Gene transfer of a β2-adrenergic receptor kinase inhibitor up-regulates the level of β2-adrenergic receptor and cAMP in the asthmatic murine lung

Objective： To investigate the effects of gene transfer of a β-adrenergic receptor（β-AR） kinase inhibitor（β ARIct） on pulmonary β2-adrenergic receptor and cAMP following β2-AR agonist treatment in asthmatic mice, and to analyze the relationship between the routes of gene delivery and the changes of β2AR and cAMP. Methods： BALB/c mice were sensitized and challenged by ovalbumin to establish the asthmatic model treated with βAR agonist （salbutamol injected intramuscularly）. The plasmid with the expression of βARKct was constructed and βARKct gene transfer was performed through intravenous injection or intratracheal instillation in asthmatic mice. The gene expression was measured with Western blot analysis, and the changes of pulmonary β-AR and cAMP evaluated by Radioimmunoassay. Results： The expression of tranfered βARKct gene was detectable in lungs and it was expressed more in the lungs of the mice receiving intratracheally plasmid than those receiving intravenously. The levels of βAR and cAMP were upregulated after using plasmid-βARKct to the asthmatic mice treated with βAR agonist. Conclusion： Our results indicated that there were down-regulation of βAR and cAMP in asthmatic mice treated with βAR agonist. Gene transfer of βARKct could inhibit the extent of the down-regulation of βAR and cAMP. The route of gene delivery could also affect the degree of up-regulation of βAR and cAMP. Gene transfer βARKct may provide a novel approach to the therapeutic strategy for asthma.     

Effects of metoprolol on β1 adrenergic receptor polymorphism and receptor density in urban Chinese patients with heart failure

β-blockers have been recommended as a standard treatment for patients with mild to moderate systolic chronic heart failure （CHF） because they not only relieve patients＇ symptoms but also decrease mortality. However, β-blockers have a variety of effects on different CHF patients. Among them, β1-adrenergic receptor （AR） gene polymorphism is probably a significant one. The purpose of this study was to investigate the effects of metoprolol on cardiac function, cardiac geometrical size and density of β1-AR of CHF patients as well as the association between two common single nucleotide polymorphisms （SNPs） of β1-AR（Gly389Arg and Ser49Gly） and the effects of metoprolol.     

Inhibition of allergic responsiveness in a murine asthma model via IFN-γ transgene expression

Objective To investigate adenoviral vector mediated exogenous gene expression in mouse lungs and the effect of mIFN-γ transgene expression on allergen-induced pulmonary eosinophil infiltration in a murine asthmatic model. Methods LacZ marker gene was transduced into CD-1 mouse airway epithelial cells by installation of a replication-deficient adenovirus with LacZ gene (AdCMVLacZ) 5×10[9]plaque forming unit (pfu) in the intratrachea or nostril.C57 mice were sensitized intraperitoneally and challenged by aerosol with ovalbumin (OVA) to produce an asthmatic model.AdCMVmIFNγ 5×10[9] pfu was administered via nostril in asthmatic mice 48 h before OVA challenge.Sera, bronchial alveolar lavage (BAL) and lungs were recovered 48 h after OVA challenge.Results After administration with AdCMVLacZ by intratracheal installation or nose-drop, the lungs revealed a high level of widespread LacZ transduction with X-gal staining, mainly along airways.IFN-γ via adenoviral vector transduction could be overexpressed both in vitro and in vivo (1624.7±1321.5 pg/ml in BAL 96 h after AdCMVIFNγ infection).In AdCMVIFNγ treated asthmatic models, histological evaluation revealed marked suppression of eosinophil peribronchial and perivascular infiltration; the recoverable percentage of eosinophils in BAL was an average of 9.00%±4.58%, which was a statistically significant decrease versus that of the positive control group (75.13%±6.85%) (P<0.001).The total cell number in BAL ((145±55.6)×10[3 cells/ml) in AdCMVmIFNγ treated mice also was tremendously reduced compared to the positive control group ((216.6±71.1)×10[3 ]cells/ml).Conclusions Adenoviral vector was able to overexpress exogenous gene in murine lungs.IFN-γ overexpression via adenoviral vector in pulmonary epithelia in vivo can abrogate allergen-induced eosinophilic infiltration in lungs in an asthmatic model, which may suggest a new preventively therapeutic method for cytokine immunogenetic transfer in allergic asthma.     

Relationship between Trp64Arg mutation in the β3-adrenergic receptor gene and metabolic syndrome: a seven-year follow-up study

Background It has been shown that the β3-adrenergic receptor （β3-AR） gene Trp64Arg mutation was closely related to obesity and insulin resistance, and may be related to the prevalence of metabolic syndrome （MS）. The aim of this study was to investigate the relationship between the 33-AR gene mutation and the prevalence of MS. Methods A seven-year follow-up study was initiated in 2000, with 496 samples of simplex obese subjects （body mass index ≥25 kg/m2） and 248 normal-weight subjects. According to the β3-AR genotypes, the subjects were classified as Trp64 homozygote group and Arg64 carrier group and after 7 years the prevalence of MS was determined. Results According to the baseline profile, there were no significant differences in the adiposity, blood pressure, lipid profile, fasting plasma glucose and fasting insulin between Trp64 homozygote group and Arg64 carrier group either in obesity or normal-weight subjects. The results of follow-up study indicated that in obese men the prevalence rate of MS was much higher in Arg64 carrier group than that in Trp64 homozygote group （54.76% vs. 40.85%, P 〈0.05）, but there was no statistical difference in women of the above groups. The prevalence rate of MS in obese men of both Trp64 homozygote group and Arg64 carrier obese group were obviously higher than that in women of the above groups （40.85% vs. 18.27% and 54.76% vs 21.28%, all P 〈0.005）. Differences were not statistically significant in the prevalence of MS for normal weight Trp64 homozygote group and normal weight Arg64 carrier group, either between men, between women, or between men and women. Comparison of populations indicated that no matter with the β3-AR gene mutation or not, the prevalence of MS in obese subjects was significantly higher than normal weight subjects （X2=28.240 and x2=15.586, all P 〈0.005）. Logistic analysis showed that the mutation of β3-AR gene was associated with the prevalence of MS in men.     

Effect of β3-adrenergic agonists on alveolar fluid clearance in hypoxic rat lungs

Background Recent research suggests that β2-adrenergic agonists increase alveolar fluid clearance （AFC） under physiologic and pathologic conditions. It is unknown whether β3-adrenergic agonists also increase AFC under pathologic conditions. The aim of this study was to investigate the effect of β3-adrenergic agonists on AFC following hypoxic lung injury and the mechanisms involved. Methods Hypoxic rats were exposed to 10% oxygen. BRL-37344 （133-adrenergic agonist） or CGP-12177 （selective β3-adrenergic agonist） alone or combined with β receptor antagonists, sodium channel blockers, or Na＋/＋~-ATPase blockers were perfused into the alveolar space of rats exposed to 10% oxygen for 48 hours. Total lung water content （TLW） and AFC were measured. Results AFC did not change for the first 24 hours but then decreased after 48-hour exposure to 10% oxygen. The perfusion of BRL-37344 or CGP-12177 significantly increased AFC in normal and hypoxic rats. The AFC-stimulating effect of CGP-12177 was lowered with amiloride （a Na＋ channel blocker） and ouabain （a Na~/K~-ATPase inhibitor） by 37% and 49%, respectively. Colchicine significantly inhibited the effect of CGP-12177. Conclusions These findings suggest that β3-adrenergic agonists can increase AFC during hypoxic lung injury in rats and accelerate the amelioration of pulmonary edema.     

Relationship between β3-AR Gene and Obesity, Type 2 Diabetes, Insulin Resistance in Chinese Han Population

Objective: To explore the relationship between the β3-adrenergic receptor(β3-AR)gene and obesity, T2DM. insulin resistance in Chinese Han population. Methods: Fifty-three healthy subjects, 105 subjects with simple obesity, 63 type 2 diabetic patients without obesity, and 114 type 2 diabetic patients with obesity were studied with the technique of PCR-RFLP in codon 64 of the exon region of β3-AR gene representing the variation Trp/Arg. Results:Compared with the subjects of Trp homozygous group, the individuals with Arg allele were more elevated in WHR,MBP,SBP,DBP,FBS,PBS, FINS,PINS, FCP,PCP and lower in ISI. Frequency of Arg allele was higher in HINS sub-group without T2DM. Cnclusion: The results indicate that the Trp/Arg variation might lead to insulin resistance, obesity and T2DM.β3-AR gene is supposed to be the candidate gene of insulin resistance, obesity and T2DM in ChineseHan population.     

Primary study on correlation between β2-adrenoceptor haplotypes and asthma in children of Han nationality in Chongqing

Objective: To investigate the correlation between β2-adrenergic receptors (β2-AR) haplotypes and asthma of Hah nationality children in Chongqing region. Methods: PCR and restriction fragment analysis were used to study 16, 27loci of the β2-AR polymorphism in 76 unrelated asthmatic children and in 100 healthy children and adults of Han nationality as control. A statistical analysis of the correlation between glycine (Gly)16 allele, Gly16/glutamine (Gln)27 haplotype and asthmatic clinical status was carried out. Results: There was no significant increase of the frequency of Gly16 and Gln27 allele in the asthmatic group as compared with the control group (P＞0.05). There was a significant increase of the frequency of Gly16 allele and Gly16/Gln27 haplotype in severe asthmatic cases than in the mild and moderate asthmatic ones (P＜0.01, 0.05). Conclusion: It is considered that asthma is not caused by Gly and Gln alleles ofβ2-AR polymorphisms. Glyl6 allele and Gly16/Gln27 haplotype are possibly correlated with the severity of the clinical manifestations in the children of Han nationality in Chongqing.     

Age-dependent changes in β-adrenoceptor function in human detrusors and possible mechanisms

Objective To study age-dependent changes in β-adrenergic responsiveness and their possible mechanisms. Methods Responsiveness to the β-adrenergic agonists isoprenaline, BRL37344, forskolin, and dibutyryl cyclic AMP (DBcAMP) was examined in samples from 10 older patients by using a cellular function test. A radioligand binding assay was performed using the non-selective β-adrenergic receptor ligand ［3H］- dihydroalprenolol (［3H］-DHA). Specimens from 10 young men were used as controls. Results There were no age-dependent changes in contractile response to KCl. The relaxation responses to isoprenaline, BRL37344, and forskolin decreased in the aged group by 15.0%, 17.6%, and 12.6%, respectively （P<0.001）. The pD2 values for isoprenaline and BRL37344 also declined significantly. There was no difference in the responsiveness to dibutyryl cyclic AMP (DBcAMP) between the two groups; the maximum binding site decreased significantly with increasing age, but the equilibrium-dissociation constant did not change. Conclusions There is an age-related decline in β-adrenergic responsiveness which might be one of the causative factors of reduced bladder compliance in the elderly. A decrease in cAMP level caused by reduced receptor density and adenylyl cyclase activity might be the underlying molecular mechanism of the changes in β-adrenergic responsiveness.     

Genetic Variation in β3-Adrenergic Receptor and Obesity, Type 2 Diabetes, Insulin Resistance in Chinese Pedigrees

Objective: To explore the association between the polymorphism of the β3-adrenergic receptor(β3-AR )gene and obesity, type 2 diabetes, insulin resistance in Chinese pedigrees. Methods: Eight pedigrees with obesity and type 2 diabetes have been detected with the technique of PCR-RFLP in codon 64 of the exon region representing the variation Trp64 Arg of β3-AR gene. Results: In pedigree linkage analysis, the maximal LOD score of β3-AR gene with obesity was 3.385109 (θ=0.000000) at the mode of autosomal dominant in pedigree 3. The maximal LOD score of β3-AR with type 2 diabetes was 0.222336 (θ=0.000000) at the mode of autosomal dominant in pedigree 2 and 0.805003(θ=0.000000) at the mode of autosomal recessive in pedigree 4. Conclusion: The results indicate that ① The Trp 64 Arg variation have a cause effect significances of some familial obesity. Pedigree linkage analysis can powerfully help to understand the action mechanism of the candidate genes, ②Better comphrehension the workings of adrenergic receptors should provide a new understanding of obesity, type 2 diabetes and insulin resistance and perhaps lead to new methods of treatment.     

β-adrenergic response modulated by κ-opioid receptor stimulation is attenuated in the cardiomyocytes of rats following chronic hypoxia

Objective: To study cross-talk between p-opioid receptor and β-adrenoceptor through determination of the intracellular calcium ( [ Ca2+ ]i ) and cAMP responses in ventricular myocytes of rats subjected to chronic hypoxia for 4 weeks. Methods: Electrically-induced [ Ca2+ ]i transient was measured in single right ventricular myocytes isolated from hearts of chronically hypoxic rats and the age-matched normoxic rats, by using a spectrofluorometric method. Results: β-adrenoceptor stimulation with isoproterenol increased the electrically-induced [ Ca + ]i transient and cAMP in myocytes of normoxic rats. U50,488H, a selective β-opioid receptor agonist, at dose (1 βmol/L) which itself had no effect on the [Ca2 + ]i transient and cAMP, significantly inhibited the effect of isoproterenol. This inhibition was completely abolished in the presence of nor-BNI, a selective K-opioid receptor antagonist. In the ventricular myocytes of chronically hypoxic rats, the inhibition of U50,488H on the increased [ Ca ]i tra     

Role of β-Adrenergic Signal Transduction Pathway on Myocardial Ischemic Preconditioning of Rats

To study the changes in every part of the β-adrenergic signal transduction pathway and their effects on ischemic preconditioning of rat myocardium in vivo. SD rats were divided into three groups： IP group, I/R group and CON group. The IP group was further divided into PC1-, 2-, 3-, and PC1＋, 2＋, 3＋ groups according to preconditioning procedure. The rats received surgical procedure and underwent left coronary artery occlusion and reperfusion. We analyzed the infarct size by TTC staining, measured serum myocardial enzymes, studied the β-AR Bmax and Kd by radioligand binding assay of receptors, checked the activity of AC and PKA by the method of biochemistry and examined the content of cAMP by radioimmunoassay. The infarct area was much smaller in the IP group than in the I/R group （P〈0. 001）, while the enzymes were significantly higher in I/R （P〈0. 001）. The Bmax of β-AR in IP was much higher than that in I/R （P〈0. 001）, but no difference in Kd could be seen between IP and I/R groups. In IP, the activity of AC and PKA and the content of cAMP were higher than those in I/R （P〈0.05, 0. 002 and 0. 001, respectively）. In the procedure of preconditioning, the content of cAMP and the activity of PKA showed the characteristic of cyclic fluctuation. Ischemic preconditioning can protect the heart from necrosis and reduce endo-enzyme leakage. The system of β-adrenergic signal transduction pathway probably takes part in the protection effect of the IP, which might be elicited by the PKA .     

Epigenetic Regulation of the ERβ Gene on the Estrogen Signal Transfection Pathway in Colon Cancer Cells

We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβ mRNA and protein was detected 72h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene ...     

Non-Fusion and Fusion Expression of β-Galactosidase from Lactobacillus bulgaricus in Lactococcus lactis

Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation pr     

The Expression of the Plasmid DNA Encoding TGF—β1 in Endothelium after Injection into the Anterior Chamber

The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection.Two days after direct injection of pMAM TGF-β1 mediated by liposome into the anterior chamber of rabbits,one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia.By means of immunohistochemical technique,the plasmid pMAM TGF-β1 expression product TGF-β1 in the endothelia was detected.Specific TGF-β1 expression was positive in the endothelia on both the paraffin slide and the single layer slide.The results showed that by direct injection into the anterior chamber,foreign plasmid DNA could be transferred into the endothelia and its expression was obtained.This may provide a foundation for further study on TGF-β1 participating in local induction of corneal immune tolerance.     

Effects of Aging and Advanced Glycation on Gene Expression in Cerebrum and Spleen of Mice

Objective To analyze the effects of aging or advanced glycation on gene expression in the cerebrum and spleen of female C57BL/6J mice. Methods The gene expression profile was determined by using cDNA expression arrays containing 588 cDNA. Results Aging and advanced glycation resulted in differential gene expression patterns of cerebrum and spleen compared with young mice. Among the 80 genes detected in cerebrum, 43 exhibited a change in mRNA ratios with aging or treatment. Thirty-four changes (79%) were common in aged and D-galactose treated mice, whereas the cerebrum from aged and AGE-lysine treated mice showed common changes in expression of 38 genes (88%). Of the 86 genes detected in spleen, 29 (34%) displayed an age-related decrease in expression, whereas 3 (3%) displayed an increase in expression levels with aging. Eighteen genes from the detectable genes exhibited expression changes in both cerebrum and spleen of mice. Conclusions The gene expression profiles of D-galactose and AGE-lysine treated     

Down-regulation of β-catenin Nuclear Localization by Aspirin Correlates with Growth Inhibition of Jurkat Cell Line

In this study, we examined the effects of aspirin on the growth rates, subcellar distribution of β-catenin protein, the expression of β-catenin/TCF signaling pathway target gene cyclinD1 mRNA, and cell cycle of Jurkat cell line （Human T-acute lymphoblastic leukemia）. Our results showed that the treatment with aspirin inhibited the growth of Jurkat cell line. Jurkat cells treated with 3 mmol/L of aspirin could significantly decrease nuclear localization of β-catenin, and at 5 mmol/L of aspirin, the nuclear localization of β-catenin was undetectable. QRT-PCR showed that the target gene cyclinDl mRNA expression was gradually decreased with the dosage of aspirin. Aspirin induced G0/G1 cell cycle arrest in Jurkat cells. We are led to conclude that aspirin acts through β-catenin-independent mechanisms. The effects of aspirin include down-regulation of β-catenin nuclear localization and G0/G1 cell cycle arrest, which might serve as a means of growth inhibition in aspirin-treated human Jurkat cell line.     

The expression of CD40 and CD40L on the surface of peripheral blood mononuclear cells in asthmatic rats and the effect of anti-CD40L McAb on Th1 and Th2 cytokines

Objective： To investigate the expression of CD40 and CD40 ligand （CD4OL） on the surface of peripheral blood mononuclear cells（PBMCs） in asthmatic rats and the effect of anti-CD40L McAb on cytokines of PBMCs. Methods： Flow cytometry and RT-PCR were used to detect the expression of CD40 and CD40L of PBMCs in asthmatic rats. After the PBMCs was treated with anti-CD40L McAb, ELISA was used to detect the IL-4 and IFN-γ levels of culture supernatants. Results： Compared with the normal control group, the expression of CD40 and CD40L of PBMCs in asthmatic rats increased （P 〈 0.05）. Compared with the untreated group, the level of IL-γ and the ratio of IL-4/IFN-γ decreased after the PBMCs was treated with anti-CD40L McAb（P 〈 0.05）. Conclusion： The expression of CD40 and CD40L on the surface of PBMCs in asthmatic rats was up-regulated. Anti-CD40L McAb can rectify the imbalance of Th1 and Th2 cytokines.