Objective To establish a micellar electrokinetic capillary chromatography method for content determination of geniposide in Biyuanshu Oral Liquid. Methods Acetaminophen was used as an internal standard, and the separation was performed on an uncoated fused silica capillary of 52 cm × 50 μm ID (42 cm effective length) with the separation voltage of 25.0 kV. The running buffer contained 50 mmol/L borax, 100 mmol/L sodium dodecyl sulfate and 15％ acetonitrile (pH=10). The sample was injected by pressure (10 s, 0.5 psi) and detected at 238 nm. Results Geniposide was in good linearity range of 15.02–320.48 μg/mL (r=0.9995). The repeatability (low, medium and high concentration of samples) and intermediate precision assays gave satisfactory RSD values of less than 1.77％and 2.01％, respectively. The average recovery of geniposide in Biyuanshu Oral Liquid was 97.50％ and the RSD was 4.43％. The contents of geniposide determined by micellar electrokinetic capillary chromatography were in accordance with the results of HPLC analysis. Conclusion The method is simple, fast, accurate and precise, which can be used for the content determination of geniposide in Biyuanshu Oral Liquid.

OBJECTIVETo evaluate clinical efficacy of suture anchors in treating acute injuries of medial collateral ligament (MCL) of knee at degree III.

METHODSTwenty-seven patients with degree III acute MCL injuries of knee were treated with suture anchors from January 2007 to June 2011. There were 15 males and 12 females, aged from 19 to 56 (averaged 32.6) years old. The time from injury to operation was 3 to 10 days, averaged 6 days. Symptoms and physical signs before and after treatment were observed, Lysholm scoring were used to evluated clinical efficacy.

RESULTSAll patients were followed up from 16 to 30 months with an average of 21.6 months. The stability of knee joints was good in all patients. Abduction stress test was negative when the knee joint was straightened at 0 degrees and flexed at 30 degrees. The average degree of flexed knee (67.00 +/- 5.80) degrees preoperatively was lower than that of postoperatively (136.50 +/- 6.30) degrees at 1 year. According to Lysholm scoring, preoperative scores ranged from 30 to 43 points, averaged 36.46 +/- 1.48; 1 year after operation ranged from 87 to 100 with an average of 91.50 +/- 3.80 and higher than postoperative. Twenty patients got an excellent results, 5 good and 2 fair.

CONCLUSIONSuture anchors in treating acute injuries of medial collateral ligament of knee at degree III has following advantages: small range of tissue dissection, easy to operate, reliable fixation and less complications.

Acute Disease ; Adult ; Collateral Ligaments ; injuries ; surgery ; Female ; Humans ; Knee Injuries ; surgery ; Male ; Middle Aged ; Suture Anchors

Objective To stratify the risks of patients with acute pulmonary embolism (APE) and pulmonary hypertension (PH) by dynamic pulmonary perfusion imaging (DPPI) and pulmonary perfusion imaging (PPI). Methods From October 2007 to February 2009, 20 healthy volunteers ( 12 males, 8 females; mean age =48.47 ±13.47 years) and 31 APE patients (21 males, 10 females; mean age =47.68 ±18.06 years; from October 2007 to July 2009) were included in the study. DPPI and PPI were performed in all subjects. Percentage of perfusion defect scores ( PPDs% ) were calculated by semi-quantitative analysis of PPI. Risk levels were defined according to PPDs% calculated from PPI: normal (PPDs% =0); very low risk (0 ＜ PPDs% ≤10% ); low risk (10% ＜ PPDs% ≤20% ); moderate risk (20% ＜ PPDs% ≤40% );high risk (40% ＜ PPDs% ≤60% ) and very high risk ( PPDs% ＞ 60% ). Lung equilibrium time (LET)was calculated on region of interest (ROI) drawn over DPPI. Clinical risk was scored by Aujesky method.The t-test, ANOVA and correlation analysis were used with SPSS 13.0 software. Results ( 1 ) LET in healthy volunteers and APE patients was ( 12.18 ± 3.28) and (32.90 ± 14.29) s respectively (t = 6. 81,P ＜ 0. 01 ). (2) The correlation coefficient, coefficient of determination between LET and PPDs% in APE patients were 0.93 and 0. 87, respectively. The correlation coefficient between LET and clinical risk score was 0.86. (3)The mean LET of APE patients in very low risk (n =5), low risk (n = 12), moderate risk (n=9), high risk (n=4) and very high risk groups (n=1) were (19.59 ±0.04), (25.03 ±0.08),(36.07 ±0. 10), (57.15 ±0.06) and (70 ±0.00) s, respectively. There was significant difference among APE patients with different risk levels (F =16. 78, P ＜0.01). Conclusions ( 1 ) DPPI was a reliable, convenient and non-invasive method for the evaluation of PH in APE. (2) Combined LET of DPPI and PPDs% of PPI was valuable for risk stratification and prognosis estimation in APE patients.

To produce antibodies capable of neutralizing botulinum neurotoxin type B(BoNT/B),We cloned the carboxy-terminal end of Hc containing the major determinants responsible for specific toxin,induced and purifed.The heavy-chain and kappa light-chain variable region gene repertoire of immunoglobulin were amplified individually from the spleen cell mRNA by RT-PCR and joined as a single-chain Fv(scFv)DNA fragment.These fragment were cloned into the phagemid pCANTAB5E and the phage display library was constructed.Results showed that the high affinity scFv was obtained after 4 rounds of panning,with its DNA sequence conforming to that of mouse antibody.

OBJECTIVETo study the effects of Naoan tablets on brain hemodynamics and cerebral microcirculation of soft membrane.

METHODCerebral blood stream flux, resistance of blood vessels, blood pressure and heart rate were used as observation indexes in hemodynamics experiment. Artery caliber and the number of capillaries with recovered blood stream were used as observation indexes in microcirculation experiment.

RESULTNaoan tablets at dose of 0.5 g x kg(-1) and 1.0 g x kg(-1) could enhance cerebral blood stream flux, decrease resistance of blood vessels, and reduce blood pressure. While no effects on heart rate. Naoan tablet at dose of 0.7 g x kg(-1) and 2.1 g x kg(-1) could increase the number of capillaries with recovered blood stream and enlarge the artery caliber of soft membrane in rats.

CONCLUSIONNaoan tablets can improve the indexes of hemodynamics and cerebral microcirculation of soft membrane.

Anesthesia ; Animals ; Brain ; blood supply ; Cerebrovascular Circulation ; drug effects ; Codonopsis ; chemistry ; Dogs ; Drug Combinations ; Drugs, Chinese Herbal ; pharmacology ; Female ; Ligusticum ; chemistry ; Male ; Microcirculation ; drug effects ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Wistar ; Tablets ; Vascular Resistance ; drug effects

OBJECTIVETo explore the effect of ricin temperature response gel on breast cancer and its regulatory effect on immune function in rats.

METHODSRicin was purified by chromatography and identified by immunoblotting. The rat subcutaneously transplanted breast cancer model was established. Forty model rats with a tumor diameter of about 3.0 cm were subjected to the study. They were randomized into four groups equally: the model group and three treated groups (blank gel, ricin, ricin-gel) were administered with blank gel, ricin, and ricin temperature response gel via percutaneous intratumor injection, respectively. The tumor was isolated 10 days later for the estimation of tumor inhibition rate (TIR) by weighing, pathologic examination, and detection of tumor apoptosis-associated genes bcl-2 and bax with semiquantitative RT-PCR. Also, peripheral blood was obtained to test T-lymphocyte subsets, the killing function of lymphocytes, and the contents of tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). The outcomes were compared between groups.

RESULTSThe TIR in the ricin-gel group was 61.8%, with the pathologic examination showing extensive tumor tissue necrosis. Compared with the model group, after ricin temperature response gel treatment, bcl-2 expression was down-regulated, bax expression was up-regulated, CD4+ lymphocytes and CD4+/CD8+ ratio in peripheral blood were increased, the killing function of lymphocytes was enhanced, and the contents of TNF-α and IL-2 were elevated (P < 0.05 or P < 0.01).

CONCLUSIONIntratumor injection of ricin temperature-responsive gel showed significant antitumor effect on breast cancer and could enhance the immune function in the tumor-bearing rat.

Animals ; Antineoplastic Agents ; administration & dosage ; Apoptosis ; drug effects ; CD4-CD8 Ratio ; Disease Models, Animal ; Female ; Gels ; therapeutic use ; Immunohistochemistry ; Immunomodulation ; drug effects ; Injections, Intralesional ; Interleukin-2 ; immunology ; metabolism ; Mammary Neoplasms, Experimental ; drug therapy ; immunology ; pathology ; Random Allocation ; Rats ; Rats, Wistar ; Ricin ; administration & dosage ; Sensitivity and Specificity ; Temperature ; Tumor Necrosis Factor-alpha ; immunology ; metabolism

OBJECTIVEHuman herpesvirus 6 (HHV-6) isolates are classified into two variants, HHV-6A and HHV-6B, based on distinct genetic, antigenic and biological characteristics. HHV-6 has been associated with encephalitis in children recently. This study aimed to establish a real time PCR assay for simultaneous detection of the two subtypes of HHV-6, and apply this new assay to children with suspected encephalitis, then analyze the relationship between the infection with HHV-6 and encephalitis in children.

METHODThe universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene (U38) of HHV-6. The 5' end of the probes for HHV-6A and HHV-6B was labeled with the fluorescein reporter tetrachloro-6-carboxyfluorescein and 6-carboxyfluorescein (6-FAM), separately, while the 3' end were quenched with 6-carboxy-tetramethylrhodamine. The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established. Then, the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 10(9) to 10(0) copies/microl, together with controls were used for testing both sensitivity and specificity of the real time PCR assay. The cerebrospinal fluid (CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced.

RESULTBoth HHV-6A (strain ZJ-159) and HHV-6B (strain GS) were positive on the real time PCR assay. There were no cross-reaction with herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus, Staphylococcus aureus, Mycoplasma pneumoniae and human DNA. A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6. The sensitivity threshold was 10 copies/microl for the real time PCR. HHV-6 positive rate by the real time PCR assay was 4.72% (21/445), including 4 cases with HHV-6A infection, 16 cases of HHV-6B infection and 1 case with mixed HHV-6A and HHV-6B infection. The new PCR assay usually took 2 to 3 hours to provide results.

CONCLUSIONThis new real time PCR assay can simultaneously detect both subtypes of HHV-6, and have high specificity and sensitivity. It will provide an early and sensitive diagnosis of HHV-6 encephalitis in children.

Adolescent ; Child ; Child, Preschool ; DNA Fingerprinting ; DNA Primers ; DNA, Viral ; Encephalitis, Viral ; cerebrospinal fluid ; diagnosis ; virology ; Female ; Fluorometry ; Genotype ; Herpesvirus 6, Human ; genetics ; isolation & purification ; Humans ; Infant ; Infant, Newborn ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo investigate the antithrombin (AT) activity (AT: A) and AT antigen (AT: Ag) level in a Chinese family with type I antithrombin (AT) deficiency, and to explore the molecular mechanism of AT deficiency.

METHODSImmuno-nephelometry and chromogenic assay were used to detect the plasma level of AT: A and AT: Ag, respectively. Genomic DNA was isolated from the peripheral blood, and all the seven exons and exon-intron boundaries of AT gene were amplified by PCR and direct sequencing.

RESULTSThe plasma levels of AT: A and AT: Ag of the proband were 45% and 97 mg/L, respectively, which led to a type I AT deficiency. A heterozygous T to A mutation was found at nucleotide 9833 in exon 5 resulting in a Tyr363Stop nonsense mutation. The sequencing results from the pedigree indicated that four other members also had this mutation.

CONCLUSIONThis heterozygous nonsense mutation of T9833A in exon 5 resulting in venous thrombosis is a novel genetic defect of hereditary AT deficiency, which has not been described before.

Antithrombin III Deficiency ; genetics ; Antithrombins ; genetics ; Blood Coagulation Tests ; Female ; Humans ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Sequence Analysis, DNA

The study was aimed to investigate the changes of T-cell subgroups in the peripheral blood (PB) of patients with aplastic anemia (AA) and the relationships between these changes and the pathogenesis of AA and the immunosuppressive therapeutic effects in AA, in order to provide a basis for selecting rational therapy of AA patients. T-cell subtype and the ratio of CD4+/CD8+ cell in the PB of 88 AA patients which had been diagnosed clearly and given conventional therapy or conventional therapy combined with immunotherapy were analyzed by tri-colour fluorescence-labeled monoclonal antibody and using multiparameter flow cytometry. The patients with AA were divided into normal type of ratio, inverted type of ratio, hypernormal type of ratio according to the ratio of CD4+/CD8+ cell in normal group, and then the relations of these subtype with patients' conditions and therapeutic effects were investigated. The results showed that the percentage of normal type of ratio in all patients was 39.8%, the percentage of inverted type of ratio in all patients was 44.3%, The percentage of hypernormal type of ratio in all patients was 15.9%. In the conventional therapy alone, there was no significant difference on therapeutic effects among these three immunological subtypes. In combined immunotherapy, total therapeutic efficacy of AA patients with inverted type of ratio and AA patients with immunologic abnormality (inverted type + hypernormal type) was 84.2% and 82.6% respectively, which were more than that in conventional therapy (45.5% and 42.8%) (p < 0.05). Total therapeutic efficacy in these patients was better than that in AA patients with normal type. It is concluded that significant abnormal ratios of CD4+/CD8+ exist in the majority of AA patients, abnormal ratios of CD4+/CD8+ both may be showed as increase or decrease, immunologic abnormality may play a role in pathogenesis of the patients with AA. The detection of PB T-cell subtype in patients with aplastic anemia contributes to evaluation of patients' condition and choice of rational treatment prescription, and enhancement of diagnostic level and therapeutic efficacy significantly, which is an important indicator for therapeutic strategy also.
Adult ; Anemia, Aplastic ; immunology ; CD4-CD8 Ratio ; Female ; Humans ; Male ; Middle Aged ; T-Lymphocyte Subsets ; cytology ; immunology ; Young Adult

OBJECTIVETo identify the fibrinogen (Fg) gene mutations in a Chinese pedigree of congenital afibrinogenemia.

METHODSThe plasma Fg activity and protein of the proband and his family members were detected. Genomic DNA was isolated from the peripheral blood mononuclear cells. All the exons and exon-intron boundaries of fibrinogen gene were amplified by PCR and sequenced thereafter.

RESULTSTwo mutations, 7972 del G in FGB and T2543A in FGG, were found in the proband.

CONCLUSIONSFGG2543 is a polymorphism site, which lead to the polymorphism of gamma144 I/K. The G deletion at base 7972 of FGB contributes to the frameshift mutation after amino acid 419, resulting in the truncated beta chain without the terminal 27 amino acids. The latter may contributes to the pathogenetic mechanisms in Chinese congenital afibrinogenemia patients. The G deletion at base 7972 of FGB is identified for the first time.

Adult ; Afibrinogenemia ; congenital ; genetics ; metabolism ; Base Sequence ; Blotting, Western ; DNA Mutational Analysis ; Exons ; genetics ; Female ; Fibrinogen ; genetics ; Humans ; Introns ; genetics ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction