技术资料

Ceramide has emerged as an important lipid messenger for many cellular processes triggered via surface receptors. In the present study,inflammatory activation of P388D1 macrophages with bacterial lipopolysaccharide (LPS) and platelet-activating factor (PAF) stimulated a transient accumulation of ceramide. Moreover,cell-permeable ceramide mimicked LPS/PAF in triggering arachidonate mobilization in these cells. LPS/PAF-induced ceramide synthesis did not result from sphingomyelinase activation but from increased de novo synthesis. Participation of this pathway in arachidonate signaling was detected since fumonisin B1,an inhibitor of de novo ceramide synthesis,was able to inhibit the LPS/PAF-induced response. These studies have uncovered a new role for sphingolipid metabolism in cellular signaling and constitute evidence that products of the sphingomyelin biosynthetic pathway may serve a specific role in signal transduction by influencing the activity of the novel Group V secretory phospholipase A2. View Publication

Retinoid induction of epidermal hyperplasia was investigated in hairless mice with synthetic ligands for the retinoic acid (RAR) and retinoid X (RXR) nuclear receptors. Induction of hyperplasia by all-trans retinoic acid and the RAR-specific retinoids TTNPB,tazarotene and AGN 190121 varied over a wide range (ED50 = 0.2-100 nmol/animal in three daily applications). Potency of induction was not directly correlated to receptor-binding affinity,but specificity of action could be demonstrated by inhibition with the high-affinity antagonist of the RARs,AGN 193109. Although RAR is functionally complexed with RXR in vivo,RXR-selective compounds have only weak potency in induction of hyperplasia. The ED50 value of the RXR-selective AGN 191701 was 600 nmol/animal compared with an ED50 value of 0.2 nmol for the structurally similar RAR-selective TTNPB. SR11237 and SR11217,also RXR-selective,each have an ED50 value of textgreater1000 nmol. Unlike RAR-specific retinoids,RXR-selective retinoids cause only very mild skin flaking at high doses. Relative potencies for cumulative topical irritation (flaking and abrasion) of both RAR and RXR ligands were well correlated with epidermal hyperplasia. These data are consistent with RXR as a silent partner in the RAR-RXR heterodimer in skin. View Publication

We compared glucocorticoid receptor binding characteristics and glucocorticoid responsiveness of human mononuclear leukocytes (HML) from hypertensive patients and matched normotensive volunteers. We also considered associations of these variables with plasma renin activity,aldosterone,cortisol,corticotropin,and electrolyte concentrations. We calculated binding affinity (Kd; nmol/L) and capacity (Bmax; sites/cell) for dexamethasone and cortisol from homologous and heterologous competition curves for specific [3H]dexamethasone binding sites on HML isolated from the blood of normotensive volunteers and subjects with essential hypertension. Glucocorticoid responsiveness of HML was evaluated as IC50 values (nmol/L) for dexamethasone and cortisol for the inhibition of lysozyme release. We measured plasma hormones by radioimmunoassay. Kd values (mean+/-SE) for cortisol in HML of hypertensive patients were higher than in control subjects (24.6+/-2.4 versus 17.5+/-1.7 nmol/L,Ptextless.04). Binding capacity (4978+/-391 versus 4131+/-321 sites/cell),Kd values for dexamethasone (6.7+/-0.5 versus 5.7+/-0.3 nmol/L),and IC50 values for dexamethasone (3.4+/-0.3 versus 3.1+/-0.2 nmol/L) and cortisol (12.2+/-1.6 versus 9.5+/-0.3 nmol/L) were not significantly different. Patients with renin values less than 0.13 ng angiotensin I/L per second were markedly less sensitive to cortisol than those with higher values. Both Kd (30.3+/-2.5 versus 19.2+/-2.4 nmol/L) and IC50 values (15.5+/-1.8 versus 8.9+/-1.2 nmol/L) for cortisol were significantly higher in patients with lower renin values (Ptextless.03). Other variables,including plasma hormone and electrolyte values and binding characteristics for dexamethasone,were not different. These data suggest that cortisol binding to glucocorticoid receptor is slightly impaired in patients with essential hypertension. In vivo,this could lead to inappropriate binding of cortisol to mineralocorticoid receptors. Hence,decreased sensitivity to cortisol is associated with renin suppression. This hypothesis is supported by evidence of hypertension and low renin activity,which others have described in patients with primary glucocorticoid resistance due to mutations of the glucocorticoid receptor. View Publication

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MUC1 mucin is expressed by normal and malignant epithelial cells and is thought to function through cell-cell interactions and transmembrane signal transduction events. Secreted cancer-associated MUC1 is immunosuppressive and inhibits human T-cell proliferation. We report here that newly synthesized MUC1 is expressed on the surface of mitogen-activated human T cells and is also found in soluble form in the supernatants from cultures of mitogen-activated human T cells. After removal of the mitogenic stimulus from the T-cell cultures,MUC1 expression is downregulated. The addition of anti-MUC1 monoclonal antibody to mitogen-activated cultures partially inhibits the T-cell proliferative response. These data suggest that MUC1 serves an immunodulatory function for human T lymphocytes. View Publication

We determined the inhibitory activities of gatifloxacin against Staphylococcus aureus topoisomerase IV,Escherichia coli DNA gyrase,and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 for S. aureus,r = 0.972 for E. coli,and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 microg/ml for S. aureus topoisomerase IV; IC50 = 0.109 microg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 microg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureus topoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However,the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones,indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases. View Publication

Prostaglandin H synthase (PGHS) is the primary enzyme responsible for the biosynthesis of prostaglandins and thromboxanes. Of the two isoenzymes of PGHS,PGHS-1 is constitutively expressed and PGHS-2 is inducible by mitogens or other inflammatory stimuli. Constitutive expression of PGHS-2 in neoplastic tissues has been implicated in carcinogenesis. Resveratrol,a lignan,was recently shown to be an anticarcinogen that selectively inhibits PGHS-1. In vitro experiments to resolve these seemingly paradoxical observations revealed that resveratrol is not only an inhibitor of PGHS-1 but also is an activator of PGHS-2. Resveratrol non-competitively inhibited PGHS-1 with a K1 of 26 +/- 2 microM but enhanced the PGHS-2 activity nearly twofold. Additionally,resveratrol did not serve as a reducing co-substrate for the peroxidase activities of either enzyme despite being an easily oxidizable phenolic compound. Resveratrol inhibited the peroxidase activity of PGHS-1 (IC50 = 15 microM) better than that of PGHS-2 (IC50 = textgreater 200 microM). Inhibition of the perxidase activity but not the cyclooxygenase activity of PGHS-2 resulted in the production of PGG2 from arachidonic acid. A plausible relationship between these observation and the anticarcinogenic activity of resveratrol is discussed. View Publication

In the adult human,mesenchymal stem cells (MSCs) resident in bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages,including cartilage. In this study,culture-expanded human MSCs (hMSCs) of 60 human donors were induced to express the morphology and gene products of chondrocytes. Chondrogenesis was induced by culturing hMSCs in micromass pellets in the presence of a defined medium that included 100 nM dexamethasone and 10 ng/ml transforming growth factor-beta(3) (TGF-beta(3)). Within 14 days,cells secreted an extracellular matrix incorporating type II collagen,aggrecan,and anionic proteoglycans. hMSCs could be further differentiated to the hypertrophic state by the addition of 50 nM thyroxine,the withdrawal of TGF-beta(3),and the reduction of dexamethasone concentration to 1 nM. Increased understanding of the induction of chondrogenic differentiation should lead to further progress in defining the mechanisms responsible for the generation of cartilaginous tissues,their maintenance,and their regeneration. View Publication

Wnt/β-catenin signalling has a variety of roles in regulating stem cell fates. Its specific role in mouse epiblast stem cell self-renewal,however,remains poorly understood. Here we show that Wnt/β-catenin functions in both self-renewal and differentiation in mouse epiblast stem cells. Stabilization and nuclear translocation of β-catenin and its subsequent binding to T-cell factors induces differentiation. Conversely,retention of stabilized β-catenin in the cytoplasm maintains self-renewal. Cytoplasmic retention of β-catenin is effected by stabilization of Axin2,a downstream target of β-catenin,or by genetic modifications to β-catenin that prevent its nuclear translocation. We also find that human embryonic stem cell and mouse epiblast stem cell fates are regulated by β-catenin through similar mechanisms. Our results elucidate a new role for β-catenin in stem cell self-renewal that is independent of its transcriptional activity and will have broad implications in understanding the molecular regulation of stem cell fate. View Publication

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence,which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here,we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity,simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. textcopyright 2013 Elsevier Inc. View Publication

Diblock copolymers consisting of poly(ethylene glycol)-block-poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-l-glutamate) (PEG-b-PVBLG-8) were synthesized and evaluated for their ability to mediate gene delivery in hard-to-transfect cells like IMR-90 human fetal lung fibroblasts and human embryonic s View Publication

Down syndrome (DS) is among the most frequent genetic causes of intellectual disability,and ameliorating this deficit is a major goal in support of people with trisomy 21. The Ts65Dn mouse recapitulates some major brain structural and behavioral phenotypes of DS,including reduced size and cellularity of the cerebellum and learning deficits associated with the hippocampus. We show that a single treatment of newborn mice with the Sonic hedgehog pathway agonist SAG 1.1 (SAG) results in normal cerebellar morphology in adults. Further,SAG treatment at birth rescued phenotypes associated with hippocampal deficits that occur in untreated adult Ts65Dn mice. This treatment resulted in behavioral improvements and normalized performance in the Morris water maze task for learning and memory. SAG treatment also produced physiological effects and partially rescued both N-methyl-d-aspartate (NMDA) receptor-dependent synaptic plasticity and NMDA/AMPA receptor ratio,physiological measures associated with memory. These outcomes confirm an important role for the hedgehog pathway in cerebellar development and raise the possibility for its direct influence in hippocampal function. The positive results from this approach suggest a possible direction for therapeutic intervention to improve cognitive function for this population. View Publication