Soluble type-I interleukin-1 receptor blocks chicken IL-1 activity

The ligand-binding domain of the chicken type-I interleukin-1 (IL-1) receptor (soluble IL-1R(I); sIL-1R(I)) was cloned into a Pichia pastoris expression system and the resulting sIL-1R(I) binding protein was used to produce antisera in rabbits (anti-IL-1R(I)). Two experiments were conducted to determine the capacity of sIL-1R(I) or anti-IL-1R(I) to block the IL-1 bioactivity (thymocyte co-stimulation) in conditioned media (CM) from HD11 chicken macrophages stimulated with lipopolysaccharide. In the first experiment, pre-incubation of CM with unpurified sIL-1R(I) significantly decreased its thymocyte co-stimulation activity by 57%. Further purification of sIL-1R(I) from other proteins secreted or shed from P. pastoris expression system by size exclusion filtration or ammonium sulfate (60%) precipitation did not influence its capacity to neutralize IL-1 bioactivity. These partially purified sIL-1R(I) preparations significantly decreased thymocyte co-stimulation activity in CM by 70.7 and 77.3%, respectively. In the second experiment, pre-incubation of thymocytes with antisera against the sIL-1R(I) decreased IL-1 activity in CM by 70% relative to control thymocyte cultures that received no antibody and by 59% relative to thymocyte cultures incubated with pre-immune sera. Presumably anti-sIL-1R(I) diminished the IL-1 bioactivity in CM by blocking IL-1 binding to its type-I receptor on thymocytes. Thus, 30% of the IL-1-like activity released by LPS-stimulated HD11 macrophages is probably due to at least one other cytokine. Our data are consistent with the type-I receptor being the primary IL-1 receptor on chicken thymocytes that is capable of providing a signal for proliferation.     

Progressive and regressive changes in the nucleus pulposus. Part I. The neonate

Magnetic resonance (MR) imaging, correlated with anatomic sections, was used to characterize the progressive and regressive changes in the nucleus pulposus in neonates. The spines of five fetuses and five full-term infants between 16 and 40 weeks old were studied. In anatomic sections, the nucleus pulposus was sharply demarcated from the anulus fibrosus, Sharpey fibers were conspicuous, and a plate of primitive notochord was evident in the equator of the disk. On long repetition time (TR)/long echo time (TE) or long TR/short TE MR images, Sharpey fibers (low signal intensity) and notochord (low signal intensity) could be differentiated from the high-signal-intensity nucleus pulposus and anulus fibrosus. The major differences between the fetal and infant spines were the amount of notochord in the disk and ossification in the vertebral body.     

A breast cancer cell microarray (CMA) as a rapid method to characterize candidate biomarkers

Tissue microarrays (TMAs) have become an invaluable tool in cancer research to evaluate expression and subcellular localization of proteins in cells and tissues. As the catalogs of candidate biomarkers and therapeutic targets become more extensive, there is a need to characterize and validate these targets and biomarkers in cell lines as a primary biological system in research laboratories. Thus, cell microarrays (CMAs) are useful as a high-throughput screening tool. Here, we constructed a CMA containing 32 publicly available immortalized breast cell lines with the goal of creating a method to rapidly screen for antigens of interest in breast cancer research in a relatively easy, rapid and cost-effective manner. As proof of concept, we performed immunocytochemical staining of the HER2 receptor, as the status of this protein is relevant to breast cancer and has previously been reported for these cell lines. We observed a complete concordance of our staining with the published status of HER2 in these cell lines. In addition, we examined the expression of CD44, epithelial markers EpCAM and E-cadherin and tyrosine phosphoproteins. The labeling of these proteins correlates with the known biology of the cell lines. Our results demonstrate the utility of our method to screen for potential biomarkers and therapeutic targets in breast cancer and we suggest that CMAs be used as a general approach in breast cancer research.     

Regulation of cellular differentiation and apoptosis by fatty acids and their metabolites.

We have reviewed the literature regarding the effects of fatty acids and their metabolites on cellular differentiation and apoptosis. Results obtained in different studies have been variable, but some generalizations can be made. Differentiation was increased by incubation of cells with arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), or leukotriene D4 (LTD4). Effects of these agents on differentiation could be magnified with the simultaneous addition of other differentiation-inducing agents like dimethylsulfoxide or retinoic acid. AA and gamma-linolenic acid increased apoptosis while the effects of n-3 fatty acids (EPA and DHA) and of eicosanoids varied from stimulation to inhibition. These inconsistencies are attributed to the differences in methods used to evaluate differentiation and apoptosis, concentrations of fatty acids and serum, exposure time and the cell models used. Studies using the physiological concentrations of the fatty acids and standardized experimental conditions need to be conducted to establish effects of fatty acids and their metabolites on these cellular processes.     

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase

Background and purpose:

Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets.

Experimental approach:

Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC).

Key results:

Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis.

Conclusion and implications:

Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.     

青蒿琥酯皮肤擦剂在小鼠和兔体内的药代动力学研究

将青蒿琥酯溶于苯二甲酸二甲酯,加适量氨酮制成皮肤擦剂。给兔脱毛后,皮肤涂抹此擦剂25mg/kg后,血药浓度达峰时间平均为2 h,峰浓度平均为1.80μg/ml。药物在兔体内平均驻留时间为3.54 h,清除半衰期约为2.46 h。给小鼠脱毛皮肤涂抹擦剂6.7,31.3和71.4 mg/kg,血药浓度在给药后0.5～4 h达高峰,峰浓度分别为0.82,2.05和7.11μg/ml,体内药物平均驻留时间为3.39,2.79及3.54 h,清除半衰期为2.35,1.93及2.45 h。可见,给兔及小鼠皮肤擦剂后,青蒿琥酯吸收良好,血药浓度维持时间较长。