6 kb of the 5' flanking sequence of the mouse TH gene or coexpression of a dominant-negative mutant of CREB prevented the stimulation of TH promoter activity by forskolin. However, neither the mutation of the CRE nor inhibition of CREB influenced basal or dexamethasone-stimulated promoter activity. Site-directed mutagenesis of the TH-GRE eliminated the response of the promoter to dexamethasone. However, the mutagenesis of a more proximal 15-bp region with a GRE-like sequence had no demonstrable effect on the ability of dexamethasone to stimulate TH promoter activity. Neither mutagenesis of the TH-GRE or the downstream GRE-like sequence had an effect on the ability of forskolin to activate this chimeric gene. Taken together, these results provide evidence that a single GRE is sufficient for maximal induction of transcriptional activity by glucocorticoids and that the CRE is not required for either partial or full activity of this upstream GRE sequence.

Animals↗
PubMed2000

Selective up-regulation of I kappaB-alpha in ischemic penumbra following focal cerebral ischemia.

Activation of transcription factor NF-kappaB plays a critical role in immune, inflammatory and cell death responses. In resting cells, NF-kappaB is sequestrated in the cytoplasm in an inactive form through its association with inhibitory proteins, I kappaB (e.g.I kappaB-alpha). In response to cell activation, I kappaB is degraded causing release of active NF-kappaB. Active NF-kappaB translocates into the nucleus leading to activation of transcription that may have a profound effect on cell survival, including that after ischemic stroke. Here, using Western blot analysis, we show that immunoreactivity to the major subunit of NF-kappaB, p65, as well as to the inhibitory subunit I kappaB-alpha is equally markedly decreased in the ischemic core after transient middle cerebral and common carotid artery occlusion in rats. In contrast, penumbral regions display no change in p65, and significant increase in I kappaB-alpha immunoreactivity, as compared to non-ischemic areas. In these penumbral regions with elevated I kappaB-alpha immunoreactivity, we find reduced cytosolic and increased nuclear I kappaB-alpha staining of neurons, as determined by immunohistochemistry. Altogether, these results suggest that an altered ratio between activating and inhibitory NF-kappaB pathways mediated through I kappaB-alpha may play an important role in survival of the ischemic penumbra.

Animals↗
PubMed2000

Combination of low dose ethanol and caffeine protects brain from damage produced by focal ischemia in rats.

Caffeine and ethanol are two commonly overused psychoactive dietary components. The purpose of this study was to assess the effects of acute, chronic, oral (p.o.) and intravenous (i.v.) caffeine, ethanol and their combination on infarct volume following focal ischemia in rats. Rats received treatment either p.o. 3 h and 1 h before, or by i.v. infusion for 2.5 h beginning 30-180 min after, ischemia. There were six acute treatment groups. (1) oral dH2O (control); (2) oral caffeine (10 mg/kg); (3) oral ethanol (0.65 g/kg total); (4) oral ethanol plus caffeine; (5) intravenous saline; and (6) intravenous ethanol (0.65 g/kg) plus caffeine (10 mg/kg) in saline. A 7th group received oral ethanol plus caffeine for three weeks prior to ischemia. After 3 h of left MCA/CCA occlusion and 24 h reperfusion, infarct volume was determined. Control animal infarct volume was 102.4+/-42.0 mm3. Oral caffeine alone had no effect (122.4+/-30.2 mm3). Oral ethanol alone exacerbated infarct volume (177.2+/-27.8 mm3). Oral caffeine plus ethanol almost entirely eliminated the damage (17.89+/-10.41 mm3). When i.v. treatment with ethanol plus caffeine was initiated at 30, 60, 90 and 120 minutes post-ischemia the infarct volume was reduced by 71.7%, 49.8%, 64.8% and 47.1%, respectively. Chronic daily oral ethanol plus caffeine prior to ischemia eliminated the neuroprotection seen with acute treatment. These studies indicate that ethanol, which by itself aggravates cerebral ischemia, and caffeine, when combined together immediately before or for 2 h after focal stroke, reduces ischemic damage.

Animals↗
PubMed2000

Effects of acute restraint stress on tyrosine hydroxylase mRNA expression in locus coeruleus of Wistar and Wistar-Kyoto rats.

Norepinephrine (NE) is thought to play a role in the stress response, and may be involved in stress-related psychopathological conditions such as depression or anxiety. Heterogeneity in individual responses to the same stressor suggest that a genetic susceptibility to the effects of stress may contribute to such pathology. To address possible mechanisms underlying this genetic aspect of the stress response, we examined acute stress-induced changes in mRNA expression for several components of the NE system in the locus coeruleus (LC) and adrenal medullae of stress-susceptible Wistar-Kyoto (WKY) rats and their parent Wistar (W) strain. Expression of tyrosine hydroxylase (TH), NE transporter (NET) and alpha(2A) receptor mRNA were measured in the LC by in situ hybridization 30 min and 2 h after the onset of 30 min restraint stress. Adrenal TH mRNA was measured by slot blots. No basal differences were observed for any measure, but in the LC, expression of TH mRNA increased by 40% in W rats at 30 min (n=8, p<0.05) and returned toward baseline by 2 h, while WKY rats showed only a non-significant 29% increase at 2 h. In contrast, adrenal TH mRNA expression increased in WKY rats at 2 h (n=3, p<0.05), with no significant change in W rats. NET and alpha(2A) mRNA were unaltered by restraint stress in both strains. Differences in the stress-reactivity of TH gene expression in the central and peripheral noradrenergic systems may be related to differences in behavioral coping strategies and autonomic responsivity to stress in these strains, and suggest that differences in noradrenergic reactivity may contribute to genetic susceptibility to stress-related pathology.

Animals↗
PubMed2000

The assessment of organ doses from plutonium for an epidemiological study of the Sellafield workforce.

An epidemiological study of mortality and cancer rates in plutonium workers at the Sellafield nuclear installation has been carried out by researchers at the London School of Hygiene and Tropical Medicine. The study required the assessment of organ-specific doses from plutonium for more than 5000 workers over a period of 40 years. This was a major undertaking as it involved the reconstruction of annual received doses from the results of some 223,000 urine samples that had been provided by the workers in the study. This paper outlines the techniques and strategies adopted in order to generate best estimates of dose from the available data, and presents summaries of these doses.

Body Burden↗
PubMed2000

Interplay between the gamma isoform of PKC and calcineurin in regulation of vulnerability to focal cerebral ischemia.

Protein phosphorylation and dephosphorylation mediated by protein kinases and protein phosphatases, respectively, represent essential steps in a variety of vital neuronal processes that could affect susceptibility to ischemic stroke. In this study, the role of the neuron-specific gamma isoform of protein kinase C (gammaPKC) in reversible focal ischemia was examined using mutant mice in which the gene for gammaPKC was knocked-out (gammaPKC-KO). A period of 150 minutes of unilateral middle cerebral artery and common carotid artery (MCA/CCA) occlusion followed by 21.5 hours of reperfusion resulted in significantly larger (P < 0.005) infarct volumes (n = 10; 31.1+/-4.2 mm3) in gammaPKC-KO than in wild-type (WT) animals (n = 12; 22.6+/-7.4 mm3). To control for possible differences related to genetic background, the authors analyzed Balb/cJ, C57BL/6J, and 129SVJ WT in the MCA/CCA model of focal ischemia. No significant differences in stroke volume were detected between these WT strains. Impaired substrate phosphorylation as a consequence of gammaPKC-KO might be corrected by inhibition of protein dephosphorylation. To test this possibility, gammaPKC-KO mice were treated with the protein phosphatase 2B (calcineurin) inhibitor, FK-506, before ischemia. FK-506 reduced (P < 0.008) the infarct volume in gammaPKC-KO mice (n = 7; 24.6+/-4.6 mm3), but at this dose in this model, had no effect on the infarct volume in WT mice (n = 7; 20.5+/-10.7 mm3). These results indicate that gammaPKC plays some neuroprotective role in reversible focal ischemia.

Animals↗
PubMed2000

Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats : functional and anatomic outcome.

BACKGROUND AND PURPOSE: Previous work by researchers in our laboratory has shown that in the rat, the exclusive use of the affected forelimb during an early critical period exaggerates lesion volume and retards functional recovery after electrolytic lesions of the forelimb sensorimotor cortex. In the present study, we examined the effects of exclusive use of the affected forelimb after middle cerebral artery occlusion (MCAO). METHODS: Ischemia of moderate severity was produced in male Long-Evans rats through 45 minutes of occlusion of the left middle cerebral and both common carotid arteries. Exclusive use of either the affected or unaffected forelimb was forced through immobilization of either the ipsilateral (MCAO+ipsi) or contralateral (MCAO+contra) forelimb, respectively, for 10 days in a plaster cast, or the animal was left uncasted (MCAO+nocast). Sham surgeries were performed, and animals were also casted for 10 days or left uncasted. Sensorimotor testing was performed during days 17 to 38. At the end of sensorimotor testing, cognitive performance was tested with use of the Morris water maze. In a separate experiment, temperatures and corticosterone levels were measured during the 10-day period after 45-minute ischemia and casting. RESULTS: The MCAO+ipsi group performed worse on sensorimotor tasks than the MCAO+contra, MCAO+nocast, and sham groups. Infarct volume was significantly larger in the MCAO+ipsi group than in the sham and MCAO+contra groups but not in the MCAO+nocast group. No group differences were found with the Morris water maze, and no group differences were found in either temperature or plasma corticosterone level. CONCLUSIONS: The exclusive use of the affected forelimb immediately after focal ischemia has detrimental effects on sensorimotor function that cannot be attributed to hyperthermia or stress.

Animals↗
PubMed1999

Disruption of the relationship between renin and atrial natriuretic peptide early in the course of ventricular dysfunction.

BACKGROUND: Plasma renin activity is normal in left ventricular dysfunction in the absence of diuretic therapy. In health there is a reciprocal relationship between renin and atrial natriuretic peptide (ANP) but a positive correlation in advanced heart failure. The relationship between renin and ANP in mild left ventricular dysfunction is unknown. METHODS AND RESULTS: Patients with left ventricular dysfunction (n = 35, 18 without diuretic therapy) were compared to 20 age-matched healthy subjects. Plasma concentrations of active renin (PARC), ANP and norepinephrine were measured after 20 min rest and 45 min after an infusion of normal saline (10 ml/kg body wt.). Basal plasma ANP was increased in patients with left ventricular dysfunction compared to healthy subjects, whether or not they were receiving diuretics. PARC was similar in healthy controls and patients untreated with diuretics but was increased in diuretic treated patients. After saline loading in healthy subjects PARC fell while ANP rose. Patients with left ventricular dysfunction had a smaller decline in PARC, that did not achieve statistical significance, but had a greater increase in plasma ANP compared to healthy subjects (P<0.05). The close reciprocal relationship between PARC and ANP observed in healthy subjects before and after saline loading (r = 0.8, P<0.001 and r = 0.6, P<0.01) was weakened in those not receiving diuretics (r = 0.4, P<0.05 and r = 0.24, ns) and lost in those receiving diuretics (r = 0.1 and r = 0.08). CONCLUSIONS: Patients with left ventricular dysfunction have a disturbance of the normal reciprocal relationship between PARC and ANP which antedates diuretic treatment. This should be taken into account when interpreting plasma neuroendocrine measurements in patients with ventricular dysfunction.

Atrial Natriuretic Factor↗
PubMed1999

Neurofilament proteolysis after focal ischemia; when do cells die after experimental stroke?

To determine the occurrence and time-course of presumably irreversible subcellular damage after moderate focal ischemia, rats were subjected to 1, 3, 6, 9, or 24 hours of permanent unilateral middle cerebral and common carotid occlusion or 3 hours of reversible occlusion followed by 3, 6, or 21 hours of reperfusion. The topography and the extent of damage were analyzed with tetrazolium staining and immunoblot using an antibody capable of detecting breakdown of neurofilament. Neurofilament proteolysis began after 3 hours in the infarct core but was still incomplete in penumbral regions up to 9 hours. Similarly, tetrazolium-staining abnormalities were observed in the core of 50% of animals after 3 hours of ischemia. At 6 hours of permanent ischemia, infarct volume was maximal, and further prolongation of occlusion to 9 or 24 hours did not increase abnormal tetrazolium staining. In contrast to permanent ischemia and in agreement with the authors' previous demonstration of "reperfusion injury" in this model, prolongation of reperfusion from 3 hours to 6 and 21 hours after 3 hours of reversible occlusion gradually augmented infarct volume by 203% and 324%, respectively. Neurofilament proteolysis initiated approximately 3 hours after ischemia was quantitatively greatest in the core and extended during reperfusion to incorporate penumbra with a similar time course to that of tetrazolium abnormalities. These data demonstrate that, at least as measured by neurofilament breakdown and mitochondrial failure, extensive cellular damage is not present in penumbral regions for up to 9 hours, suggesting the potential for rescuing these regions by appropriate and timely neuroprotective strategies.

Animals↗
PubMed1999

Clearance kinetics of solutes used to measure glomerular filtration rate.

Agents used to measure glomerular filtration rate (GFR) give a biexponential plasma disappearance curve on multiple peripheral venous sampling between 20 min and 4 h after intravenous injection. These two exponentials are generally regarded to represent equilibration of agent throughout the extracellular fluid (ECF) space and renal clearance, respectively. In seven subjects undergoing diagnostic arteriography, arterial and antecubital venous plasma samples were obtained up to 60 min in five and up to 120 min in two following simultaneous intravenous injection of 99Tcm-diethylene triamine pentaacetate (99Tcm-DTPA) and inulin. The count rate from 99Tcm was simultaneously recorded over the calf with a collimated scintillation probe in five subjects up to 60 min post-injection. The arterial and venous time-concentration curves were interpolated and subtracted to give a curve of the arterio-venous (A-V) concentration difference, which was then integrated. Arterial time-concentration curves display three exponentials, the first of which has the largest amplitude and disappears by about 20 min. The A-V concentration difference becomes zero at about the same time. The integral of the A-V concentration difference, which represents activity in the interstitial space of the forearm, has a time course consistent with the second compartment of a model of two compartments in series (the first being plasma) and a time course that is reciprocally similar to the first exponential of the triexponential arterial plasma curve. The curve externally recorded by scintillation probe has a shape consistent with a signal that is the composite of interstitial 99Tcm-DTPA and plasma 99Tcm-DTPA activities. The arterial plasma clearance curve of GFR agents is triexponential; the first exponential reflects equilibration of agent between plasma and the interstitial space of carcass tissue (mainly muscle and skin). The second exponential is minor compared with the first; it is not clear what it represents. The third exponential reflects renal clearance.

Adult↗
PubMed1999

Spur cell anaemia and hepatic iron stores in patients with alcoholic liver disease undergoing orthotopic liver transplantation.

BACKGROUND: Following orthotopic liver transplantation (OLT) histological examination of explant livers from patients with alcoholic liver disease (ALD) sometimes shows extensive iron deposits in a distribution suggestive of homozygous haemochromatosis. AIMS: To use haemochromatosis gene (HFE) assays to distinguish between ALD with notable siderosis and hereditary haemochromatosis. To evaluate the possible influence of spur cell haemolytic anaemia on hepatic iron loading. PATIENTS: Thirty seven patients with ALD were abstinent for at least six months prior to OLT. Twenty three patients had transferrin saturations greater than 55%, 16 also had increased serum ferritin (>350 micrograms/l). Eight of 37 (22%) explant livers had grade 3 or 4 hepatic iron deposition, predominantly in hepatocytes. Of these, four had a hepatic iron index greater than 1. 9 and most seemed to have spur cell haemolytic anaemia. METHODS: Mutation analysis for C282Y and H63D mutations was performed on DNA extracts from peripheral blood or explant liver. Spur cell haemolytic anaemia was diagnosed when the haemoglobin was 105 g/l in the presence of notable acanthocytosis. RESULTS: None of the eight patients with grade 3 or 4 hepatic iron had evidence of the C282Y mutation. Two of the eight were heterozygous for H63D. None of the remaining 28 patients tested showed homozygous HFE mutations. Spur cell anaemia was present in six of the eight patients with heavy iron deposition and only one of the remaining patients. CONCLUSIONS: The HFE mutation was not present in these patients with advanced ALD and heavy iron loading. Spur cell haemolytic anaemia provides an alternative potential mechanism for the heavy iron loading.

Adult↗
PubMed1999

Nuclear factor-kappaB and cell death after experimental intracerebral hemorrhage in rats.

BACKGROUND AND PURPOSE: Nuclear factor-kappaB (NF-kappaB) is a ubiquitous transcription factor that, when activated, translocates to the nucleus, binds to DNA, and promotes transcription of many target genes. Its activation has been demonstrated in chronic inflammatory conditions, cerebral ischemia, and apoptotic cell death. The present study evaluated the presence and activation of NF-kappaB in relation to cell death surrounding intracerebral hemorrhage (ICH). METHODS: Striatal ICH was induced in rats by the double blood injection method. Animals were killed 2, 8, and 24 hours and 4 days after ICH. To examine changes in NF-kappaB protein, Western blot was performed on brain extract. We determined NF-kappaB activity using electrophoretic mobility shift assay (EMSA) and immunohistochemistry, using an antibody that only recognizes active NF-kappaB. DNA fragmentation was detected with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) staining. RESULTS: Western blot analysis of the NF-kappaB p65 subunit showed that there was no difference in p65 protein levels in the control, 2-hour, 8-hour, or 24-hour groups. However, ipsilateral perilesional samples from the 4-day group revealed a 1.8- to 2.5-fold increase compared with the contralateral hemisphere. Western blotting showed no differences in the inhibitor of NF-kappaB, IkappaBalpha, in any group. EMSA showed 1.3-, 2.1-, and 3.6-fold increased NF-kappaB activation in the ipsilateral striatum from the 8-hour, 24-hour, and 4-day groups, respectively, compared with the contralateral hemisphere. Immunohistochemistry, in which an activation-dependent anti-NF-kappaB antibody was used, demonstrated perivascular NF-kappaB activation as early as 2 hours after ICH with more generalized activation at 8 hours, in agreement with the EMSA results. NF-kappaB activation colocalized to cells containing fragmented DNA measured by TUNEL. CONCLUSIONS: The present study suggests a relationship between NF-kappaB and the pathobiology of perilesional cell death after ICH.

Animals↗
PubMed1998

Transcriptional and posttranscriptional control of tyrosine hydroxylase gene expression during persistent stimulation of pituitary adenylate cyclase-activating polypeptide receptors on PC12 cells: regulation by protein kinase A-dependent and protein kinase A-independent pathways.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates catecholamine release and biosynthesis in sympathetic postganglionic cells. Moreover, PACAP receptor activation in cultured adrenal chromaffin and superior cervical ganglion cells has been reported to increase the expression of the gene coding for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. However, the relative contribution of transcriptional and posttranscriptional mechanisms to the effects of PACAP on TH gene expression has not been evaluated. Therefore, in this study we compared the temporal effects of PACAP on TH gene transcription with the duration of its effects on TH mRNA levels. We had previously shown that vasoactive intestinal polypeptide, peptide histidine isoleucine, and secretin, peptides closely related to PACAP, induce TH gene expression through a cyclic AMP (cAMP)-dependent pathway. Therefore, using a mutant PC12 cell line deficient in cAMP-dependent protein kinase II (PKA), we also evaluated the role of the cAMP pathway in the effect of PACAP on TH gene expression. Continuous treatment of wild-type PC12 cells with PACAP (1 nM) increased TH mRNA levels maximally by 12 h and maintained TH mRNA at near maximal levels for at least 2 days. In contrast, the rate of TH gene transcription, as measured by a nuclear run-on assay, was maximal by 1 h and returned to basal levels by 3 h. The fact that a new steady-state level of TH mRNA was achieved and maintained for days in the absence of a sustained increase in TH gene transcription supports the involvement of posttranscriptional mechanisms. Removal of PACAP after 12 h, a time at which TH gene transcription was at basal levels, resulted in a subsequent return of TH mRNA to unstimulated levels within 36 h. Thus, continuous PACAP stimulation is required to maintain sustained increases in TH mRNA levels in the absence of a sustained elevation of transcription. To examine the role of the cAMP pathway in these effects, we compared the effects of PACAP in wild-type PC12 cells and in a mutant PC12 cell line (A126-1B2) that is deficient in PKA. PACAP failed to stimulate either TH mRNA levels or TH gene transcription in the mutant cells. In contrast to the effects of PACAP, dexamethasone increased TH mRNA levels by the same magnitude in both cell lines. It is noteworthy that stimulation of the PKA-deficient mutant cells with a combination of PACAP and dexamethasone (1 microM) produced a synergistic increase in TH mRNA levels, which was nearly twice that induced by dexamethasone stimulation alone. This synergistic effect was not transcriptionally mediated. The effect of the combined treatment on TH gene transcription was identical to the effect of dexamethasone alone. Taken together, these data indicate that PACAP regulates TH gene expression through a transcriptional mechanism requiring an intact cAMP pathway and through posttranscriptional mechanisms under the control of a cAMP-independent pathway(s).

Adenylyl Cyclases↗