Objective To determine the pharmacokinetic parameters of P-methoxybenzyl alcohol from Gastrodiae Rhizoma in plasma of rats by HPLC. Methods Gavage and intravenous injection were employed for administration. HPLC was used to determine the concentrations of P-methoxybenzyl alcohol from Gastrodiae Rhizoma in plasma of rats in different time points. The pharmacokinetic parameters were computed by DAS3.0. Results The linear range of P-methoxybenzyl alcohol in plasma was 0.63-321.17 μg/mL, r 2=0.994 5. Intra-day accuracy, inter-day accuracy, absolute recovery and stability were in specified range. Conclusion The method is simple and accurate for the determination of the pharmacokinetic parameters of P-methoxybenzyl alcohol from Gastrodiae Rhizoma in plasma of rats.

To purify the recombinant human BMP-6 protein and to establish its in vitro bioassay method. The cDNA encoding the mature peptide of hBMP-6 protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR), using human placental mRNA as template, and subcloned into the high-expression vector pET-15b under the control of T7 lac promoter. The resulting construct, pET-BMP6, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant hBMP-6 protein (rhBMP-6). After 4 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), rhBMP-6 (approximately 15kD) was expressed and formed inclusion bodies, contributing up to 10% of the total bacterial protein. The inclusion bodies were isolated and redissolved in 8mol/L urea, and the denatured rhBMP-6 was purified to 95% purity by CM-Cellulose 32 ion exchange chromatography (IEC). The osteoinductivity of rhBMP-6 was measured by the expression of some of the osteoblast differentiation marker genes in rhBMP-6-treated C3H10T1/2 cells as reflected by determinations of alkaline phosphatase (ALPase) activity and semi-quantitative RT-PCR. At the end of the purification process, about 80% of rhBMP-6 formed disulphide-linked homodimers after refolding during renaturation. The apparent size of the protein was 30kD on non-reducing SDS-PAGE, similar to that of the native form of hBMP-6. The enzyme assays showed that the ALPase activity was increased in a dose-dependent manner with the treatment of rhBMP-6. After the addition of 300ng/mL of rhBMP-6, the ALPase activity of C3H10T1/2 cells increased significantly. The activity of rhBMP-6 used was comparable to about 70% of that of the standard hBMP-6 derived from eukaryotic cells. RNA extraction data also showed rhBMP-6 stimulated expression of osteoblast marker genes, including type I collagen, osterix, and osteocalcin in a time-dependent manner. After 5 days of treatment, their level of expression was increased to 3 times that of controls. Bone morphogenetic protein (BMP)-6, a member of the 60A subgroup of the bone morphogenetic protein (BMPs) family, plays a pivotal role in bone formation. Previous evidence showed that BMP-6 is selectively up-regulated by estrogen, suggesting its potential role in the treatment of osteoporotic fractures, especially for menopausal osteoporosis. Our present study demonstrates that the recombinant hBMP-6 produced in Escherichia coli is able to induce pre-osteoblastic cells to differentiate into osteoblasts in vitro, and analysis of mRNA expression of type I collagen, osterix, and osteocalcin can be also a method for measuring the osteoinductivity of BMP. This provides the basis for further studies on ectopic bone formation in the body and for the development of auxiliary drugs for the treatment of osteoporotic fractures.
Alkaline Phosphatase ; metabolism ; Animals ; Biological Assay ; methods ; Bone Morphogenetic Protein 6 ; genetics ; isolation & purification ; metabolism ; pharmacology ; Cell Line ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme Activation ; drug effects ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; drug effects ; genetics ; Humans ; Isopropyl Thiogalactoside ; pharmacology ; Mice ; Plasmids ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo develop a double antibody sandwich ELISA assay for quantitative determination of recombinant human interferon alpha1b.

METHODSMouse monoclonal antibodies with different binding site on rIFN-alpha1b were screened to select optimized candidates as coating and HRP-labeled index antibodies respectively. And a double antibodies sandwich ELISA was assembled; the reliable lower detection limit, specificity, accuracy and reproducibility were evaluated and validated.

RESULTSThe quantitative sandwich ELISA had a reliable lower detection limit of 10 ng/ml, with a liner detection range 10-100 ng/ml (R2 = 0.992), variation coefficient inter-plates is less than 10%.

CONCLUSIONThe developed sandwich ELISA was a sensitive and specific, accuracy and reproducibility method for quantitative determination of recombinant human interferon alpha1b in final product.

Animals ; Antibodies, Monoclonal ; analysis ; Enzyme-Linked Immunosorbent Assay ; instrumentation ; methods ; Humans ; Interferon-alpha ; blood ; Mice ; Mice, Inbred BALB C

OBJECTIVETo observe the specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad5F35-LMP2 in rhesus monkeys.

METHODSSixteen rhesuses were immunized with Ad5F35-LMP2 through intra-muscular injection in three groups: high dosage group (1.5 x 10(10) TCID(50)/rhesus), medium dosage group (1.5 x 10(9)TCID(50)/rhesus), low dosage group (1.5 x 10(8)TCID50/rhesus) and the last group was control (PBS 4 ml/rhesus). They were totally immunized three times at intervals of one month. The EBV-LMP2 specific cellular immune responses were tested during the 0, 4, 8, 12 weeks by Elispot after immunization respectively. And the titers of anti-LMP2 antibody were tested by EIA at the same time.

RESULTSEBV-LMP2 specific cellular and humoral immune responses which were induced by recombinant adenovirus Ad5F35-LMP2 can be found in all the three dosage groups. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response.

CONCLUSIONThe recombinant adenovirus Ad5F35-LMP2 could elicit LMP2 specific cellular and humoral immune responses in rhesus.

Adenoviruses, Human ; genetics ; Animals ; Cell Differentiation ; Herpesvirus 4, Human ; genetics ; Immunity, Cellular ; immunology ; Immunization ; methods ; Macaca mulatta ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Matrix Proteins ; genetics ; immunology

OBJECTIVETo explore the characteristics of 3.0T dynamic contrast-enhanced magnetic resonance (DCE-MR) imaging of lung cancer and its correlation with microvessel density (MVD).

METHODSThirty-seven patients with pathologically proven lung cancer underwent DCE-MR with liver acquisition with volume acceleration sequence. DCE-MR images were acquired intermittently for a total of 4 minutes on a 3.0T MR scanner. The relative enhancing percentage (SI%) at each time point was measured. The shapes of T-SI% curves were defined as A (rapidly ascending followed by a descending branch) and B (rapidly ascending branch followed by a plateau). The early peak enhancement (SIEP%), early peak time (TEP), maximum enhancement (SIpeak%), and peak time (Tpeak) were recorded and compared according to different dimensions, locations, histological types, and differentiation grades of lung cancer. Tumour specimens were immunostained for CD31 and CD34 in ten patients who had undergone surgical resections. The enhancement values were correlated with MVD. Results The SIEP% and SIpeak% of tumors with smaller dimensions (< or = 5 cm) were significantly higher than those with larger dimensions (> 5 cm) (P = 0.014, P = 0.024). The SIEP% and SIpeak% were positively correlated with the tumor MVD. Conclusion The SIEP% and SIpeak% of lung cancer correlate with tumor dimension and can reflect MVD in tumor.

Adenocarcinoma ; blood supply ; diagnosis ; Adult ; Aged ; Carcinoma, Squamous Cell ; blood supply ; diagnosis ; Contrast Media ; Female ; Humans ; Image Enhancement ; methods ; Lung Neoplasms ; blood supply ; diagnosis ; Magnetic Resonance Imaging ; methods ; Male ; Microvessels ; pathology ; Middle Aged ; Neovascularization, Pathologic ; diagnosis

OBJECTIVETo observe the growth and cell cycle changes of nasopharyngeal carcinoma cell 5-8F in response to silencing of the expression of epidermal growth factor receptor (EGFR) with RNA interference (RNAi), and explore the possible relationships between EGFR and the occurrence, differentiation and progression of nasopharyngeal carcinoma.

METHODThree EGFR-specific small interfering RNAs (siRNAs) were obtained by in vitro transcription and synthesis and were transiently transfected into 5-8F cells. The EGFR expression levels in the transfected cells were detected by reverse transcription (RT)-PCR and Western blotting, respectively. After EGFR expression silencing, the growth and cell cycle changes of the cells were observed.

RESULTSEGFR mRNA contents and protein levels decreased by approximately 67.5% and 77%, respectively, after RNAi of 5-8F cells, and the cell proliferation decreased and cell cycle arrest at G1 phase occurred in association with EGFR silencing.

CONCLUSIONEGFR silencing by RNAi can reduce the proliferation of nasopharyngeal carcinoma cells and induce cell cycle arrest at G1 phase, which sheds light on the possible use of RNAi for further investigation of the pathogenesis and gene therapy of nasopharyngeal carcinoma.

Cell Line, Tumor ; Cell Proliferation ; Genetic Therapy ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, Epidermal Growth Factor ; biosynthesis ; genetics ; Transfection

Objective To investigate the underlying mechanisms of Miller-Fisher syndrome (MFS) and Bickerstaff' s brainstem encephalitis (BBE) by studying their clinical and electrophysiological characteristics.Methods The clinical and electrophysiological characteristics of 13 MFS and 7 BBE cases in Peking Union Medical College Hospital between 2000 and 2011 were retrospectively analyzed.The electrophysiological parameters included sensory and motor nerve conduction,electromyography,F wave,sympathetic skin response and brainstem auditory evoked potential and blink reflex.Results MFS and BBE had similar clinical characteristics:respiratory symptoms were the most common infectious symptoms before disease onset; Ophthalmoplegia,facial palsy and bulbar symptoms were common; They both had cerebrospinal fluid albuminocytological dissociation and positive serum anti-GQ1b antibody.However,BBE had more central nervous system lesion signs clinically such as conscious disturbance,positive Babinski' s sign and central facial palsy.Electrophysiologically,MFS and BBE also had similar electrophysiological features:sensory nerve abnormality ratios were 6/13,2/7 respectively,with prominently reduced sensory nerve active potential amplitude,normal or slightly slowed sensory conduction velocity; Motor nerves abnormality ratios were 2/13,1/7 respectively,with slightly prolonged distal motor latency and normal compound muscle action potential; Electromyography abnormality ratios were 1/7,0/4 respectively; F wave frequency abnormality ratios were 3/13,5/7 respectively,and in some cases,F wave frequency would restore; Sympathetic skin response abnormality ratios were 1/2,1/3 respectively; Blink reflex abnormalityratios were 1/2,1/1 respectively,with central involvement in BBE; Brainstem auditory evoked potential abnormality ratios were 3/5,1/4 respectively,with wave Ⅰ latency or amplitude abnormality.Conclusion The similarities of clinical and electrophysiological features suggest that MFS and BBE have the same mechanism and they form a continuous spectrum with variable central nervous system and peripheral nervous system involvement.

The secondary structure of Capsid protein was predicted by the methods of Chou-Fasman,Garnier-Robson and Karplus-Schultz based on the sepuence of capsid protein gene of Swine Vesicular Disease Virus (SVDV) and hydrophilicity. Surface probility plot and antigenic index for capsid protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-wolf, respectively, Combining the results according to these methods, the B cell epitopes for capsid protein of SVDV were predicted. The results showed that there are much flexible region such as coil region and turn region in capsid protein of SVDV, there are more predominant B cell epitopes in VP1 than in VP2 and VP3. This study would be helpful for identification of B cell epitopes for capsid protein using experimental methods and research of reverse vaccine of SVDV.

A novel rapid method for detection of the illicit beta2-agonist additives in health foods and traditional Chinese patent medicines was developed with the desorption corona beam ionization mass spectrometry (DCBI-MS) technique. The DCBI conditions including temperature and sample volume were optimized according to the resulting mass spectra intensity. Matrix effect on 9 beta2-agonists additives was not significant in the proposed rapid determination procedure. All of the 9 target molecules were detected within 1 min. Quantification was achieved based on the typical fragment ion in MS2 spectra of each analyte. The method showed good linear coefficients in the range of 1-100 mg x L(-1) for all analytes. The relative deviation values were between 14.29% and 25.13%. Ten claimed antitussive and antiasthmatic health foods and traditional Chinese patent medicines from local pharmacies were analyzed. All of them were negative with the proposed DCBI-MS method. Without tedious sample pretreatments, the developed DCBI-MS is simple, rapid and sensitive for rapid qualification and semi-quantification of the illicit beta2-agonist additives in health foods and traditional Chinese patent medicines.
Adrenergic beta-2 Receptor Agonists ; analysis ; Drugs, Chinese Herbal ; chemistry ; Food, Organic ; analysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Substance Abuse Detection ; methods ; Tandem Mass Spectrometry

OBJECTIVETo summarize the electroclinical characteristics of myoclonic atonic epilepsy (MAE) in children.

METHODThe clinical data, video electroencephalogram (EEG) and simultaneous electromyography (EMG) of MAE patients were analyzed. The treatment and its effects were followed up.

RESULTIn 47 MAE patients, 25 had a history of febrile seizures (FS), 20 had a family history of FS or epilepsy. All patients had a normal development before the illness. The age of afebrile seizure onset was between 1.4 years to 5.8 years. The first seizure was generalized tonic-clonic seizure (GTCS) in 41 patients (87.2%). All patients had multiple seizure types, including 47 GTCS (97.9%), 34 myoclonic atonic seizures (72.3%), 47 myoclonic seizures (100%), 32 atonic seizures (68.1%), 36 atypical absences (76.6%) and 3 tonic seizures (6.4%). EEG backgrounds were slow or parietal θ rhythm, interictal EEG showed 1-4 Hz (predominant 2-3 Hz) generalized spike and wave or poly spike and wave discharges in all cases. Seizures were controlled by antiepileptic drugs (AEDs) in 41 patients (87.2%). Valproate was used in 37. Lamotrigine was used in 26. Mild mental retardation was observed in 10 children after the onset of the illness.

CONCLUSIONThe clinical features of MAE included the following: the development was normal before the onset of the illness; the onset of seizure type was often GTCS. All patients had multiple generalized seizure types. Myoclonic atonic seizure was its characteristic seizure type. EEG showed generalized discharges. Early diagnosis and rational choice of AEDs are important for getting a better prognosis.

Child ; Child, Preschool ; Electroencephalography ; Epilepsies, Myoclonic ; diagnosis ; physiopathology ; therapy ; Epilepsy, Generalized ; diagnosis ; physiopathology ; therapy ; Female ; Humans ; Infant ; Male