OBJECTIVETo determine the regulatory role and mechanism of nitric oxide (NO) in the development and hatching of mouse blastocysts.

METHODSThe Kunming female mice were superovulated and then mated with mature male mice. On the day 2.5 of their pregnancy, morulae were flushed from their uterine horns with culture media. Morulae were cultured in different concentrations of N-nitro-L arginine methyl ester (L-NAME), sodium nitroprusside (SNP), or the combination of L-NAME and SNP in culture media for 48 hours. The development and hatching of blastocysts were examined on day 4 and day 5 and the total numbers of blastocyst cells and cysteinyl aspartate specific proteinase 3 (caspase 3) were observed under confocal laser scanning microscope.

RESULTSWith the increase of the concentration of L-NAME or SNP, the hatching rate of blastocysts and the total number of blastocyst cells were significantly reduced. The addition of 10 nmol/L SNP in culture media with 5 mmol/L L-NAME significantly increased the development of blastocysts and promoted hatching of blastocysts. However, with increase of SNP concentration in culture media with 5 mmol/L L-NAME, the development and hatching rates of blastocysts were significantly decreased. L-NAME had no obvious effect on the expression of active caspase 3 in blastocyst cells. However,when being above 500 nmol/L,SNP significantly increased the expression of caspase 3 in blastocyst cells.

CONCLUSIONSNO plays an important role in development and hatching of mouse blastocysts. Excessively high or low NO can damage the division of blastomeres, resulting in the failure of the blastocyst development and hatching. Also, excessively high NO can lead to the apoptosis of the blastocyst cells.

Animals ; Arginine ; analogs & derivatives ; Blastocyst ; Culture Media ; Female ; Humans ; Male ; Mice ; Nitric Oxide ; Nitroprusside ; Pregnancy ; Uterus

OBJECTIVES: To study the change in asymmetric dimethylarginine (ADMA) in the circulation system of full-term infants with persistent pulmonary hypertension of the newborn (PPHN) and its association with treatment response, as well as the possibility of ADMA as a therapeutic target and a marker for treatment response. METHODS: A prospective study was performed. A total of 30 full-term neonates who were diagnosed with PPHN within 3 days after birth were enrolled as the PPHN group, and the neonates without PPHN, matched for gestational age and age, who were treated or observed in the department of neonatology were enrolled as the control group. Serum samples were collected on days 1, 7, and 14 of treatment. The high-performance liquid chromatography-tandem mass spectrometry was used to measure the serum concentrations of L-arginine, ADMA, and its isomer symmetric dimethylarginine (SDMA). RESULTS: For the neonates in the control group, the serum concentrations of ADMA and L-arginine continuously increased and the serum concentration of SDMA continuously decreased within the first 14 days of treatment. On days 1 and 14, there was no significant difference in the serum concentration of ADMA between the control and PPHN groups (P>0.05). On day 7, the PPHN group had a significantly higher serum concentration of ADMA than the control group (P<0.05), while there were no significant differences in serum concentrations of SDMA or L-arginine (P>0.05). Moreover, after 7 days of treatment, the PPHN neonates with a systolic pulmonary arterial pressure (sPAP) of >35 mmHg had a significantly higher serum concentration of ADMA than those with an sPAP of ≤35 mm Hg. CONCLUSIONS There are continuous increases in the ADMA concentration and the ADMA/SDMA ratio in the circulation system of full-term infants within the first 2 weeks after birth, and this process is accelerated by the pathological process of PPHN, suggesting that ADMA may be involved in the pathologic process of PPHN. A high level of ADMA is associated with the resistance to PPHN treatment, suggesting that inhibition of ADMA might be a potential target of drug intervention to improve the treatment response of PPHN.
Arginine/analogs & derivatives* ; Biomarkers ; Humans ; Hypertension, Pulmonary/drug therapy* ; Infant, Newborn ; Prospective Studies

OBJECTIVETo screen the asymmetric dimethyl arginines (ADMA)-containing proteins which could combine with protein arginine methyltransferase 1 (PRMT1).

METHODSWestern blot was adopted to identify the expression of PRMT1 and the proteins with ADMA in glioma cell lines and normal brain tissues, and then to detect the changes of ADMA level after knock-down of PRMT1 with RNAi transfection in U87MG cells. Co-Immunoprecipitation (Co-IP), western blot, and sliver staining were employed to screen the candidate binding proteins of PRMT1. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the binding proteins of PRMT1.

RESULTSThe expression of PRMT1 and some levels of ADMA were higher in glioma cell lines than in normal brain tissues. After knocking down PRMT1, some ADMA levels were found declined. After screening the binding proteins of PRMT1 with Co-IP and LC-MS/MS, 26 candidate binding proteins were identified. Among them, 6 candidate proteins had higher ions scores (> 38) and bioinformation analysis predicted that SEC23-IP, ANKHD1-EIF4EBP3 protein, and 1-phosphatidylinositol-3-phosphate 5-kinase isoform 2 had possible methylated aginine sites.

CONCLUSIONSThe high expression of PRMT1 in glioma may induce the change of ADMA levels. Altogether 26 candidate proteins were identified, which contain ADMA and specifically bind with PRMT1.

Arginine ; analogs & derivatives ; analysis ; Cell Line, Tumor ; Chromatography, Liquid ; Glioma ; chemistry ; Humans ; Immunoprecipitation ; Protein-Arginine N-Methyltransferases ; analysis ; physiology ; Repressor Proteins ; analysis ; physiology ; Substrate Specificity ; Tandem Mass Spectrometry

OBJECTIVETo compare plasma asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and cystatin C levels in patients with or without coronary artery disease (CAD).

METHODSWe recruited 87 CAD patients (39 with acute myocardial infarction and 48 with unstable angina pectoris) and 51 non-CAD controls. Plasma ADMA was measured by HPLC, cystatin C by particle-enhanced immunonephelometric assay (N Latex cystatin C, Dade Behring) with nephelometer (BNII, Dade Behring). CAD patients were further divided into low cystatin C group (< 1.0 mg/L, 36 cases) and high cystatin C group (> 1.0 mg/L, 51 cases).

RESULTS(1) The plasma levels of ADMA [(0.47 ± 0.15) µmol/L vs. (0.37 ± 0.15) µmol/L], SDMA [(0.39 ± 0.19) µmol/L vs. (0.28 ± 0.12) µmol/L] and cystatin C [(1.16 ± 0.32) mg/L vs. (0.73 ± 0.16) mg/L] were significantly higher in CAD patients than in controls (all P < 0.05). The plasma L-Arg was significantly lower in CAD patients than in controls [(59.4 ± 19.4) µmol/L vs. (83.7 ± 19.6) µmol/L, P < 0.05]. (2) Plasma ADMA was similar in CAD patients with low cystatin C level and controls [(0.42 ± 0.12) µmol/L vs. (0.39 ± 0.15) µmol/L, P = 0.251] and Plasma ADMA was significantly higher in CAD patients with high cystatin C level than in controls [(0.50 ± 0.17) µmol/L vs. (0.39 ± 0.15) µmol/L, P < 0.05].

CONCLUSIONADMA levels were significantly increased only in CAD patients with elevated cystatin C levels but not in CAD patients with normal renal function. The reported relationship between coronary heart disease and ADMA may not be direct, but could be secondary due to reduced renal function.

Aged ; Arginine ; analogs & derivatives ; blood ; Case-Control Studies ; Coronary Disease ; blood ; Cystatin C ; blood ; Female ; Humans ; Male ; Middle Aged

OBJECTIVETo study changes of plasma ADMA levels of patients with non-ST elevation myocardial infarction (NSTEMI) undergoing percutaneous coronary intervention (PCI) and to explore the effect of Salvia Miltiorrhiza (SM) on them.

METHODSTotally 52 patients with confirmed NSTEMI undergoing PCI were randomly assigned to the SM treated group and the control group, 26 in each group. Patients in the SM treated group received the conventional therapy plus SM (1 g each time, three times per day till one month after PCI). Those in the control group only received the conventional therapy. Plasma ADMA levels were measured before PCI, and at day 1 and 30 after PCI.

RESULTSPlasma ADMA levels in both group obviously decreased at day 30 after PCI with statistical difference (P < 0.01). The decrement was more obviously seen in the SM treated group, with statistical difference when compared with the control group (P < 0.01).

CONCLUSIONSPatients with NSTEMI undergoing PCI could have plasma ADMA levels decreased. Administration of SM just before PCI might be associated with negative regulating plasma ADMA levels.

Arginine ; analogs & derivatives ; blood ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Myocardial Infarction ; metabolism ; Percutaneous Coronary Intervention ; Salvia miltiorrhiza

OBJECTIVE: To examine the prognostic value of serum levels of asymmetric dimethylarginine (ADMA) in patients with stable coronary heart disease (CHD) thus explore a potential biomarker of "toxin syndrome" in CHD. METHODS: In this prospective nested case-control study, 36 of 1,503 Chinese patients with stable CHD experienced at least 1 recurrent cardiovascular event (RCE) during 1-year follow-up. Serum levels of ADMA at the start of follow-up were compared between these 36 cases and 36 controls which matched to cases in terms of gender, age, history of hypertension, and myocardial infarction. RESULTS: Based on the crude model, subjects in the 2 highest ADMA quartiles showed significantly higher risk of developing RCE than those in the lowest ADMA quartile [odds ratio (OR) 4.09, 95% confidence interval (CI) 1.01 to 16.58; OR 6.76, 95% CI 1.57 to 29.07]. This association was also observed in the case-mix model (OR 5.51, 95% CI 1.23 to 24.61; OR 7.83, 95% CI 1.68 to 36.41) and multivariable model (OR 6.64, 95% CI 1.40 to 31.49: OR 13.14, 95% CI 2.28 to 75.71) after adjusting for confounders. The multivariable model which combined ADMA and high-sensitivity C-reactive protein (hsCRP) showed better predictive power with areas under the receiver operator characteristic curves (0.779) than the model of either ADMA (0.694) or hsCRP (0.636). CONCLUSION Serum ADMA level may be a potential biomarker of "toxin syndrome" in CHD which shows favorable prognostic value in predicting 1-year RCE in patients with stable CHD. [The registration number is ChiCTR-PRNRC-07000012].
Arginine ; analogs & derivatives ; blood ; Biomarkers ; blood ; Coronary Disease ; blood ; Humans ; Odds Ratio ; ROC Curve ; Recurrence ; Risk Factors ; Syndrome

OBJECTIVETo observe the effect of curcumin on nitric oxide (NO) in plasma of atherosclerotic rabbits, activity of constitutive nitric oxide synthase (cNOS) and asymmetric dimethylarginine (ADMA), and discuss curcumin's effect against AS and its correlation with ADMA.

METHODThirty-eight male Japanese white rabbits were randomly divided into four groups: the control group (eight rabbits fed with standard diets), the model group (ten rabbits fed with high-fat diets), the low dose curcumin group (ten rabbits fed with high-fat diets and 100 mg . kg-1 d -1 ) and the high dose curcumin group (ten rabbits fed with high-fat diets and 200 mg kg-1 d-1 curcumin). At the end of the 12th week, their plasmas were tested for TC, LDL-C, NO, endothelin (ET) , ADMA and activity of aortic cNOS. Aortic tissues were collected for histological examinations.

RESULTThe three groups fed with high-fat diets showed higher plasma ADMA and ET than the control group (P <0. 01) , but with decrease in plasma NO concentration and arterial cNOS activity (P <0. 01). Compared with the model group (P <0. 05) , the curcumin groups showed lower plasma ADMA and ET (P <0. 05), but higher plasma NO concentration and arterial cNOS activity than the model group (P <0. 01). There was no significant difference between the two curcumin groups.

CONCLUSIONCurcumin may play an important protective role in AS process by reducing plasma ADMA level. [Key words] atherosclerosis; asymmetric dimethylarginine; crucumin; nitric oxide; nitric oxide synthase

Animals ; Arginine ; analogs & derivatives ; blood ; Atherosclerosis ; blood ; drug therapy ; metabolism ; Curcumin ; therapeutic use ; Endothelium, Vascular ; drug effects ; Male ; Nitric Oxide Synthase ; metabolism ; Rabbits

OBJECTIVETo investigate the effects of a novel tripeptide analog of arginine on isoproterenol (ISO), a beta-adrenergic receptor agonist, induced the change in concentration transient cytosolic free calcium in cultured neonatal rat cardiac myocytes (CMs).

METHODSThe expression of inducible nitric oxide synthase (iNOS) was induced in cultured neonatal rat CMs by stimulation with lipopolysaccharide (LPS) and interleukin-6 (IL-6) for 24 h, and quantitatively measured using Western blotting. The CMs were incubated in the presence of the new arginine analog for 6 h and the changes of fluorescence signal of free calcium in response to isoproterenol (ISO) treatment were measured under laser scanning confocal microscope.

RESULTSIncubation of the CMs for 24 h in the presence of IL-6 and LPS resulted in significantly increased nitric oxide (NO) production, and also in increased iNOS protein accumulation in the cells. Specific inhibition of iNOS by the new arginine analog substantially inhibited NO production and increased the peak value of ISO-induced Ca2+ transient.

CONCLUSIONThe new arginine analog strongly inhibits IL-6 and LPS-induced NO production and increases beta-adrenergic responsiveness in cultured neonatal rat CMs.

Animals ; Animals, Newborn ; Arginine ; analogs & derivatives ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Isoproterenol ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Oligopeptides ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the protective effect of neferine against damages of endothelial cells induced by lysophos-phatidylcholine (LPC) and the relationship with asymmetric dimethylarginine (ADMA).

METHODThe human umbilical vein endothelial cells (HUVEC-12) were treated with LPC (10 mg x L(-1)) for 24 h to establish the model of endothelial cells damages; HUVECs were prior exposed to neferine (0.1, 1.0 or 10.0 micromol x L(-1) ) for 1 h, and then exposed to LPC in the presence of the neferine for 24 h. At the end of the experiment, the cultured medium was collected for measuring the concentration of nitric oxide (NO), aleic dialdehyde (MDA) as well as ADMA and the cells were collected for measuring the level of intracellular reactive oxygen species (ROS).

RESULTCompared with control group, exposure of endothelial cells to LPC (10 mg x L(-1)) for 24 h significantly increased the concentration of MDA and ADMA in the medium and the level of intracellular ROS and coinstantaneously significantly decreased the concentration of NO in the medium. Neferine (0.1, 1.0 or 10.0 micromol x L(-1)) significantly inhibited the elevated concentration of MDA, ADMA as well as the level of intracellular ROS and coinstantaneously significantly attenuated the decreased level of NO induced by LPC.

CONCLUSIONNeferine can protect the endothelial cells against damages induced by LPC and the protective effect is related to the decrease of the concentration of ADMA.

Arginine ; analogs & derivatives ; metabolism ; Benzylisoquinolines ; pharmacology ; Cell Line ; Endothelial Cells ; drug effects ; metabolism ; Humans ; Lysophosphatidylcholines ; pharmacology ; Malondialdehyde ; metabolism ; Nitric Oxide ; metabolism ; Reactive Oxygen Species ; metabolism

OBJECTIVETo evaluate the effects of arginine modified chitosan or hexadecylated modified chitosan as gene carriers on the cellular uptake by vascular smooth muscle cells and its in vitro cytotoxicity. METHODS Plasmid DNA was labeled with alpha-32P-dATP and complexed with the modified chitosans or unmodified chitosan to form nanoparticle complexes by complex coacervation method. Uptake of all kinds of chitosan/ DNA nanoparticle complexes (CNC) by A10 cells was measured by beta-liquid scintillation counting. The in vitro cytotoxicity of the CNC was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay.

RESULTSThe diameters of the CNC ranged from 55.9-174.9 nm and the zeta potentials were from 10. 8 mV for the arginine modified chitosan/DNA nanoparticle complexes (ACNC) to 1.8 mV for the hexadecylated chitosan/DNA nanoparticle complexes (HCNC). The cellular uptake of the modified chitosan/ DNA nanoparticle complexes (MCNC) by A10 cells increased significantly when compared with the unmodified chitosan/DNA nanoparticle complexes (UCNC) (P < 0.05), with the HCNC at N/P ratio of 1:1 and the ACNC at ratio of 8:1 showing the highest cellular uptake (1.3 fold higher than UCNC, P < 0.05). MCNC were much less cytotoxic when compared with Lipofectamine 2000-DNA nanoparticles.

CONCLUSIONDNA nanoparticle complexes prepared with either arginine or hexadecylated modified chitosan can improve the cellular uptake of the DNA, while the in vitro cytotoxicity of both of the modified chitosan is much less than that of Lipofectamine 2000.

Animals ; Antigen-Antibody Complex ; Arginine ; pharmacology ; Chitosan ; chemistry ; pharmacology ; Citric Acid ; analogs & derivatives ; pharmacology ; Cytotoxicity, Immunologic ; DNA ; pharmacology ; Genetic Vectors ; Nanoparticles ; Rats