[Show abstract][Hide abstract] ABSTRACT: Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca2+-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction.
The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion
enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with
KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited
by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of
the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling
pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is
not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient
for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular
contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.
Full-text · Article · Jun 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Lymphatic absorption is a highly regulated process driven by both an extrinsic mechanism (external force) and an intrinsic mechanism (lymphatic vessel contractility). The lymphatic muscle is a specialized smooth muscle with unique mechanical properties. To understand the molecular mechanism and relative contribution of smooth muscle contraction in lymphatic absorption, we analyzed mice with a smooth muscle-specific deletion of Mylk, a critical gene for smooth muscle contraction. Interestingly, the knockout mice were significantly resistant to anesthesia reagents. Upon injection in the feet with FITC-dextran, the mutant mice displayed a 2-fold delay of the absorption peak in the peripheral circulation. Examining the ear lymphatic vessels of the mutant mice revealed a reduction in the amount of fluid in the lumens of the lymphangions, suggesting an impairment of lymph formation. The Mylk-deficient lymphatic muscle exhibited a significant reduction of peristalsis and of myosin light chain phosphorylation in response to depolarization. We thus concluded that MLCK and myosin light chain phosphorylation are required for lymphatic vessel contraction. Lymphatic contractility is not an exclusive requirement for lymphatic absorption, and external force appears to be necessary for absorption.
Full-text · Article · May 2014 · The International Journal of Biochemistry & Cell Biology
[Show abstract][Hide abstract] ABSTRACT: TNF α plays a central role in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and murine acute liver injury induced by injection of D-galactosamine and subsequent LPS. Recombinant Fc-fused soluble TNF receptor II (sTNFRII-Fc) has been used in the treatment of rheumatoid arthritis for a decade. We have recently constructed a novel fusion protein sTNFRII-gAD, which is composed of a soluble TNF receptor II and a globular domain of adiponectin. Utilizing the inclination of gAD to form homologous trimer naturally, we sought to explore TNFα antagonism of the novel trimerized sTNFRII-gAD and meantime compare TNFα-neutralizing effects in vitro and in vivo between sTNFRII-Fc and sTNFRII-gAD. Here, we evaluated the TNFα-antagonizing activity of sTNFRII-gAD with TNFα-induced L929 cytotoxicity assay. Furthermore, sTNFRII-Fc or sTNFRII-gAD was administered simultaneously with d-galactosamine 1h prior to LPS injection in the murine model of acute liver injury. Serum TNFα and TNFα-sTNFRII-gAD complex were measured by ELISA and the liver injury was assessed through alanine transaminase measurement and liver histological analysis. sTNFRII-gAD was shown to have higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in the L929 cytotoxicity assay. With a significant attenuation of murine lethality (p<0.05), sTNFRII-gAD showed more protective effects than sTNFRII-Fc in the murine model of acute liver injury. These results demonstrated that sTNFRII-gAD was more efficacious than sTNFRII-Fc as a TNFα antagonist, highlighting the potential of sTNFRII-gAD for the treatment of diseases associated with excessive TNFα.
Full-text · Article · Mar 2012 · International immunopharmacology
[Show abstract][Hide abstract] ABSTRACT: In situ gene therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) was demonstrated to successfully inhibit tumour cell growth in a mouse orthotopic bladder cancer model, but suffered from several disadvantages, such as limited efficiency for gene delivery, low expression efficiency of the transgene and the safety concern resulting from viral vector. In order to address the limits, a novel immunotherapy was developed attentively through immobilization of streptavidin-tagged bioactive GM-CSF on the biotinylated mucosal surface of bladder wall on the basis of both the unique property of streptavidin (SA) to bind rapidly and almost irreversibly to any biotin-linked molecule and the outstanding ability of biotin to be incorporated easily into the proteins on the cell surface. The mouse orthotopic model of MB49 bladder cancer was used to evaluate the feasibility and efficacy of the novel immunotherapy performed twice a week for 3 weeks. Briefly, 1 day after intravesical implantation of 1 x 10(6) MB49 tumour cells in C57BL/6 mouse, 100 microl of 1 mg/ml NHS-PEO4-biotin was instilled and allowed to incubate in the bladder for 30 min., followed by intravesical instillation of 100 microl of 0.15 mg/ml SA-GM-CSF bifunctional fusion protein and incubation for 1 hr. SA-GM-CSF fusion protein was shown to be immobilized efficiently and durably on the biotinylated mucosal surface of bladder wall. The bladder cancer incidence was dramatically decreased from 100% in the control group to 37.5% in the SA-GM-CSF group. Importantly, 70% of the SA-GM-CSF-cured mice were protected against a second intravesical wild-type MB49 tumour challenge, indicating that an effective anti-tumour immunity was generated against MB49 bladder cancer. Thus, the novel immunotherapy may be an attractive therapeutic alternative and should be evaluated in bladder cancer patients.
Full-text · Article · Aug 2009 · Journal of Cellular and Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: To establish a simple and efficient method for establishing a mouse model of orthotopic superficial bladder cancer.
C57BL/6 mice were anesthetized with sodium pentobarbital and catheterized with modified IV catheter (24 G). The mice were intravesically pretreated with HCl and then with NaOH, and after washing the bladders with phosphate-buffered saline (PBS), 100 microl (1 x 10(7)) MB49 cells were infused and allowed to incubate in the bladder for 2 h followed intravesical mitomycin C (MMC) administration. The tumor formation rate, survival, gross hematuria, and bladder weight were determined as the outcome variables, and the pathology of the bladders was observed.
Instillation of MB49 tumor cells resulted in a tumor formation rates of 100% in all the pretreated groups while 0% in the control group without pretreatment. MMC significantly reduced the bladder weight as compared to PBS.
We have successfully established a stable, reproducible, and reliable orthotopic bladder cancer model in mice.
Full-text · Article · May 2009 · Nan fang yi ke da xue xue bao = Journal of Southern Medical University