1.Association between mRNA level of Pde4d and Alox5ap and hypertensive stroke as well as hypertension in rats.
Xiao-wei XU ; Wen-li SHENG ; Liu-Lin
Chinese Journal of Medical Genetics 2007;24(5):491-494
OBJECTIVETo evaluate whether mRNA levels of Pde4d and Alox5ap were associated with hypertensive stroke and hypertension in stroke-prone renovascular hypertensive rats (RHRSP) which could simulate human being's hypertensive cerebral stroke.
METHODSFive groups were established: normotensive group, gradient hypertensive groups I, II and III(with contractive pressure of 140-159 mmHg, 160-179 mmHg and 180-199 mmHg respectively) and spontaneous stroke group. RNA from leukocytes in peripheral blood of each rat underwent real time PCR after reversed.
RESULTSThe mRNA levels of Pde4d and Alox5ap of spontaneous stroke group were statistically higher than that of the other groups. Expression of Pde4d of hypertensive group I was a bit higher than that of normotensive group and hypertensive groups II and III; as for Alox5ap, there was no statistical difference between normotensive group and all gradient hypertensive groups.
CONCLUSIONAnimal experiments come to conclusions that over-expression of Pde4d and Alox5ap are associated with hypertensive stroke but not with hypertension. Therefore, the two genes confer the risk of hypertensive stroke independent of traditional risk factors. It is speculated that over-expression of Pde4d and Alox5ap can motivate onset of hypertensive cerebral stroke by participating in inflammation of arterial walls.
5-Lipoxygenase-Activating Proteins ; Animals ; Carrier Proteins ; genetics ; Cyclic Nucleotide Phosphodiesterases, Type 3 ; genetics ; Cyclic Nucleotide Phosphodiesterases, Type 4 ; Gene Expression Regulation ; Hypertension ; complications ; genetics ; Membrane Proteins ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Stroke ; complications ; genetics
2.The effects of antisenes oligodeoxynucleotide on the cyclic nucleotide monophosphates in smooth muscle cells of human corpus cavernosum.
Wen-Jun BAI ; Shu-Kun HOU ; Xiao-Feng WANG ; Zheng YAN ; Pei-Ying HE ; Qing-Ping DENG ; Xiao-Peng HU ; Kao-Peng GUAN
National Journal of Andrology 2002;8(2):88-91
OBJECTIVESTo investigate the effects of antisense oligodeoxynucleotide(ASON) on the cyclic nucleotide monophosphates (cNMP) in smooth muscle cells of human corpus cavernosum, and provide experimental groundwork for the gene therapy of erectile dysfunction.
METHODSPDE5 gene ASON(containing exon 1) was transfected into the corpus cavernosum smooth muscle cells with the presence of liposome DOTAP. Another sense oligodeoxynucleotide(SON) and 1% of bovine serum were also transducted into the cells as controls. Two of cNMP, cAMP and cGMP, were probed and measured by ELISA at 1, 2, 4, 6, 10, 24 and 48 h after transfection.
RESULTSAfter transfection, the level of cGMP(1-6 h) in human corpus cavernosum smooth muscle cells was significantly higher than that in controls(P < 0.01).
CONCLUSIONSThe PDE5 gene ASON had been showed to manifest stimulative effect on the cGMP in smooth muscle cells of human corpus cavernosum in vitro, and it provides experimental groundwork for the gene therapy of erectile dysfunction.
3',5'-Cyclic-GMP Phosphodiesterases ; antagonists & inhibitors ; genetics ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Humans ; Male ; Muscle, Smooth ; drug effects ; metabolism ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Penis ; cytology
3.Expression of human phosphodiesterase 3A gene using baculovirus expression system in insect cell.
Yong-Min YAN ; Wen-Rong XU ; Hai-Bo ZHU ; Ling ZHANG ; Ren-Ren FEI ; Xiao-Liang WANG ; Hui QIAN ; Shun-Zu HAO ; Wen-Bing WANG
Acta Pharmaceutica Sinica 2005;40(9):810-813
AIMTo investigate the expression of recombinant human phosphodiesterase 3A (HPDE3A) using baculovirus expression system in Tn cell line.
METHODSThe HPDE3A cDNA was recombined with baculovirus, and then the recombinant was transfected into Tn cell line. The expression of HPDE3A in Tn cell line was detected and identified by the RT-PCR, SDS-PAGE and Western blotting.
RESULTSThe recombinant HPDE3A protein was stably expressed in Tn cell line and detected by the distinct morphological changes of Tn cell, RT-PCR, SDS-PAGE and Western blotting using polyclonal antibody. The M(w) of the recombinant protein was about 120 kD.
CONCLUSIONRecombinant HPDE3A can be expressed in Tn cell line using the baculovirus expression system, and thus provided the basic material for studying its bioactivity and application in screening for HPDE3A inhibitor.
3',5'-Cyclic-AMP Phosphodiesterases ; genetics ; metabolism ; Animals ; Baculoviridae ; genetics ; Cell Line ; Cyclic Nucleotide Phosphodiesterases, Type 3 ; Electrophoresis, Polyacrylamide Gel ; Moths ; cytology ; enzymology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection
4.Construction of an antisense RNA recombinant adenovirus vector of the human PDE5A1 gene promoter.
Yong-Ping ZHAO ; Wen-Jun BAI ; Zheng YAN ; Guo-Qiang CHEN ; Shi-Jun LIU ; Qing-Ping DENG ; Xiao-Feng WANG
National Journal of Andrology 2005;11(5):331-334
OBJECTIVETo construct an antisense RNA recombinant adenovirus vector of the human PDE5A1 promoter gene.
METHODSA cDNA fragment containing the human PDE5A1 promoter and the human PDE5A1-specific exon was determined according to the gene bank. The antisense RNA fragment was synthesized artificially and subcloned into the pENTR. Then, the sequence of pENTR fragment was detected, and the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A1 was constructed by LR reaction with the Gateway expression system. The identified recombinant adenovirus plasmid was digested with Pac I and transformed into 293A cells to package recombinant adenovirus particles. The recombinant adenovirus particles were tested with PCR technique and purified after acquisition by CsCl density gradient ultracentrifugation.
RESULTSThe sequencing result showed a 145 bp fragment in pENTR, which was proved to be the domain of the antisense RNA fragment. PCR confirmed that the antisense RNA fragment was cloned into the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A1 successfully and could infect 293A cells. The titer of virus stocks was up to 10(8) - 10(10)/microl after purification.
CONCLUSIONWith the Gateway expression system, the culturing and reproduction of 293A cells can reproduce recombinant adenovirus pAd/CMV/V5-DEST successfully, and the recombinant adenovirus vector can meet the need of in vivo gene transfection in laboratory studies.
3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; Adenoviridae ; genetics ; Cell Line ; Cloning, Molecular ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Genetic Vectors ; Humans ; Plasmids ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; RNA, Antisense ; genetics ; Recombination, Genetic
5.Phosphodiesterase type 5 siRNA increases cGMP in the smooth muscle cells of human corpus cavernosum.
Guo-qiang CHEN ; Wen-jun BAI ; Xiao-feng WANG ; Shi-jun LIU ; Pei-ying HE ; Shu-kun HOU
National Journal of Andrology 2006;12(11):979-984
OBJECTIVETo investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on the cGMP in the smooth muscle cells of human corpus cavernosum, and to provide an experimental groundwork for the gene therapy of erectile dysfunction (ED).
METHODSSmall interfering RNAs targeting PDE5 gene were synthesized by using web design software provided by Ambion, three siRNAs and a control siRNA were synthesized by Ambion. siRNAs were transfected into the smooth muscle cells of human corpus cavernosum by using siPORT Lipid reagent. cGMP was detected by ELISA at different times (24, 48, 72 and 96 h) after transfection.
RESULTSThe cGMP levels of the siRNA1, siRNA2 and siRNA3 groups were significantly higher than those of the siRNA control and blank control groups (P < 0.05), and so was it in the siRNA1 group than the siRNA2 and siRNA3 groups (P < 0.05), with significant difference between the siRNA control and the blank control group (P > 0.05).
CONCLUSIONThe synthesized siRNAs in vitro are capable of increasing the level of cGMP in the smooth muscle cells of human corpus cavernosum, different siRNAs with different capabilities. The siRNA technique could provide not only an extremely powerful tool for the functional analysis of genome but also a new approach to ED gene therapy.
3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Gene Silencing ; Humans ; Male ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Penis ; metabolism ; RNA, Small Interfering ; pharmacology ; Transfection
6.Effect of berberine on the mRNA expression of phosphodiesterase type 5 (PDE5) in rat corpus cavernosum.
Yan TAN ; Qiang TANG ; Benrong HU ; Jizhou XIANG
National Journal of Andrology 2004;10(12):890-893
OBJECTIVETo further investigate the action mechanisms of berberine (Ber) and to assess the effects of Ber on the mRNA expression of phosphodiesterase type 5 (PDE5) in rat corpus cavernosum.
METHODSAfter incubating with Ber for 1 or 3 h respectively, we examined the levels of PDE5 mRNA by reverse transcription polymerase chain reaction (RT-PCR).
RESULTSThere were PDE5A1 and PDE5A2 mRNA expressions in the rat corpus cavernosum with PDE5A2 as the dominant isoform. Ber could obviously inhibit the mRNA expression of the two isoforms in the rat penis and bring on a pronounced decrease in PDE5A2 (P < 0.01).
CONCLUSIONThe present study indicates that the inhibitory effect of Ber on PDE5 mRNA expression, especially on PDE5A2, might account for its molecular mechanism for treating ED.
3',5'-Cyclic-GMP Phosphodiesterases ; biosynthesis ; genetics ; Animals ; Berberine ; pharmacology ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Male ; Penis ; drug effects ; metabolism ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction
7.Gene therapy and erectile dysfunction: the current status.
David H W LAU ; Sashi S KOMMU ; Emad J SIDDIQUI ; Cecil S THOMPSON ; Robert J MORGAN ; Dimitri P MIKHAILIDIS ; Faiz H MUMTAZ
Asian Journal of Andrology 2007;9(1):8-15
Current available treatment options for erectile dysfunction (ED) are effective but not without failure and/or side effects. Although the development of phosphodiesterase type 5 (PDE5) inhibitors (i.e. sildenafil, tadalafil and vardenafil) has revolutionized the treatment of ED, these oral medications require on-demand access and are not as effective in treating ED related to diabetic, post-prostatectomy and severe veno-occlusive disease states. Improvement in the treatment of ED is dependent on understanding the regulation of human corporal smooth muscle tone and on the identification of relevant molecular targets. Future ED therapies might consider the application of molecular technologies such as gene therapy. As a potential therapeutic tool, gene therapy might provide an effective and specific means for altering intracavernous pressure "on demand" without affecting resting penile function. However, the safety of gene therapy remains a major hurdle to overcome before being accepted as a mainstream treatment for ED. Gene therapy aims to cure the underlying conditions in ED, including fibrosis. Furthermore, gene therapy might help prolong the efficacy of the PDE5 inhibitors by improving penile nitric oxide bioactivity. It is feasible to apply gene therapy to the penis because of its location and accessibility, low penile circulatory flow in the flaccid state and the presence of endothelial lined (lacunar) spaces. This review provides a brief insight of the current role of gene therapy in the management of ED.
3',5'-Cyclic-GMP Phosphodiesterases
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antagonists & inhibitors
;
Cyclic Nucleotide Phosphodiesterases, Type 5
;
Erectile Dysfunction
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drug therapy
;
genetics
;
therapy
;
Gene Transfer Techniques
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Genetic Therapy
;
adverse effects
;
Humans
;
Male
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Phosphodiesterase Inhibitors
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therapeutic use
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Vasodilator Agents
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therapeutic use
8.Phosphodiesterase 4D Gene and Risk of Noncardiogenic Ischemic Stroke in a Korean Population.
Myeong Kyu KIM ; Jun Tae KIM ; Sung Min CHOI ; Seung Han LEE ; Man Seok PARK ; Ki Hyun CHO
Journal of Korean Medical Science 2009;24(2):307-310
Recently published studies from different populations provide apparently conflicting evidence on the association between the phosphodiesterase 4D (PDE4D) gene and ischemic stroke. The relationship between a representative PDE4D genotype and ischemic stroke was explored in a case-control study of 205 consecutive Korean patients with noncardiogenic ischemic stroke and 103 healthy controls who were neurologically and radiologically proven to be stroke-free. We selected and genotyped a PDE4D single nucleotide polymorphism (SNP 41, rs152312) as a candidate marker for susceptibility to ischemic stroke because SNP 41 has shown the most significant association with stroke in both a meta-analysis and the original Icelandic study of the PDE4D gene. No significant difference was observed between the cases and controls in the distribution of the PDE4D SNP 41 genotypes. The results from the adjusted conditional logistic regression analysis (adjusted for age, hypertension, diabetes and smoking status) showed no significant association between PDE4D SNP 41 genotypes and an increased risk of noncardiogenic ischemic stroke. The PDE4D gene is not a major risk factor for noncardiogenic ischemic stroke in a Korean population, which supports the recent evidence suggesting that the causative genetic variants of ischemic stroke may differ across populations.
Aged
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Asian Continental Ancestry Group/*genetics
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Brain Ischemia/diagnosis/*genetics
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Case-Control Studies
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Cyclic Nucleotide Phosphodiesterases, Type 3/*genetics
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Cyclic Nucleotide Phosphodiesterases, Type 4/*genetics
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Female
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Genetic Predisposition to Disease
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Genotype
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Humans
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Korea
;
Magnetic Resonance Angiography
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Magnetic Resonance Imaging
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Male
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Middle Aged
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Odds Ratio
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Polymorphism, Single Nucleotide
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Regression Analysis
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Risk
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Stroke/diagnosis/*genetics
9.Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats.
Wu-Jiang LIU ; Zhong-Cheng XIN ; Hua XIN ; Yi-Ming YUAN ; Long TIAN ; Ying-Lu GUO
Asian Journal of Andrology 2005;7(4):381-388
AIMTo investigate the effect of icariin on erectile function and the expression of nitric oxide synthase (NOS) isoforms in castrated rats.
METHODSThirty-two adult male Wistar rats were randomly divided into one sham-operated group (A) and three castrated groups (B, C and D). One week after surgery, rats were treated with normal saline (groups A and B) or oral icariin (1 mg/[kg.day] for group C and 5 mg/[kg.day] for group D) for 4 weeks. One week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure (ICP) during electrostimulation of the cavernosal nerve. The serum testosterone (ST) levels, the percent of smooth muscle (PSM) in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and phosphodiesterase V (PDE5) in corpus cavernosum (CC) were also evaluated.
RESULTSICP, PSM, ST and the expression of nNOS, iNOS, eNOS and PDE5 were significantly decreased in group B compared with those in group A (P 0.01). However, ICP, PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B (P 0.05). Changes in ST and the expression of eNOS and PDE5 were not significant (P 0.05) in groups C and D compared with those in group B.
CONCLUSIONOral treatment with icariin ( 98.6 % purity) for 4 weeks potentially improves erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.
3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; metabolism ; Animals ; Blood Pressure ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Drugs, Chinese Herbal ; pharmacology ; Erectile Dysfunction ; drug therapy ; metabolism ; Flavonoids ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Male ; Muscle, Smooth ; drug effects ; physiology ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type I ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Orchiectomy ; Penile Erection ; drug effects ; Penis ; drug effects ; enzymology ; Pressure ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Testosterone ; blood