搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study,we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs,characterized by transcriptional repression of IL-1α and β,IL-1R1,and vascular endothelial growth factor A,and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs,therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated,at least in part,by increases in EPC/CAC proliferation,by decreases in EPC/CAC apoptosis,and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs,and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis,but not anti-neutrophil cytoplasmic Ab-positive vasculitis,showed this pathway to be operational in vivo,with increased IL-1R antagonist,downregulation of vascular endothelial growth factor A,and glomerular and blood vessel decreased capillary density,compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease. View Publication

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators,such as Smad proteins,the p38 mitogen-activated protein kinase (MAPK),and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate,Smad3,and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM,respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However,the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production,the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA,indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast,SB-431542,but not the selective p38 MAPK inhibitor SB-242235,inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e.,FN),whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e.,col Ialpha1). View Publication

We have recently shown that proteasome inhibitor PS-341 induces apoptosis in drug-resistant multiple myeloma (MM) cells,inhibits binding of MM cells in the bone marrow microenvironment,and inhibits cytokines mediating MM cell growth,survival,drug resistance,and migration in vitro. PS-341 also inhibits human MM cell growth and prolongs survival in a SCID mouse model. Importantly,PS-341 has achieved remarkable clinical responses in patients with refractory relapsed MM. We here demonstrate molecular mechanisms whereby PS-341 mediates anti-MM activity by inducing p53 and MDM2 protein expression; inducing the phosphorylation (Ser15) of p53 protein; activating c-Jun NH(2)-terminal kinase (JNK),caspase-8,and caspase-3; and cleaving the DNA protein kinase catalytic subunit,ATM,and MDM2. Inhibition of JNK activity abrogates PS-341-induced MM cell death. These studies identify molecular targets of PS-341 and provide the rationale for the development of second-generation,more targeted therapies. View Publication

In this study,in an attempt to identify neuroblastoma-associated surface antigens,we generated mAbs against the ACN neuroblastoma cell line. A mAb was selected (5B14) that reacted with all neuroblastoma cell lines analyzed and allowed detection of tumor cell infiltrates in bone marrow aspirates from neuroblastoma patients. In cytofluorimetric analysis,unlike anti-disialoganglioside mAb,5B14 mAb did not display reactivity with normal bone marrow hematopoietic cell precursors,thus representing a highly specific marker for identifying neuroblastoma cells. Molecular analysis revealed that the 5B14 mAb-reactive surface glycoprotein corresponded to the recently identified 4Ig-B7-H3 molecule. Remarkably,mAb-mediated masking of the 4Ig-B7-H3 molecule on cell transfectants or on freshly isolated neuroblastoma cells resulted in enhancement of natural killer-mediated lysis of these target cells. These data suggest that 4Ig-B7-H3 molecules expressed at the tumor cell surface can exert a protective role from natural killer-mediated lysis by interacting with a still undefined inhibitory receptor expressed on natural killer cells. View Publication

Metformin is an anti-diabetic drug with anorexigenic properties. The precise cellular mechanisms of its action are not entirely understood. Adipose tissue has recently been recognized as an important endocrine organ that is pivotal for the regulation of insulin resistance and energy homeostasis. Due to its thermogenic capacity brown adipose tissue contributes to the regulation of energy metabolism and is an attractive target tissue for pharmacological approaches to treating insulin resistance and obesity. Leptin is the prototypic adipocyte-derived hormone inducing a negative energy balance. We investigated effects of metformin on adipocyte metabolism,signalling,and leptin secretion in a brown adipocyte model. Metformin acutely stimulated p44/p42 mitogen-activated protein (MAP) kinase in a dose- (3.2-fold at 1 mmol/l,Ptextless 0.05) as well as time-dependent (3.8-fold at 5 min,Ptextless 0.05) manner. This stimulation was highly selective since phosphorylation of intermediates in the stress kinase,janus kinase (JAK)-signal transducer and activator of transcription (STAT),and phosphatidylinositol (PI) 3-kinase signalling pathways such as p38 MAP kinase,STAT3,and Akt was unaltered. Furthermore,chronic metformin treatment for 12 days dose-dependently inhibited leptin secretion by 35% and 75% at 500 mumol/l and 1 mmol/l metformin respectively (Ptextless 0.01). This reduction was not caused by alterations in adipocyte differentiation. Moreover,the impairment in leptin secretion by metformin was reversible within 48 h after removal of the drug. Pharmacological inhibition of p44/p42 MAP kinase prevented the metformin-induced negative effect on leptin secretion. Taken together,our data demonstrate direct acute effects of metformin on adipocyte signalling and endocrine function with robust inhibition of leptin secretion. They suggest a selective molecular mechanism that may contribute to the anorexigenic effect of this antidiabetic compound. View Publication

Because osteoblasts and marrow adipocytes are derived from a common mesenchymal progenitor,increased adipogenesis may occur at the expense of osteoblasts,leading to bone loss. Our previous in vitro studies indicated that activation of the proadipogenic transcription factor peroxisome proliferator-activated receptor isoform gamma 2 with rosiglitazone suppressed osteoblast differentiation. Here,we show that 5-month-old Swiss-Webster mice receiving rosiglitazone for 28 d exhibited bone loss associated with an increase in marrow adipocytes,a decrease in the ratio of osteoblasts to osteoclasts,a reduction in bone formation rate,and a reduction in wall width--an index of the amount of bone formed by each team of osteoblasts. Rosiglitazone had no effect on the number of early osteoblast or osteoclast progenitors,or on osteoblast life span,but decreased the expression of the key osteoblastogenic transcription factors Runx2 and Osterix in cultures of marrow-derived mesenchymal progenitors. These effects were associated with diversion of bipotential progenitors from the osteoblast to the adipocyte lineage,and suppression of the differentiation of monopotential osteoblast progenitors. However,rosiglitazone had no effect on osteoblastic cells at later stages of differentiation. Hence,rosiglitazone attenuates osteoblast differentiation and thereby reduces bone formation rate in vivo,leading to bone loss. These findings provide a mechanistic explanation for the recent evidence that peroxisome proliferator-activated receptor isoform gamma activation is a negative regulator of bone mass and suggest that the increased production of oxidized fatty acids with age may indeed be an important mechanism for age-related osteoporosis in humans. View Publication

The transcription factor p53 coordinates cell cycle arrest and apoptosis in response to DNA damage. Pifithrin-alpha (PFT-alpha) is a small molecule inhibitor of p53 activity that is frequently used in cell culture studies of p53 function. Here we report an investigation of the stability of this compound. PFT-alpha rapidly converts to a planar tricyclic derivative,with a half-life of 4.2 h under physiological conditions. This spontaneous conversion greatly alters the structural and physicochemical properties of the drug. PFT-alpha has a pKa of 9.11 and is an ionic species in physiological medium,whereas the tricyclic derivative has a pKa of 4.36 and exists as the neutral free base at pH 7. The tricyclic derivative is very hydrophobic,with a log P of 4.26. Although PFT-alpha is generally used at 10-30 microM concentration,the aqueous solubility of its derivative is only 0.2 microM,and it can form a visible precipitate under conditions of typical use. The conversion of PFT-alpha proceeds via an intramolecular cyclization reaction involving the imine and carbonyl groups. Modification of the carbonyl function creates a stable analogue of PFT-alpha that remains soluble indefinitely. These results provide a strategy for the rational design of PFT-alpha analogues that exhibit predictable stability,hydrophobicity,and aqueous solubility. View Publication

Numerous proteins undergo modification by palmitic acid (S-acylation) for their biological functions including signal transduction,vesicular transport and maintenance of cellular architecture. Although palmitoylation is an essential modification,these proteins must also undergo depalmitoylation for their degradation by lysosomal proteases. Palmitoyl-protein thioesterase-1 (PPT1),a lysosomal enzyme,cleaves thioester linkages in S-acylated proteins and removes palmitate residues facilitating the degradation of these proteins. Thus,inactivating mutations in the PPT1 gene cause infantile neuronal ceroid lipofuscinosis (INCL),a devastating neurodegenerative storage disorder of childhood. Although rapidly progressing brain atrophy is the most dramatic pathological manifestation of INCL,the molecular mechanism(s) remains unclear. Using PPT1-knockout (PPT1-KO) mice that mimic human INCL,we report here that the endoplasmic reticulum (ER) in the brain cells of these mice is structurally abnormal. Further,we demonstrate that the level of growth-associated protein-43 (GAP-43),a palmitoylated neuronal protein,is elevated in the brains of PPT1-KO mice. Moreover,forced expression of GAP-43 in PPT1-deficient cells results in the abnormal accumulation of this protein in the ER. Consistent with these results,we found evidence for the activation of unfolded protein response (UPR) marked by elevated levels of phosphorylated translation initiation factor,eIF2alpha,increased expression of chaperone proteins such as glucose-regulated protein-78 and activation of caspase-12,a cysteine proteinase in the ER,mediating caspase-3 activation and apoptosis. Our results,for the first time,link PPT1 deficiency with the activation of UPR,apoptosis and neurodegeneration in INCL and identify potential targets for therapeutic intervention in this uniformly fatal disease. View Publication

Angiotensin II (Ang II) type 2 (AT2) receptors are abundantly expressed not only in the fetal brain where they probably contribute to brain development,but also in pathological conditions to protect the brain against stroke; however,the detailed mechanisms are unclear. Here,we demonstrated that AT2 receptor signaling induced neural differentiation via an increase in MMS2,one of the ubiquitin-conjugating enzyme variants. The AT2 receptor,MMS2,Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1),and newly cloned AT2 receptor-interacting protein (ATIP) were highly expressed in fetal rat neurons and declined after birth. Ang II induced MMS2 expression in a dose-dependent manner,reaching a peak after 4 h of stimulation,and this effect was enhanced with AT1 receptor blocker,valsartan,but inhibited by AT2 receptor blocker PD123319. Moreover,we observed that an AT2 receptor agonist,CGP42112A,alone enhanced MMS2 expression. Neurons treated with small interfering RNA of MMS2 failed to exhibit neurite outgrowth and synapse formation. Moreover,the increase in AT2 receptor-induced MMS2 mRNA expression was enhanced by overexpression of ATIP but inhibited by small interfering RNA of SHP-1 and overexpression of catalytically dominant-negative SHP-1 or a tyrosine phosphatase inhibitor,sodium orthovanadate. After AT2 receptor stimulation,ATIP and SHP-1 were translocated into the nucleus after formation of their complex. Furthermore,increased MMS2 expression mediates the inhibitor of DNA binding 1 proteolysis and promotes DNA repair. These results provide a new insight into the contribution of AT2 receptor stimulation to neural differentiation via transactivation of MMS2 expression involving the association of ATIP and SHP-1. View Publication

Depletion of CD4+CD25+ regulatory T cells (Treg) by treatment with alphaCD25 antibody synergizes with vaccination protocols to engender protective immunity in mice. The effectiveness of targeting CD25 to eliminate Treg is limited by the fact that CD25,the low-affinity interleukin-2 receptor,is up-regulated on conventional T cells. At present,foxp3 is the only product known to be exclusively expressed in Treg of mice. However,foxp3 is not expressed on the cell surface and hence cannot be targeted with antibodies. In this study,we tested the hypothesis that vaccination of mice against foxp3,a self-antigen expressed also in the thymus,is capable of stimulating foxp3-specific CTL that will cause the depletion of Treg and enhanced antitumor immunity. Vaccination of mice with foxp3 mRNA-transfected dendritic cells elicited a robust foxp3-specific CTL response and potentiated vaccine-induced protective immunity comparably with that of alphaCD25 antibody administration. In contrast to alphaCD25 antibody treatment,repeated foxp3 vaccination did not interfere with vaccine-induced protective immunity. Importantly,foxp3 vaccination led to the preferential depletion of foxp3-expressing Treg in the tumor but not in the periphery,whereas alphaCD25 antibody treatment led to depletion of Treg in both the tumor and the periphery. Targeting foxp3 by vaccination offers a specific and simpler protocol for the prolonged control of Treg that may be associated with reduced risk of autoimmunity,introducing an approach whereby specific depletion of cells is not limited to targeting products expressed on the cell surface. View Publication

The sirtuins are members of the histone deacetylase family of proteins that participate in a variety of cellular functions and play a role in aging. We identified a potent inhibitor of sirtuin 2 (SIRT2) and found that inhibition of SIRT2 rescued alpha-synuclein toxicity and modified inclusion morphology in a cellular model of Parkinson's disease. Genetic inhibition of SIRT2 via small interfering RNA similarly rescued alpha-synuclein toxicity. Furthermore,the inhibitors protected against dopaminergic cell death both in vitro and in a Drosophila model of Parkinson's disease. The results suggest a link between neurodegeneration and aging. View Publication

OBJECTIVE Metformin is an antidiabetic drug commonly used to treat type 2 diabetes. The aim of the study was to determine whether metformin regulates hepatic gluconeogenesis through the orphan nuclear receptor small heterodimer partner (SHP; NR0B2). RESEARCH DESIGN AND METHODS We assessed the regulation of hepatic SHP gene expression by Northern blot analysis with metformin and adenovirus containing a constitutive active form of AMP-activated protein kinase (AMPK) (Ad-AMPK) and evaluated SHP,PEPCK,and G6Pase promoter activities via transient transfection assays in hepatocytes. Knockdown of SHP using siRNA SHP was conducted to characterize the metformin-induced inhibition of hepatic gluconeogenic gene expression in hepatocytes,and metformin-and adenovirus SHP (Ad-SHP)-mediated hepatic glucose production was measured in B6-Lep(ob/ob) mice. RESULTS Hepatic SHP gene expression was induced by metformin,5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR),and Ad-AMPK. Metformin-induced SHP gene expression was abolished by adenovirus containing the dominant negative form of AMPK (Ad-DN-AMPK),as well as by compound C. Metformin inhibited hepatocyte nuclear factor-4alpha-or FoxA2-mediated promoter activity of PEPCK and G6Pase,and the inhibition was blocked with siRNA SHP. Additionally,SHP knockdown by adenovirus containing siRNA SHP inhibited metformin-mediated repression of cAMP/dexamethasone-induced hepatic gluconeogenic gene expression. Furthermore,oral administration of metformin increased SHP mRNA levels in B6-Lep(ob/ob) mice. Overexpression of SHP by Ad-SHP decreased blood glucose levels and hepatic gluconeogenic gene expression in B6-Lep(ob/ob) mice. CONCLUSIONS We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP. View Publication