Restoration of human beta-globin gene expression in murine and human IVS2-654 thalassemic erythroid cells by free uptake of antisense oligonucleotides

Correct human beta-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with beta-globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/beta(E) genotype. This was accomplished in a dose- and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in beta-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human beta-globin mRNA and hemoglobin A in patients' erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2-654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2-654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.     

Clinical observation on YiSuiShengXueGranule on treating 156 patients with beta-thalassemia major and the molecular mechanism study

OBJECTIVE: To investigate the clinical effects and security of YiSuiShengXueGranule (YSSXG) on treating 156 patients with beta-thalassemia major. Methods: YSSXG was given orally to 156 patients with beta-thalassemia in GuangXi Autonomous Region (the high incidence area of beta-thalassemia in China) for 3 months as one therapeutic course, 3 times a day, 10 g each time (for children, the dose should be reduced properly according to their body weight and age), and no blood transfusion used during the course. Clinical symptoms and levels of hemoglobin (Hb), red blood cell (RBC), reticulocyte (Ret) and hemoglobin F (HbF) were observed before and after treatment, and side-effects were observed during the course. A 3-6 months follow up study was performed after withdrawal of YSSXG. And systemic gene analysis was conducted with PCR, SSCP-PCR, RT-PCR and DNA sequences analysis and mRNA differently expression technique, in order to study the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation. RESULTS: Levels of Hb, RBC, Ret and HbF obviously elevated, and clinical symptoms markedly ameliorated in patients after treated with YSSXG from the 1st to 3rd month (all p<0.01). Dynamical observation showed that the improvement of symptoms kept accordance with the elevation of hemorrheological indexes. The treatment was effective in 145 patients and ineffective in 11, and the total effective rate was 92.9%, without any adverse reaction founded. Follow-up studies showed the therapeutic effect could sustain for 3 to 4 months after drug-withdrawal. The molecular mechanism study showed: YSSXG did not change the genetic mutation type, but could obviously increase gamma/(beta+gamma) globin ratio, both gamma-globin mRNA and GM-CSF mRNA expression were significantly enhanced so as to induce HbF synthesis increasing after treated with YSSXG. CONCLUSION: YSSXG had obvious effects in treating beta-thalassemia by unlocking gamma-gene, increasing the gamma-globin expression and enhancing HbF synthesis so as to compensate for the gene defect. This study has provided a new path for the treatment of beta-thalassemia with Traditional Chinese Medicine.     

Induction of erythroid differentiation of human K562 cells by cisplatin analogs

Human leukemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including hemin, butyric acid, 5-azacytidine, and cytosine arabinoside. Differentiation of K562 cells is associated with an increase in the expression of embryo-fetal globin genes, such as the zeta-, epsilon-, and gamma-globin genes. Therefore, the K562 cell line has been proposed as a very useful in vitro model system for determining the therapeutic potential of new differentiating compounds as well as for studying the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation that stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper, we analyzed the effects of a series of cisplatin analogs on both cell growth and differentiation of K562 cells. Among seven cisplatin analogs studied, three were found to be potent inducers of erythroid differentiation. Erythroid differentiation was associated with an increase in the accumulation of (a) hemoglobins Gower 1 and Portland and (b) gamma-globin mRNA.     

Inhibition of IgE-mediated phosphorylation of FcepsilonRIgamma protein by antiallergic drugs in rat basophilic leukemia (RBL-2H3) cells: a novel action of antiallergic drugs

We examined the effect of antiallergic drugs, azelastine and epinastine, on the expression of FcepsilonRIalpha, beta, and gamma chains and phosphorylation of the gamma chains in rat basophilic leukemia (RBL-2H3) cells. The cells were cultured for 24 h with IgE treatment in the presence of azelastine or epinastine at the concentration of 10(-5) M. The FcepsilonRIalpha mRNA expression was determined by northern blot analysis. The protein level of FcepsilonRI expressed on the plasma membrane was examined following IgE treatment by immunoprecipitation with anti-IgE light chain, followed by western blot analysis with anti-gamma chain of FcR. Azelastine and epinastine had no effect on the FcepsilonRIalpha, beta and gamma mRNA levels. Although the amount of gamma chain assembled into IgE-bound FcepsilonRI was not changed by treatment with azelastine nor epinastine, phosphorylation levels of gamma chains of IgE-bound FcepsilonRI were inhibited by azelastine. The inhibitory effect of azelastine on the IgE-mediated expression of FcepsilonRIgamma protein is not due to their inhibition of mRNA and protein expression, but due to abrogating phosphorylation of the gamma chains, which is important for initiation of FcepsilonRI signaling cascade elicited by IgE interaction.     

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.     

Propranolol causes a paradoxical enhancement of cardiomyocyte foetal gene response to hypertrophic stimuli

BACKGROUND AND PURPOSE: Pathological cardiac hypertrophy is associated with the expression of a gene profile reminiscent of foetal development. The non selective beta-adrenoceptor antagonist propranolol is able to blunt cardiomyocyte hypertrophic response in pressure-overloaded hearts. It remains to be determined whether propranolol also attenuates the expression of hypertrophy-associated foetal genes. EXPERIMENTAL APPROACH: To address this question, the foetal gene programme, of which atrial natriuretic peptide (ANP), the beta-isoform of myosin heavy chain (beta-MHC), and the alpha-skeletal muscle isoform of actin (skACT) are classical members, was induced by thoracic aortic coarctation (TAC) in C57BL/6 mice, or by phenylephrine, a selective alpha(1)-adrenoceptor agonist, in cultured rat neonatal cardiomyocytes. KEY RESULTS: In TAC mice, the left ventricular weight-to-body weight (LVW/BW) ratio increased by 35% after 2 weeks. Levels of ANP, beta-MHC and skACT mRNA in the left ventricles increased 2.2-fold, 2.0-fold and 12.1-fold, respectively, whereas alpha-MHC and SERCA mRNA levels decreased by approximately 50%. Although propranolol blunted cardiomyocyte growth, with approximately an 11% increase in the LVW/BW ratio, it enhanced the expression of ANP, beta-MHC and skACT genes (10.5-fold, 27.7-fold and 22.7-fold, respectively). Propranolol also enhanced phenylephrine-stimulated ANP and beta-MHC gene expression in cultured cardiomyocytes. Similar results were obtained with metoprolol, a selective beta(1)-adrenoceptor antagonist, but not with ICI 118551, a beta(2)-adrenoceptor antagonist. CONCLUSIONS AND IMPLICATIONS: Propranolol enhances expression of the hypertrophy-associated foetal genes mainly via the beta(1)-adrenoceptor blockade. Our results also suggest that, in pressure-overloaded hearts, cardiomyocyte growth and foetal gene expression occur as independent processes.     

2-aroylindoles and 2-aroylbenzofurans with N-hydroxyacrylamide substructures as a novel series of rationally designed histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors are considered to be drugs for targeted cancer therapy and second-generation HDIs are currently being tested in clinical trials. Here, we report on the synthesis and biological evaluation of a novel HDAC inhibitor scaffold with the hydroxamate Zn(2+) complexing headgroup, selected from the 2-aroylindol motif. Inhibition of nuclear extract HDAC and recombinant HDAC 1 as well as induction of histone H3K(9+14) hyperacetylation mediated by E-N-hydroxy-(2-aroylindole)acrylamides or E-N-hydroxy-(2-aroylbenzofuran)acrylamides were studied. Moreover, the cytotoxic activity, the effects on the cell cycle, and histone H3S(10) phosphorylation of selected compounds were determined. By use of a panel of 24 different human tumor cell lines, mean IC(50) values of the most potent analogues 6c and 7b were 0.75 and 0.65 microM, respectively. The novel compounds were shown to be no substrates of the P-glycoprotein drug transporter. Comparable to N(1)-hydroxy-N(8)-phenyloctanediamide "2 (SAHA)", cells in the S phase of the cell cycle are depleted, with partial arrest in G1 and G2/M and finally induction of massive apoptosis.     

Modulation of Inflammatory Cytokines and Mitogen-activated Protein Kinases by Acetate in Primary Astrocytes

Acetate supplementation attenuates neuroglia activation in a rat model of neuroinflammation by a mechanism associated with an increase in brain acetyl-CoA, an alteration in histone acetylation, and reduction of interleukin (IL)-1β expression. We propose that reduced astroglial activation occurs by disrupting astrocyte-derived inflammatory signaling and cytokine release. Using primary astroglial cultures, we found that LPS (0–25 ng/ml, 4 h) increased tumor necrosis factor (TNF-α) and IL-1β in a concentration-dependent manner, which was reduced by treatment with sodium acetate (12 mM). LPS did not alter H3K9 acetylation or IL-6 levels, whereas acetate treatment increased H3K9 acetylation by 2-fold and decreased basal levels of IL-6 by 2-fold. Acetate treatment attenuated the LPS-induced increase in TNF-α mRNA, but did not reverse the mRNA levels of other pro-inflammatory cytokines. By contrast, LPS decreased TGF-β1 and IL-4 protein and TGF-β1 mRNA, all of which was reversed with acetate treatment. Further, we found that acetate treatment completely reversed LPS-induced phosphorylation of MAPK p38 and decreased basal levels of phosphorylated extracellular signal-regulated kinases1/2 (ERK1/2) by 2-fold. Acetate treatment also reversed LPS-elevated NF-κB p65, CCAAT/enhancer-binding protein beta protein levels, and reduced basal levels of phosphorylated NF-κB p65 at serine 536. These results suggest that acetate treatment has a net anti-inflammatory effect in LPS-stimulated astrocytes that is largely associated with a disruption in MAPK and NF-κB signaling.     

Bcl-xL and Mcl-1 are involved in prevention of in vitro apoptosis in rat late-stage erythroblasts derived from bone marrow

Apoptosis controls erythroid homeostasis by balancing survival and death of erythroid cells. The mitochondrial pathway of apoptosis involves regulation of apoptotic events caused by the Bcl-2 family proteins, including the anti-apoptotic and pro-apoptotic members. However, little has been reported on the role of the anti-apoptotic Bcl-2 family members in rat late-stage erythroblasts that are no longer erythropoietin (EPO)-dependent. In the present study, to investigate this we analyzed changes in apoptosis-related factors that occurred in vitro. EPO stimulation resulted in reduced apoptotic cell death of the late-stage erythroblasts accompanied by decreased caspase-3 and caspase-9 activities, which is indicative of the induction of apoptosis through the mitochondrial pathway. Analysis of mRNA expression of the Bcl-2 family proteins demonstrated that EPO stimulation up-regulated the Bcl-xL mRNA, resulting in decreases in the mRNA ratios of Bak, Bax, and Bad to Bcl-xL. Also, the mRNA ratios of Bak and Noxa to Mcl-1 were decreased, mainly due to up-regulation of Mcl-1 mRNA. These results showed a close association between reduced apoptotic cell death and increased mRNA levels of Bcl-xL and Mcl-1 in the presence of EPO. Thus, the present study suggests that Bcl-xL may be an important anti-apoptotic factor of rat late-stage erythroblasts as has been reported in murine erythroblasts. Moreover, the results also indicate the possibility that Mcl-1 may act on the rat late-stage erythroblasts as an anti-apoptotic factor.     

GABAA receptor subunit mRNA levels are differentially influenced by chronic FG 7142 and diazepam exposure.

Levels of mRNA for the alpha 1, gamma 2 and beta 1 subunits of the GABAA receptor complex were examined in rats maintained on a chronic, continuous schedule of exposure to the benzodiazepine inverse agonist FG 7142. The effect of chronic exposure to the benzodiazepine agonist diazepam was also examined on levels of gamma 2 subunit mRNA. FG 7142 (2 mg/ml of 100% dimethyl sulfoxide (DMSO) or vehicle (100% DMSO) was administered continuously for 8 days in the right ventricle via an osmotic minipump. At the end of the eighth day of exposure, the brain was removed and cerebral cortex, cerebellum and hippocampus were dissected and mRNA prepared from each region. Levels of GABAA alpha 1 and gamma 2 subunit mRNA were examined by Northern blot analysis with cDNA probes specific for these subunits. A significant increase in alpha 1 mRNA was measured in both cortex and hippocampus, but not in cerebellum, of rats chronically exposed to FG 7142 relative to vehicle-treated rats. A significant increase in gamma 2 subunit mRNA in cortex was also evident in drug-treated rats; however, no change in gamma 2 subunit mRNA was observed in either the hippocampus or cerebellum. Examination of GABAA beta 1 subunit mRNA by solution hybridization using a beta 1 riboprobe revealed no effect of chronic FG 7142 treatment on this subunit in either cortex, hippocampus or cerebellum. In rats chronically exposed to diazepam (21 days via silastic implants), levels of gamma 2 subunit mRNA were significantly decreased in cortex, but not changed in either hippocampus or cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential induction of hepatic microsomal epoxide hydrolase by alkyl sulphides and alkyl ethers in rat

1. Previous studies have shown that the expression of certain cytochromes P450 and major glutathione S-transferases (GSTs) is differentially modulated by alkyl sulphide treatment. The current study was designed to examine the influence of several alkyl sulphides and alkyl ethers on the induction of hepatic microsomal epoxide hydrolase (mEH) in rat. 2. Northern blot analysis has shown that treatment of rats with allyl disulphide (ADS) or allyl sulphide (AS) (50?mg/kg/day, 7 days) resulted in 5-6-fold increases in mEH mRNA levels, whereas allyl ether failed to stimulate mEH gene expression. Rats treated with propyl disulphide (PDS) or propyl sulphide (PS) exhibited increases in mEH mRNA levels, although the relative increases were less than those produced by ADS or AS. Propyl ether failed to increase hepatic mEH mRNA levels. Immunoblot analyses confirmed that allyl and propyl sulphides induce hepatic mEH protein, with the agents containing allyl moiety being more effective. 3. Treatment of rats with ADS at 100?mg/kg resulted in 9-14-fold increases in mEH mRNA levels at 12 and 24?h post-treatment respectively, whereas AS caused a maximal increase at the 24-h time point. Animals treated with PDS or PS exhibited time-related increases in mEH mRNA levels, resulting in 2-6-fold elevations at 72-h post-treatment. The changes in mEH mRNA levels after ADS or AS treatment appeared to be more rapid than those after PDS or PS. The rank order for increases in mEH mRNA levels was ADS > AS > PS > PDS. The result of immunoblot analysis was consistent with that of Northern blot analysis. 4. Rats treated with isopropyl sulphide (IPS),butyl sulphone, tert-butyl sulphide,secbutyl sulphide or butyl sulphide (100?mg/kg/day) showed 3-5-fold increases in the mEH mRNA levels at 3 days post-treatment. IPS treatment caused a similar increase in the mRNA level to that of PS, whereas the induction efficacy of PDS was lower than that of PS or IPS. Treatment with butyl sulphone as well as the structural isomers of butyl sulphides caused approximately 3-fold elevations in mEH mRNA levels. 5. These results provide evidence that alkyl sulphides, but not alkyl ethers, are capable of increasing mEH mRNA levels and that the organosulphur compounds with the allyl moiety are more effective in stimulating the mEH gene than are saturated alkyl sulphides. The comparable increase in mEH mRNA by butyl sulphone as well as the necessity of sulphur atom suggests that sulphone metabolites from the alkyl sulphides might be the ultimate inducers of mEH.     

3-HMG-Coenzyme A reductase inhibition and extracellular matrix gene expression in the pressure-overloaded rat heart

The purpose of this study was to determine whether 3-HMG-Coenzyme A (HMG-CoA) reductase inhibition would attenuate the early pressure overload-induced activation of extracellular matrix genes in the left ventricle (LV) of the heart. Sprague-Dawley rats were randomized to 1 of 4 treatment groups: sham-operation+vehicle (SH-V), aortic constriction+vehicle (AC-V), AC+rosuvastatin (RSV, 2 mg/kg; AC-LO), and AC+RSV (10 mg/kg; AC-HI). Rats were injected with normal NaCl (V) or RSV once daily, beginning 1 day before surgery, and killed 1 or 3 days after surgery. Hemodynamic measurements were made in the open-chest anesthetized state. LV levels of transforming growth factor beta1 (TGF-beta1), procollagen 1 (C1), and fibronectin (FN) mRNA were measured by Northern blotting. AC induced a approximately 25% increase in LV weight after 3 days that was not altered by RSV treatment. LV expression of TGF-beta1, C1, and FN mRNA was approximately 2-fold, approximately 2.5-fold, and approximately 5-fold greater, respectively, in hearts of AC-V compared to SH-V rats 3 days post-operation, and was not significantly decreased by either dose of RSV. Inhibition of HMG-CoA reductase does not attenuate the pronounced aortic constriction-induced increases in the early expression of TGF-beta1, C1, and FN in this model of acute pressure overload of the rat heart.     

High affinity central benzodiazepine receptor ligands: synthesis and biological evaluation of a series of phenyltriazolobenzotriazindione derivatives

A series of 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones (PTBTs), VII, were prepared and tested at the central benzodiazepine receptor (BzR). The skeleton of these compounds was designed by formally combining the N-C=O moieties of the known BzR ligands, triazoloquinoxalines (IV) and triazinobenzimidazoles (ATBIs) (VI). Most of the PTBTs displayed submicromolar/nanomolar potency at the BzR. The 9-chloro derivatives (45-49) were generally found to be more potent than their 9-unsubstituted counterparts (37-44). Compound 45 turned out to be the most potent of the PTBTs (K(i) 2.8 nM). A subset of compounds (37, 42, 45, 49), when tested for their affinity on recombinant rat alpha1beta2gamma2, alpha2beta2gamma2, and alpha5beta3gamma2 GABA(A)/Bz receptor subtypes, showed enhanced affinities for the alpha1beta2gamma2 isoform, with compounds 45 and 49 exhibiting the highest selectivity. Moreover, compounds 45 and 49 were found to display a full agonist efficacy profile at alpha1 and alpha2 receptor subtypes, and an antagonist efficacy at alpha5-containing receptors.     

针刺和神经生长因子治疗对脑性瘫痪幼鼠智力及Casp3、BAX、BCL-2 mRNA的影响

目的：探讨针刺与神经元生长因子( NGF)治疗脑瘫幼鼠对其智力及Casp3、BAX、BCL-2基因mRNA的影响。方法选取8窝40只新生SD健康幼鼠,每窝各选择1只作为正常组(8只)、假手术组(8只),剩余24只采用缺血缺氧法制备脑瘫SD幼鼠模型,随机分为模型组、针刺组和针刺+NGF组,每组8只,分组后给予各组幼鼠对应的干预措施。于造模后第18 d采用Y迷宫实验对各组幼鼠的智力情况进行评估,采用荧光定量PCR法检测各组幼鼠大脑海马组织的Casp3、BAX、BCL-2基因mRNA水平,采用TUNEL染色法检测各组幼鼠大脑皮质神经细胞凋亡情况。结果模型组、针刺组、针刺+NGF组的Y迷宫实验正确次数均显著低于正常组和假手术组( P<0．05),在Y迷宫实验正确次数组间比较模型组<针刺组<针刺+NGF组,差异均有统计学意义( P<0．05)。模型组、针刺组、针刺+NGF组大脑海马组织Casp3、BAX基因mRNA水平均显著高于正常组和假手术组、BCL-2基因mRNA水平显著低于正常组和假手术组(P<0．05),Casp3、BAX基因mRNA水平组间比较模型组>针刺组>针刺+NGF组(P<0．05),BCL-2基因mRNA水平组间比较模型组<针刺组<针刺+NGF组(P<0．05)。模型组、针刺组、针刺+NGF组幼鼠大脑皮质神经细胞凋亡指数均显著高于正常组和假手术组(P<0．05),神经细胞凋亡指数组间比较模型组>针刺组>针刺+NGF组(P<0．05)。结论针刺、NGF对脑瘫幼鼠模型均有一定的治疗作用,联合应用效果更佳,其机制可能与影响Casp3、BAX、BCL-2的表达有关。