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

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

To confirm the existence of heme oxygenase (HO)- carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HT-MCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.
Carbon Monoxide/*metabolism ; Cells, Cultured ; Cyclic GMP/*biosynthesis ; Cyclic GMP/genetics ; Heme Oxygenase (Decyclizing)/*biosynthesis ; Heme Oxygenase (Decyclizing)/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Signal Transduction ; Trabecular Meshwork/cytology ; Trabecular Meshwork/*metabolism

OBJECTIVETo investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on cyclic guanosine monophosphatethe (cGMP) in the smooth muscle cells of human corpus cavernosum, and to provide laboratory evidence for the application of the RNA interference (RNAi) technique for the treatment of erectile dysfunction.

METHODSThe recombinant adenovirus rAd5-shRNA-PDE5A3 expressing three pairs of specific shRNA was constructed successfully. The smooth muscle cells of human corpus cavernosum were divided into an experimental, a negative control and a blank control group, and transfected respectively with rAd5-shRNA-PDE5A3, adenovirus rAd5-mock and phosphate buffered saline. The concentration of cGMP was measured by radioimmunoassay at 24, 48 and 72 hours after transfection, and the effect of rAd5-shRNA-PDE5A3 was detected on the cGMP in the smooth muscle cells of the corpus cavernosum.

RESULTSThe cGMP level in the smooth muscle cells of the corpus cavernosum was significantly higher in the rAd5-shRNA-PDE5A3 group than in the rAd5-mock control and blank control groups (P < 0.05), most significantly at 72 hours after transfection.

CONCLUSIONThe rAd5-shRNA-PDE5A3 can obviously increase the cGMP level in the smooth muscle cells of human corpus cavernosum, and enhance the inhibition of the PDE5 gene.

Adenoviridae ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; genetics ; Erectile Dysfunction ; genetics ; Genetic Vectors ; Humans ; Male ; Myocytes, Smooth Muscle ; metabolism ; Penis ; cytology ; RNA, Small Interfering

OBJECTIVE: This study aimed to investigate the regulation of histone-like nucleoid structuring protein (H-NS) on biofilm formation and cyclic diguanylate (c-di-GMP) synthesis in Vibrio parahaemolyticus RIMD2210633. METHODS: Regulatory mechanisms were analyzed by the combined utilization of crystal violet staining, quantification of c-di-GMP, quantitative real-time polymerase chain reaction, LacZ fusion, and electrophoretic-mobility shift assay. RESULTS: The deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V. parahaemolyticus RIMD2210633. H-NS can bind the upstream promoter-proximal DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979 to repress their transcription. These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism. CONCLUSION One of the mechanisms by which H-NS represses the biofilm formation by V. parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.
Bacterial Proteins/metabolism* ; Biofilms ; Cyclic GMP/analogs & derivatives* ; Gene Expression Regulation, Bacterial ; Gentian Violet ; Histones/metabolism* ; Vibrio parahaemolyticus/genetics*

Escherichia coli biofilm is a complex membrane aggregation produced by the adhesion and secretion of extracellular polymeric substances by E. coli cells aggregated on specific media. Pathogenic E. coli will evade the immune system and the impact of various harmful factors in the environment after the formation of biofilm, causing sustained and even fatal damage to the host. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger ubiquitous in bacteria and plays a crucial role in regulating biofilm formation. This paper reviewed the recent studies about the role of c-di-GMP in the movement, adhesion, and EPS production mechanism of E. coli during biofilm formation, aiming to provide a basis for inhibiting E. coli biofilm from the perspective of c-di-GMP.
Bacterial Proteins/genetics* ; Biofilms ; Cyclic GMP/analogs & derivatives* ; Escherichia coli/metabolism* ; Escherichia coli Proteins/metabolism* ; Gene Expression Regulation, Bacterial

To confirm the existence of heme oxygenase (HO)- carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HT-MCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.
Carbon Monoxide ; metabolism ; Cells, Cultured ; Cyclic GMP ; biosynthesis ; genetics ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Trabecular Meshwork ; cytology ; metabolism

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

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
Animals ; Carbon Monoxide ; metabolism ; Cell Hypoxia ; Cyclic GMP ; metabolism ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; PC12 Cells ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Up-Regulation

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg(559) through a negatively charged β-turn at its RA domain. Moreover, the three Glu residues (180-182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu(180-182) and Lys(140), other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.
Animals ; Calcium ; metabolism ; Cattle ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide-Gated Cation Channels ; chemistry ; genetics ; metabolism ; GRB10 Adaptor Protein ; genetics ; metabolism ; Humans ; Kidney ; cytology ; metabolism ; Models, Molecular ; Protein Conformation ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Static Electricity