We demonstrate in vivo that act-D, completely cancelled the IP-induced cardioprotection. The influence of act-D on cardioprotection, transcription factors, and activities of ERKs and JNKs indicates a possible transcriptional role of these MAPKs signal transduction pathways during ischemia and in IP.
Analysis of gene transcription patterns in complex tissues with multiple cell types is a major challenge. Examination of cellular subpopulations for molecular expression patterns requires their isolation from other surrounding cells. We performed single-cell mRNA analysis to study gangliogliomas obtained from patients with pharmacoresistant epilepsy (n ¼ 6), in order to characterize CD34 expressing cells found in these tumors. Fresh-frozen biopsy tissue was analyzed by initial in situ-reverse transcription (in situ-RT) with oligonucleotides, subsequent immunohistochemistry (IHC) to identify specific cell types, and laser-capture microdissection (LCM, herein termed immuno-LCM) to obtain antigen-expressing cell subpopulations. Isolated complementary DNAs (cDNAs) were then quantified by real time-polymerase chain reaction (RT-PCR). We found that short-vs long-term incubation time for the IHC step did not adversely affect cDNA abundance obtained by subsequent RT-PCR, either for high-abundance (glyceraldehyde dehydrogenase; GAPDH), medium-abundance (glial fibrillary acidic protein; GFAP), or low abundance (neurofilament; NFM) gene transcripts. We also determined that the cellular specificity of capture was excellent, as determined by lack of contamination between different immuno-LCM cell isolates. We were therefore able to examine the lineage expression markers of isolated CD34-expressing cells. We observed coexpression of CD34 and NFM, suggesting neuronal differentiation of the CD34 expressing cellular elements in gangliogliomas. Expression markers for other cellular types (myelin basic protein for oligodendroglia; GFAP for astrocytes) were negative. Our findings support the hypothesis that gangiogliomas contain neuronal elements with compromised or atypical differentiation. We consider that this in situ-RT/immuno-LCM protocol is of general applicability, whereby virtually any primary antibody can be used to facilitate capture of individual cells in tissue sections for molecular analysis.
In ischaemic porcine myocardium, the growth of collateral vessels by angiogenesis is observed in clusters in the vicinity of focal necroses. Because mitosis of endothelial cells is a prerequisite for angiogenesis, the purpose of this study has been to evaluate the time course of mitosis as an indicator of vascular growth in a porcine model of coronary microembolization. Ischaemia was induced by injection of 25-microm microspheres in the left circumflex artery, followed by tissue collection from non-ischaemic and ischaemic areas of the same heart after 24, 72 or 168 h microembolization. Tissue was studied by histone H3 in-situ hybridization, PCNA/cyclin immunohistochemistry and electron microscopy. The number of blood vessels in ischaemic myocardium was compared with that in normal control tissue. Capillary growth started as early as 24 h after microembolization, as indicated by increasing numbers of proliferating, histone H3- and PCNA/cyclin-positive cells in the necrotic inflammatory foci of the ischaemic area. At 72 h and 168 h, the number of blood vessels was significantly higher in ischaemic than in normal myocardium, whereas at 168 h, mitosis of cells was, as in normal myocardium, a rare event. Coronary microembolization of porcine myocardium thus leads to an increased cellular proliferation rate between 24 h and less than 7 days after the onset of microembolization, followed by enhanced capillary growth. In-situ hybridization with histone H3 and PCNA/cyclin immunohistochemistry seem to be reliable markers for proliferation and vascular growth in non-cancerogenic tissue.
Comparison of the gene expression profiles of pre- and post-mortem human brains suggests that post-mortem human brain samples are suitable for investigating general gene-expression patterns.
This article is available online at http://dmd.aspetjournals.org ABSTRACT:The bladder spasmolytics propiverine was shown to induce hepatic cytochrome P450 (P450) and aminopyrine and aniline oxidation in rats. To characterize the type of enzyme induction and its dose dependence, activities of seven hepatic microsomal P450-dependent monooxygenases were measured in 72 male LEW1A albino rats (body weight 236-295 g) after oral treatment with 0.5, 2, 6, and 60 mg/kg of propiverine hydrochloride for 5 days and compared with the effects of 40 mg/kg -naphthoflavone, 10 mg/kg phenobarbital, and 20 mg/kg dexamethasone (each group, n ؍ 8). CYP2B expression was measured by Western blotting. Furthermore, the inhibitory potency of propiverine on P450 enzymes was evaluated in competition assays with three most specific monooxygenases. Results show that Propiverine induced several monooxygenases and CYP2B expression dose dependently. The effects were well comparable with a phenobarbital-type inducer with 60 mg/kg being equipotent to 10 mg/kg phenobarbital. Furthermore, propiverine in low concentrations inhibited pentylresorufin O-dealkylase (for CYP2B) in vitro. In conclusion, propiverine is a phenobarbital-type inducer on hepatic P450 enzymes in rats in doses about 100-times above the therapeutic doses in man.The benzilic acid derivative, propiverine 1 [(2,2-diphenyl-2(1-propoxy) acetic acid (1-methylpiperid-4-yl) ester] (MICTONORM, Apogepha, Dresden), has been shown in controlled clinical trials to be effective in the treatment of children, adults, and elderly suffering from detrusor hyperreflexia and symptoms of an overactive bladder. Pharmacodynamic investigations showed anticholinergic and additional effects on calcium influx and calcium homeostasis in urinary bladder preparations, thus proving the dual mode of action of propiverine in relaxing detrusor smooth muscle. The drug is rapidly absorbed from the gastrointestinal tract, widely distributed, and highly bound to plasma proteins. Incomplete oral bioavailability is mainly caused by intensive first-pass metabolism. Propiverine undergoes N-oxidation of the piperidine moiety and dealkylation of the propyl side chain by enzymes of the hepatic microsomal drug-oxidizing system (for review, Madersbacher and Mürtz, 2001).There is evidence from former animal studies that propiverine at higher doses increases the content of cytochrome P450 (P450) and the activities of aniline hydroxylase and aminopyrine demethylase in rat liver (Borchert et al., 1986;Wengler et al., 1989;Yamashita et al., 1990). Since most patients suffering from symptoms of overactive bladder are over 60 years and consumers of two and more concomitant drugs, information on potential enzyme-inducing or -inhibiting properties of a drug, which is subjected for chronic treatment, is required from studies in animals and man. Therefore, the influence of repeated oral administration of propiverine on the most important hepatic microsomal P450-dependent monooxygenases was measured in rats to evaluate its influence on...
Benchmark sng thrombin (Sigma Chemical, St. Louis, MO, USA) in a total volume of 20 µ L for 1 h at 37°C. The buffer used was 50 mM sodium citrate, pH 6.5, 150 mM NaCl. Samples incubated with or without thrombin were analyzed by electrophoresis on a 15% polyacrylamide gel. After electrophoresis, a Western blot was performed to detect the sizes and thrombin susceptibilities of each GlpR (thrombin cleavage was also detected on the Coomassie blue-stained SDS gel). As shown in Figure 2, GlpR encoded by pGZ117 was cleaved by thrombin to yield a product that co-migrated with authentic GlpR. The GlpR variants encoded by pGZ118 or pGZ119 were slightly larger than authentic GlpR and were not cleaved by thrombin. Since the thrombin site encoded by pGZ117 is fused in the same reading frame predicted by the nucleotide sequence, the results prove that the reading frame predicted for glpRis correct as deduced from its DNA sequence and that cysteine is in fact the C-terminal amino acid residue of the glprepressor.This method has proven to be an effective tool for verification of the translational reading frame predicted by DNA sequence analysis. The approach is adaptable to any gene. Other restriction enzyme pairs, such as Nar I and Kas I, can be used to create translational frameshifts. In most cases, the construct with the correct reading frame will yield a gene product that can be purified in a single step using immobilized metal affinity chromatography (2).
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