Simultaneous Determination of Six Compounds in Rat Plasma by Ultra‑Performance Liquid Chromatography with Tandem Mass Spectrometry: Application in the Pharmacokinetic Study of Qing Gan‑Shu Yu‑Fang

A rapid and high selective ultra?performance liquid chromatography (UPLC) with tandem mass spectrometry method for simultaneous determination of six compounds including albiflorin, paeoniflorin, picroside I, picroside II, saikosaponin A, and saikosaponin D in rat plasma was developed and validated using butyl p-hydroxybenzoate as an internal standard. One-step direct protein precipitation with acetonitrile was used to extract the compounds from the rat plasma samples. Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) at a flow rate of 0.4 mL/min, using gradient mode containing 0.1% formic acid in water and acetonitrile were used as the Mobile phase A and B. Electrospray ionization in negative ion mode and multiple reaction monitoring were used to identify and quantify active components. Calibration curves showed good linearity (R2 > 0.9908) over a wide concentration range for all compounds. The intra- and interday precision (relative standard deviation) ranged 2.4%–7.0% and 2.6%–8.0%, respectively. The accuracy (relative error) was from ?13.0% to 13.2% at all quality control levels. The recovery ranged from 81.1% to 92.5%. The validated method was successfully applied to pharmacokinetic study in rats after oral administration of Qing Gan?Shu Yu?Fang. The results show that one can draw a conclusion that these six active ingredients can be quickly absorbed and play a pharmacodynamic role rapidly in vivo.     

乳房原发性鳞状细胞癌

乳房原发性鳞状细胞癌极为罕见,文献中有确切证据者迄今仅21例。本文报道两例。病例1,44岁,经产妇。1981年4月入院。主诉左乳有一黄豆大小肿块两年。近二月来明显增大,偶有乳头溢液。体格检查,左乳中内象限触及5.5×4.5cm 肿块,质坚有轻     

Activation of the lama2 gene in muscle regeneration: abortive regeneration in laminin alpha2-deficiency

Mutations in laminin alpha2, a subunit of the basement membrane protein laminin-2/merosin, cause merosin-deficient congenital muscular dystrophy. To gain insight into the molecular mechanism of disease, we generated and used a mutant mouse, dyW, in which the lacZ gene was inserted into the lama2 gene so that beta-galactosidase would be expressed in place of laminin alpha2. Heterozygous and homozygous mutant mice are normal at birth, but homozygous mice develop muscular dystrophy at 2 to 3 weeks of age. The lama2/lacZ gene was highly expressed in muscle in the early stages of embryonic myogenesis, but was down-regulated at later stages in both heterozygous and homozygous mice. No beta-galactosidase activity was detected in skeletal muscle after birth in adult heterozygous mice. In contrast, high beta-galactosidase activity was detected in postnatal homozygous mice. Induction of injury in heterozygous mice resulted in intense reexpression of beta-galactosidase in the injured muscle early in regeneration, with a decline in enzyme activity as repair of the tissue progressed. Although the initial response to injury was similar in heterozygous and homozygous mice with abundant beta-galactosidase-positive, mononucleated cells in the injured area, repair was rarely completed in the homozygous mice, evidently caused by excessive death of cells associated with immature myofibers. The defect in muscle repair was very efficiently corrected in homozygous dyW mice expressing a human LAMA2 transgene in skeletal muscle. The data show the importance of laminin alpha2 in muscle regeneration and suggest that a major contributor to disease in muscular dystrophy is abortive regeneration.     

Changes in systemic and splanchnic hemodynamics after orthotopic liver transplantation in cirrhotic rats

Ｐｒｅｖｉｏｕｓｓｔｕｄｉｅｓｈａｖｅｓｈｏｗｎｔｈａｔｓｙｓｔｅｍｉｃａｎｄｓｐｌａｎｃｈｎｉｃｈｅｍｏｄｙｎａｍｉｃａｂｎｏｒｍａｌｉｔｉｅｓｐｅｒｓｉｓｔｉｎｃｉｒｒｈｏｔｉｃｐａｔｉｅｎｔｓｗｉｔｈｐｏｒｔａｌｈｙｐｅｒｔｅｎｓｉｏｎａｆｔｅｒｏｒｔｈｏｔｏｐｉｃｌｉｖｅｒｔｒａｎｓｐｌａｎｔａｔｉｏｎ(ＯＬＴ),ｄｅｓｐｉｔｅｔｈｅｒｅｔｕｒｎｏｆｐｏｒｔａｌｐｒｅｓｓｕｒｅｔｏｎｏｒｍａｌＴｈｅｅｌｕｃｉｄａｔｉｏｎｏｆｔｈｅｍｅｃｈａｎｉｓｍｒｅｍａｉｎｓｃｏｎｔｒｏｖｅｒｓｉａｌ15…     

手术在桥本氏病治疗中的地位

目的探讨桥本氏病(Hashimoto'sdisease,HD)合并有其它甲状腺疾病的手术应用和指征。方法总结我院1993年1月～1997年12月甲状腺手术病例986例,病理证实为HD的31例,对合并症和误诊原因进行分析。结果合并甲状腺癌5例;合并甲状腺机能亢进4例;有气管压迫症状4例;误诊为结节性甲状腺肿7例。结论应注意对无症状而伴有双侧结节性甲状腺肿的HD患者的误诊,HD可合并腺癌;HD如伴有孤立的结节或压迫症状,抑制治疗无改善则应积极手术治疗。     

Simultaneous Determination and Pharmacokinetics of Tetrandrine, Fangchinoline, and Cyclanoline in Rat Plasma by Ultra-High Performance Liquid Chromatography-Mass Spectrometry after Oral Administration of Stephaniae Tetrandrae Radix Extract

Objective: The objective of the study wasto develop a rapid and sensitive ultra?performance liquid chromatography?tandem massspectrometric method for the determination of tetrandrine, fangchinoline, and cyclanoline in rat plasma and to investigate their pharmacokinetics after oral administration of Stephaniae Tetrandrae Radix extracts. Methods: Sample pretreatment involved methanol pretreatment and liquid–liquid extraction of ethyl acetate from plasma with methanol. Tramadol was used as the internal standard. The analysis was performed using an high strength silica T3 column (100 mm × 2.1 mm, 1.8 μm) and a gradient elution method consisting of mobile phase solution A (0.1% formic acid in water) and B (acetonitrile) at a flow rate of 0.4 mL/min. The detection was performed using a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode and using an electrospray ionization source in the positive ionization mode. Results: High efficiency was achieved with an analysis time of 4 min/sample. The calibration curve linear in the concentration range of 1250 ng/ml (R2 ≥ 0.9900) and the lower limit of quantification is 1 ng/ml. The intraday and interday precision (relative standard deviation) values were lower than 9.4. Accuracy (relative error) was within 10.3% at all three quality control levels. Conclusions: This method was successfully applied in pharmacokinetics of tetrandrine, fangchinoline, and cyclanoline in rats after oral administration of Stephaniae Tetrandrae Radix extracts. The maximum plasma concentration (Cmax) of tetrandrine, fangchinoline, and cyclanoline was 124.71 ± 16.08, 84.56 ± 3.28, and 57.61 ± 6.26 ng/ mL, respectively. The time to reach Cmax was 10.39 ± 3.04 for tetrandrine, 10.17 ± 3.04 for fangchinoline, and 6.40 ± 3.16 for cyclanoline. The pharmacokinetic results might help further guide the clinical application of Stephaniae Tetrandrae Radix.     

Dysfunctional CD4+,CD25+ regulatory T cells in untreated active systemic lupus erythematosus secondary to interferon-alpha-producing antigen-presenting cells

OBJECTIVE: To explore whether there are extrinsic factors that impair the suppressive function of CD4+,CD25+ regulatory T cells in patients with untreated active systemic lupus erythematosus (SLE). METHODS: We studied 15 patients with untreated active SLE, 10 patients with SLE in remission, and 15 healthy control subjects. Percentages of CD4+,CD25+,FoxP3+ Treg cells and levels of forkhead box P3 (FoxP3) protein were analyzed by flow cytometry. Expression of messenger RNA (mRNA) for FoxP3 in purified Treg cell populations was assessed by real-time polymerase chain reaction analysis. Experiments examining Treg cell function in SLE were designed to distinguish primary from secondary T cell dysfunction. Levels of interferon-alpha (IFNalpha) in supernatants from the function assays were determined with an IFN-stimulated response element-luciferase reporter assay. RESULTS: The percentage of CD4+,CD25+, FoxP3+ cells in peripheral blood was significantly increased in SLE patients as compared with controls (mean +/- SEM 9.11 +/- 0.73% versus 4.78 +/- 0.43%; P < 0.0001). We found no difference in FoxP3 expression at either the mRNA or protein level in any CD4+,CD25+ T cell subset from SLE patients as compared with controls. Antigen-presenting cells (APCs) from SLE patients were responsible for decreased Treg cell activity and could also render dysfunctional Treg cells from healthy control subjects. CD4+,CD25+ Treg cells from SLE patients exhibited normal suppressive activity when cultured with APCs from healthy controls. A partial Treg cell blockade effect was induced by the high levels of IFNalpha derived from SLE patient APCs. CONCLUSION: We suggest that blockade of Treg cell-mediated suppression by IFNalpha-producing APCs in SLE patients may contribute to a pathogenic loss of peripheral tolerance in this disease.     

Production of recombinant orange-spotted grouper (Epinephelus coioides) follicle-stimulating hormone (FSH) in single-chain form and dimer form by Pichia pastoris and their biological activities

FSH is a key regulator of steroidogenesis and gonadal growth in teleosts. However, function of FSH is elusive in grouper due to the lack of purified and native FSH. In the present study, we reported production of bioactive orange-spotted grouper (Epinephelus coioides) FSH in dimer form and single-chain form by Pichia pastoris. Dimer form of recombinant grouper FSH (rgFSHba) was accomplished by co-expressing mature FSHb-subunit and a-subunit genes. Fusion of mature FSHb-subunit and a-subunit genes together linking with a polypeptide (4×(Gly-Ser)-Gly-Thr) gene generated single-chain form of recombinant grouper FSH (rgFSHb-a). Recombinant grouper common α-subunit (rgCga) and FSHb-subunit (rgFSHb) were also separately produced. Recombinant proteins were verified by Western blot and mass spectrometry assays, and characterized by deglycosylation analysis. Deglycosylation assay suggested that glycosylation of recombinant FSH mainly occurred on common a-subunit. Bioactivities of recombinant proteins were initially evaluated by activating grouper FSH receptor, and further demonstrated by incubating ovarian fragments of adult grouper and intraperitoneal injection in juvenile female grouper. Two forms of recombinant FSH presented similar biological activities of activating FSH receptor and stimulating in vitro testosterone (T) and estradiol-17β (E2) secretion, though the dimer form functioned slightly weaker than the single-chain form. However, injections of rgFSHb-a or rgFSHba could significantly increase serum T and E2 levels, induce early ovarian development, reduce hypothalamic gnrh1 mRNA level, and increase hypothalamic cyp19a1b mRNA level. Data in this study suggested that recombinant gonadotropin could be produced in dimer form or single-chain form by P. pastoris, and FSH could regulate steroidogenesis and early ovarian development in juvenile grouper.