-
[show abstract]
[hide abstract]
ABSTRACT: To establish a new quantitative method for simultaneous determination of multi-coumarins in Fraxini Cortex by using one chemical reference substance, and validate its feasibilities.
The new quality evaluation method, quantitative analysis of multi-components by singer-marker (QAMS), was established and validated with Fraxini Cortex. Four main coumarins were selected as analytes to evaluate the quality and their relative correlation factors (RCF) were determined by HPLC-DAD. Within the linear range, the values of RCF at 340 nm of aesculin to asculetin, fraxin and fraxetin were 1.771, 0.799, 1.409, respectively. And the contents of aesculin in samples of Fraxini Cortex were authentically determined by the external standard method, and the contents of the three other coumarins were calculated by their RCF. The contents of these four coumarins in all samples were also determined by the external standard method.
Within a certain range, the RCF had a good reproducibility (RSD 2.5%-3.9%). Significant differences were not observed between the quantitative results of two methods.
It is feasible and suitable to evaluate the quality of Fraxini Cortex and its Yinpian by QAMS.
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica 07/2011; 36(13):1782-9.
-
[show abstract]
[hide abstract]
ABSTRACT: AimsP2X3 (ATP-gated receptors) in nociceptive neurons of dorsal root ganglion (DRG) participate in transmission of pain signals from the periphery to the spinal cord. However, the role of P2X3 receptors in chronic prostate pain and continued intractable pain remains unclear.Materials and Methods
We examined ATP-evoked responses and P2X3 expression in DRG neurons isolated from rats with prostatic inflammation induced by injection of complete Freund's adjuvant (CFA) into the prostate. Neurons were dissociated from the L6–S1 DRG. The effect of ATP on the excitability of DRG neurons was determined using whole-cell patch clamp. P2X3 receptor expression was determined with Western blot on the 3rd and 10th days after irritation of the prostate.ResultsAlthough application of ATP induced both fast- and slow-inactivating currents and caused depolarization in control and inflamed neurons, compared to the control group, the increase in ATP responses gave rise to large depolarization that exceeded the threshold of action potentials in inflamed DRG neurons. The affinity of P2X3 receptor for ATP increased significantly and inflammation enhanced the expression of P2X3 receptor in inflamed neurons.ConclusionsP2X3 receptor upregulation could account for neuronal hypersensitivity and contribute to abnormal pain responses associated with chronic prostatitis. These results suggest that P2X3 receptors are useful targets for the treatment of pain in chronic prostatitis. 30:612–618, 2011. © 2011 Wiley-Liss, Inc.
Neurourology and Urodynamics 03/2011; 30(4):612 - 618. · 2.96 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: P(2)X(3) (ATP-gated receptors) in nociceptive neurons of dorsal root ganglion (DRG) participate in transmission of pain signals from the periphery to the spinal cord. However, the role of P(2)X(3) receptors in chronic prostate pain and continued intractable pain remains unclear.
We examined ATP-evoked responses and P(2)X(3) expression in DRG neurons isolated from rats with prostatic inflammation induced by injection of complete Freund's adjuvant (CFA) into the prostate. Neurons were dissociated from the L(6)-S(1) DRG. The effect of ATP on the excitability of DRG neurons was determined using whole-cell patch clamp. P(2)X(3) receptor expression was determined with Western blot on the 3rd and 10th days after irritation of the prostate.
Although application of ATP induced both fast- and slow-inactivating currents and caused depolarization in control and inflamed neurons, compared to the control group, the increase in ATP responses gave rise to large depolarization that exceeded the threshold of action potentials in inflamed DRG neurons. The affinity of P(2)X(3) receptor for ATP increased significantly and inflammation enhanced the expression of P(2)X(3) receptor in inflamed neurons.
P(2)X(3) receptor upregulation could account for neuronal hypersensitivity and contribute to abnormal pain responses associated with chronic prostatitis. These results suggest that P(2)X(3) receptors are useful targets for the treatment of pain in chronic prostatitis.
Neurourology and Urodynamics 01/2011; 30(4):612-8. · 2.96 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the structure and function of nonstructural (NS) protein 2B of the dengue serotype 2 virus (DV2) during infection, polyclonal antibodies (Abs) against DV2 NS2B were prepared by immunisation with NS2B protein or by DNA immunisation. The full-length NS2B gene was cloned and inserted into the prokaryotic expression vector pQE31, resulting in a vector, named pQE-NS2B, or into the eukaryotic expression vector pCAGGS-P7, resulting in the vector pCAG-NS2B. The pQE-NS2B vector was transfected into Escherichia coli JM109, and recombinant NS2B protein was obtained by Ni(2+)-NTA agarose affinity chromatography. Vero cells transfected with pCAG-NS2B showed that NS2B protein can be expressed in eukaryotic cells. Finally, mice were immunised with the recombinant NS2B protein or pCAG-NS2B. Anti-NS2B sera from the immunised mice could specifically react with DV2 NS2B proteins, as visualised by fluorescence staining and Western blotting. Immunisation with NS2B protein induced a higher titre of the antibody than that induced by DNA immunisation. These data indicate that our antisera against DV2 NS2B can recognise both the natural and denatured NS2B protein. Based on these results, the polyclonal Abs could be used as a tool for studying the role of NS2B in the pathogenesis of DV2.
Journal of virological methods 06/2009; 163(1):10-6. · 2.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are highly infectious diseases caused by dengue virus (DV). DV non-structural protein 3 (NS3) is known to possess ATPase, helicase, and protease activity that is a constitutive part of the replication complex of DV. In this study, we discuss the cloning, expression, and purification of the DV-2 NS3 protein to immunize mice by intrasplenic injection and then to generate a monoclonal antibody (MAb). One MAb, named 4F5, was obtained and it was specific to NS3 of DV-2. Immunofluorescence show that 4F5 recognizes the native protein in infected ECV304 cells. Likewise, C6/36-infected lysates were used in Western blot analysis, and we observed the specific characteristic band that defines NS3. We conclude that MAb 4F5 may be a useful tool, not only to study the replicative process of DV, but also to generate specific diagnostic tools for DV infection.
Hybridoma (2005) 01/2009; 27(6):467-71. · 0.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Adiponectin, an adipocyte-derived polypeptide hormone, plays an important role in regulating fatty acid oxidation. beta-oxidation of fatty acids supplies most of the cardiac energy and carnitine palmitoyltransferase (CPT)-1 serves as a key regulator during this process. To characterize the potential effects of adiponectin on CPT-1, we incubated rat neonatal cardiomyocytes with globular adiponectin (gAd). Results showed that gAd promoted the activity and mRNA expression of CPT-1. The underlying signal pathway involved in this modulatory effect was further investigated. Inhibition of AMP-activated protein kinase (AMPK) with adenine 9-beta-d-arabinofuranoside (AraA) completely abrogated gAd-mediated AMPK and acetyl coenzyme A carboxylase (ACC) phosphorylation and suppressed the promotion of CPT-1 activity. gAd also induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and peroxisome proliferator-activated receptor (PPAR)-alpha, which was inhibited by AraA. SB202190, a p38MAPK inhibitor, blocked gAd-stimulated PPAR-alpha phosphorylation. When AMPK and/or p38MAPK was inhibited, gAd-enhanced mRNA expression of CPT-1 was partially reduced. In conclusion, our study suggests that the activation of AMPK signaling cascade participates in the promotion effect of gAd on CPT-1.
Regulatory Peptides 04/2007; 139(1-3):72-9. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The role of protein kinase C (PKC) and its cross talk with extracellular signal-regulated kinase (ERK) cascade in angiotensin II (AngII)-elicited vascular smooth muscle cell (VSMC) proliferation are still unclear. In this study, the PKC pathway of AngII to activate ERK1/2 and induce cell proliferation was investigated in rat aortic smooth muscle cells. The proliferation of VSMCs was tested by [3H]-thymidine incorporation assay. Phosphorylated and non-phosphorylated PKCzeta, ERK1/2, Elk-1, and mitogen-activated ERK-activating kinase (MEK) were estimated by Western blot analysis. The interactions of signal molecules were examined by immunoprecipitation. AngII-induced VSMC proliferation and activation of ERK1/2 and nuclear transcription factor Elk-1 were all down-regulated by PKC non-specific inhibitor (staurosporine) and PKCzeta pseudosubstrate inhibitor (PS-PKCzeta). Dominant negative Ras transfection into VSMCs decreased AngII-induced PKCzeta and ERK1/2 phosphorylation. AngII stimulated the association of PKCzeta with Ras. AngII-induced MEK phosphorylation was inhibited by PKCzeta pseudosubstrate inhibitor and the PKCzeta-MEK complex was detected by immunoprecipitation. These results suggest that PKCzeta isoform is involved in VSMC proliferation and Elk-1 activation. AngII can activate ERK1/2 by Ras/PKCzeta/MEK pathway, which may be one of the important signal transduction pathways in AngII-induced VSMC proliferation.
Regulatory Peptides 06/2005; 128(1):43-50. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The envelope glycoprotein (E) of dengue (DENV) viruses mediates viral attachment and entry through membrane fusion. In this study, three fragments (domain 3a, domain 12 and domain 123a) of DENV envelope protein were inserted into plasmid pGEX-6p-1 to express glutathione's transferase (GST) fusion proteins in Escherichia coli (E. coli) strain BL21. High yields of soluble recombinant proteins were obtained as follows: 20 mg/L of GST-E3a protein, 5 mg/L of GST-E12 protein, and 3 mg/L of GST-E123a protein. Although they all failed to protect vero cells from DENV infection in virus-binding blocking assay, three proteins could be recognized by mouse serum against DENV in western blot analysis. Then, polyclonal antibodies against either GST-E12 or GST-E3a were raised in New Zealand rabbits, and used in immunofluorescence staining. The infected vero cells showed typical cytoplasmatic fluorescence near the nucleus where the virus replication took place. The study indicated that soluble GST fusion E proteins might lack some biological activities of natural dengue E protein. But polyclonal antibodies against GST-E3a and GST-E12 might be helpful tools for further study of DENV pathogenesis in vitro.
-
[show abstract]
[hide abstract]
ABSTRACT: Purpose: Evidence shows that adenosine triphosphate (ATP) is involved in the transmission of multiple chronic pain via P2X7 receptor. This study was to investigate the P2X7 and microglial cells in the chronic prostatitis pain. Materials and Methods: Rats were divided into control group and chronic prostatitis group (n = 24 per group). A chronic prostatitis animal model was established by injecting complete Freund's adjuvant (CFA) to the prostate of rats, and the thermal withdrawal latency (TWL) was detected on days 0, 4, 12 and 24 (n = 6 at each time point in each group). Animals were sacrificed and the pathological examination of the prostate, detection of mRNA expression of P2X7 and ionized calcium binding adaptor molecule 1 (IBA-1) and measurement of content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the dorsal horn of L5-S2 spinal cord were performed on days 0, 4, 12 and 24. In addition, the content of TNF-α and IL-1β in the dorsal horn of L5-S2 spinal cord was measured after intrathecal injection of inhibitors of microglial cells and/or P2X7 for 5 days. Results: The chronic prostatitis was confirmed by pathological examination. The expression of P2X7 and IBA-1 and the content of TNF-α and IL-1β in rats with chronic prostatitis were significantly higher than those in the control group. On day 4, the expressions of pro-inflammatory cytokines became to increase, reaching a maximal level on day 12 and started to reduce on day 24, but remained higher than that in the control group. Following suppression of microglial cells and P2X7 receptor, the secretion of TNF-α and IL-1β was markedly reduced. Conclusion: In chronic prostatitis pain, the microglial cells and P2X7 receptor are activated resulting in the increased expression of TNF-α and IL-1β in the L5-S2 spinal cord, which might attribute to the maintenance and intensification of pain in chronic prostatitis.
International braz j urol: official journal of the Brazilian Society of Urology 39(2):276-285.
