Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains Differentially Modulates Src Kinase Activity in Brain Maturation

Src family kinases (SFK) control multiple processes during brain development and function. We show here that the phosphoprotein associated with glycosphigolipid-enriched microdomains (PAG)/Csk binding protein (Cbp) modulates SFK activity in the brain. The timing and localization of PAG expression overlap with Fyn and Src, both of which we find associated to PAG. We demonstrate in newborn (P1) mice that PAG negatively regulates Src family kinases (SFK). P1 Pag1-/- mouse brains show decreased recruitment of Csk into lipid rafts, reduced phosphorylation of the inhibitory tyrosines within SFKs, and an increase in SFK activity of >/ = 50%. While in brain of P1 mice, PAG and Csk are highly and ubiquitously expressed, little Csk is found in adult brain suggesting altered modes of SFK regulation. In adult brain Pag1-deficiency has no effect upon Csk-distribution or inhibitory tyrosine phosphorylation, but kinase activity is now reduced (−20–30%), pointing to the development of a compensatory mechanism that may involve PSD93. The distribution of the Csk-homologous kinase CHK is not altered. Importantly, since the activities of Fyn and Src are decreased in adult Pag1-/- mice, thus presenting the reversed phenotype of P1, this provides the first in vivo evidence for a Csk-independent positive regulatory function for PAG in the brain

Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-Resorptive Effects

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function. [Methods]: For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model. [Results]: Dasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2-5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1-2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVβ3 integrin and CCR1 expression. [Conclusions]: Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease. © 2012 Garcia-Gomez et al.This work was supported by grants from the Spanish Ministry of Science and Innovation – ISCIII (PI081825); Mutua Madrileña Medical Research Foundation (AP27262008); Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León, Consejería de Sanidad JCyL – ISCIII; the Cooperative Research Thematic Network in Cancer (RTICC; RD06/0020/0006 and RD03/0020/0041); and Spanish FIS (PS09/01897). AG-G and CS are supported by the Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León Project.Peer Reviewe

STUDY OF THE MOLECULAR-DYNAMICS FROM DEUTERIUM ZEEMAN AND QUADRUPOLAR NMR RELAXATION OF 4,4'-DI-N-HEPTYLAZOXYBENZENE IN THE NEMATIC AND SMECTIC-A PHASES

Abstract The deuterium spin-lattice (T1Z) and quadrupolar (R1Q) relaxation times of the liquid crystal 4,4´-Heptyl-azoxybenzene (HAB), deuterated on the aromatic rings and the first methylene group of the chains, have been measured by means of the Jeener-Broekaert pulse sequence at two different frequencies (10 and 46.04 M Hz) and throughout the nematic and the smectic A phase. By applying suitable models, we have determined rotational and reorientational diffusion coefficients and evalu­ated the contribution of the different motions (internal, molecular and collective) to the relaxation. In particular we have found a non negligible contribution from order director fluctuations. A comparison between the T1Z and T1Q measurements performed by means of Jeener-Broekaert and Wimperis pulse sequences is also shown.</jats:p

Spin labeling study of human serum albumin in reverse micelles

Human serum albumin (HSA), spin labeled at the sulfhydryl group by the reagent 3-maleimidoproxyl (3MAL), was studied in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The electron spin resonance spectra were recorded at different water contents and analyzed by computer simulations. In order to obtain agreement between experimental and calculated spectra, the use of an anisotropic model of reorientational diffusion for 3MAL-HSA in reverse micelles was necessary. An isotropic reorientational motion was suitable to simulate the 3MAL-HSA spectrum in aqueous solution. This result suggests that conformational changes of the protein which modify the label environment occur in reverse micelles. The rotational correlation times for 3MAL-HSA in reverse micelles were strongly dependent on the water content: the protein experienced a more hindered environment for rotational diffusion as the water pool size decreased

Study of the orientational order and dynamics in the nematic and smectic phases of p'-hexyloxybenzyliden-p-fluoroaniline by means of H-2-NMR

Measurements of orientational order parameters and spectral densities of motion in the nematic, smectic A and smectic B phases of p'- hexyloxybenzyliden-p-fluoroaniline,fully deuterated in the chain and partially deuterated in the aniline ring, are reported. The deuterium spin-lattice (Tlz) and quadrupolar ( T ~ Qr)elaxation times have been measured at 46.04 MHz using a broadband multiple pulse Jeener-Broekaert sequence. The orientational order of the C-D bonds and the spectral densities determined suggest that, on entering the highly ordered smectic phases, the internal motions of the first two methylene groups in the chain slow down sensibly. These effects are discussed within the framework of the models used to describe molecular and internal dynamics in mesomorphicphases