Picrosides I and II,selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.     

Rational design and improvement of the dimerization‐disrupting peptide selectivity between ROCK‐I and ROCK‐II kinase isoforms in cerebrovascular diseases

Human rho‐associated coiled‐coil forming kinases (ROCKs) ROCK‐I and ROCK‐II have been documented as attractive therapeutic targets for cerebrovascular diseases. Although ROCK‐I and ROCK‐II share a high degree of structural conservation and are both present in classic rho/ROCK signaling pathway, their downstream substrates and pathological functions may be quite different. Selective targeting of the two kinase isoforms with traditional small‐molecule inhibitors is a great challenge due to their surprisingly high homology in kinase domain (~90%) and the full identity in kinase active site (100%). Here, instead of developing small‐molecule drugs to selectively target the adenosine triphosphate (ATP) site of two isoforms, we attempt to design peptide agents to selectively disrupt the homo‐dimerization event of ROCK kinases through their dimerization domains which have a relatively low conservation (~60%). Three helical peptides H1, H2, and H3 are split from the kinase dimerization domain, from which the isolated H2 peptide is found to have the best capability to rebind at the dimerization interface. A simulated annealing (SA) iteration method is used to improve the H2 peptide selectivity between ROCK‐I and ROCK‐II. The method accepts moderate degradation in peptide affinity in order to maximize the affinity difference between peptide binding to the two isoforms. Consequently, hundreds of parallel SA runs yielded six promising peptide candidates with ROCK‐I over ROCK‐II (I over II [IoII]) calculated selectivity and four promising peptide candidates with ROCK‐II over ROCK‐I (II over I [IIoI]) calculated selectivity. Subsequent anisotropy assays confirm that the selectivity values range between 13.2‐fold and 83.9‐fold for IoII peptides, and between 5.8‐fold and 21.2‐fold for IIoI peptides, which are considerably increased relative to wild‐type H2 peptide (2.6‐fold for IoII and 2.0‐fold for IIoI). The molecular origin of the designed peptide selectivity is also analyzed at structural level; it is revealed that the peptide residues can be classified into conserved, non‐conserved, and others, in which the non‐conserved residues play a crucial role in defining peptide selectivity, while conserved residues confer stability to kinase‐peptide binding.     

Different coexpressions of arthritis-relevant genes between different body organs and different brain regions in the normal mouse population

Structural changes in different parts of the brain in rheumatoid arthritis (RA) patients have been reported. RA is not regarded as a brain disease. Body organs such as spleen and lung produce RA-relevant genes. We hypothesized that the structural changes in the brain are caused by changes of gene expression in body organs. Changes in different parts of the brain may be affected by altered gene expressions in different body organs. This study explored whether an association between gene expressions of an organ or a body part varies in different brain structures. By examining the association of the 10 most altered genes from a mouse model of spontaneous arthritis in a normal mouse population, we found two groups of gene expression patterns between five brain structures and spleen. The correlation patterns between the prefrontal cortex, nucleus accumbens, and spleen were similar, while the associations between the other three parts of the brain and spleen showed a different pattern. Among overall patterns of the associations between body organs and brain structures, spleen and lung had a similar pattern, and patterns for kidney and liver were similar. Analysis of the five additional known arthritis-relevant genes produced similar results. Analysis of 10 nonrelevant-arthritis genes did not result in a strong association of gene expression or clearly segregated patterns. Our data suggest that abnormal gene expressions in different diseased body organs may influence structural changes in different brain parts.     

Recent advances in quantitative colocalization analysis: Focus on neuroscience

Quantitative colocalization analysis is an advanced digital imaging tool to characterize the spatial expression of molecules of interest in immunofluorescence images obtained using confocal microscopes. It began from simple pixel counting and, with introduction of specialized algorithms, transformed into a powerful image analyzing technique capable of identifying the exact locations of various molecules in tissues and cells and describing their subtle changes in dynamics. Applications of quantitative colocalization in the field of neuroscience proved to be particularly informative by helping to obtain observations not otherwise achievable using other techniques. In this article, we review the background and applicability of quantitative colocalization with special focus on neuroscience research.