PURPOSE: To compare the effects of the barrier function in human trabecular meshwork (TM) cells monolayer and the production of nitric oxide (NO) between trabecular outflow drugs, Rho-associated kinase (ROCK) inhibitors, adenosine, and statin. METHODS: Primary cultured TM cells were exposed to 10 or 25 µM Y-27632, 0.1 or 1 µM N6-cyclohexyladenosine (CHA), or 15 or 30 µM simvastatin for 24 hours. NO production and expression of endothelial nitric oxide synthase mRNA were measured by Griess assay and reverse transcription polymerase chain reaction, respectively. Barrier functions of the TM cell monolayer were measured by carboxyfluorescein and trans-endothelial electrical resistance. The expression of matrix metalloproteinase-2 mRNA was assessed with reverse transcription polymerase chain reaction. RESULTS: In TM cells, treatment with each drug increased endothelial nitric oxide synthase mRNA expression. Treatment with 25 µM Y-27632 and 1.0 µM CHA increased NO production significantly (p = 0.035 and p = 0.043, respectively). Treatment with each drug increased the permeability (all p = 0.001) and decreased the trans-endothelial electron resistance of the TM cell monolayer. Treatment with 0.1 µM and 1.0 µM CHA significantly increased matrix metalloproteinase-2 mRNA expression, but simvastatin inhibited its expression. CONCLUSIONS: Since treatment with ROCK inhibitor more greatly increased NO production and permeability than did adenosine or statin, ROCK inhibitor seems to be more effective for lowering intraocular pressure.
Adenosine ; Electric Impedance ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; Intraocular Pressure ; Matrix Metalloproteinase 2 ; Nitric Oxide Synthase Type III ; Nitric Oxide* ; Permeability* ; Polymerase Chain Reaction ; Reverse Transcription ; rho-Associated Kinases ; RNA, Messenger ; Simvastatin ; Trabecular Meshwork*

nc886 (=vtRNA2-1, pre-miR-886, or CBL3) is a newly identified non-coding RNA (ncRNA) that represses the activity of protein kinase R (PKR). nc886 is transcribed by RNA polymerase III (Pol III) and is intriguingly the first case of a Pol III gene whose expression is silenced by CpG DNA hypermethylation in several types of cancer. PKR is a sensor protein that recognizes evading viruses and induces apoptosis to eliminate infected cells. Like viral infection, nc886 silencing activates PKR and induces apoptosis. Thus, the significance of the nc886:PKR pathway in cancer is to sense and eliminate pre-malignant cells, which is analogous to PKR's role in cellular innate immunity. Beyond this tumor sensing role, nc886 plays a putative tumor suppressor role as supported by experimental evidence. Collectively, nc886 provides a novel example how epigenetic silencing of a ncRNA contributes to tumorigenesis by controlling the activity of its protein ligand.
Apoptosis ; Carcinogenesis ; DNA ; Epigenomics ; Immunity, Innate ; Protein Kinases ; RNA Polymerase III ; RNA, Untranslated*

OBJECTIVETo analyze the association of micoRNA-related genes DROSHA single nucleotide polymorphisms (SNP) rs10719 and rs6877842, DICER1 rs3742330and GEMIN4 rs3744741 with prognosis of T-cell lymphoma.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to determine the genotypes of the above 4SNPs and their associations with complete remission (CR) rate and overall survival (OS) in 163 patients with TCL.

RESULTSPatients carrying the rs6877842 CG genotype had a significantly higher CR rate compared with those carrying the CC genotype (OR=0.07, 95% CI 0.01-0.72, P=0.026); the same for patients carrying the DICER1 rs3742330 GG genotype compared with those carrying the GA genotype (OR=0.15, 95% CI 0.02-0.97, P=0.047) or the AA genotype (OR=0.11, 95% CI 0.02-0.71, P=0.020). In addition, patients with the DICER1 rs3742330 GG genotype had a significantly improved OS compared with those carrying the GA (HR=9.02, 95% CI 1.22-66.92, P=0.031) or AA genotype (HR=8.77, 95% CI 1.19-64.67, P=0.033). The other two SNPs of rs10719 and rs3744741 had no significant association with CR or OS.

CONCLUSIONDROSHA rs6877842 and DICER1 rs3742330 were independent factors for TCL CR, and DICER1 rs3742330 was also an independent prognostic factor for TCL OS.

DEAD-box RNA Helicases ; genetics ; Genetic Predisposition to Disease ; Genotype ; Humans ; Lymphoma, T-Cell ; diagnosis ; genetics ; MicroRNAs ; genetics ; Minor Histocompatibility Antigens ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Prognosis ; Ribonuclease III ; genetics ; Ribonucleoproteins, Small Nuclear ; genetics

OBJECTIVETo screen potential serum marker proteins of osteosarcoma, and to make a preliminary bioinformatics analysis of RNA polymerase III polypeptide F (POLR3F).

METHODSGene chip and SELDI-TOF MS was used to screen genes differentially expressed in osteosarcoma. The associations of potential biomarkers from SELDI data and microarray analysis were further inferred by link-test to identify biomarkers that could likely be used for diagnosis. MATLAB was used to search transcription factors binding site in the promoter region and miRNAs binding site in 3'-UTR of POLR3F, respectively.

RESULTS653 differentially expressed genes were found in osteosarcoma cells, while six differentially expressed protein peaks with significant statistical significances were detected by SELDI-TOF MS in patient's serum. 13 potential biomarkers for early diagnosis of osteosarcoma were screened by link-test. A conserved STAT3 binding site and a miRNA target site were found in proximal promoter regions and 3'-UTR region of POLR3F, respectively.

CONCLUSIONSLink-test is a effective method to identify osteosarcoma biomarkers from both microarray and SELDI-TOF MS database. The results confirmed that POLR3F may be a promising biomarker for early diagnosis and a therapy target of osteosarcoma.

Adolescent ; Adult ; Binding Sites ; Biomarkers, Tumor ; blood ; Blood Proteins ; analysis ; Bone Neoplasms ; genetics ; metabolism ; Child ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Oligonucleotide Array Sequence Analysis ; Osteosarcoma ; genetics ; metabolism ; Peptides ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; Protein Binding ; Protein Subunits ; genetics ; metabolism ; RNA Polymerase III ; genetics ; metabolism ; RNA, Messenger ; genetics ; STAT3 Transcription Factor ; metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Young Adult

BACKGROUND: The safety of plasma derivatives has been reinforced since 1980s by variable pathogen inactivation or elimination techniques. Nucleic acid amplification test (NAT) for the source plasma has also been implemented worldwide. Recently nanofiltration has been used in some country for ensuring safety of plasma derivatives to eliminate non-enveloped viruses such as parvovirus B19 (B19V) and hepatitis A virus (HAV). We evaluated the efficacy of nanofiltration for the elimination of B19V and HAV. METHODS: To verify the efficacy of nanofiltration, we adopted a 20 nm Viresolve NFP (Millipore, USA) in the scaling down (1:1,370) model of the antithrombin III production. As virus stock solutions, we used B19V reactive plasma and porcine parvovirus (PPV) and HAV obtained from cell culture. And 50% tissue culture infectious dose was consumed as infectious dose. The methods used to evaluate the virus-elimination efficacy were reverse-transcriptase polymerase chain reaction for B19V and the cytopathic effect calculation after filtration for PPV and HAV. RESULTS: B19V was not detected by RT-PCR in the filtered antithrombin III solutions with initial viral load of 6.42x10(5) IU/mL and 1.42x10(5) IU/mL before filtration. The virus-elimination efficacy of nanofiltration for PPV and HAV were > or =10(3.32) and > or =10(3.31), respectively. CONCLUSIONS: Nanofiltration would be an effective method for the elimination of B19V and HAV. It may be used as a substitute for NAT screening of these viruses in source plasma to ensure safety of plasma derivatives in Korea.
Antithrombin III/isolation & purification ; DNA, Viral/analysis ; Filtration/*methods ; Hepatitis A virus/genetics/*isolation & purification ; Humans ; Nanotechnology/*methods ; Parvovirus B19, Human/genetics/*isolation & purification ; RNA, Viral/analysis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVE: To compare the effect of intermittent and constant high glucose media on the synthesis of vascular relaxing factor nitric oxide (NO) in cultured human umbilical vein endothelial cells (HUVECs), and to investigate its mechanism. METHODS: HUVECs were exposed to 5.5 mmol/L glucose (normal control), 20 mmol/L glucose (constant high glucose), and 5.5 mmol/L alternating with 20 mmol/L glucose (intermittent high glucose) for 7 days. The NO level was measured using Griess Reaction in cell culture supernatants; the expressions of PI3K, PKB and eNOS mRNA and protein were measured with RT-PCR or Western blot. RESULTS: NO level in the intermittent high glucose group was significantly lower than that in the constant high glucose group (P<0.05). The expression of PI3K, PKB, and eNOS in the intermittent high glucose group was significantly lower than that in the constant high glucose group (P<0.05). CONCLUSION Intermittent high glucose could be more deleterious to endothelial cells than constant high glucose, and may lead to decrease synthesis of NO by inhibiting PI3K/PKB/eNOS pathway activation.
Cells, Cultured ; Culture Media ; Endothelial Cells ; cytology ; metabolism ; Glucose ; pharmacology ; Humans ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Umbilical Veins ; cytology

BACKGROUND: The up-regulation of the nitric oxide (NO)-cGMP pathway might be involved in the change of vascular reactivity in rats 3 days after they suffer acute myocardial infarction. However, the underlying mechanism for this has not been clarified. MATERIAL AND METHOD: Acute myocardial infarction (AMI) was induced by occluding the left anterior descending coronary artery (LAD) for 30 min (Group AMI), whereas the sham-operated control rats were treated similarly without LAD occlusion (Group SHAM). The concentration-response relationships for phenylephrine (PE), KCl, acetylcholine (Ach) and sodium nitroprusside (SNP) were determined in the endothelium intact E(+) and endothelium denuded E(-) thoracic aortic rings from the rats 3 days after AMI or a SHAM operation. The concentration-response relationships of PE in the E(+) rings from the AMI rats were compared with those relationships in the rings pretreated with nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) or the cyclooxygenase inhibitor indomethacin. The plasma nitrite/nitrate concentrations were checked via a Griess reaction. The cyclic GMP content in the thoracic aortic rings was measured by radioimmunoassay and the endothelial nitric oxide synthase (eNOS) mRNA expression was assessed by real time PCR. RESULT: The mean infarct size (%) in the rats with AMI was 21.3+/-0.62%. The heart rate and the systolic and diastolic blood pressure were not significantly changed in the AMI rats. The sensitivity of the contractile response to PE and KCl was significantly decreased in both the E(+) and E(-) aortic rings of the AMI group (p<0.05). L-NAME completely reversed these contractile responses whereas indomethacin did not (p<0.05). Moreover, the sensitivity of the relaxation response to Ach was also significantly decreased in the AMI group (p<0.05). The plasma nitrite and nitrate content (p<0.05), the basal cGMP content (p<0.05) and the eNOS mRNA expression (p=0.056) in the AMI rats were increased as compared with the SHAM group. CONCLUSION: Our findings indicate that the increased eNOS activity and the up-regulation of the NO-cGMP pathway can be attributed to the decreased contractile or relaxation response in the rat thoracic aorta 3 days after AMI.
Acetylcholine ; Animals ; Aorta, Thoracic ; Blood Pressure ; Coronary Vessels ; Cyclic GMP ; Endothelium ; Heart Rate ; Indomethacin ; Myocardial Infarction ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type III ; Nitroprusside ; Phenylephrine ; Plasma ; Prostaglandin-Endoperoxide Synthases ; Radioimmunoassay ; Rats ; Real-Time Polymerase Chain Reaction ; Relaxation ; RNA, Messenger ; Salicylamides ; Up-Regulation

PURPOSE: It is well known that metformin, an oral biguanide insulinsensitizing agent, targets AMP-activated protein kinase (AMPK), which activates endothelial nitric oxide synthase (eNOS), which may be associated with erectile dysfunction. The aim of this study was to evaluate whether metformin activates eNOS in the penile tissue of a genetically obese rat model. MATERIALS AND METHODS: We measured phospho-eNOS levels and eNOS expression in the penile tissue of Otsuka Long Evans Tokushima Fatty (OLETF) rats after 3 days of treatment with metformin (150 or 300 mg) to evaluate whether metformin activates eNOS in the penile tissue of this genetically obese rat model. Seven-month-old OLETF rats were used, and eNOS expression was analyzed by real-time polymerase chain reaction (PCR). The experimental groups were compared with use of the Kruskal-Wallis or Mann-Whitney test. RESULTS: Seven-month-old OLETF rats had severe visceral fat deposition and elevated serum leptin concentrations. eNOS expression analyzed by real-time PCR was lower in the penile tissue of OLETF rats than in Long Evans Tokushima Otsuka (LETO) rats. Short-term treatment with metformin did not change visceral fat mass or serum leptin levels. However, metformin treatment increased eNOS mRNA expression in the penile tissue as determined by real-time PCR. The levels of phospho-eNOS and phospho-AMPK (pAMPK) in penile tissue revealed a dose-dependent tendency to increase with metformin treatment; however, there was no statistical difference by Kruskal-Wallis test among the experimental groups. The pAMPK level was dose-dependently elevated in the soleus by metformin treatment. There was no significant change in pSTAT3 by metformin treatment in the soleus. CONCLUSIONS: Metformin activates eNOS mRNA expression in the penile tissue of OLETF rats. Further study on the relation between erectile function and eNOS levels is needed for clinical application.
AMP-Activated Protein Kinases ; Animals ; Erectile Dysfunction ; Intra-Abdominal Fat ; Leptin ; Male ; Metformin ; Nitric Oxide ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type III ; Penis ; Rats ; Rats, Inbred OLETF ; Real-Time Polymerase Chain Reaction ; RNA, Messenger

OBJECTIVEThe etiology of developmental dislocation of the hip (DDH) remains uncertain, but some research has shown that this disorder is closely related to hip joint laxity. This study examined the expression of collagens type I and III mRNA and protein in the hip capsule of children with DDH in order to investigate the roles of collagens type I and III in hip joint laxity.

METHODSNine children with DDH and nine age and gender-matched normal children (control group) were enrolled. Semiquantitative RT-PCR method was used to detect mRNA expression of COL1a1 and COL3a1 in the hip capsule. Western-Blot method was used to detect protein expression of COL1a1 and COL3a1 in the hip capsule. The quantitative analysis of the COL1a1 and COL3a1 was performed by professional image software and the results were analyzed with standard statistical methods.

RESULTSmRNA and protein expression of COL1a1 in the DDH group was significantly lower than that in the control group (P<0.01). Compared with the control group, COL1a3 mRNA expression in the DDH group decreased significantly (P<0.01), but COL1a3 protein expression was not significantly different.

CONCLUSIONSThe decreased collagen I mRNA and protein expression in the hip capsule might contribute to hip joint laxity in children with DDH. Collagen type III may not be associated with hip joint laxity in DDH.

Blotting, Western ; Child ; Child Development ; Child, Preschool ; Collagen Type I ; analysis ; genetics ; Collagen Type III ; analysis ; genetics ; Female ; Hip Dislocation ; metabolism ; Humans ; Infant ; Male ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDOpioid preconditioning (PC) reduces anoxia/reoxygenation (A/R) injury to various cells. However, it remains unclear whether opioid-induced delayed PC would show anti-apoptotic effects on pulmonary artery endothelial cells (PAECs) suffering from A/R injury. The present study was conducted to elucidate this issue and to investigate the potential mechanism of opioid-induced delayed PC.

METHODSCultured porcine PAECs underwent 16-hour anoxia followed by 1-hour reoxygenation 24 hours after pretreatment with saline (NaCl; 0.9%) or morphine (1 micromol/L). To determine the underlying mechanism, a non-selective K(ATP) channel inhibitor glibenclamide (Glib; 10 micromol/L), a nitric oxide (NO) synthase blocker NG-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L), and an opioid receptor antagonist naloxone (Nal; 10 micromol/L) were given 30 minutes before the A/R load. The percentage of apoptotic cells was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. eNOS mRNA level was measured by real-time polymerase chain reaction (PCR). NO content of PAECs supernatants was measured with the Griess reagent.

RESULTSCompared to the A/R PAECs, morphine-induced delayed PC significantly reduced PAECs apoptosis ((18.1 +/- 1.9)% vs (5.5 +/- 0.3)%; P < 0.05), increased NO release ((11.4 +/- 1.3) micromol/L vs (20.5 +/- 2.1) micromol/L, P < 0.05), and up-regulated eNOS gene expression nearly 9 times (P < 0.05). The anti-apoptosis effect of morphine was abolished by pretreatment with Glib, L-NAME and Nal, but the three agent-selves did not aggravate the A/R injury. Furthermore, L-NAME and Nal offset the enhanced release of NO caused by pretreatment with morphine.

CONCLUSIONSMorphine-induced delayed PC prevents A/R injury of PAECs. This effect may be mediated by activation of K(ATP) channel via opioid receptor and NO signaling pathways.

Analgesics, Opioid ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Glyburide ; pharmacology ; In Situ Nick-End Labeling ; Morphine ; pharmacology ; NG-Nitroarginine Methyl Ester ; pharmacology ; Naloxone ; pharmacology ; Narcotic Antagonists ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; antagonists & inhibitors ; genetics ; metabolism ; Oxygen ; pharmacology ; Pulmonary Artery ; cytology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Swine