Epigenetic silencing of the dual-role signal mediator, ANGPTL4 in tumor tissues and its overexpression in the urothelial carcinoma microenvironment.

Urothelial carcinoma (UC) carcinogenesis has been hypothesized to occur through epigenetic repression of tumor-suppressor genes (TSGs). By quantitative real-time polymerase chain reaction array, we found that one potential TSG, angiopoietin-like 4 (ANGPTL4), was expressed at very low levels in all bladder cancer cell lines we examined. Previous studies had demonstrated that ANGPTL4 is highly expressed in some cancers, but downregulated, by DNA methylation, in others. Consequently, owing to these seemingly conflicting functions in distinct cancers, the precise role of ANGPTL4 in the etiology of UC remains unclear. In this study, using methylation-specific PCR and bisulfite pyrosequencing, we show that ANGPTL4 is transcriptionally repressed by DNA methylation in UC cell lines and primary tumor samples, as compared with adjacent noncancerous bladder epithelium. Functional studies further demonstrated that ectopic expression of ANGPTL4 potently suppressed UC cell proliferation, monolayer colony formation in vitro, and invasion, migration, and xenograft formation in vivo. Surprisingly, circulating ANGPTL4 was significantly higher in plasma samples from UC patients than normal control, suggesting it might be secreted from other cell types. Interestingly, our data also indicated that exogenous cANGPTL4 could promote cell proliferation and cell migration via activation of signaling through the Erk/focal adhesion kinase axis. We further confirmed that mouse xenograft tumor growth could be promoted by administration of exogenous cANGPTL4. Finally, immunohistochemistry demonstrated that ANGPTL4 was downregulated in tumor cells but overexpressed in tumor adjacent stromal tissues of muscle-invasive UC tissue samples. In conclusion, our data support dual roles for ANGPTL4 in UC progression, either as a tumor suppressor or oncogene, in response to microenvironmental context.

Research on the role of GLP-2 in the central nervous system EPK signal transduction pathway of mice with vascular dementia.

OBJECTIVE: To investigate the role of Glucagon-like peptide-2 (GLP-2) in the central nervous system eukaryotic protein kinase (EPK) signal transduction pathway of mice with vascular dementia. MATERIALS AND METHODS: We take the 3-week-old mice raised in the laboratory as the object of study in this research and then divide them into four groups in random, including sham operation group, control group, GLP-2 group, and GLP-2+ERK (extracellular-signal-regulated kinase) inhibitor intervention group, with 30 in each group. The step-down test, water-maze test, electron microscopy observation, immunohistochemical method, and Western-blotting are adopted to investigate the role of GLP-2 in the central nervous system EPK signal transduction pathway of mice with vascular dementia. RESULTS: The step-down test, as well as the water-maze learning and memory test, shows that the mice injected with GLP-2 in the experiment group have their learning and memory ability improved significantly when compared with other three groups, which is greatly different from that of other three groups (p < 0.05); the electron microscopy observation shows that the injection of GLP can partially reverse the reduction of vesicles while ERK inhibitor removes the said protection; the immunohistochemical result and image analysis result show that the EPK expression quantity in GLP-2 group has no significant difference from that of other three groups (p > 0.05). However, the content of pi-EPK in the GLP-2 group is significantly higher than that in VD group and GLP-2+PD98095 group and is significantly different from them (p < 0.05), and such result is in line with the result of Western-blotting. CONCLUSIONS: GLP can influence the change in hippocampus cells extensions and finally influence their cognitive function by activating the EPK signal transduction pathway of hippocampal neuron in the central nervous system.

Protein binding of brequinar in the plasma of healthy donors and cancer patients and analysis of the relationship between protein binding and pharmacokinetics in cancer patients.

The protein binding of weakly acidic and basic drugs has been shown to be altered in cancer patients. Brequinar is a weakly acidic, low-clearance, and highly protein-bound (> 98% bound) antitumor agent. The pharmacokinetic parameters of brequinar are subject to large interpatient variability. This large interpatient variability may be related to brequinar's plasma protein-binding capacity (assuming no change in the intrinsic clearance of the unbound drug). The objectives of this study, therefore, were (a) to characterize brequinar's protein binding in the plasma of healthy donors and cancer patients and (b) to examine the relationships between brequinar's plasma protein binding and its pharmacokinetics in patients. Brequinar protein binding was determined in human serum albumin (HSA) solution, drug-free donor plasma, and brequinar-free, predose plasma samples obtained from a phase I cancer trial. Pharmacokinetic results from this study were used to examine relationships between plasma protein binding and drug disposition. In HSA solution and healthy donor plasma, brequinar's protein binding as determined using spiked samples was concentration-dependent. The unbound brequinar fraction increased by a factor of 3 (from 0.3% to 0.9% free) in 4% HSA solution and by a factor of 4 (from 0.4% to 1.6% free) in donor plasma as the brequinar concentrations increased from 0.1 to 2.3 mM in the HSA solution and from 0.076 to 1.5 mM in the donor plasma. Analysis of brequinar binding characteristics using the binding ratio and Rosenthal binding plots showed that albumin was the primary protein for brequinar binding in human plasma. The addition of various concentrations of alpha 1-acid glycoprotein to 4% HSA solution did not affect the protein binding of brequinar to HSA. The protein binding determined in the plasma of cancer patients was not quantitatively different, except for variability, from that observed in the plasma of healthy donors. Examination of relationships between the unbound brequinar fraction and pharmacokinetics suggested that plasma protein binding was not a major determinant of brequinar disposition in cancer patients.

Expression of N-methyl-D-aspartate receptor subunit mRNAs in the rat pheochromocytoma cell line PC12.

The expression of the N-methyl-D-aspartate (NMDA) receptor subunit mRNAs NMDAR1 and NMDAR2A-D was characterized in undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells using Northern blotting, RNase protection assays (RPA) and polymerase chain reaction (PCR). PC12 cells expressed predominately the splice variant NMDAR1-4a and smaller amounts of NMDAR1-1a, NMDAR1-2a and NMDAR1-3a. No splice isoforms containing exon 5 were detected. The NMDAR2C subunit was detected in PC12 cells by Northern blotting and trace amounts of NMDAR2A, B and D were detected by PCR. PC12 cells may be a useful model system for the study of the transcriptional and post-transcriptional regulation of expression of the NMDA receptor subunit genes, including the alternative splicing of NMDAR1 pre-mRNAs.

Another look at differences in the susceptibility of Escherichia coli and Klebsiella pneumoniae to cephalothin and cefazolin.

The significance of in vitro susceptibility tests on Enterobacteriaceae to cephalothin and cefazolin has not been exactly defined in the guidelines of the National Committee for Clinical Laboratory Standards. In the hope of clarifying this confusion, we provide additional information from an ancillary study of the Taiwan Surveillance of Antimicrobial Resistance 1998 (TSAR I). There were 505 Escherichia coli and 227 Klebsiella pneumoniae isolates susceptible to cephalothin, reported by 42 participating hospitals. The susceptibility of these isolates were re-tested at the Microbial Infections Reference Laboratory using cefazolin, with the result that 72% of the 252 cephalothin-resistant E. coli isolates and 24% of the 41 cephalothin-resistant K. pneumoniae isolates were found to be susceptible to cefazolin. We further surveyed the availability of cephalothin and cefazolin in Pharmacy Departments; all of the TSAR I hospitals had cefazolin available in their pharmacies. The resistance rate of E. coli was significantly lower for 12 hospitals that had cefazolin in both pharmacy and laboratory compared with 11 hospitals that had cefazolin available in pharmacy but cephalothin in laboratory. In addition, for all the hospitals that had cephalothin available for clinical use, the resistance rate was twice as low in two hospitals reporting cefazolin susceptibility as in the seven hospitals reporting cephalothin susceptibility. Our findings suggest that inappropriate selection of cephalothin and cefazolin for susceptibility testing contribute to inaccurate indications of in vivo activity for first generation cephalosporins in the treatment of E. coli infections.

Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed.

The Drosophila sprouty gene encodes an antagonist of FGF and EGF signaling whose expression is induced by the signaling pathways that it inhibits. Here we describe a family of vertebrate Sprouty homologs and demonstrate that the regulatory relationship with FGF pathways has been conserved. In both mouse and chick embryos, Sprouty genes are expressed in intimate association with FGF signaling centers. Gain- and loss-of-function experiments demonstrate that FGF signaling induces Sprouty gene expression in various tissues. Sprouty overexpression obtained by infecting the prospective wing territory of the chick embryo with a retrovirus containing a mouse Sprouty gene causes a reduction in limb bud outgrowth and other effects consistent with reduced FGF signaling from the apical ectodermal ridge. At later stages of development in the infected limbs there was a dramatic reduction in skeletal element length due to an inhibition of chondrocyte differentiation. The results provide evidence that vertebrate Sprouty proteins function as FGF-induced feedback inhibitors, and suggest a possible role for Sprouty genes in the pathogenesis of specific human chondrodysplasias caused by activating mutations in Fgfr3.

Developmental and regional expression pattern of a novel NMDA receptor-like subunit (NMDAR-L) in the rodent brain.

A novel NMDA receptor-like (NMDAR-L) cDNA was isolated that contained an open reading frame coding for a predicted polypeptide of 1115 amino acids that shares approximately 27% identity with NMDA receptor subunits. In situ hybridization experiments indicated that NMDAR-L mRNA was expressed in the developing rodent CNS. On postnatal day 1 (P1), NMDAR-L mRNA expression was pronounced in the entorhinal cortex, the subiculum and the thalamus, in layer V of the developing neocortex, in the superior and inferior colliculi, and various regions of the hindbrain, excluding the cerebellum. On P5, NMDAR-L mRNA was expressed in layer V of the neocortex, in the entorhinal cortex, in the subiculum, and in the thalamus. On P14, NMDAR-L mRNA was expressed in layers II-VI of the neocortex, in the entorhinal and piriform cortex, in the subiculum and CA1 field, and in the nucleus of the lateral olfactory tract. In the adult brain, NMDAR-L mRNA was detected predominately in the nucleus of the lateral olfactory tract. Injection of NMDAR-L cRNA into Xenopus oocytes did not lead to the expression of homomeric glutamate-activated channels. However, coinjection of the triple combination of NMDAR-L with NMDAR1 and NMDAR2B cRNAs led to a striking decrease in the current magnitude compared to currents obtained after coexpression of the double combination of NMDAR1 with NMDAR2B. While the function of NMDAR-L remains to be established, its developmental and regional expression pattern suggests that NMDAR-L may influence axonal outgrowth and synaptogenesis during brain development.