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Objective:To explore the disturbance of metal element balance in mice after exposure to radon.Methods:Mice were randomly divided into control group, radon exposure of 30 WLM group, 60 WLM group and 120 WLM groups, with 10 mice in each group. After radon exposure with the cumulative dose, the lung function of mice was detected by a non-invasive pulmonary function testing instrument. Mice blood was taken from eyeballs. The lungs, heart, liver, kidney and spleen were also collected. HE staining was used to observe the pathological changes of lung tissue. Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect the content of metal elements, including essential trace elements in the body: chromium (Cr), molybdenum (Mo), cobalt (Co), selenium (Se), copper (Cu), zinc (Zn), manganese (Mn), nickel (Ni), and potentially toxic elements: arsenic (As), tin (Sn), lead (Pb), aluminum (Al), mercury (Hg), cadmium (Cd), and silver (Ag).Results:Compared with the control group, lung ventilation function of the radon-exposed mice was decreased, alveolar structure was destroyed, and the contents of pulmonary metal elements Cr, Al, Pb, Sn( F=0.34, 0.66, 3.14, 1.16, P<0.05) and essential trace elements Mn, Cr, Zn, and Mo in the blood were decreased( F=0.65, 1.44, 0.97, 2.08, P<0.05), while the elements of Cu, Mo, Se and As in the lungs were increased( F=1.31, 1.26, 0.81, 2.04, P<0.05), and the element contents in other tissues also fluctuated. Conclusions:Inhalation of a certain cumulative dose of radon can reduce the lung ventilation function of mice and induce lung inflammation, as well reduce the content of essential trace elements in the lung and blood so that the content of metal elements in the body fluctuates.
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PEP06 is a novel endostatin-Arg-Gly-Asp-Arg-Gly-Asp(RGDRGD)30-amino-acid polypeptide featuring a terminally fused RGDRGD hexapeptide at the N terminus.The active endostatin fragment of PEPO6 directly targets tumor cells and exerts an antitumoral effect.However,little is known about the kinetics and degradation products of PEP06 in vitro or in vivo.In this study,we investigated the in vitro metabolic stability of PEP06 after it was incubated with living cells obtained from animals of different species;we further identified the degradation characteristics of its cleavage products.PEP06 underwent rapid enzymatic degradation in multiple types of living cells,and the liver,kidney,and blood play important roles in the metabolism and clearance of the peptides resulting from the molecular degradation of PEP06.We identified metabolites of PEP06 using full-scan mass spectrometry(MS)and tandem MS(MS2),wherein 43 metabolites were characterized and identified as the degradation metabolites from the parent peptide,formed by successive losses of amino acids.The metabolites were C and N terminal truncated products of PEP06.The structures of 11 metabolites(M6,M7,M16,M17,M21,M25,M33,M34,M39,M40,and M42)were further confirmed by comparing the retention times of similar full MS spectrum and MS2 spectrum information with reference standards for the synthesized metabolites.We have demonstrated the metabolic stability of PEP06 in vitro and identified a series of potentially bioactive downstream metabolites of PEP06,which can support further drug research.
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Recombinant humanized anti-ricin monoclonal antibody (MIL50) is a recombinant humanized monoclonal antibody targeting ricin. In this study, an ELISA method was used to establish a method for the determination of MIL50 in macaque serum, and a cross design method was used. Twelve rhesus monkeys were intravenously injected 1 mg·kg-1 test preparation (MIL50 freeze-died powder injection) and reference preparation (MIL50 liquid preparation) to determine the plasma concentration of MIL50 at different time points, and the pharmacokinetic parameters were analyzed to compare the pharmacokinetic characteristics of MIL50 liquid preparation and freeze-died powder injection in rhesus monkeys. Animal welfare and experimental procedures follow the regulations of the Animal Ethics Committee of the Chinese Academy of Medical Sciences and Use of Laboratory Animals and the regulations derived by the Animal Care and Welfare Committee of the Institute of Radiation Medicine, Academy of Military Medical Sciences (IACUC-DWZX-2020-503). The results showed that there was no significant difference between Cmax and AUC0-5d in the two groups. The liquid preparation was the reference preparation, with Cmax ratio of 101.6% and AUC0-5d ratio of 101.9%, the 90% confidence interval of Cmax was 79.42%-129.92%, and the 90% confidence interval of AUC0-5d was 85.72%-121.18%. These results suggested that different dosage forms of MIL50 had certain differences in the changes of blood drug concentration in rhesus monkeys.
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OBJECTIVE@#To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology.@*METHODS@#shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins.@*RESULTS@#The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G/G phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex.@*CONCLUSION@#Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.
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Humans , Apoptosis , Cell Proliferation , Gene Knockdown Techniques , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Nuclear ProteinsABSTRACT
OBJECTIVE@#To investigate the effect of baicalin derivative 02-036 on proliferation and apoptosis human Burkitt lymphoma cell line CA46 and its related mechanisms.@*METHODS@#The MTT assay and cell colony formation assay were used to measure the growth inhibition of CA46 cells after 02-036 treatment. The flow cytometry with AnnexinV-FITC/PI double staining was employed to detect the apoptosis induction effect of 02-036 on CA46 cells. Cell cycle distribution of CA46 cells was estimeted by using DNA ploid analysis. Western blot was used to determine the changes of apoptosis-related proteins, including C-MYC, BCL-2, Procaspase-9, Procaspase-3, PARP and Cleaved-PARP.@*RESULTS@#Baicalin derivative 02-036 obviously inhibited the proliferation of CA46 cells, with dose- and time-dependent manner (r=0.963, r=0.992). The averaged IC value of CA46 cells was (6.04±0.11) μmol/L after 48-hour treatment. Low concentration of 02-036 could significantly inhibit the colony formation of CA46 cells. Flow cytometry analysis confirmed that 02-036 could effectively induce CA46 cell apoptosis. The apoptosis rate correlated with drug concentrations (r=0.959). Also, DNA ploid analysis showed that the cell cycle of CA46 was arrested in the S phase. The expression levels of BCL-2, Pro-caspase-9, Pro-caspase-3, PARP and C-MYC proteins decreased with a 02-036-dose dependent manner (r values were -0.990, -0.939, -0.971 and -0.967, respectively). In contrast, the expression level of cleaved-PARP increased with the same manner (r=0.920).@*CONCLUSION@#Baicalin derivative 02-036 can effectively inhibit the proliferation and induce apoptosis of CA46 cells, and its related mechanisms may be correlated with the down-regulation of apoptosis-related molecule expression levels, such as BCL-2, Pro-caspase-9, Pro-caspase-3, PARP and C-MYC.
Subject(s)
Humans , Apoptosis , Burkitt Lymphoma , Cell Line, Tumor , Cell Proliferation , FlavonoidsABSTRACT
Objective At present, there are few studies on the application of intensity-modulated radiation therapy (IMRT) combined with chemotherapy in the treatment of non-small cell carcinoma (NSCLC). The article aimed to analyze the efficacy of chemotherapy combined with IMRT on patients with locally advanced NSCLC and the impact on life quality.Methods From January 2012 to December 2014, 160 patients with locally advanced NSCLC were treated in our department of radiotherapy. The patients were divided into IMRT group(chemotherapy of paclitaxel or gemcitabine combined with cisplatin, IMRT) and control group(chemotherapy of paclitaxel or gemcitabine combined with cisplatin) according to different treatments, 80 patients in each group. The patients′ treatment efficacy along with its impact on the patients′ life quality were compared between two groups.Results The effective rate of IMRT group was higher than that of control group(78.75% vs 47.50%, P0.05). Conclusion Chemotherapy combined with IMRT can significantly improve the therapeutic effect in patients with locally advanced NSCLC, featuring less side effects, high safety, improved life quality and lengthened survival time. Therefore, the treatment is worthy of clinical application.
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Objective Transplantation of bone marrow mesenchymal stem cells (BMSCs) has a good prospect of application for cerebral infarction,but the environment and the inflammatory response to ischemia and hypoxia after cerebral infarction are not con-ducive to the survival of transplanted cells. This article investigated the effects of Shuxue Tongmai capsule(SXTM) combined with BM-SCs transplantation on the improvement of cerebral ischemic injury in rats. Methods A model of middle cerebral artery occlusion was es-tablished in Sprague-Dawley(SD) rats using thread method and these 15 SD rats were randomly divided into model group,BMSCs group and combination therapy group (BMSCs transplantation combined with SXTM treatment). At 24h after modeling,rats in combination therapy group were given tail vein injection of 1 mL BMSCs suspension (2× 109 per/L) and gavage administration of SXTM 0. 64 g/kg. Rats in BMSCs group were given tail vein injection of 1 mL BMSCs suspension (2×109 per/L) and gavage administration of equal volume of sa-line. For model group,the rats were given tail vein injection of equal volume of PBS and gavage administration of equal volume of sa-line. Neurologic function was assessed before cell transplantation and at 3,7,14,28 days after cell transplantation to check the injury of neurologic function. At 28 days after transplantion,the rats were decapitated after anesthesia to take brain tissues for immunohisto-chemical detection of vascular endothelial growth factor(VEGF) and brain-derived neurotrophic factor(BDNF) protein expression. Mor-phological changes of the brain tissue and apoptosis in cortical neurons were observed and detected by hematoxylin-eosin staining and TUNEL,respectively. Results At 7,14,28 days after transplantation,the neurological defect score in combination therapy group was significantly lower than those of model group and BMSCs group(P<0.05). In each group,the neurological defect score at 3 days after transplantation was significantly decreased compared with those before transplantation(P<0.05). In the same group,the neurologi-cal defect scores at 14,28 days after transplantation were significantly decreased compared with those at 7 days after transplantation (P<0.05). The neurological defect scores at 14,28 days after transplantation were significantly decreased compared with those at 7 days after transplantation(P<0.05). The neurological defect score at 28 day after transplantation was significantly decreased compared with that at 7 day after transplantation(P<0.05). At 28 day after transplantation,the number of apoptotic cells in combination therapy group (51.40±4.04) was significantly fewer than those of model group (74.80±5.31) and BMSCs group (67.20±4.66) and the num-ber of apoptotic cells in BMSCs group was significantly decreased compared with model group(P<0.05). The results of immunohisto-chemistry showed that the VEGF and BDNF positive cells in the cerebral ischemic region of rats were brownish or sepia in color. Com-pared with model group,the expression levels of VEGF and BDNF protein in BMSCs group and combination therapy group were signifi-cantly increased (P<0.05),and that of combination therapy group was significantly increased compared with BMSCs(P<0.05). Conclusion SXTM combined with BMSCs transplantation can promote neurological recovery from cerebral ischemia by increasing the protein expression of VEGF and BDNF and reducing neuronal apoptosis.
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Objective To establish a quantitative analysis method for determining 99mTc-HYNIC-PEG4-E[PEG4-c(RGDfK)]2 (99mTc-3PRGD2,a radioactive tumor agent)byγcounter, and to investigate the distribution of 99mTc-3PRGD2 in mice bearing with lung carcinoma xenograft. Methods The mice were divided into 4 normal groups and one blocking peptide group(control group). The 99mTc-3PRGD2(8μg/kg)was injected to mice bearing with lung carcinoma xenograft through the tail intravenous administration. Tissues of the normal mice were taken at 0.5,1,2 and 4 h. The control group were treated by 3PRGD2 and 99mTc-3PRGD2. The control mice were injected with the 3PRGD2 saline solution(2.5 mg/ml,0.2 ml)at 0.5 h earlier before the injection of 99mTc-3PRGD2. The tu?mor and organ tissues of the control mice were taken at 2 h. The radioactivity was detected by Gamma Counter. Results The radioac?tivity of 99mTc-3PRGD2 detected was high in the tumor and very low in brain. In addition,high radioactivity in kidneys and bladder sug?gested that the drug excreted by renal. Conclusion The results proved that the blocking peptide can competitively inhibit the combi?nation of 99mTc-3PRGD2 and integrinαvβ3 receptors.
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Objective To establish a rapid LC-MS/MS method for the simultaneous quantitative determination of lopinavir (LPV)and ritonavir(RTV)in human plasma. Methods Plasma samples were prepared by protein precipitation and separated by a Thermo Hypersil GOLD column(2.1 mm×100 mm,5 mm)with the mobile phase consisting of methanol and water(0.1%formic acid) at a flow rate of 0.2 ml/min. Detection of LPV,RTV and the internal standard(IS)MS 275 was performed using selected reaction moni?toring(SRM)of the transitions m/z 629.3→155.0,m/z 721.3→268.0 and m/z 377.1→359.2 in positive ion mode,respectively. Re?sults The calibration curve was linear in the range of 20-1000 ng/ml(r>0.994)for LPV and RTV. The intra and inter-day precision and accuracy values met the set acceptance criteria. Conclusion The method is rapid,sensitive and accurate for the therapeutic drug monitoring of LPV and RTV simultaneously in clinic and pharmacokinetic studies.
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Objective To establish a chemiluminescent immunoassay method for the determination of cathelicidin-BF(BF-30)in rat serum,to provide a fast and simple analytical method for its pharmacokinetic studies,then to investigate the pharmacokinet?ic characteristics of BF-30 in vivo. Methods A chemiluminescent immunoassay method was developed and the matrix effect,preci?sion,accuracy,stability and the dilution effect were evaluated,then the method was used to determine the concentration of BF-30 in rat serum. Results The linear range of BF-30 in rat serum was 0.5-40 ng/ml,and the lowest limit of determination was 0.5 ng/ml. No matrix effect was observed. The RSD of inter-and intra-day precisions were 8.76%-14.15%and 4.91%-11.11%,respectively,and ac?curacy was-1.27%--7.71%. The stability of samples for 13 days and stock solution for 30 days at-20℃were good,and the stability of serum samples after 2 cycles of freeze-thaw met the requirements. There was no dilution effect noted after 40 times sample dilution. Conclusion We heve developed a simple,accurate and reliable method which can be applied to the determination of BF-30 in rat se?rum and following pharmacokinetic study.
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As a widespread phenomenon in living system, molecular self-assembly has become the meeting point of multidisciplinary research including chemistry, biology, materials science and medicine. In recent years, the rapid development in molecular self-assembly of peptide technology is showing a great potential in the application of tissue engineering, drug delivery, bionic medicine, cosmetology field, optical and electronic product development, etc. Especially, the remarkable hemostatic effect of self-assembling peptides (SAP) on organs, nerves and brain wounds successfully promoted its application to the material science and clinical medicine. This review focuses on the hemostatic effects and characteristics of SAP on different bleeding wound models, action mechanism, its benefits and limitations as well as its adrancing trends.
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Humans , Drug Delivery Systems , Hemostasis , Peptides , Tissue EngineeringABSTRACT
Objective To establish an ultra-high-performance liquid chromatograph(UHPLC)method for the determination of paclitaxel (PTX)in polydipeptide paclitaxel (PDP)preparation. Methods PDP preparation was dissolved in deionized water (DIW) and degraded by 2.0 mol/L sodium hydroxide solution. The concentration of paclitaxel was calculated indirectly by its degradation product. The separation was achieved on an Agilent SB C18 column(2.1 mm × 50 mm,1.8μm). Elution was carried out using a mobile phase consisting of acetonitrile-water (10 ∶ 90,V/V)at the flow rate of 0.2 ml/min. UV detection wavelength was performed at 240 nm and reference wavelength was 360 nm. The temperatures of autosampler and column were thermostated at 15℃(± 0.5℃)and 40℃(± 0.5℃),respectively. The injection volume was 2 μl. Results The relationship between the concentration of paclitaxel (0.31-5.00 mg/ml)and the peak area of its degradation product was in good linearity (r=0.9992,n=5). Total amount of paclitaxel in different batches of PDP preparation was in the range of 26.77-33.19 mg per vial. Conclusion The method is accurate, rapid,reproducible and suitable for the analysis of paclitaxel in PDP preparation.
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A rapid, sensitive and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous determination of yogliptin and its metabolite in Wistar rat plasma. Linagliptin and dexamethasone were chosen as the internal standards of yogliptin and its metabolite, (R)-8-(3-hydroxypiperidine- -yl)-7-(but-2-yn-1-yl)-1-((5-fluorobenzo[d]thiazol-2-yl)methyl)-3-methyl- H-purine-2, 6 (3H, 7H)-dione, respectively. After a simple protein precipitation using acetonitrile as the precipitating solvent, both analytes and ISs were separated on a Grace Altima HP C18 column (2.1 mm x 50 mm, 5 microm) with gradient elution using methanol (containing 0.1% formic acid, 4 mmol x L(-1) ammonium acetate)-0.1% formic acid (containing 4 mmol x L(-1) ammonium acetate) as the mobile phase. A chromatographic total run time of 4.4 min was achieved. Mass spectrometric detection was conducted with electrospray ionization under positive-ion and multiple-reaction monitoring modes. Linear calibration curves for yogliptin and its metabolite were over the concentration range of 0.5 to 500 ng x mL(-1) with a lower limit of quantification of 0.5 ng x mL(-1). The intra- and inter- assay precisions were all below 14%, the accuracies were all in standard ranges. The method was used to determine the concentration of yogliptin and M1 in Wistar rat plasma after a single oral administration of yogliptin (27 mg x kg(-1)). The method was proved to be selective, sensitive and suitable for pharmacokinetic study of yogliptin and M1 in Wistar rat plasma.
Subject(s)
Animals , Rats , Chromatography, Liquid , Dexamethasone , Blood , Dipeptidyl-Peptidase IV Inhibitors , Blood , Pharmacokinetics , Linagliptin , Blood , Rats, Wistar , Tandem Mass SpectrometryABSTRACT
A rapid, sensitive and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous determination of yogliptin and its metabolite in Wistar rat plasma. Linagliptin and dexamethasone were chosen as the internal standards of yogliptin and its metabolite, (R)-8-(3-hydroxypiperidine- -yl)-7-(but-2-yn-1-yl)-1-((5-fluorobenzo[d]thiazol-2-yl)methyl)-3-methyl- H-purine-2, 6 (3H, 7H)-dione, respectively. After a simple protein precipitation using acetonitrile as the precipitating solvent, both analytes and ISs were separated on a Grace Altima HP C18 column (2.1 mm x 50 mm, 5 microm) with gradient elution using methanol (containing 0.1% formic acid, 4 mmol x L(-1) ammonium acetate)-0.1% formic acid (containing 4 mmol x L(-1) ammonium acetate) as the mobile phase. A chromatographic total run time of 4.4 min was achieved. Mass spectrometric detection was conducted with electrospray ionization under positive-ion and multiple-reaction monitoring modes. Linear calibration curves for yogliptin and its metabolite were over the concentration range of 0.5 to 500 ng x mL(-1) with a lower limit of quantification of 0.5 ng x mL(-1). The intra- and inter- assay precisions were all below 14%, the accuracies were all in standard ranges. The method was used to determine the concentration of yogliptin and M1 in Wistar rat plasma after a single oral administration of yogliptin (27 mg x kg(-1)). The method was proved to be selective, sensitive and suitable for pharmacokinetic study of yogliptin and M1 in Wistar rat plasma.
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Healthy Beagle dogs were administrated with batroxobin by intravenous infusion at high, medium and low doses. The study of pharmacodynamics and pharmacokinetics was intended to clarify the relevance of them and provided strong evidence for clinical use of batroxobin. The blood samples were collected after injection based on the time schedule and samples were tested by ELISA method to get the concentration of batroxobin. At the same time, changes of prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimmer were tested. The results showed that the concentration of D-D increased significantly after administration compared with that of before administration. The main pharmacokinetic parameters were as follows: t1/2 were (2.27 +/- 0.42) h, (10.65 +/- 2.19) h and (11.01 +/- 3.51) h; C(max) were (11.9 +/- 1.72) ng x mL(-1), (154.53 +/- 12.38) ng x mL(-1) and (172.14 +/- 47.33) ng x mL(-1); AUC(last) were (29.38 +/- 3.69) ng xh x mL(-1), (148.43 +/- 72.85) ng x h x mL(-1) and (599.22 +/- 359.61) ng x h x mL(-1). The elimination of batroxobin was found to be in accord with linear kinetics characteristics. The results of pharmacodynamics showed that D-dimmer level increased significantly after the administration of batroxobin, which was similar with the changes of batroxobin plasma concentration. Simultaneously, Fib concentrations in Beagle dog blood decreased significantly after the iv administration of batroxobin, while recovered to base level after 48 hours. PT, TT and APTT significantly became longer after administration, which returned to normal level after 48 hours. Especially, the D-dimmer levels and the batroxobin concentration in plasma after intravenous infusion of the drug were synchronized in Beagle dogs. Changes between PD/PK results had obvious correlation, and the D-dimmer levels in plasma can be one of the important monitoring indicators of batroxobin in thrombolytic medication.
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Healthy Beagle dogs were administrated with batroxobin by intravenous infusion at high, medium and low doses. The study of pharmacodynamics and pharmacokinetics was intended to clarify the relevance of them and provided strong evidence for clinical use of batroxobin. The blood samples were collected after injection based on the time schedule and samples were tested by ELISA method to get the concentration of batroxobin. At the same time, changes of prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimmer were tested. The results showed that the concentration of D-D increased significantly after administration compared with that of before administration. The main pharmacokinetic parameters were as follows: t1/2 were (2.27 +/- 0.42) h, (10.65 +/- 2.19) h and (11.01 +/- 3.51) h; C(max) were (11.9 +/- 1.72) ng x mL(-1), (154.53 +/- 12.38) ng x mL(-1) and (172.14 +/- 47.33) ng x mL(-1); AUC(last) were (29.38 +/- 3.69) ng xh x mL(-1), (148.43 +/- 72.85) ng x h x mL(-1) and (599.22 +/- 359.61) ng x h x mL(-1). The elimination of batroxobin was found to be in accord with linear kinetics characteristics. The results of pharmacodynamics showed that D-dimmer level increased significantly after the administration of batroxobin, which was similar with the changes of batroxobin plasma concentration. Simultaneously, Fib concentrations in Beagle dog blood decreased significantly after the iv administration of batroxobin, while recovered to base level after 48 hours. PT, TT and APTT significantly became longer after administration, which returned to normal level after 48 hours. Especially, the D-dimmer levels and the batroxobin concentration in plasma after intravenous infusion of the drug were synchronized in Beagle dogs. Changes between PD/PK results had obvious correlation, and the D-dimmer levels in plasma can be one of the important monitoring indicators of batroxobin in thrombolytic medication.
Subject(s)
Animals , Dogs , Male , Area Under Curve , Batroxobin , Blood , Pharmacokinetics , Pharmacology , Enzyme-Linked Immunosorbent Assay , Fibrin Fibrinogen Degradation Products , Metabolism , Fibrinogen , Metabolism , Fibrinolytic Agents , Blood , Pharmacokinetics , Pharmacology , Infusions, Intravenous , Partial Thromboplastin Time , Prothrombin Time , Thrombin TimeABSTRACT
A 59-year-old female farmer presented with left painful swollen eye for 1 week after being stung by a rice black bug ( Scotinophara sp. ). It was associated with acute progressive blurring of vision. On examination of the left eye, there was a marked periorbital swelling with proptosis and complete ptosis. The extraocular movements were restricted in all the directions. The cornea was hazy with large epithelial defect. Fundoscopy showed combined features of both central retinal vein and artery occlusions with swollen optic disc and ischaemia of the macular area. CT scan and MRI of orbit and brain showed evidence of orbital soft tissue inflammation. Patient was diagnosed with left orbital cellulitis, keratouveitis and central retinal vein and artery occlusions. The periorbital swelling and proptosis were improved after treatment with systemic and topical antibiotics. However, the vision remained no perception of light(NPL)and limitation of ocular movements persisted. The potential ophthalmic insults by Scotinophara sp. Can be severe and permanent. Awareness of the debilitating insults by Scotinophara sp. To human eye should be instilled timely especially in its prone areas.
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This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.
Subject(s)
Humans , Cloning, Molecular , Complement C3 , Genetics , Metabolism , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Macrophage-1 Antigen , Genetics , Metabolism , Receptors, Complement 3b , Genetics , Recombinant Proteins , GeneticsABSTRACT
Objective:To investigate the inhibitory effect of breast cancer-associated antigen 1(BRCAA1)gene silencing on gastric cancer MGC-803 cells and the related mechanism.Methods:Plasmid shRNA-BRCAA1 and shRNA-N were constructed and transfected with FuGene HD into gastric cancer cell line MGC-803.The transfection efficiency was examined using fluorescent microscope 24 h later.The total RNAs was extracted 48 h 'after transfection and the expression of BRCAA1 and GAPDH gene were analyzed by real-time PCR.The cell proliferation was assessed by MTT assay 24 h,48 h,and 72 h after transfection.The cell apoptosis was determined by Annexin V-PE/TAAD.The expression of Rb,Bax, Bcl-2 and BRCAA1 proteins was analyzed by Western blotting 48 h after transfection.Results:We found that the transfection efficiency of shRNA-BRCAA1 was(81.2?2.6)%24 h after transfection.Forty-eight hours after transfection with shRNA-BRCAA1 the expression of BRCAAI mRNA decreased by 61.4%;the inhibition rate of MGC-803 cells growth was 45.0%.The cell apoptosis rate of shRNA-BRCAA1 transfection group was significantly higher than those of untransfected group and mock plasmid transfected group([14.4?1.6]%vs[5.4?2.0]%,[4.4?2.5]%,P