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Primary immune thrombocytopenia (ITP) is a bleeding disorder commonly encountered in clinical practice. The International Working Group (IWG) on ITP has published several landmark papers on terminology, definitions, outcome criteria, bleeding assessment, diagnosis, and management of ITP. The Chinese consensus reports for diagnosis and management of adult ITP have been updated to the 4th edition. Based on current consensus positions and new emerging clinical evidence, the thrombosis and hemostasis group of the Chinese Society of Hematology issued Chinese guidelines for management of adult ITP, which aim to provide evidence-based recommendations for clinical decision making.

Nowadays, network pharmacology-based methods were increasing proposed to screen synergistic or combinatorial compounds from herbal medicines (HMs), while these researches mainly focused on structural prediction or experiment-based interaction between single compound and target protein. The proportion of each chemical in the nature and their metabolic process was ignored, which might decide an optimized composition for their synergistic effect. To exact the effective combination of HMs, a metabolic distribution-oriented network regulation strategy was developed for the identification of effective combination. Firstly, comprehensive chemical profiling and metabolic exposure of HMs in a pathological state were conducted. Then the effective combination for HMs was screened by combining network regulation and the metabolic exposure level of HMs. Finally, with the extract of Ginkgo biloba (EGB) as a case, a combination of 12 active compounds was found for treating ischemia stroke, showing bioactivity equivalence with original herb. The results also indicated that beside the well-known ginkgolides and flavonoids, trace compounds might also play an important role of the holistic effect of EGB. This method can be used as an alternative for effective combination screening., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Thrombopoietin receptor agonists (TPO-RAs) have been clinically used in primary immune thrombocytopenia (ITP) with favorable outcomes, while their effect on cytokine regulation in ITP remains unknown. In the present study, plasma and mRNA expression levels of interleukin (IL)-2, interferon gamma (IFN-γ), IL-4, IL-17A, and transforming growth factor-β1 (TGF-β1) were determined by ELISA and real-time quantitative PCR in 26 corticosteroid-resistant/relapsed ITP patients receiving eltrombopag or rhTPO therapy and 15 healthy controls (HCs). Results showed that plasma and mRNA levels of IL-2, IFN-γ, IL-4, and IL-17A in ITP patients did not change significantly after TPO-RA treatment, whereas TGF-β1 levels increased remarkably. The pre- and post-treatment plasma and mRNA levels of IFN-γ and IL-2 were significantly higher, while the pre- and post-treatment IL-4 levels as well as the pre-treatment TGF-β1 levels were remarkably lower in ITP patients compared with HCs. There was no significant difference in TGF-β1 levels between TPO-RA-treated ITP patients and HCs. No statistical difference was found in plasma levels of IL-17A between ITP patients before or after treatment and HCs. However, the pre- and post-treatment mRNA expression of IL-17A and retinoic orphan receptor (ROR) γt in ITP patients were higher than that in HCs. Overall, these findings indicated that TPO-RA treatment could promote the secretion of TGF-β1, while it could not correct the Th1 and Th17 polarization in ITP patients. This study might improve our understanding of the mechanism of action of TPO-RAs and provide important information for optimizing therapeutic strategies for ITP., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.

B-cell acute lymphoblastic leukemia (B‑ALL) is an aggressive hematological malignancy and a leading cause of cancer-related mortality in children and young adults. The molecular mechanisms involved in the regulation of its gene expression has yet to be fully elucidated. In the present study, we performed large scale expression profiling of microRNA (miRNA) and transcription factor (TF) by Illumina deep‑sequencing and TF array technology, respectively, and identified 291 differentially expressed miRNAs and 201 differentially expressed TFs in adult B‑ALL samples relative to their controls. After integrating expression profile data with computational prediction of miRNA and TF targets from different databases, we construct a comprehensive miRNA‑TF regulatory network specifically for adult B‑ALL. Network function analysis revealed 25 significantly enriched pathways, four pathways are well‑known to be involved in B‑ALL, such as PI3K‑Akt signaling pathway, Jak‑STAT signaling pathway, Ras signaling pathway and cell cycle pathway. By analyzing the network topology, we identified 28 hub miRNAs and 19 hub TFs in the network, and found nine potential B‑ALL regulators among these hub nodes. We also constructed a Jak‑STAT signaling sub‑network for B‑ALL. Based on the sub‑network analysis and literature survey, we proposed a cellular model to discuss MYC/miR‑15a‑5p/FLT3 feed-forward loop (FFL) with Jak‑STAT signaling pathway in B‑ALL. These findings enhance our understanding of this disease at the molecular level, as well as provide putative therapeutic targets for B-ALL.

Phosphatidylinostitol-3-kinase (PI3K) is the potential anticancer target in the PI3K/Akt/ mTOR pathway. Here we reviewed the ATP-competitive small molecule PI3K inhibitors in the past few years, including the pan Class I PI3K inhibitors, the isoform-specific PI3K inhibitors and/or the PI3K/mTOR dual inhibitors.

Soluble fibrinogen-like protein 2 (sFGL2) is the soluble form of fibrinogen-like protein 2 belonging to the fibrinogen-related protein superfamily. It is now well characterized that sFGL2 is mainly secreted by regulatory T cell (Treg) populations, and exerts potently immunosuppressive activities. By repressing not only the differentiation and proliferation of T cells but also the maturation of dendritic cells (DCs), sFGL2 acts largely as an immunosuppressant. Moreover, sFGL2 also induces apoptosis of B cells, tubular epithelial cells (TECs), sinusoidal endothelial cells (SECs), and hepatocytes. This mini-review focuses primarily on the recent literature with respect to the signaling mechanism of sFGL2 in immunomodulation, and discusses the clinical implications of sFGL2 in transplantation, hepatitis, autoimmunity, and tumors.

In this retrospective cohort study conducted in 63 children with idiopathic thrombocytopenic purpura (ITP) in China; petechiae, bruises and bleeding were the major presentations. Most cases required therapy with one/more treatment options.

Phellodendrine, a quaternary ammonium alkaloid extracted from the dried bark of Phellodendrom chinensis Schneid and Phellodendrom amurense Rupr, has the effect of suppressing cellular immune response, reducing blood pressure and antinephritis. However, few investigations have been conducted for the pharmacokinetic study of phellodendrine. Thus, a rapid, simple and reliable ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-QQQ MS/MS) method has been established for quantification of phellodendrine in rat plasma and tissues by using magnoflorine as internal standard. The chromatographic separation was achieved on an Agilent ZORBAX SB-C18 column (4.6mm×50mm, 1.8μm) by gradient elution using 0.1% aqueous formic acid (A) and methanol (B). Triple quadrupole mass detection with multiple reaction monitoring mode was used to monitor the ion transitions, at m/z 342.20→192.20 for phellodendrine and m/z 342.20→58.20 for internal standard, respectively. The developed method was fully validated and successfully applied to the pharmacokinetics and tissue distribution study of phellodendrine after intravenous administration. The lower limits of quantification were 0.5ng/mL for plasma samples, 2.5ng/g for brain and 1ng/g for other tested tissues. Precisions and accuracy values were within the Food and Drug Administration acceptance criteria, the recovery and matrix effects were between 87.8-113.5%. The area under the curve (AUC0-t) ranged from 15.58 to 57.41mg/L min and Cmax were between 1.63-4.93mg/L. The results showed that phellodendrine was eliminated in 120min in plasma and most of tissues and the highest concentrations of phellodendrine were found in the kidney. This study may provide a basis for the further study of phellodendrine., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Elevated expression of the activating Fcγ receptor (FcγR) I and FcγRIIa together with decreased expression of the inhibitory FcγRIIb are involved in the pathogenesis of primary immune thrombocytopenia (ITP). Thrombopoietin receptor agonists (TPO-RAs) have been used clinically for the management of ITP; however, little is known about the effect of TPO-RAs on FcγR modulation in ITP. In this prospective study, we measured the alteration in monocyte FcγR expression from 21 corticosteroid-resistant/relapsed patients with chronic ITP receiving eltrombopag therapy. Results showed that the mRNA and protein levels of FcγRIIb were significantly elevated after 6-week eltrombopag treatment. Concurrently, FcγRI and IIa levels decreased remarkably, whereas FcγRIII expression did not change. In vitro phagocytosis assays indicated that a shift in the balance of FcγR toward inhibitory FcγRIIb on monocytes was accompanied with a considerable decrease in monocyte/macrophage phagocytic capacity. The response to eltrombopag therapy in patients with ITP was associated with FcγR phenotype and functional changes of monocytes/macrophages. Moreover, the plasma transforming growth factor-β1 (TGF-β1) concentrations increased significantly in eltrombopag responders. Modulation of monocyte FcγR balance by TPO-RAs was also found in a murine model of ITP established by transferring splenocytes from immunized CD61 knockout mice into CD61(+) severe combined immunodeficient mice. Romiplostim administration in ITP mice significantly upregulated inhibitory FcγRII expression and downregulated activating FcγRI expression. These findings showed that recovery of platelet counts after TPO-RA treatment in ITP is associated with the restoration of FcγR balance toward the inhibitory FcγRIIb on monocytes, and suggested that thrombopoietic agents have a profound effect on immune modulation in ITP. This study is registered at ClinicalTrials.gov as #NCT01864512., (© 2016 by The American Society of Hematology.)

Primary immune thrombocytopenia (ITP) is a complex autoimmune disorder in which the patient's immune system reacts with platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. Corticosteroids can induce sustained remission rates in 50% to 75% of patients with active ITP. For these patients who are unresponsive to glucocorticoids, or relapsed after an initial response, multiple second-line treatment modalities can be chosen. However, how to make an optimal therapeutic strategy for a specific patient still remains a major challenge. As the pathogenetic heterogeneity of the ITP is increasingly identified, pathogenesis-oriented approach might offer an opportunity to improve the outcome of corticosteroid-resistant or relapsed ITP., Competing Interests: Conflict of interest statement: Authors state no conflict of interest.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. In this study, the MDSC population was evaluated in adult patients with primary immune thrombocytopenia (ITP), where cell-mediated immune mechanisms are involved in platelet destruction. Our data demonstrated that both the numbers and suppressive functions of MDSCs were impaired in the peripheral blood and spleens of patients with ITP compared with healthy control patients. High-dose dexamethasone (HD-DXM) treatment rescued MDSC numbers in patients with ITP. And DXM modulation promoted the suppressive function of MDSCs induced in vitro. Moreover, the expression of interleukin 10 and transforming growth factor β was significantly upregulated in DXM-modulated MDSCs compared with the unmodulated cultures. DXM-modulated MDSCs inhibited autologous CD4(+)T-cell proliferation and significantly attenuated cytotoxic T lymphocyte-mediated platelet lysis, further indicating enhanced control over T-cell responses. Elevated expression of the transcription factor Ets1 was identified in DXM-modulated MDSCs. Transfection of Ets-1 small interfering RNA efficiently blocked regulatory effects of MDSCs, which almost offset the augmentation of MDSC function by DXM. Meanwhile, splenocytes from CD61 knockout mice immunized with CD61(+)platelets were transferred into severe combined immunodeficient (SCID) mouse recipients (C57/B6 background) to induce a murine model of severe ITP. We passively transferred the DXM-modulated MDSCs induced from bone marrow of wild-type C57/B6 mice into the SCID mouse recipients, which significantly increased platelet counts in vivo compared with those receiving splenocyte engraftment alone. These findings suggested that impaired MDSCs are involved in the pathogenesis of ITP, and that HD-DXM corrected MDSC functions via a mechanism underlying glucocorticoid action and Ets1., (© 2016 by The American Society of Hematology.)

Screening of high potent enzyme inhibitors from herbal medicines is always lacking of efficiency due to the complexity of chemicals. The constituents responsible for the holistic effect may be trace-level chemicals, but these chemicals were covered by highly abundant compositions. To challenge this bottleneck, a strategy for screening minor bioactive compounds was proposed. It generally included four steps, (1) preliminarily find the enzyme binders by ultrafiltration; (2) optimise and predict the potential inhibitors by docking analysis; (3) selectively identify and prepare trace compounds by segment and exposure approach; (4) validate the activity and summarize the structure-activity relationship. As a case study, α-glucosidase (AGH) and Ginkgo biloba extract were used as the experimental materials. By comprehensive screening, 11 trace flavones were screened out and identified as strong AGH inhibitors. Among them, AGH inhibitory activities of syringetin and sciadopitysin were reported for the first time. Their IC50 values were 36.80 and 8.29μM, respectively, which were lower than that of a positive control acarbose. In addition, the AGH inhibitory activities of the flavonoids could be ranked, based on a decreased order, as biflavone, flavone, flavone glycoside, flavone biglycoside. The strategy is expected to be practical and useful for screening bioactive molecules from herbal medicines, especially for the minor compounds, which will definitely accelerate the discovery of new drug candidates., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To characterize and identify multiple constituents in Danhong injection (DHI), a fast ultra-high performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-QTOF/MS) method was established and validated in the present study. A total of 63 compounds, including 33 phenolic acids, 2 C-glycosyl quinochalcones, 6 flavonoid O-glycosides, 4 iridoid glycosides, 6 organic acids, 5 amino acids, and 3 nucleosides, were identified or tentatively characterized. In conclusion, the UHPLC-ESI-QTOF/MS method is useful and efficient for in-depth structural elucidation of chemical compounds in complex matrices of herbal medicines such as DHI., (Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

An ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) approach was developed and validated for the determination of ginkgolide L (GL) in rat plasma and tissues using diazepam as internal standard (IS). Detection was performed on a triple quadrupole MS system using multiple reaction monitoring (MRM) mode in positive mode. Sample preparation was carried out through a liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved by using an Agilent ZORBAX SB-Aq column with a mobile phase of 0.5% aqueous formic acid (A) and methanol (B). The monitored transitions were set at m/z 391.14→271.10 for GL and m/z 285.08→193.10 for IS, respectively. The validated method was successfully applied to the pharmacokinetic and tissue distribution study of GL in rats after intravenous administration. Good linearity was found between 2.5-2000ng/mL (r>0.996) for plasma samples, and calibration curves were also linear for other tissue samples over a wide range. The results indicated that GL has linear pharmacokinetic properties after intravenous administration at three doses. GL could distribute to tissues quickly and the major distribution tissue of GL in rats was liver. This was the first report of pharmacokinetic and tissue distribution data for GL., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recently, screening of bioactive compounds by on-line ultra-high performance liquid chromatography (UHPLC) draws increasing attentions for the advantages of rapidity and intuition. Nevertheless, most on-line methods were limited to the shortcoming like low resolution and peak capacity, which could interfere the active ingredient identification. Comprehensive two-dimensional UHPLC (LC×LC) has revealed to be a powerful tool to separate complex mixtures. Herein, a strategy based on LC×LC analysis coupled with pre-column 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was proposed to screen the antioxidants from the extract of Ginkgo biloba (EGB). A total of 61 compounds were identified in EGB, and 25 of them showed appreciable radical scavenging capacity. This work may offer pharmacodynamics base for further research about EGB, also the strategy is likelihood to be applied in screening antioxidant in other herbal medicine., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rheumatoid arthritis (RA) is the most common inflammatory arthritis and a major cause of disability. Presently, the clinical therapeutic medicines for inflammatory and arthritic diseases are unsatisfactory due to severe adverse effects or ineffectiveness. The Guge Fengtong formula (GGFT), containing the standardized extracts of Dioscoreae Nipponicae Rhizoma, Spatholobi Caulis, and Zingiberis Rhizoma, has long been used for RA treatment by Chinese doctorsin China. However, the detailed anti-inflammatory and anti-arthritic activity of GGFT has not been reported so far. In the present work, we aimed to evaluate the anti-inflammatory and anti-arthritic effects of GGFT using three in vivo animal models, and tried to uncover its preliminarythe underlying mechanism of action mechanism in RAW 264.7 macrophages. The obtained results indicated that GGFT significantly attenuated ear edema, decreased carrageenan-induced paw edema, reduced the arthritis score, and reversed the weight loss of the complete Freund's adjuvant (CFA)CFA-injected rats. Additionally, marked decrease of in synovial inflammatory infiltration and synovial lining hyperplasia in the joints and decline of inflammatory factors (TNF-α and IL-1β) in the serum were observed in the GGFT-treated rats. In lipopolysaccharide-activated RAW264.7 macrophages, GGFT reduced the production of NO, PGE2, and IL-6, and inhibited the expression of iNOS, COX-2, and NF-κB expression. Our results demonstrated that GGFT possessed considerable anti-inflammatory activity and have had potential therapeutic effects on adjuvant induced arthritis in rats, which provided providing experimental evidences for its traditional application in the treatment of RA and other inflammatory diseases., (Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Qi-Fu-Yin (QFY), a classical traditional Chinese medicine formula, is proven to have significant neuroprotective effects by modern pharmacological studies. However, the chemical constituents of QFY have not been fully explored. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF MS) was developed for comprehensive analysis of chemical constituents in QFY. By using characteristic ions and fragmentation rules, a reliable identification of 156 compounds was described here, including 69 triterpene saponins, 23 oligosaccharide esters, 22 flavanoids, 9 alkaloids, 9 phenolic acids, 8 phthalides, 7 phenylethanoid glycosides, 3 xanthones, 3 sesquiterpene lactones, 2 ionones and 1 iridoid glycoside. Twenty-six major compounds were then determined in a single run by UHPLC coupled with triple quadrupole tandem mass spectrometry (QQQ MS) with fast positive/negative polarity switching. It allows for the acquisition of MS data in both ionization modes from a single run. The proposed method was then validated in terms of linearity, accuracy, precision and recovery. The overall recoveries for 26 analytes ranged from 91.35% to 109.58%, with RSDs ranging from 0.82% to 4.83%. In addition, the content of 26 analytes in QFY prepared by five batches of herbal materials was also analyzed. These results demonstrated that our present method was effective and reliable for comprehensive quality evaluation of QFY. Meanwhile, the study might provide the chemical evidence for revealing the material basis of its therapeutic effects., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Flavonoids are the main active constituents in Ginkgo biloba L., which have been suggested to have broad-spectrum free-radical scavenging activities. This review summarizes the recent advances in the chemical analysis of the flavonoids in G. biloba and its finished products (from 2009 to 2014), including chemical composition, sample preparation, separation, detection and different quality criteria. More than 70 kinds of flavonoids have been identified in this plant. In this review, various analytical approaches as well as their chromatographic conditions have been described, and their advantages/disadvantages are also compared. Quantitative analyses of Ginkgo flavonoids applied by most pharmacopeias start with an acidic hydrolysis followed by determination of the resulting aglycones using HPLC. But increasing direct assay of individual flavonol glycosides found that many adulterated products were still qualified by the present tests. To obtain an authentic and applicable analytical approach for quality evaluation of Ginkgo and its finished products, related suggestions and opinions in the recent publications are mainly discussed in this review. This discussion on chemical analyses of Ginkgo flavonoids will also be found as a significant guide for widely varied natural flavonoids., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: IL-17A G197A and IL-17F A7488G gene polymorphisms are found to be associated with the risk of several diseases, however few studies have focused on their correlation with risk of liver cirrhosis (LC)., Aims: The aim of this study was to assess the impact of IL-17A G197A and IL-17F A7488G gene polymorphisms on development of LC from chronic hepatitis B (CHB) in Chinese patients., Methods: A total of 163 HBV-related LC patients and 168 CHB patients were enrolled in the present study. IL-17A and IL-17F gene polymorphisms were analyzed using a polymerase chain reaction (PCR) restriction fragment length polymorphism assay (PCR-RFLP)., Results: Frequencies of IL-17A G197A genotype AA and allele A were significantly higher in LC patients compared with that in CHB patients (AA 42.33% vs 27.98%, P = 0.032; A 56.34% vs 46.15%, P=0.011, respectively); While no significant difference in frequencies of genotypes and alleles of IL-17F A7488G was found between the two groups. The AA genotype of IL-17A G197A significantly increased LC risk (OR 4.186, 95% CI: 1.479-11.844), while A allele carriers were also associated with an increased LC risk (OR 1.856, 95% CI: 1.161-2.967)., Conclusion: IL-17A G197A is a candidate gene that confers the genetic susceptibility for LC development from CHB in Chinese population.

The aim of this study was to develop a convenient method without pretreatments for nontarget discovery of interested compounds. The segment and exposure strategy, coupled with two mass spectrometer data acquisition methods was firstly proposed for screening the saponins in extract of Panax notoginseng (Sanqi) via high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS). By gradually removing certain major or moderate interference compounds, the developed segment and exposure strategy could significantly improve the detection efficiency for trace compounds. Moreover, the newly developed five-point screening approach based on a modified mass defect filter strategy and the visual isotopic ion technique was verified to be efficient and reliable in picking out the interested precursor ions. In total, 234 ginsenosides including 67 potential new ones were characterized or tentatively identified from the extract of Sanqi. Particularly, some unusual compounds containing the branched glycosyl group or new substituted acyl groups were firstly reported. The proposed integrated strategy held a strong promise for analyses of the complex mixtures., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The present study was designed to characterize the chemical constituents of Guge Fengtong Tablet (GGFTT). Based on the chromatographic retention behavior, fragmentation pathways of chemical components and the published literatures, a diagnostic ion filtering strategy with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-Q-TOF/MS) was established to identify the multiple bioactive constituents of GGFTT. The rapid identification of forty-seven components, including 18 phenolic acids, 8 saponins, 14 gingerol-related compounds, and 7 diarylhepatonoids, was accomplished using this newly developed method. The coupling of HPLC-ESI-Q-TOF/MS with the diagnostic ion filtering strategy was useful and efficient for the in-depth structural elucidation of chemical compounds of GGFTT., (Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Ginkgolide K (GK), a derivative compound of ginkgolide B, has been recently isolated from the leaves of Ginkgo biloba. It is a powerful natural platelet activate factor (PAF) antagonist, and also has obvious protect effects for cerebral ischemia. However, no reports have been described for the pharmacokinetic study of GK. In this study, a simple, sensitive and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the determination of GK in rat plasma and tissues. Biological samples were pretreated by an efficient liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on an Agilent ZORBAX SB-Aq column (4.6 mm × 50 mm, 1.8 μm) with a mobile phase of 0.5% aqueous formic acid (A)-menthol (B). Quantitation was carried out on a triple quadruple mass spectrometry using positive electrospray ionization in multiple reaction monitoring mode. Diazepam was used as internal standard (IS). The ion transitions monitored were set at m/z 407.10 → 389.20 and m/z 285.08 → 193.10 for GK and IS, respectively. The developed method was fully validated and successfully applied to the pharmacokinetics and tissue distribution study of GK after intravenous administration. The current results have indicated that pharmacokinetic parameters of GK vary in a dose-dependent manner with rapid elimination in 4h. The major distribution tissues of GK in rats were liver and kidney. This study would provide critical information to promote the future study of GK., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: To investigate the effect of changed ratio of polyunsaturated fatty acids (PUFA) on dextran sulfate sodium (DSS)-induced colitis in mice., Methods: Thirty-two male BALB/c mice were randomly divided into two groups: control group and PUFA group, PUFA group was continuously divided into 3 sub-groups: PUFA ω-3/ω-6 1:3 group, PUFA ω-3/ω-6 1:15 group and PUFA ω-3/ω-6 1:30 group. According to the difference in the sub-groups, PUFA group mice were fed with the corresponding modified diet. The control group was fed with the common diet, whose ratio of PUFA ω-3/ω-6 was 1:15. After eight weeks of different diets, experimental colitis in the three sub-groups of PUFA group was induced by DSS exposure. The mice were placed on three five-day cycles of 30 g/L DSS with ten days of recovery after each cycle, then were sacrificed after the final ten-day period. Overall symptomatic score and histopathological score were evaluated. And levels of mucosal prostaglandin E2 (PGE2) in the proximal and distal colon were measured respectively by enzyme immunoassay., Results: The changed ratio of PUFA ω-3/ω-6 had no effect on the weight gain of the growing mice. Although there were no significant differences among the PUFA groups from the three separate aspects: weight gain, stool character and blood in the stool, there were significant differences among the three groups in overall symptomatic scores. A further comparison showed the overall symptomatic score of 1:3 group was significantly lower than that of the 1:30 group (P<0.05). There were significant differences among the PUFA groups in the histopathological score. The following comparison between the sub-groups showed the histopathological score of the 1:3 group was significantly lower than that of the 1:30 group (P<0.05). One mouse in the 1:30 group died of severe hemorrhage and one mouse also in this group had a huge dysplastic adenomatous polyp. The mucosal PGE2 which could reflect the level of intestinal inflammation showed that in the distal colon, the inflammations were obvious, and the levels of mucosal PGE2 of the distal colon in the 1:15 group [(153.0 ± 49.4) ng/g tissue] and the 1:30 group [(192.4 ± 94.0) ng/g tissue] were significantly higher than that of the control group [(43.2 ± 13.4) ng/g tissue, P<0.05], but there was no significant difference between the 1:3 group [(43.4 ± 8.2) ng/g tissue] and the control group. Although the mucosa damages were sparing in proximal colon, the level of mucosal PGE2 of the proximal colon in 1:30 group [(97.4 ± 64.8) ng/g tissue] markedly increased as compared with the control group [(21.6 ± 16.0) ng/g tissue, P<0.01], there were no differences among the 1:3 group [(36.6 ± 4.6) ng/g tissue], the 1:15 group [(18.8 ± 6.4) ng/g tissue] and the control group., Conclusion: The colonic inflammatory severity and the level of mucosal PGE2 in the experimental colitis mice were affected by the changed ratio of PUFA ω-3/ω-6 in the feed. Increased ratio of PUFA ω-3/ω-6 in the feed had a protective effect on the intestinal mucosa in the experimental colitis mice, otherwise had hazards. Before the inflammation happened, changed ratio of PUFA ω-3/ω-6 firstly altered the local inflammatory factors, such as PGE2, and then affected the inflammatory severity.

Aim: To evaluate potential risk factors in the development of ulcerative colitis (UC) in China., Methods: A total of 1308 patients with UC and 1308 age-matched and sex-matched controls were prospectively studied in China. The UC cases were collected from 17 hospitals in China from April 2007 to April 2010. Uniform questionnaires were designed to investigate risk factors including smoking, appendectomy, stress, socio-economic conditions, nonsteroidal anti-inflammatory drugs (NSAIDs), oral contraceptives, diet, breastfeeding, infections and family sanitary conditions. Group comparisons by each factor were done using simple logistic regression analysis. Conditional logistic regression was used for multivariate analysis., Results: By univariate analysis, the variables predictive of UC included feeling stress, light and heavy alcoholic drinking, spicy food, sugar consumption and infectious diarrhea, while heavy tea intake and tap water consumption were protective against UC. On multivariate analysis, the protective factor for UC was tap water consumption [odds ratios (OR) = 0.424, 95%CI: 0.302-0.594, P < 0.001]; while the potential risk factors for UC were heavy sugar consumption (OR = 1.632, 95%CI: 1.156-2.305, P < 0.001), spicy food (light intake: OR = 3.329, 95%CI: 2.282-4.857, P < 0.001; heavy intake: OR = 3.979, 95%CI: 2.700-5.863, P < 0.001), and often feeling stress (OR = 1.981, 95%CI: 1.447-2.711, P < 0.001). Other factors, such as smoking habit, appendectomy, breastfeeding, a history of measles, rural or urban residence, education, oral contraceptives, and NSAID use have not been found to have a significant association with the development of UC in the present study., Conclusion: Our study showed tap water consumption was a protective factor for UC, while spicy food, heavy sugar consumption and often feeling stress were risk factors for UC in this Chinese population.

Objective: To assess the expression of TNFα, IL-1β and the serotonin transporter (SERT) in adult rats of chronic visceral hypersensitivity induced by colon irritation during postnatal development, and to provide evidence to clarify the complex relationship between inflammatory cytokines and SERT among visceral hypersensitivity., Methods: Sixteen neonatal male Sprague-Dawley rats were randomly separated into two groups undergoing different treatments. The irritated group (n = 8) was received intracolonic injections of acetic acid (0.5%) between postnatal days 8 and 21 and another group (n = 8) was received colonic infusion with 0.9% saline served as control. When they became adults (ages 7 weeks), the threshold intensity for a visually identifiable contraction of the abdominal wall and body arching were recorded during rectal distention (RD) to evaluate visceral hypersensitivity. Histological evaluation and MPO activity assay were performed to quantify the inflammation. The expression of cytokine of colon was assessed through immunohistochemistry. The expression of SERT was examined by Western blot., Results: Histological examination of the tissues showed no significant structural damage or loss of crypts. The MPO levels in both groups were similar [(0.497 ± 0.570) unit/g vs (0.623 ± 0.739) unit/g, P = 0.724]. The threshold to elicit a distinctive abdominal muscle contraction in response to RD decreased from (0.40 ± 0.14) ml in the control group to (0.19 ± 0.06) ml in the irritated group. And the threshold for bodying arching decreased from (0.91 ± 0.26) ml in the control group to (0.47 ± 0.13) ml in the irritated group (P < 0.01). Cytokine immunoreactivity was increased in the irritated group when compared to the control group (TNFα: 0.194 ± 0.001 vs 0.182 ± 0.001, P < 0.01; IL-1β: 0.196 ± 0.002 vs 0.185 ± 0.001, P < 0.01), while SERT expression were reduced in the irritated group (0.298 ± 0.038 vs 0.634 ± 0.200, P < 0.05)., Conclusion: There is an increase in the proinflammatory cytokines and a decrease in the SERT expression associated with the presence of chronic visceral hypersensitivity, both of them may play an important role in the pathogenesis of visceral hypersensitivity.

A-type lamins are encoded by LMNA and a major component of the nuclear lamina, a dynamic meshwork located just under the nuclear envelope, providing essential mechanical support. In addition, A-type lamins associate with chromatin both directly and indirectly and have been suggested to play important roles in chromatin organization, transcription, DNA replication, and apoptosis. Mutations of LMNA or its encoding proteins can cause a series of human hereditary diseases which are collectively referred to as laminopathies. To approach functions of A-type lamins and their roles in laminopathies, this review summarizes several parts of A-type lamins, including its structure, type, modification, assembly, dynamics, interaction proteins and laminopathies.

Background & Objective: Protein kinase CK2, a highly conserved protein serine/threonine kinase that is ubiquitously distributed in eukaryotes, has a close relationship with human leukemia. Mitoxantrone is an effective drug used for acute leukemias. This study was to observe the effects of mitoxantrone on the activity of recombinant holoenzyme of human protein kinase CK2 and proliferation in human leukemia cell line HL-60., Methods: The CK2 holoenzyme composed of alphao and beta subunits was recombined in vitro. Subsequently, CK2 was treated with mitoxantrone at various concentrations, followed by addition of reacting liquid containing [gamma-32P] ATP. CK2 activity was measured by detecting the radioactivity of 32P transferred onto the substrates of CK2. The effect of mitoxantrone on the proliferation of HL-60 cells was detected by the trypan blue dye exclusion assay; the changes in the cell cycle were analyzed by flow cytometry (FCM); apoptosis was analyzed by FCM and DNA agarose gel electrophoresis; effects of the drugs on the intrinsic CK2 activity were measured by a specific CK2 peptide substrate., Results: Mitoxantrone strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 (IC50=0.66 micromol/L). The inhibition of CKZ by mitoxantrone was competitive with respect to ATP (Ki 0.25 micromol/L) and mostly non-competitive with respect to casein (Ki 0.66 micromol/L). Mitoxantrone exerted strong cytotoxicity to HL-60 cells. When treated with 0.1 micromol/L mitoxantrone for 12 h, the apoptotic rate of HL-60 cells was 9.3%. Mitoxantrone did not affect intracellular CK2 activity., Conclusions: Mitoxantrone is a strong inhibitor of recombinant human protein kinase CK2 in vitro. Apoptosis induced by mitoxantrone in HL-60 cells has no correlation to intracellular CK2 activity.

Objective: To establish a method for detecting platelet-associate autoantibodies against platelet-specific receptors using cytometric bead array, and compare the clinical usefulness of this method and modified indirect monoclonal antibody immobilization of platelet antigen technique (MAIPA) in the differential diagnosis of idiopathic thrombocytopenic purpura (ITP) from non-immune thrombocytopenic purpura (non-ITP)., Methods: The microbeads were coated with monoclonal antibodies against glycoprotein IIb/IIIa (CD41a), platelets were isolated from blood samples, then platelet lysate was incubated with the coated microbeads, and R-Phycoerythrin-conjugated goat-antihuman IgG polyclonal antibodies, finally analyzed with flow cytometry. GP IIb/IIIa autoantibodies in sample plasma were measured by modified indirect MAIPA at the same time., Results: The individual fluorescence level was calculated as the ratio to the three controls. The mean ratios were 3.36 (range 0.84 - 22.94) in the ITP group, 1.16 (range 0.67 - 5.59) in the non-ITP patient and 1.08 (range 0.72 - 1.76) in the healthy controls. There was a highly significant difference (P <0.01) between the ITP patients and either the non-ITP patients or the normal controls. If the up limit of healthy controls was set as cutoff value, ratio of greater than 1.76 was considered positive. Cytometric bead array had a sensitivity of 71.43%, a specificity of 94.28% and a positive predictive value of 95.24% for the diagnosis of ITP, the sensitivity being higher than that of modified indirect MAIPA' s (5179%) (chi2 = 4.57, P <0.05). The ROC curve showed the discriminative validity of cytometric bead array was 0.916., Conclusion: Flow cytometric bead method for detection of platelet-associate autoantibodies against platelet-specific receptors is a more rapid, better reproducibility and higher sensitivity than modified MAIPA, and has a potential value in promoting the diagnosis of ITP and guiding treatment.

Objective: To investigate the content and distribution of serotonin in the ileocecal mucosa of rats under partial restraint stress (PRS) and to study the effect of tegaserod on the content of serotonin in the positive cells., Methods: Male Wistar rats (n = 24) were divided into three test groups. After sham-stress (control group) or PRS had been applied to the rats for two hours, 1-methyl-2-pyrrolidinone (solvent) or tegaserod (a partial 5-HT(4) receptor agonist) was administered intra-peritoneally to the corresponding groups. The cellular distribution and quantitative analysis of 5-HT in the ileocecal mucosa were achieved with immunohistochemical method and computerized image system. The 5-HT content of individual enterochromaffin cells (EC) in tegaserod group and PRS group were observed with immunoelectron microscopic technique., Results: Serotonin immunoreactivity was mainly found in the epithelium and lamina propria of the mucosa. As compared with the control group, the optical density of serotonin-immunoreactive cells in ileocecal mucosa decreased in the PRS rats (0.326 +/- 0.035 vs 0.362 +/- 0.042, P < 0.001), the optical density of serotonin-immunoreactive cells in ileocecal mucosa in the tegaserod group (0.364 +/- 0.033) was much higher than in the PRS rats (P < 0.001). Immunoelectron microscopic study showed that in the tegaserod group, EC in the epithelium contained more serotonin-positive secretary granules than in the PRS rats., Conclusions: The increased 5-HT release of positive cells in gut mucosa may be partly responsible for visceral hypersensitivity. The effect of tegaserod on visceral hypersensitivity, at least in part, is likely to act through the pathway involving regulation of gastrointestinal 5-HT release.

Objective: To verify 5-HT(4) receptor mRNA expression in intestinal mucosa mast cells (IMMC)., Methods: IMMC was isolated from the whole intestines of normal rats by collagenase digestion and purified by percoll. In situ hybridization was performed to detect the expression of 5-HT(4) receptor mRNA in IMMC., Results: Evidently positive staining was shown in the cytoplasm of IMMC., Conclusion: We have verified the expression of 5-HT(4) receptor mRNA in IMMC for the first time and provided evidence for further research.

Objective: To study the clinical features of primary biliary cirrhosis (PBC) in order to facilitate cognition of the disease., Methods: Clinical data of 42 patients clinically and/or histologically diagnosed with PBC were reviewed. Anti mitochondrial antibody (AMA) negative/positive patients as well as the patients who were/were not associated with Sjogren Syndrome (SS) were compared in terms of clinical, biochemical and immunological features., Results: Among the 42 patients, 78.6% (33/42) of the cases were females; the mean age at diagnosis was (61.1+/-10.8) years. The most frequent symptoms were fatigue. Serum alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (gamma-GT) and total bile acid (TBA) levels were markedly elevated in the majority of the patients, whereas ALT and AST levels were mildly to moderately elevated. Thirty-one patients had a total bilirubin (TBil) level above normal. The levels of TBil and prothrombin time had positive correlationship with years of the course (P=0.000, r=0.696; P=0.005, r=0.424), whereas serum albumin level had negative correlationship with years of the course (P=0.002, r=-0.462). Thirty-seven patients had elevated serum IgM and 34 patients were AMA/AMA-M(2) positive. AMA negative and AMA positive patients were similar in terms of clinical manifestations and liver biochemistries findings. Serum IgM and IgA levels were significantly lower, whereas total cholesterol level was higher in AMA negative patients when compared with AMA positive cases. Fifteen cases were associated with SS, which were similar in terms of clinical, biochemical and immunological features when compared with the PBC patients were not associated with SS., Conclusion: PBC is mostly found in middle aged and old women. Elevated serum ALP, TBA and gamma-GT levels together with positive AMA/AMA-M(2) can help to diagnose PBC. AMA negative PBC patients are characterized by relatively lower serum IgM and IgA levels and higher total cholesterol level. PBC patients who are associated with SS have not substantial differences in the clinical, biochemical and immunological spectra of the disease.

Background & Objective: Recently, changes in composition, structure, and function of nuclear matrix proteins (NMPs) in generation and development of tumors evoked more and more attention. Separation and identification of tumor-related NMPs is a new way to search for tumor specific biomarkers, and to study tumor pathogenesis. This study was to analyze differential expression of STRBP8, one of esophageal carcinoma specific NMPs, in cancerization of immortalized human esophageal epithelial cells., Methods: NMPs were extracted from immortalized human esophageal epithelial cell line SHEE and malignantly transformed esophageal carcinoma cell line SHEEC. Differential expression of STRBP8 was detected by two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS), and reverse transcription-polymerase chain reaction (RT-PCR). STRBP8 cDNA obtained by RT-PCR was linked to pGEM-T easy vector, and introduced into TOP10F' E.coli competent cells. Positive clones were sequenced and analyzed with BLAST., Results: STRBP8 was only detected in SHEEC cells by 2-DE, MALDI-TOF-MS, and RT-PCR. The sequence of positive clones contained STRBP8 cDNA was identical to that in GenBank database., Conclusion: STRBP8, as a candidate oncogene, might relate to cancerization of esophageal epithelial cells, which might be a specific biomarker of esophageal carcinoma, and probe into the pathogenesis of esophageal carcinoma.

Background & Objective: Protein kinase CK2, a kind of ubiquitous eukaryotic messenger-independent protein serine/threonine kinase, plays a vital role in cell differentiation and proliferation, signal transduction and procession. Activity of CK2 in hematopoietic cells is 2-8 folds higher than that in relevant normal tissues, moreover changes of CK2 activity are correlated to tumor growth. In order to investigate the mechanism of its effect on hematopoietic cells, we used yeast two-hybridization screening the proteins interacting with protein kinase CK2alpha' subunit from HL-60 cells cDNA library., Methods: The target CK2alpha' cDNA was obtained by amplifying recombinant plasmid pTHCK2A', containing human protein kinase CK2alpha' subunit cDNA, through polymerase chain reaction (PCR). Pst I/Nde I-digested PCR products were directionally cloned into DNA-BD vector pGBKT7, which had also been digested by Pst I/Nde I. The recombinant plasmid was named yeast two-hybridization BD bait plasmid, and confirmed by DNA sequencing, and auto-activated experiments. Total RNA of HL-60 cells was extracted, CLONTECH switching mechanism at 5' end of RNA transcript method was used to construct a cDNA library in yeast cells. Library plasmid was named AD plasmid. BD plasmid and AD plasmid were co-transformed into competent yeast AH109. Yeast two-hybridization was used to screen positive clones., Results: Six proteins, interacting with human protein kinase CK2alpha' subunit, were screened. DNA sequencing and homology comparison showed that one of the proteins was highly homologous with ubiquitin/ribosomal protein S27a (99.8%)., Conclusion: Using yeast two-hybridization system could screen out ubiquitin/ribosomal protein S27a, which may interact with human protein kinase CK2alpha' subunit, from HL-60 cells.

Background & Objective: Adenosine plays an essential role in cells signal transduction, including inhibiting the activity of phosphatidylinositol 4-kinase, up-regulating the activity of adenylate cyclase and the concentration of cAMP, and inhibiting platelet actin polymerization. Adenosine analogues have the same bioactivities as adenosine, but are slowly deaminated by adenosine deaminase. This study was to observe the effects of 2-chloro-adenosine(2-ClAdo), 2-chloro-2'-deoxyadenosine (2-CldAdo), and 2'-deoxyadenosine (2'-dAdo) on invasion of human ovarian cancer cell line HO-8910PM., Methods: HO-8910PM cells were treated with 2-ClAdo, 2-CldAdo, and 2'-dAdo, Trypan blue dye exclusion assay was used to examine cytotoxities of these 3 adenosine analogues to HO-8910PM cells, 5-fluorouracil (5-FU) was used as sensitive control. Reconstituted basement membrane invasion assay was used to evaluate invasive, adhesive, and kinetic activities of the cells. PAGE substrate zymography was used to analyze secretion,and activity of matrix metalloproteinase-9 (MMP-9). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyze mRNA levels of mta1 and nm23H1., Results: The 50% inhibition concentrations (IC(50)) of 2-ClAdo, and 2-CldAdo at 72 h were 5.80, and 2.61 micromol/L; while inhibitory effect of 2'-dAdo on the cells was not significant, its IC(50) was more than 100 micromol/L. Inhibition rates of 6.0 micromol/L of 2-ClAdo, and 2-CldAdo on cells invasion were (42.5+/-1.5)%, and (9.9+/-0.5)%; those on cells migration were (36.9+/-2.1)%,and (17.1+/-0.4)%; and those on cells adhesion were (32.2+/-2.3)%, and (19.5+/-3.1)%. All these inhibition rates were significantly higher than those of control (P< 0.05). While 2'-dAdo had no significant effect on cells invasion. All 3 chemicals could not inhibit the production and activity of MMP-9 in HO-8910PM cells. 2-ClAdo down-regulated mRNA level of mta1, but neither 2-ClAdo nor 2-CldAdo affected mRNA level of nm23H1., Conclusion: Of these 3 adenosine analogues, 2-ClAdo might be a potential chemical inhibiting tumor invasion and metastasis because of its weaker cytotoxity but stronger inhibitive effect on invasion of tumor cells.

Background & Objective: Protein kinase CK2 is a ubiquitous and pleiotropic Ser/Thr protein kinase in eukaryotic cells. CK2 activity has been shown to be markedly elevated in solid tumors and leukemia cells. Its alpha or alpha' gene is a protooncogene. CK2 is attracting increasing attention as a potential target for anti-neoplastic. This study was to search specific CK2 inhibitors in tumor cells through observation of the inhibitory effects of baicalein on recombinant human protein kinase CK2 holoenzyme and its kinetics in vitro., Methods: Recombinant human protein kinase CK2 alpha' and beta subunits were cloned and expressed by gene engineering, and purified to homogeneous. These 2 subunits were mixed at equal molar ratio to reconstitute CK2 holoenzyme. The CK2 activity was evaluated by detecting radioactivity of 32P of [gamma-32P]ATP which was incorporated into the substrate in various conditions., Results: Baicalein was shown to strongly inhibit the holoenzyme activity of CK2 with IC50 of 2.54 micromol/L. Kinetic studies of baicalein on CK2 showed that baicalein acted as an inhibitor of noncompetitive with ATP(KI=7.73 micromol/L) and mixed types with casein(KI=3.07 micromol/L)., Conclusion: Baicalein is an effective inhibitor of protein kinase CK2 in vitro.

Objective: Duodenogastric reflux (DGR) is an important factor causing gastric mucosal damage, but whether short-term DGR cause gastric damage and its characteristics remain unclear. The aim of the study is to investigate the damage of gastric mucosa and its characteristics due to short-term DGR in a rat model., Methods: Healthy, male SD rats were divided into three groups: a DGR group, a pylorus ligation group and a control group. The rats were sacrificed three weeks after the operation. The damage of gastric mucosa was observed macroscopically and microscopically. The pH value and bilirubin level of the gastric fluid were examined. The tight junctions between antral mucosal cells were evaluated under transmission electronmicroscopy. The serum gastrin concentration was investigated with radioimmune assay. Myeloperoxidase (MPO) was examined with chemical method., Results: Macroscopically, there were significant mucosal lesions such as erosions, ulcers and bleeding spots in the DGR group. Microscopically, short-term DGR can cause faveolar hyperplasia, but no apparent intestinal metaplasia and atrophy were observed. There was scarcely presence of acute and chronic inflammatory cell infiltration and MPO activity remained unchanged. The pH value and bilirubin level of gastric fluid increased significantly and the serum gastrin concentrations did not change a lot. Short-term DGR can cause destruction of tight junction, which was shorter in duration, less in deformation and might even disappear at the time of examination., Conclusions: It is suggested that short-term DGR can induce gastric damage, faveolar hyperplasia and tight junction destruction without inflammatory cell infiltration and MPO activity changes.

Objective: To investigate relationship between the cold-restraint stress induced colonic circular muscle contractility disorder and calcium-handling properties alteration of the affected muscle in rats., Methods: Stress was conducted on rats to induce the fecal pellet output increased. Contractile responses to acetylcholine (Ach), KCl, and NaF were determined for colonic circular muscle. To investigate Ca(2+) mobilization, effects of Ca(2+)-free solution, high concentration of extracellular Ca(2+), and pinaverium bromide on the contractility of colon were determined. Prepared sarcoplasmic reticular (SR) of colonic muscle by differential centrifugation and measured Ca(2+)-ATPase activity with colorimetric assay., Results: In stressed rats, contractile responses of colonic circular muscle to Ach, KCl, and NaF increased significantly; the contraction was more sensitive to high concentration of extracellular Ca(2+) than control. In Ca(2+)-free solution, contractions were significantly suppressed in both groups. Pinaverium dose-dependently blocked contractions acting with different sensitivity on the two preparations. The Ca(2+)-ATPase activity of the SR in stress was lower than control., Conclusions: Cold- restraint stress caused a marked increase in the contractility of the colonic circular muscle in rats. Stressed rats' colonic muscle shows an alteration in the homeostasis of intracellular Ca(2+), with appears to be the consequence of increased influx of extracellular Ca(2+), decreased release of intracellular stores of Ca(2+) and a decreased Ca(2+)-ATPase activity of the SR.

Background & Objective: Emodin inhibited the activity of TPK and CK2 and the degradation of I-kappaB. Apigenin inhibited the activity of MAPK and PI(3)K. In this study the authors observed the effect of emodin and apigenin on the invasion of HO-8910PM cells in vitro., Methods: Trypan blue dye exclusion assay was used to examine the cytotoxity of emodin and apigenin. Reconstituted basement membrane invasion assay was utilized to evaluate the invasive activity. Type IV collagenase production was analyzed by PAGE substrate zymography., Results: Emodin had weaker cytotoxity on HO-8910PM cells than apigenin, their IC(50) after treatment with the chemicals for 48 hours were (35.30+/-3.50) micromol/L, and (28.92+/-2.60)micromol/L, while emodin significantly inhibited membrane invasion and adhesion and migration of HO-8910PM cells. Their inhibition rates after treated with the chemical of 40 micromol/L were (45.31+/-3.10)%,(25.42+/-1.70)%, and (41.59+/-1.90)%. Emodin inhibited the production but not activity of MMP-9. Apigenin inhibited migration and adhesion of HO-8910PM cells, their inhibition rates after treated with the chemical of 40 micromol/L were (29.04+/-1.70)% and (30.80+/-3.00)%, while weakly inhibited membrane invasion (the inhibition rate only was 12.1%) and inhibited neither production nor activity of MMP-9., Conclusion: Both emodin and apigenin had cytotoxicity on HO-8910PM cells. Emodin was a potential agent inhibiting tumor invasion and metastasis.

Background & Objective: Protein kinase CK2 is a highly conserved and ubiquitous eukaryotic serine/threonine kinase that is elevated and can serve as an oncogene in many tumor cells. To further research the structure and function of CK2, this study was designed to construct, express, and preliminarily identify a recombinant expression plasmid which contains the cDNA encoding mouse protein kinase CK2 alpha subunit., Methods: The aimed cDNA was obtained from NIH 3T3 mouse fibroblasts by RT-PCR. Nde I/BamH I-digested PCR product was directly cloned into pT7-7 expression vector which had been digested by Nde I/BamH I and dephosphorylated by calf intestinal alkaline phosphatase in advance. After E. coli DH5 alpha was transformed with the recombinant DNA by CaCl2 method, transformants were obtained. The positive clones were screened out primarily by gel electrophoresis, and then analyzed by digesting with restriction enzyme. Four positive clones were selected at random and sequenced respectively. The correct recombinant plasmid was transformed into E. coli BL21(DE3) and then expressed by inducing with IPTG. The products were identified with Western blotting., Results: The positive rate of transformants was 100%. The results of restriction analysis indicated that DNA band size of the insert fragment and recombinant plasmid were consistent with theoretically predicated values. The sequencing results showed one of the four clones possessed the cDNA sequence which has no mutation in the processing of PCR, which was termed as pTMCKA. One protein with molecular mass of 42 kDa was overexpressed by inducing with IPTG. The Western blot results confirmed that the recombinant product could specially react with antibody against human CK2 alpha subunit., Conclusions: The authors have successfully cloned and expressed recombinant mouse protein kinase CK2 alpha subunit in this experiment.

Aim: To study the direct effect of tyrphostin AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline] on recombinant human protein kinase CK2 holoenzyme and its kinetics., Methods: Recombinant human protein kinase CK2 alpha and beta subunits were mixed at equal molar ratio and CK2 holoenzyme were reconstituted. The CK2 activity was assayed by detecting incorporation of [gamma-32P]ATP or [gamma-32P]GTP into substrates in various conditions., Results: These results demonstrated that the recombinant human CK2 was a second messengers (Ca2+, cAMP, and cGMP)-independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG1478 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with IC50 of 25.9 micromol/L, the inhibition is very close to that of N-(2-aminoethyl)-5-chloronaphthalene-1-sulfonamide (A3), but less potent than that of 5,6-dichloro-1- beta-D-ribofuranosylbenzimidazole (DRB), known as CK2 special inhibitors with IC50 of 25.5 micromol/L and 10.4 micromol/L respectively. Kinetic studies of AG1478 on recombinant human CK2 showed that inhibitions were competitive with both GTP and casein, thus AG1478 was as bisubstrate inhibitor., Conclusion: The present study indicates that AG1478 is not only an effective inhibitor of protein tyrosine kinases of epidermal growth factor receptor (EGFR), but also a novel potent inhibitor of protein kinase CK2. The recombinant human protein kinase CK2 might be used as a molecular target for simpler screening and development of more effective inhibitors of CK2.

Aim: To study the direct effect and kinetics of sodium quercetin-7,4'-disulphate (SQDS) on recombinant human protein kinase CK2 holoenzyme., Methods: The recombinant human CK2 holoenzyme activity was assayed by detecting incorporation of 32P of [gamma-32P] ATP into the substrate in various conditions., Results: The recombinant human CK2 was a second messenger (Ca2+, cAMP and cGMP) independent protein kinase. The characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. SQDS was shown to strongly inhibit the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 4.4 mumol.L-1, which was more effective than DRB and A3, known CK2 special inhibitors. Kinetic studies of SQDS on recombinant human CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein., Conclusion: SQDS is a potent inhibitor of protein kinase CK2. This study provide experimental basis for the development of more effective inhibitors of CK2 and for clinical application of SQDS in the future.

Background & Objective: Protein kinase CK2 is an evolutionarily conserved serine/threonine kinase that is upregulated and can serve as an oncogene in many cancer cells. Targeted inhibition of CK2 could be useful in the treatment of many cancers. To search specific CK2 inhibitors we investigated the direct effect of tyrphostin AG1109 on recombinant human protein kinase CK2 holoenzyme and its kinetics., Methods: Recombinant human protein kinase CK2 alpha and beta subunits were cloned and expressed by gene engineering, and purified to homogeneous. The two subunits were mixed at the same molar ratio and reconstituted CK2 holoenzyme, which exerted the maximum biological activity. The CK2 activity was assayed by detecting incorporation of 32P of [gamma-32P]ATP or [gamma-32P]GTP into the substrate in various conditions., Results: The recombinant human CK2 was the second messengers (Ca2+, cAMP, and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG1109 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 9.7 mumol/L, which was slightly more effective than 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and N-(2-aminoethyl)-5-chloronaphthalene-1-sulfonamide (A3), which were both known CK2 special inhibitors. Kinetic studies of AG1109 on recombinant human CK2 showed that the inhibition was mixed (competitive is dominant) with GTP and noncompetitive with casein., Conclusions: AG1109 is an effective inhibitor of recombinant human protein kinase CK2 holoenzyme. The recombinant human protein kinase CK2 might be used as a molecular target for simpler screening and development of more effective inhibitors of CK2.