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

AIMTo establish an HPLC-fluorescent spectrometric method for the determination of mexiletine hydrochloride in plasma after derivatization with fluram.

METHODSFluram acetone solution was added to the deproteinized plasma with acetone to obtain the derivative of mexiletine. The HPLC method was performed on a column of Allitima C18 (150 mm x 4.6 mm, 5 microns) with the mobile phase of methanol-water-diethylamine-phosphoric acid buffer (2.4 mol.L-1, pH 4.0) (70:28:2), and the detective wavelength were set at Ex 392 nm and Em 480 nm.

RESULTSMexiletine has a liner range over the concentration range from 0.100-6.400 mg.L-1. The lowest detectable concentration of this method was 5 micrograms.L-1 (S/N > or = 4). The intra-day and inter-day RSDs were 1.34%-5.31%, respectively.

CONCLUSIONThis method is simple, selective and can be used for therapeutic drug monitoring (TDM) and pharmacokinetic studies of mexiletine.

Anti-Arrhythmia Agents ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; methods ; Fluorescamine ; chemistry ; Humans ; Mexiletine ; blood ; pharmacokinetics

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

S1 nuclease (from Aspergillus oryzae) is a specific enzyme to degrade single stranded DNA or RNA molecules. It has been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions, but this function has not been well explored. In order to use the action of S1 nuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs, the pUC19 was used to investigate the relationship between the linearization efficiency of S1 nuclease and the amount of enzyme used. By this way the optimal conditions for linearization of circular DNAs by S1 nuclease would be determined. 0.3u to 17u S1 nuclease per 100ng pUC19 DNA was added into a 25 microL system, respectively, to perform the reaction. The effectiveness of enzyme digestion was realized by electrophoresis in a 1.2% agarose gel. The results showed that along with the increase in enzyme amount from 0.3u to 17u a gradual decrease in the superhelical form, a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u S1 nuclease per 100ng DNA. Besides, electrophoretic mobility of the S1 nuclease-linearized pUC19 was the same as that of the linear form produced by restriction enzyme digestion. According to the result of phiX174 digested by S1 nuclease it has been proposed that the enzyme cleaves first randomly on one site of one strand, thus converting the superhelical molecules into open circle form, and then on the same site of the complementary strand to produce the linear form. Therefore, the S1 nuclease-linearized DNA molecules are intact in the sense of their length and can be used for cloning. The plasmid-like DNA pC3 from cucumber mitochondria is a double stranded circular DNA molecule with about 550bp and the smallest known plasmid-like DNA in eukaryotic mitochondria. Many attempts have been made to linearize the molecule by using restriction enzymes but failed. Therefore, S1 nuclease was used to linearize pC3 based on the results obtained with pUC19. The linearized pC3 DNA molecules formed a very sharp band in a 2.5% agarose gel after electrophoresis. They were then recovered from the gel, added an "A" tail and ligated with T-vector. After transformation into E. coli JM109 cells, the positive clones were, screened by the blue-white selection. The insert was then cut using restriction enzymes EcoRI and Pst I. The result of electrophoresis shows that the electrophoretic mobility of the insert is just the same as that predicted. A 32 P-labled probe was synthesized using pC3 as the template and Southern blot analysis was carried out. The result shows that the inserted DNA is hybridized to the probe, which indicates that the cloned DNA fragment is from pC3. The sequence information of the insert shows that the plasmid-like DNA pC3 was 537bp in length. The nucleotide sequence was deposited in the GenBank (the accession number is AF522195).
Blotting, Southern ; Cloning, Molecular ; methods ; DNA, Circular ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Single-Strand Specific DNA and RNA Endonucleases ; genetics ; metabolism

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