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The study was designed to establish a 2D UPLC-QTOF method to extrapolate the structure of an unknown substance in carboplatin injection and its relationship with the excipient. By using phenyl-hexyl column (250 mm×4.6 mm, 5 μm) with mobile phase consisting of tetrabutylammonium sulfate buffer (pH 7.5) and acetonitrile in gradient elution mode, an unknown impurity in carboplatin injection was found and quantitatively determined. Then a 2D UPLC-QTOF, HSS T3 column (100 mm×2.1 mm, 1.7 μm) was employed to confirm the molecular weight and the structure of the unknown impurity (electrospray ionization source, positive ion mode, MSE mode) with mobile phase consisting of 0.1% formic acid and acetonitrile. The relationship among impurities, API and excipient was investigated by accelerated stability test with ICP-MS/MS, ICP-AES. Results showed that disodium edetate in the formulation interacted with carboplatin producing an unknown impurity containing platin, and induced the increase of 1,1-cyclobutanedicarboxylic acid. The research should be done on the rationality of the addition of disodium edetate in such injections containing heavy metals.
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Objective To investigate the effects and mechanisms of perilipin-5 (Plin5) on the apoptosis of mouse cardiac microvascular endothelial cells induced by high fat and high glucose.Methods The mouse cardiac microvascular endothelial cells (MCMECs) cultured with high glucose medium were respectively given 0,100,300 and 500μmol/L palmitic acid for 24 hours.In order to explore the effects and mechanisms of Plin5 on MCMECs injuries induced by high fat and high glucose,MCMECs exposed to 300μmol/L palmitic acid for 24 hours were divided into control group,Scra siRNA group and Plin5 siRNA group.The control group was only treated with transfection reagent,the Scra siRNA group was given treatment of transfection reagent and garbled RNA,the Plin5 siRNA group was given treatment of transfection reagent and Plin5 specific siRNA.In order to further confirm the specific mechanism of Plin5 in high fat/glucose inducing MCMECs injury,MCMECs in Plin5 siRNA group were divided into vehicle group and N-acetyl cysteine (NAC) group,and given the same intervention of high fat.The apoptotic rate was detected by flow cytometry,qRT-PCR and Western blotting were respectively used to detect the mRNA and protein expression of Plin5,and the intracellular reactive oxygen species (ROS) level was tested by DHE staining and ELISA kit.Results The apoptotic rate of MCMECs was increased in a fat concentration-dependent manner (P<0.05).Compared with 0μmol/L palmitic acid group,the intracellular ROS content and the expression of Plin5 increased significantly in 300μmol/L palmitic acid group (P<0.05).Compared with the control group and the Scra siRNA group,the intracellular ROS content and apoptotic rate increased significantly in Plin5 siRNA group under the action of 300μmol/L palmitic acid (P<0.05).Compared with the vehicle group,the intracellular ROS content and apoptotic rate decreased remarkably in NAC group (P<0.05).Conclusion With inhibition of oxidative stress,Plin5 may reduce the apoptosis of MCMECs induced by high fat and high glucose.
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Objective To investigate the role of mitochondrial calcium uptake 1 (MICUI) in myocardial hypertrophy of mice and underlying mechanism.Methods The model of myocardial hypertrophy was established via incubation of mouse cardiac myocytes (MCM) with 300nmol/L angiotensin Ⅱ (Ang Ⅱ) for 48 hours in vitro.After that,MICU1 specific small interfering RNA (siRNA) was delivered to knockdown MICU1 levels in MCM.On the other hand,adenovirus-mediated over-expression of MICU 1 was transfected into MCM.Accordingly,the expressions of ANP and BNP in myocardial cells were measured by qRT-PCR.Mitochondrial membrane potential and ATP contents were detected byJC-1 assay kit and ATP assay kit,respectively.Then,Western blotting and qRT-PCR were used to detect the levels of MICU1 in myocardial cells.The mitochondrial Ca2+ contents were measured via atomic absorption flame spectroscopy.The size of myocardial cells was determined by α-actinin staining.Results Mitochondrial membrane potential and ATP contents in hypertrophic cardiomyocytes induced by Ang Ⅱ were both decreased.Meanwhile,myocardial hypertrophy significantly increased mitochondrial Ca2+ contents but decreased MICU1 levels.With the method of genetic intervention,we found that MICUI deficiency exacerbated mitochondrial Ca2+ overload,increased cell surface and elevated the expression of BNP.Conversely,the overexpression of MICU1 obviously decreased mitochondrial Ca2+ overload,cell surface of MCM and expressions of ANP and BNP.Conclusion MICU1 alleviates Ang Ⅱ-induced myocardial hypertrophy via inhibiting mitochondrial Ca2+ overload.
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As nanomedicines are developing fast in both academic and market areas, building up suitable methods for nanomedicine analysis with proper techniques is an important subject, requiring further research. The techniques, which could be employed for grain size analysis of nanomedicines, were reviewed. Several key techniques were discussed with their principles, scope of applications, advantages and defects. Their applications to nanomedine analysis were discussed according to the properties of different nanomedicines, with the purpose of providing some suggestions for the control and administration of nanomedicines.
Subject(s)
Drug Delivery Systems , Light , Microscopy, Electron , Methods , Microscopy, Scanning Probe , Methods , Nanoparticles , Chemistry , Classification , Particle Size , Scattering, Radiation , Scattering, Small Angle , Spectrum Analysis, Raman , Methods , X-Ray Diffraction , MethodsABSTRACT
<p><b>OBJECTIVE</b>To explore the relationship between heavy metals exposure and neurobehavioral function impairment in welders.</p><p><b>METHODS</b>The metals exposure in 82 welders and 51 operators were investigated with blood Pb, Cd and Mn via AAS, and the nervous impairment was evaluated with neurobehavioral core test battery (NCTB).</p><p><b>RESULTS</b>Pb [(115.49 +/- 79.22) microg/L] and Cd [(3.67 +/- 3.19) microg/L] in welders were significantly higher than operators [(69.32 +/- 50.79) and (0.83 +/- 0.76) microg/L respectively] (P < 0.05). Welders had worse standard scores of NCTB 13 items such as depression-dejection than non-welders (P < 0.05). Significant difference of confusion-bewilderment and forward digit span in welders only existed in different groups of Pb and Mn, respectively. A dose-effect relationship was found between forward digit span and serum Mn level in welders. General linear regression analysis indicated that Pb exposure, Mn exposure and alcohol consume had negative relation with the loss of nervous system function.</p><p><b>CONCLUSION</b>The nervous impairment in welders is attributed to occupational exposure to Pb and Mn, concomitantly.</p>
Subject(s)
Adult , Female , Humans , Male , Young Adult , Air Pollutants, Occupational , Central Nervous System Diseases , Cross-Sectional Studies , Metals, Heavy , Neuropsychological Tests , Occupational Exposure , WeldingABSTRACT
<p><b>OBJECTIVE</b>To investigate the genetic relationships of Erigeron breviscapus at the molecular biology level.</p><p><b>METHOD</b>Thirty seven germplasm resources of E. breviscapus which collected from Yunnan, Sichuang and Guizhou province in 2005 were analyzed by Random amplified polymorphic DNA (RAPD) and cluster analysis based on NTSYS2.</p><p><b>RESULT</b>A total of 10 primers were screened, and 107 bands were amplified, among which 94 (87.85%) bands were found to be polymorphic. Thirty seven germplasm resources of E. breviscapus were clustered into 3 groups at genetic distance 0.36, the I group include in 9 germplasm resources collected from Mile, Qiubei, Luxi, Gejiu, and Yanshan of south east of Yunnan province; the II group included 8 germplasm resources collected from Gucheng, and shangrila of north west of Yunnan province, and Mile and Qiubei of south east of Yunnan province; the III group included in 20 germplasm resources collected from the center of Yunnan province, north east of Yunnan province, Sichuan province, and Guizhou province.</p><p><b>CONCLUSION</b>There were abundant genetic diversity in the germplasm resources of E. breviscapus, and the genetic relationships are closely related to geographical distance where they were collected.</p>
Subject(s)
Cluster Analysis , Erigeron , Cell Biology , Genetics , Genetic Variation , Polymerase Chain Reaction , Random Amplified Polymorphic DNA TechniqueABSTRACT
<p><b>OBJECTIVE</b>To ascertain the optimal harvest time of Erigeron breviscapus.</p><p><b>METHOD</b>The dry matter weight accumulation of different organs in growth process and contents of scutellarin and coffeic acid ester in whole plant of E. breviscapus were determined.</p><p><b>RESULT</b>The number of leaves per plant, the dried weight of single leaf and dry matter weight of whole plant and different organs reached the highest after seedling 130-140 d. The content of scutellarin gradually decreased with growth period, and sharply decreased after seedling 140 d. The content of coffeic acid ester varied irregularly with growth period.</p><p><b>CONCLUSION</b>The optimal harvest time of E. breviscapus is in early bloom period after seedling 130 d.</p>
Subject(s)
Apigenin , Biomass , Erigeron , Chemistry , Gardening , GlucuronatesABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of angiotensin II (AngII) receptor (AT(1), AT(2)) antagonists on myocardial matrix metalloproteinases (MMPs) and fibronectin (FN) in rats with myocardial infarction (MI).</p><p><b>METHODS</b>Rat MI was induced by permanent ligation of the left coronary artery. Placebo, AT(1) receptor antagonist valsartan (10 mgxkg(-1)xd(-1)) or AT(2) receptor antagonist PD123319 (30 mgxkg(-1)xd(-1)) were given 7 days prior MI surgery. On the 1st, 3rd and 7th day after MI, Expressions of MMP-2, 3, 9, tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) and FN at protein level were determined by Western blot in left ventricular free wall (LVFW), interventricular septum (IS) and right ventricular (RV). Myocardial FN distribution was also assayed by immunofluorescence.</p><p><b>RESULTS</b>Typical myocardial remodeling was shown in IS and LVFW 7 days after MI. MMP-2, 3, 9 expressions at protein level were significantly increased whereas TIMP-1 and FN expressions significantly decreased in IS 1, 3, 7 days post MI in a time-dependent manner compared to that of sham operated hearts. MMP-2, 3, 9 expressions was significantly increased and TIMP-1 and FN expression significantly decreased in LVFW at the 1st post MI day and maintained up to 7th post MI day compared to that of sham operated hearts. Up-regulated expressions of MMP-2, 3, 9 and down-regulated TIMP-1 and FN expressions in IS and LVFW could be significantly attenuated by valsartan but not by PD123319. Valsartan but not PD123319 also significantly reduced MI sizes (40.4% +/- 2.1% vs 49.5% +/- 2.1%, P < 0.05).</p><p><b>CONCLUSION</b>AT(1) receptor antagonist involves in the pathology procession of myocardial remodeling and might lead to the development and progression of congestive heart failure by the increasing expressions of MMP-2, 3, 9, which contribute to degradative extracellular matrix FN in myocardium.</p>
Subject(s)
Animals , Male , Rats , Angiotensin II Type 1 Receptor Blockers , Therapeutic Uses , Fibronectins , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 3 , Matrix Metalloproteinase 9 , Myocardial Infarction , Drug Therapy , Pathology , Rats, WistarABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of aluminum chloride on dissociated Ca(2+) in hippocampus neuron cells in mice and the relationship to the learning and memory.</p><p><b>METHODS</b>Male ICR mice in the three intoxicated groups were administered with the double distilled water containing AlCl(3) (10, 50, 300 mg.kg(-1).d(-1)) while those in the control group were administered with the double distilled water for 100 days. The methods of behavior toxicology such as Morris swim maze were used for studying the effect of aluminum chloride on the changes of learning and memory in mice. With calcium sensitive fluorescence indicator Fura-2 as the fluorescent probe, the influence of the subchronic exposure to Al on the dissociated Ca(2+) in hippocampus neuron cells was observed.</p><p><b>RESULTS</b>The dissociated Ca(2+) in hippocampus neuron cells in the middle dosage group and the high dosage group [(412.25 +/- 53.20), (467.37 +/- 32.85) times] was lower than those in the control group [(293.91 +/- 32.21) times] respectively (P < 0.01), and correlated positively with the dose and dissociated Ca(2+) (r = 0.861, P < 0.01). Compared with the control group, the latent period was lengthened (P < 0.05) in the middle dosage and the high dosage group.</p><p><b>CONCLUSION</b>The subchronic exposure to AlCl(3) in mice affects the dissociated Ca(2+) in hippocampus neuron cells. The increase of dissociated Ca(2+) in hippocampus neuron cells may be correlated with the disfunction of cognition in the aluminium intoxicated mice.</p>
Subject(s)
Animals , Male , Mice , Aluminum Compounds , Pharmacology , Toxicity , Calcium , Metabolism , Chlorides , Pharmacology , Toxicity , Dose-Response Relationship, Drug , Hippocampus , Cell Biology , Metabolism , Learning , Memory , Mice, Inbred ICR , Neurons , MetabolismABSTRACT
<p><b>AIM</b>To investigate the relation among myocardial angiotension II receptor (AT1/AT2) expression, calpain and cardiac function in patients with congestive heart failure (CHF).</p><p><b>METHODS</b>Message RNA (mRNA) expression of AT1/AT2 receptor in myocardial tissue of 39 patients with CHF due to valvular heart disease and 8 control subjects were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR). Immunoprecipitation was used to assay the protein expression of u-calpain and m-calpain.</p><p><b>RESULTS</b>Pathological changes of myocardial tissue in CHF due to valvular heart disease showed typical myocardial remodeling. AT1 receptor mRNA expression was slightly increased in the patients with mild CHF than in the control subjects, but decreased in the moderate and severe CHF patients. No difference was observed in AT2 receptor mRNA expression among all the groups, but the ratio of AT1/AT2 decreased. The protein expression of u-calpain and m-calpain were positively correlated to the levels of cardiac function in patient with CHD.</p><p><b>CONCLUSION</b>The expression of AT1 receptor is down-regulated in moderate and severe CHF, the dominant receptor subtype is transformed to AT2. Cardiomyocyte apoptosis or death may be induced via AT2 receptor to activate calpain system, leading the deterioration of cardiac function.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Young Adult , Calpain , Metabolism , Case-Control Studies , Heart Failure , Metabolism , Myocardium , Metabolism , Receptor, Angiotensin, Type 2 , Metabolism , Ventricular RemodelingABSTRACT
<p><b>OBJECTIVE</b>To study the effect of aluminum chloride on motor and species-typical behaviors in mice.</p><p><b>METHODS</b>Male ICR mice were administered with drinking double distilled water only containing AlCl(3) (10, 50, 300 mg x kg(-1) x d(-1)), and control group with drinking double distilled water only for 100 days. Spontaneous activity test, grip strength, beam traversal, tightrope task, food hoarding, and nest construction were used to study the effect of chloride aluminum on motor and species-typical behaviors in mice.</p><p><b>RESULTS</b>The frequencies of spontaneous activity in low dose group, medium dose group and high dose group [(81.53 +/- 8.97), (71.67 +/- 8.37), (66.73 +/- 6.96) times respectively] were lower than that in control [(106.46 +/- 8.21) times] (P < 0.01), and were negatively correlated with doses (r(s) = -0.42, P < 0.01). Grip strength scores in medium dose group (19.19 +/- 1.48) and high dose group (13.36 +/- 1.46) respectively were lower than that in control (24.31 +/- 1.43) (P < 0.05, P < 0.01). Food hoarding was greater in high dose group [96.10 (90.20-99.00) g] than that in control group [84.00 (78.00-90.00) g (P < 0.05)]. The rest of parameters were of no statistical significance.</p><p><b>CONCLUSION</b>Subchronic exposure to AlCl(3) in mice may diminish motor activity and grip strength, but motor coordination was not impaired; alteration in food hoarding suggests damage to hippocampus cell.</p>
Subject(s)
Animals , Male , Mice , Aluminum Compounds , Toxicity , Behavior, Animal , Chlorides , Toxicity , Dose-Response Relationship, Drug , Mice, Inbred ICR , Motor ActivityABSTRACT
<p><b>OBJECTIVE</b>To investigate the regulation of calcium sensitive signal substance calpain in signal transduction of myocardial remodeling in patients with congestive heart failure.</p><p><b>METHODS</b>All 39 congestive heart failure (CHF) patients with rheumatic mitral valve stenosis disease were selected and 38 cases of healthy persons were included as controls. Cardiac function parameters were measured by echocardiography. The concentration of angiotension II (AngII) in plasma and myocardial tissues was determined by radio immunoassay (RIA). Western blot was used to assay the protein expression of calpain, cain/cabin 1, cain/cabin 1Delta, and calcineurin (CaN) phosphorylation.</p><p><b>RESULTS</b>The AngII concentrations in the plasma and myocardial tissues in patients with CHF were higher than those in the control group. Meanwhile the AngII concentrations positively correlated to the parameters of the cardiac dilation respectively but negatively correlated to the parameters of cardiac function. Pathological changes of myocardial tissues in CHF with valvular heart disease showed typical myocardial remodeling. The hypertrophy was dominant at early stage of CHF, while at the end stage the characteristics include disordered alignment of the myocytes, the discontinuity and dissolving of cardiomyofibrills, destroyed subcellular organs, and the hyperplasia of interstitial tissue. Compared to the control group, u-calpain, m-calpain, and cain/cabin 1Delta protein expression, CaN phosphorylation in myocardial tissues in CHF groups were highly expressed and their expressions were positively correlated to the severity of CHF. The expression of cain/cabin1 deceased and its expression was negatively correlated to the severity of CHF.</p><p><b>CONCLUSION</b>The degradation of cain/cabin 1 by calpain may play an important role by causing the activation of CaN signal pathway in myocardial remodeling mediated by renin angiotension system in CHF.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Adaptor Proteins, Signal Transducing , Angiotensin II , Blood , Calcineurin , Metabolism , Calpain , Metabolism , Heart Failure , Metabolism , Myocardium , Metabolism , Pathology , Signal Transduction , Ventricular RemodelingABSTRACT
To explore the role of platelet derived growth factor-AA (PDGF-AA) and PDGFR-alpha expression in the proliferation and hypertrophy of vascular smooth muscle cells (VSMCs) in spontaneously hypertension rats (SHR), protein expression of PDGF-AA, PDGFR-alpha and PDGFR-beta in SHR/Wistar-Kyoto (WKY)-VSMC was observed by Western blot. Proliferation and hypertrophy of SHR-VSMCs induced by PDGF-AA were observed by measurement of PCNA and [(3)H] incorporation. PDGF-AA and PDGFR-alpha expression was markedly increased in SHR-VSMCs compared with that in WKY-VSMCs, but PDGFR-beta was not different in SHR and WKY-VSMCs. PDGF-AA induced PCNA expression and [(3)H] incorporation was increased in a dose-dependent manner in SHR, but not in WKY. It is suggested that an enhancement of PDGF-AA and PDGFR-alpha in SHRs may be one of the important factors for vascular modeling.
Subject(s)
Animals , Male , Rats , Cell Division , Cells, Cultured , Hypertension , Muscle, Smooth, Vascular , Cell Biology , Metabolism , Platelet-Derived Growth Factor , Pharmacology , Rats, Inbred SHR , Rats, Inbred WKY , Receptor, Platelet-Derived Growth Factor alphaABSTRACT
Objective To evaluate the role of transfected angiotensinⅡ(Ang Ⅱ) receptor AT1 anti-sense nucleotide (AT1A) in the expression of subtypes of AngⅡ receptor mRNA, synthesis of protein and nucleic acid in cardiomyocytes. Methods AT1 cDNA sequence (476 bp) was cloned with RT-PCR and reversely inserted into PcDNA3.1 (5.4 kb) to construct an intact plasmid containing AT1A (PAT1A). The plasmid was then transfected into the cultured cardiomyocytes and identified with RT-PCR and Western blot. The synthesis of protein and nucleic acid identified by 3H-Leu and 3H-TdR incorporation, and expressions of AT1 and AT2 mRNA by RT-PCR, were compared between transfected and nontransfected cardiomyocytes after being stimulated with 10-7 mol/L AngⅡ for 24 h. Results The plasmid PAT1A were successfully constructed. The AT1 mRNA and its protein were expressed significantly less in the transfected cardiomyocytes than in the control (P<0.01). In the transfected cardiomyocytes, AT1 mRNA expression was markedly decreased, but that of AT2 mRNA obviously increased (P<0.01) when compared with the nontransfected cardiomyocytes after stimulation for 24 h with AngⅡ 10-7 mol/L; no significant difference was found in 3H-Leu and 3H-TdR incorporation between them. Conclusion After the cardiomyocytes was tranfected with AT1A, the expression of AT1 mRNA was markedly suppressed,while AT2 mRNA up-regulated at the same time. Our results indicate that AT1A blocking can not effectively interrupt the Ang Ⅱ-induced synthesis of the protein and nucleic acid in cardiomyocytes.
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Objective To evaluate the role of transfected angiotensinⅡ(Ang Ⅱ) receptor AT1 anti-sense nucleotide (AT1A) in the expression of subtypes of AngⅡ receptor mRNA, synthesis of protein and nucleic acid in cardiomyocytes. Methods AT1 cDNA sequence (476 bp) was cloned with RT-PCR and reversely inserted into PcDNA3.1 (5.4 kb) to construct an intact plasmid containing AT1A (PAT1A). The plasmid was then transfected into the cultured cardiomyocytes and identified with RT-PCR and Western blot. The synthesis of protein and nucleic acid identified by 3H-Leu and 3H-TdR incorporation, and expressions of AT1 and AT2 mRNA by RT-PCR, were compared between transfected and nontransfected cardiomyocytes after being stimulated with 10-7 mol/L AngⅡ for 24 h. Results The plasmid PAT1A were successfully constructed. The AT1 mRNA and its protein were expressed significantly less in the transfected cardiomyocytes than in the control (P<0.01). In the transfected cardiomyocytes, AT1 mRNA expression was markedly decreased, but that of AT2 mRNA obviously increased (P<0.01) when compared with the nontransfected cardiomyocytes after stimulation for 24 h with AngⅡ 10-7 mol/L; no significant difference was found in 3H-Leu and 3H-TdR incorporation between them. Conclusion After the cardiomyocytes was tranfected with AT1A, the expression of AT1 mRNA was markedly suppressed,while AT2 mRNA up-regulated at the same time. Our results indicate that AT1A blocking can not effectively interrupt the Ang Ⅱ-induced synthesis of the protein and nucleic acid in cardiomyocytes.