Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.     

Two new antibacterial sesquiterpenoids from Centipeda minima

Two new guaiane-type sesquiterpene lactones, compounds 1 and 2, along with three known guaianolide- or pseudoguaianolides, were isolated from Centipeda minima (whole plant). Their structures were identified by spectroscopic and mass-spectrometric analyses. The configuration at C5 of the guaiane framework of 1 was rationalized by quantum-mechanical calculations (Table 2). All compounds were found to be active against eight different microbial pathogens (Table 3), with MIC values in the range of 6.25-100 microg/ml.     

The S100 proteins for screening and prognostic grading of bladder cancer

The S100 gene family, which is composed of at least 24 members carrying the Ca2+ binding EF-hand motif, has been implicated in both intracellular and extracellular functions, including enzyme activities, immune responses, cytoskeleton dynamics, Ca2+ homeostasis, cell growth and cell differentiation. Altered S100 protein levels are associated with a broad range of diseases, including cardiomyopathy, inflammatory and immune disorders, neurodegenerative disorders and cancer. Although the precise role of S100 protein in carcinogenesis is poorly understood, it seems that formation of homo- and hetero-dimers, binding of Ca2+ and interaction with effector molecules are essential for the development and progression of many cancers. Several studies have suggested that S100 proteins promote cancer progression and metastasis through cell survival and apoptosis pathways. In animal models of bladder cancer, several S100 proteins are differentially expressed in bladder tumors relative to normal urothelium. In human bladder cancer, overexpression of S100A4, S100A8 or S100A11 are associated with stage progression, invasion, metastasis and poor survival. This review summarizes these findings and evaluates their implications for human bladder cancer management.     

Enhancement of Astragalus polysaccharide on the immune responses in pigs inoculated with foot-and-mouth disease virus vaccine

The effects of Astragalus polysaccharides (APS) on the immune response in pigs immunized with foot-and-mouth disease virus (FMDV) vaccine were investigated. Fifteen pigs were randomly divided into five groups. Four groups were vaccinated with a FMDV inactivated vaccine. Pigs in three experimental groups were administered varying doses of APS (APS1, 5 mg/kg; APS2, 10 mg/kg; APS3, 20 mg/kg). The influence of APS on the number of CD3⁺CD4⁻CD8⁺ cytotoxic T cells, CD3⁺CD4⁺CD8⁺ T helper memory cells, and CD3⁻CD4⁻CD8⁺ natural killer cells among peripheral blood lymphocytes (PBL) in the three APS groups were significant compared to the vaccine group. In vitro stimulation of PBL by Con A and LPS in APS groups induced a stronger proliferative response at 2 and 6 weeks post-inoculation (PI). APS markedly increased the titer of FMDV-specific antibody in a dose-dependent manner, and up-regulated mRNA expression of IFN-γ and IL-6. APS could potentially be used as an immunomodulator for a FMDV vaccine and provide better protection against FMDV.     

d-Ribose glycates β(2)-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β(2)-Microglobulin (β(2)M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β(2)M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β(2)M in vitro. We find that the glycating agent d-ribose interacts with human β(2)M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β(2)M molecules are highly oligomerized compared with unglycated β(2)M, and have granular morphology. Furthermore, such ribosylated β(2)M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β(2)M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β(2)M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β(2)M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β(2)M-associated diseases such as HAA.     

Characterization of the in vivo and in vitro metabolic profile of PAC-1 using liquid chromatography-mass spectrometry

In the present study, the metabolic profile of PAC-1, a potential anticancer drug, was investigated using liquid chromatography-mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers--a quadrupole time-of-flight (Q-TOF) mass spectrometer and an ion trap (IT) mass spectrometer--were employed to acquire structural information on PAC-1 metabolites. A gradient liquid chromatographic system composed of 0.2% formic acid in methanol and 0.2% formic acid in water was used for metabolite separation on an Agilent TC-C(18) column. A total of 16 metabolites were detected. The corresponding product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites thereby confirming the proposed structures of these metabolites. Phase I metabolic changes were predominantly observed, including debenzylation, dihydrodiol formation, hydroxylation, and dihydroxylation. The detected phase II metabolites included PAC-1 and hydroxylated PAC-1 glucuronide conjugates. Based on metabolite analysis, several PAC-1 metabolic pathways in rat were proposed.     

Mesangial cells of lupus-prone mice are sensitive to chemokine production

Infectious antigens may be triggers for the exacerbation of systemic lupus erythematosus. The underlying mechanism causing acceleration and exacerbation of lupus nephritis (LN) is largely unknown. Bacterial lipopolysaccharide (LPS) is capable of inducing an accelerated model of LN in NZB/W mice, featuring diffuse proliferation of glomerular resident cells. We hypothesized that mesangial cells (MCs) from LN subjects are more responsive to LPS than normal subjects. Cultured primary NZB/W and DBA/W (nonautoimmune disease-prone strain with MHC class II molecules identical to those of NZB/W) MCs were used. Monocyte chemoattractant protein-1 (MCP-1) and osteopontin (OPN) expressions either in the baseline (normal culture) condition or in the presence of LPS were evaluated by real-time PCR, ELISA, or western blot analysis. NF-κB was detected by ELISA, electrophoresis mobility-shift assay, and immunofluorescence. First, either in the baseline condition or in the presence of LPS, NZB/W MCs produced significantly higher levels of MCP-1 and OPN than the DBA/W MC controls. Second, NZB/W MCs expressed significantly higher levels of Toll-like receptor 4, myeloid differentiation factor 88, and NF-κB than the DBA/W MC controls, both receiving exactly the same LPS treatment. In conclusion, NZB/W MCs are significantly more sensitive than their normal control DBA/W MCs in producing both MCP-1 and OPN. With LPS treatment, the significantly elevated levels of both chemokines produced by NZB/W MCs are more likely due to a significantly greater activation of the Toll-like receptor 4-myeloid differentiation factor 88-associated NF-κB pathway. The observed abnormal molecular events provide an intrarenal pathogenic pathway involved in an accelerated type of LN, which is potentially infection triggered.