Clean & Fast Amide Couplings in Water

EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) is a carbodiimide that is well known reagent for the amide coupling reactions between an amine and carboxylic acid in the presence of a base. It is the commonly used coupling agent in industries because it produces a water-soluble urea byproduct, which can be easily removed by an aqueous work-up. However, our methodology, the desired amide product precipitates out of the reaction mixture, and no additional work-up is required.3 Using aqueous micelles of PS-750-M as a solvent dramatically increases the rate of reaction.2 The filtrate recovered can be recycled to perform several coupling runs after post-reaction manipulation

PYK2 selectively mediates signals for growth versus differentiation in response to stretch of spontaneously active vascular smooth muscle.

Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium-dependent signals. We studied the role of the calcium- and integrin-activated proline-rich tyrosine kinase 2 (PYK2) in stretch-induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr-402 was increased both by a 10-min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF-4594755 (0.5-1 μmol/L), while still sensitive to stretch. In 3-day organ culture, PF-4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high-K(+) (60 mmol/L) or to α1-adrenergic stimulation by cirazoline. Basal and stretch-induced PYK2 phosphorylation in culture were inhibited by PF-4594755, closely mimicking inhibition of non-voltage-dependent calcium influx by 2-APB (30 μmol/L). In contrast, the L-type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch-induced but not basal PYK2 phosphorylation. Stretch-induced Akt and ERK1/2 phosphorylation was eliminated by PF-4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch-sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 μmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage- and non-voltage-dependent calcium influx. A small-molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle

Differential effects of RGS proteins on Gαq and Gα11 activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human Gαq and Gα11 spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of Gα-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both Gαq and Gα11 but, in contrast, RGS5 and RGS16 were much less effective against Gα11 than Gαq. Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active Gαq QL/Gα11 QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-Gα specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of Gαq/Gα11 enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both Gα subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential Gα activity, further highlighting that RGS proteins can discriminate between two very closely related Gα subunits

Plantar fasciopathy: revisiting the risk factors

Background Plantar fasciopathy is the most common cause of acquired sub-calcaneal heel pain in adults. To-date, research of this condition has mainly focused on management rather than causal mechanisms. The aetiology of plantar fasciopathy is likely to be multifactorial, as both intrinsic and extrinsic risk factors have been reported. The purpose of this review is to critically reevaluate risk factors for plantar fasciopathy. Methods A detailed literature review was undertaken using English language medical databases. Results No clear consensus exists as to the relative strength of the risk factors reported. Conclusions To-date numerous studies have examined various intrinsic and extrinsic risk factors implicated in the aetiology of plantar fasciopathy. How these factors interact may provide useful data to establish an individuals’ risk profile for plantar fasciopathy and their potential for response to treatment. Further research is indicated to rank the relative significance of these risk factors

Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast terminal differentiation

Autophagy is a conserved lysosomal degradation process that has important roles in both normal human physiology and disease. However, the function of autophagy in bone homeostasis is not well understood. Here, we report that autophagy is activated during osteoblast differentiation. Ablation of focal adhesion kinase family interacting protein of 200 kD (FIP200), an essential component of mammalian autophagy, led to multiple autophagic defects in osteoblasts including aberrantly increased p62 expression, deficient LC3‐II conversion, defective autophagy flux, absence of GFP‐LC3 puncta in FIP200‐null osteoblasts expressing transgenic GFP‐LC3, and absence of autophagosome‐like structures by electron microscope examination. Osteoblast‐specific deletion of FIP200 led to osteopenia in mice. Histomorphometric analysis revealed that the osteopenia was the result of cell‐autonomous effects of FIP200 deletion on osteoblasts. FIP200 deletion led to defective osteoblast terminal differentiation in both primary bone marrow and calvarial osteoblasts in vitro. Interestingly, both proliferation and differentiation were not adversely affected by FIP200 deletion in early cultures. However, FIP200 deletion led to defective osteoblast nodule formation after initial proliferation and differentiation. Furthermore, treatment with autophagy inhibitors recapitulated the effects of FIP200 deletion on osteoblast differentiation. Taken together, these data identify FIP200 as an important regulator of bone development and reveal a novel role of autophagy in osteoblast function through its positive role in supporting osteoblast nodule formation and differentiation. © 2013 American Society for Bone and Mineral Research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/100319/1/jbmr1971.pd

The retinoid anticancer signal: mechanisms of target gene regulation

Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARβ2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARβ2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARβ2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARβ2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARβ2, DUSP6, and RGS16

Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2)

Snai2-deficient cells are radiosensitive to DNA damage. The function of Snai2 in response to DNA damage seems to be critical for its function in normal development and cancer. Here, we applied a functional genomics approach that combined gene-expression profiling and computational molecular network analysis to obtain global dissection of the Snai2-dependent transcriptional response to DNA damage in primary mouse embryonic fibroblasts (MEFs), which undergo p53-dependent growth arrest in response to DNA damage. Although examination of the response showed that overall expression of p53 target gene expression patterns was similarly altered in both control and Snai2-deficient cells, we have identified and validated candidate Snai2 target genes linked to Snai2 gene function in response to DNA damage. This work defines for the first time the effect of Snai2 on p53 target genes in cells undergoing growth arrest, elucidates the Snai2-dependent molecular network induced by DNA damage, points to novel putative Snai2 targets, and suggest a mechanistic model, which has implications for cancer management

Abrogation of Cbl–PI3K Interaction Increases Bone Formation and Osteoblast Proliferation

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl–PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (CblYF/YF, YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745–36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl–PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation