Arachidonic acid inhibits adipocyte differentiation of 3T3-L1 cells via a prostaglandin synthesis-dependent pathway. Here we show that this inhibition requires the presence of a cAMP-elevating agent ...during the first two days of treatment. Suppression of protein kinase A activity by H-89 restored differentiation in the presence of arachidonic acid. Arachidonic acid treatment led to a prolonged activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 activity by the addition of U0126 rescued differentiation. Upon induction of differentiation, expression of cyclooxygenase-2 (COX-2) was transiently induced and then declined, whereas COX-1 expression declined gradually as differentiation progressed. Treatment with arachidonic acid led to sustained expression of COX-1 and COX-2. Omission of a cAMP-elevating agent or addition of H-89 or U0126 prevented sustained expression of COX-2. Unexpectedly, we observed that selective COX-1 or COX-2 inhibitors rescued adipocyte differentiation in the presence of arachidonic acid as effectively as did the nonselective COX-inhibitor indomethacin.
De novo fatty acid synthesis, diacylglycerol acyltransferase (DGAT) activity, and triacylglycerol accumulation were repressed in cells treated with arachidonic acid. Indomethacin restored DGAT activity and triacylglycerol accumulation without restoring de novo fatty acid synthesis, resulting in an enhanced incorporation of arachidonic acid into cellular triacylglycerols.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The irrational and prolonged use of antibiotics in orthopaedic infections poses a major threat to the development of antimicrobial resistance. To combat antimicrobial resistance, researchers have ...implemented various novel and innovative modalities to curb infections. Nanotechnology involves doping ions/metals onto the scaffolds to reach the target site to eradicate the infective foci. In this connotation, we reviewed silver nanoparticle technology in terms of mechanism of action, clinical applications, toxicity, and regulatory guidelines to treat orthopaedic infections.
CCAAT/enhancer-binding protein β (C/EBPβ) is expressed early during the adipocyte differentiation program and plays an important role in this process. In an attempt to identify novel proteins that ...interact with C/EBPβ, we performed a yeast two-hybrid screen with a preadipocyte cDNA library and identified a new co-regulator, delta-interacting protein A (DIPA). DIPA mRNA is expressed during adipocyte differentiation of clonal cell lines. DIPA interacts with C/EBPβ and -δ proteins in intact cells and inhibits their transcriptional activity but not that of C/EBPα. Stable overexpression of DIPA in preadipocytes partially inhibits adipocyte differentiation, whereas its gene silencing enhances this process. DIPA and C/EBPβ co-localize in the nucleus, and overexpression of DIPA in preadipocytes results in a partial inhibition of the mitotic clonal expansion which is critical for differentiation. Thus, DIPA is a novel partner of C/EBPβ that down-regulates early events of adipogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The retinoblastoma protein (pRB) is an important regulator of development, proliferation, and cellular differentiation. pRB was recently shown to play a pivotal role in adipocyte differentiation, to ...interact physically with adipogenic CCAAT/enhancer-binding proteins (C/EBPs), and to positively regulate transactivation by C/EBPβ. We show that PPARγ-mediated transactivation is pRB-independent, and that ligand-induced transactivation by PPARγ1 present in RB+/+and RB−/− mouse embryo fibroblasts is sufficient to bypass the differentiation block imposed by the absence of pRB. The differentiated RB−/− cells accumulate lipid and express adipocyte markers, including C/EBPα and PPARγ2. Interestingly, adipose conversion of pRB-deficient cells occurs in the absence of compensatory up-regulations of the other pRB family members p107 and p130. RB+/+ as well asRB−/− cells efficiently exit from the cell cycle after completion of clonal expansion following stimulation with adipogenic inducers. We conclude that ligand-induced activation of endogenous PPARγ1 in mouse embryo fibroblasts is sufficient to initiate a transcriptional cascade resulting in induction of PPARγ2 and C/EBPα expression, withdrawal from the cell cycle, and terminal differentiation in the absence of a functional pRB.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
One of the major characteristics of Type 2 Diabetes is insulin resistance, which is often treated by insulin sensitizing drugs such as thiazolidinediones (TZDs). The primary target for the TZDs is ...the peroxisome proliferator-activated receptor (PPAR) γ. However, critical side effects of TZDs can occur, as they are full PPARγ agonists. Partial PPARγ agonists are associated with fewer side effects but still may maintain the effect on insulin resistance. Recently, a general screening for potential partial PPARγ agonists was carried out on a number of plant extracts. This screening resulted in a 60% hit rate among the tested plant species and one of the most promising of these was purple coneflower (
Echinacea purpurea
) 1. Based on this general screening a large-scale bioassay-guided fractionation of a hexane extract of the flowers of purple coneflower was carried out. The fractionations were done by flash column chromatography and reverse phase semi-preparative HPLC for isolation of active constituents. Bioactivity of fractions and purified compounds were assessed using a PPARγ transactivation assay. Mouse embryonic fibroblasts were used for this assay and each sample was tested in triplicate in three different concentrations. Activation of PPARγ for each sample was compared to both vehicle (DMSO) and the TZD rosiglitazone, as a positive control. Two of the compounds being able to activate PPARγ were isolated and identified as linoleic acid and linolenic acid, but also some of the isolated alkamides were shown to activate PPARγ. Hence, the PPARγ activating properties of purple coneflower also seem to depend on the presence of these specific alkamides. This suggest a whole new field of application for purple coneflower which is one of the most extensively used medicinal plants worldwide for treatment of the common cold.
References: 1. Christensen, K.B. et al. 2008, unpublished.
Pref-1 is a highly glycosylated Delta-like transmembrane protein containing six epidermal growth factor-like repeats in the extracellular domain. Pref-1 is abundantly expressed in preadipocytes, but ...expression is down-regulated during adipocyte differentiation. Forced expression of Pref-1 in 3T3-L1 cells was reported to inhibit adipocyte differentiation. Here we show that efficient and regulated processing of Pref-1 occurs in 3T3-L1 preadipocytes releasing most of the extracellular domain as a 50-kDa heterogeneous protein, previously isolated and characterized as FA1. Unexpectedly, we found that forced expression of the soluble form, FA1, or full-length Pref-1 did not inhibit adipocyte differentiation of 3T3-L1 cells when differentiation was induced by standard treatment with methylisobutylxanthine, dexamethasone, and high concentrations of insulin. However, forced expression of either form of Pref-1/FA1 in 3T3-L1 or 3T3-F442A cells inhibited adipocyte differentiation when insulin or insulin-like growth factor-1 (IGF-1) was omitted from the differentiation mixture. We demonstrate that the level of the mature form of the IGF-1 receptor is reduced and that IGF-1-dependent activation of p42/p44 mitogen-activated protein kinases (MAPKs) is compromised in preadipocytes with forced expression of Pref-1. This is accompanied by suppression of clonal expansion and terminal differentiation. Accordingly, supplementation with insulin or IGF-1 rescued p42/p44 MAPK activation, clonal expansion, and adipocyte differentiation in a dose-dependent manner.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The biological functions of liver X receptors (LXRs) α and β have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation ...in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRα+/+/LXRβ+/+), LXRα knock-out mice (LXRα-/-/LXRβ+/+), LXRβ knock-out mice (LXRα+/-/LXRβ-/-), and LXR double knock-out mice (LXRα-/-/LXRβ-/-) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRα or a dominant negative version of LXRα, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-κB activity, since expression of a dominant negative version of IκBα prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic ...inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity. Ras-dependent signaling plays a significant, although incompletely understood, role in adipocyte differentiation, because activated Ras has been reported to either promote or inhibit adipogenesis depending on the cellular context. In various cell types activation of Ras leads to activation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein kinase B (PKB)/Akt, which exert opposing effects on adipogenesis, with ERK1/2 inhibiting and PKB/Akt promoting terminal differentiation. Here we report that the levels of activated ERK1/2 and PKB/Akt are significantly increased in pRB-deficient MEFs both before and after the addition of adipogenic inducers. Consistently, we detected higher levels of activated Ras in MEFs lacking pRB. Suppression of ERK1/2 activation by the MEK inhibitor UO126 restored the ability of pRB-deficient MEFs to undergo adipocyte differentiation, as manifested by expression of adipocyte marker genes and lipid accumulation. Furthermore and reflecting the elevated levels of activated PKB/Akt in the pRB-deficient MEFs, differentiation proceeded in an insulin-independent manner. In conclusion, we suggest that pRB plays a pivotal role in adipogenesis by suppressing MAPK activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
BACKGROUNDSchwannoma is a benign, encapsulated and slowly growing tumor originating from Schwann cells and is rarely seen in the peripheral nerve system. Typical symptoms are soreness, radiating pain ...and sensory loss combined with a soft tissue mass. AIMTo evaluate pre- and postoperative symptoms in patients operated for schwannomas in the extremities and investigate the rate of malignant transformation. METHODSIn this single center retrospective study design, all patients who had surgery for a benign schwannoma in the extremities from May 1997 to January 2018 were included. The location of the tumor in the extremities was divided into five groups; forearm, arm, shoulder, thigh and leg including foot. The locations of the tumor in the nerves were also categorized as either; proximal, distal, minor or major nerve. During the pre- and postoperative clinical evaluation, symptoms were classified as paresthesia, local pain, radiating pain, swelling, impairment of mobility/strength and asymptomatic tumors that were found incidentally (with magnetic resonance imaging). The patients were evaluated after surgery using the following categories: Asymptomatic or symptomatic patients (radiating and/or local pain) and those with complications. The follow up period was from the time of surgery until last examination of the particular physician. Multivariate logistic regression analysis was performed to identify independent prognostic factors for postoperative significant symptoms at follow-up. RESULTSWe identified 858 cases from the institutional pathology register. We excluded cases with duplicate diagnoses (n = 407), pathology not including schwannomas (n = 157), lesions involving the torso, spine and neck (n = 150) leaving 144 patients for further analysis. In this group 99 patients underwent surgery and there were five complications recorded: 2 infections (treated with antibiotics) and 3 nerve palsies (2 involving the radial nerve and one involving the median nerve) that recovered spontaneously. At the end of follow-up, 1.4 mo (range 0.5-76) postoperatively, we recorded a post-operative decrease in clinical symptoms: Local pain 76% (6/25), radiating pain 97% (2/45), swelling 20% (8/10). Symptoms of paresthesia increased by 2.8% (37/36) and there was no change in motor weakness before and after surgery 1% (1/1). Multivariate analysis showed that tumors located within minor nerves had a significantly higher prevalence of postoperative symptoms compared with tumors in major nerves (odds ratio: 2.63; confidence intervals: 1.22-6.42, P = 0.029). One patient with schwannoma diagnosed by needle biopsy was diagnosed to have malignant transformation diagnosed in the surgically removed tumor. No local recurrences were reported. CONCLUSIONSurgery of schwannomas can be conducted with low risk of postoperative complications, acceptable decrease in clinical symptoms and risk of malignant transformation is low.
A simple ELISA test to detect antibodies against scabby mouth virus (SMV) has been developed. Native whole virions and subunits of SMV generated by boiling the virus in the presence of sodium dodecyl ...sulphate (SDS) detergent and β-mercaptoethanol were compared as ELISA assay reagents using naive and hyperimmune sera from sheep and rabbits. Approximately 2 × 10
4 intact virus particles per microtiter well were required to generate a positive to negative signal of 0.8:0.3 ELISA O.D. units when the serum was used at a dilution of
1
100
. In contrast, total subunit antigen generated by disrupting and coupling of 250–500 virions per well provided a signal ratio of 1.58:0.3 ELISA O.D. units at a serum dilution of
1
250
. Total subunit antigens were therefore 400 times more economical to use than intact virions. In addition, subunit antigens could be readily bound to microtiter plates without the need for removal of the SDS. Secondly, it was not necessary to block non-specific binding sites on the plate with blockers such as gelatin and skim-milk, thereby shortening the time needed to complete the ELISA assay. The total subunit antigen ELISA test was used to detect seroconversion in new born lambs where there was an occurrence of SMV infection in housed sheep. Three bleeds were taken at fortnightly intervals and the ELISA results showed that 9 out of 15 lambs were seropositive for all bleed points. Four of the lambs showed a sequential rise in titer while only one lamb failed to seroconvert. The subunit ELISA test can therefore provide a very economical and sensitive test for the detection of antibody reactivity against SMV.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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