Effects of RXRα gene silencing on the porcine adipocyte differentiation in vitro

The elucidation of the molecular mechanism of porcine adipocyte differentiation is important not only to domestic animal industry, but also to medical science because of the similarities in the trait of body lipid deposition between human and pigs. In this study, we investigated the roles of RXRα in porcine (Sus scrofa) adipocyte differentiation by using RNAi method and primary cultured porcine adipocytes. With the transfection of RXRα-siRNA, the gene and protein expressions of RXRα were successfully inhibited in differentiating porcine adipocytes. By the RXRα gene silencing, the adipocyte differentiation, as characterized by lipid accumulation and GPDH activity, was suppressed. Furthermore, in association with the decrease in RXRα gene expression level, PPARγ and C/EBPα genes expression level also decreased, suggesting that RXRα is involved in their expression. Thus, our study clearly demonstrated the pivotal roles of RXRα in porcine adipocyte differentiation.     

(±)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis

Praeruptorin A is a coumarin compound naturally occurring in the roots of Peucedanum praeruptorum Dunn., a commonly used traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. Although previous studies indicated the relaxant effects of (±)-praeruptorin A on tracheal and arterial preparations, little is known about the functional characteristics of the enantiomers. In the present study, the two enantiomers were successfully isolated and identified by using a preparative Daicel Chiralpak AD-H column, and their relaxant effects on aorta rings were observed and compared. (+)-Praeruptorin A showed more potent relaxation than (?)-praeruptorin A against KCl- and phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. Removal of the endothelium remarkably reduced the relaxant effect of (+)-praeruptorin A but not that of (?)-praeruptorin A. Pretreatment of aortic rings with N^ω-nitro-l-arginine methyl ester (l-NAME, an inhibitor of nitric oxide synthase) or methylene blue (MB, a soluble guanylyl cyclase inhibitor) resulted in similar changes of the relaxant effects of the two enantiomers to endothelium removal. Molecular docking studies also demonstrated that (+)-praeruptorin A was in more agreement to nitric oxide synthase pharmacophores than (?)-praeruptorin A. On the other hand, the two enantiomers of praeruptorin A could slightly attenuate the contraction of rat aortic rings induced by internal Ca²⁺ release from sarcoplasmic reticulum (SR). These findings indicated that (+)-praeruptorin A and (?)-praeruptorin A exerted distinct relaxant effects on isolated rat aorta rings, which might be mainly attributed to nitric oxide synthesis catalyzed by endothelial nitric oxide synthase.     

Characterization of muscarinic acetylcholine receptors on intact neuroblastoma × glioma NG108-15 cell upon induced differentiation

Summary Studies have established that major increases in muscarinic acetylcholine receptor (mAchR) binding in the brain appear to coincide with synaptogenesis. The neuroblastoma × glioma hybrid NG108-15 cell line has been demonstrated to possess numerous functional characteristics of intact neurons, including synapse formation with myotubes. The present study examines and characterizes the mAchR on the hybrid NG108-15 cells during differentiation, induced by 1 mM dBcAMP. Specific binding of [³H]-QNB for differentiated cells increases gradually to a final level of 130% (P < 0.05) over the control undifferentiated cells during the first 24 hr of incubation. Further, this increase of receptor sites appears to correlate proportionately to the degree of neurite extension of the differentiating cells. The dissociation rate constant at equilibrium (K_d) and maximum binding capacity (B_max) have been determined to be 5.6 nM and 920 fmol/10⁶ cells, respectively, for differentiated cells, and 4.4 nM and 400 fmol/10⁶ cells, respectively, for undifferentiated cells. Computer analyses of the data obtained from saturation experiments reveal a single class of binding sites for [³H]-QNB on both differentiated and undifferentiated cells. The Hill plot analysis of the QNB-binding indicates a Hill coefficient (nH) of 1.0 and 0.91 for differentiated and undifferentiated cells, respectively, suggesting the unity of receptor sites with no co-operativity. Our results depict that increases of mAchRs on intact cells correlate with the degree of cellular differentiation.     

Atherogenic effects of TNF-α and IL-6 via up-regulation of scavenger receptors

Patients with chronic inflammatory disorders such as rheumatoid arthritis (RA) have a high risk of developing cardiovascular disease. We evaluated the effects of TNF-α and IL-6 on foam cell formation, a pivotal process in atherogenesis. Accumulation of intracellular oxidized LDL (oxLDL) was induced when THP-1/macrophages were stimulated with TNF-α or IL-6. TNF-α induced the expressions of scavenger receptors SR-A and LOX-1, and IL-6 induced SR-A expression. Inhibition of the NF-κB signaling markedly decreased TNF-α-induced foam cell formation and SR-A expression. Serum from RA patients, but not healthy subjects, induced foam cell formation, which was partially reversed by either IL-6 or TNF-α blockade in conjunction with inhibiting the induction of scavenger receptors. The present study clearly showed that in patients with chronic inflammation mediated by TNF-α and IL-6, these cytokines are directly implicated in atherosclerotic plaque formation.     

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E₂), testosterone, and dihydrotestosterone (DHT). Mechanistically, E₂ and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E₂ signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E₂ enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E₂ treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E₂ and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E₂ and DHT membrane-associated PKC activity in costochondral cartilage cells.     

Umbelliferone derivatives exert neuroprotective effects by inhibiting monoamine oxidase A,self-amyloidβ aggregation,and lipid peroxidation

Umbelliferone has been demonstrated to have a wide range of biological activities. However, the effect of incorporating a formyl moiety in the umbelliferone scaffold has not been investigated. In this paper, we investigated the inhibitory activity of six coumarins, namely umbelliferone (1), 6-formyl umbelliferone (2), 8-formyl umbelliferone (3), umbelliferone-6-carboxylic acid (4), esculetin (5), and scopoletin (6) against human monoamine oxidases (hMAOs), self-amyloid β (Aβ) aggregation, and lipid peroxidation. We found that all compounds had high selectivity for hMAO-A in comparison with hMAO-B. Among the compounds, 2 exhibited the highest hMAO inhibitory activity with an IC₅₀ value of 3.23 µM for hMAO-A and 15.31 µM for hMAO-B. Enzyme kinetic analysis showed that 2 and 3 were competitive hMAO inhibitors. In silico hydrated molecular docking simulations revealed that the coumarins interacted with substrate-binding site residues of the enzymes and the isoalloxazine ring of FAD. In addition, formyl coumarins 2 and 3 significantly inhibited lipid peroxidation in rat brain homogenates and self-Aβ_25-35 aggregation compared to other derivatives. These represent the first experimental and modelling data for hMAO-A/B inhibition by umbelliferone derivatives. Together, the data suggest that introduction of a formyl moiety in the 7-hydroxycoumarin scaffold, especially at the 6 position, plays an important role in the inhibition of hMAOs, Aβ self-aggregation, and lipid peroxidation. Umbelliferone derivative 2 is a promising therapeutic lead scaffold for developing anti-neuropsychiatric disorder drugs that function via selective hMAO-A inhibition.     

The effect of interferon-β on mouse neural progenitor cell survival and differentiation

Interferon-β (IFN-β) is a mainstay therapy for relapse-remitting multiple sclerosis (MS). However, the direct effects of IFN-β on the central nervous system (CNS) are not well understood. To determine whether IFN-β has direct neuroprotective effects on CNS cells, we treated adult mouse neural progenitor cells (NPCs) in vitro with IFN-β and examined the effects on proliferation, apoptosis, and differentiation. We found that mouse NPCs express high levels of IFNα/β receptor (IFNAR). In response to IFN-β treatment, no effect was observed on differentiation or proliferation. However, IFN-β treated mouse NPCs demonstrated decreased apoptosis upon growth factor withdrawal. Pathway-specific polymerase chain reaction (PCR) arrays demonstrated that IFN-β treatment upregulated the STAT 1 and 2 signaling pathway, as well as GFRA2, NOD1, Caspases 1 and 12, and TNFSF10. These results suggest that IFN-β can directly affect NPC survival, possibly playing a neuroprotective role in the CNS by modulating neurotrophic factors.     

Protective effects of salusin-α and salusin-β on renal ischemia/reperfusion damage and their levels in ischemic acute renal failure

Salusin-α and salusin-β are expressed in many tissues including the central nervous system, vessels and kidneys; they have been shown to decrease endoplasmic reticulum stress during heart ischemia/reperfusion (I/R) and to decrease apoptosis. We investigated the relation of salusin-α and salusin-β levels to acute ischemic renal failure. We also investigated whether these peptides are protective against renal I/R damage. Fifty-three rats were divided into six groups: control, I/R, I/R + salusin-α1, I/R + salusin-α10, I/R + salusin-β1 and I/R + salusin-β10. After removing the right kidney, the left kidney was subjected to ischemia for 1 h and reperfusion for 23 h. The treatment groups were injected subcutaneously at the beginning of ischemia with 1 or 10 μg/kg salusin-α, and 1 or 10 μg/kg salusin-β. Histopathology was assessed at the end of the experiment. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX) activity and malondialdehyde (MDA) levels were measured in the kidney tissue. Serum levels of blood urea nitrogen (BUN), creatinine (Cre), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) also were measured. Levels of salusin-α and salusin-β were measured in the serum and kidney tissues of the control and I/R groups. SOD, CAT and GSH-PX activities were decreased and the levels of MDA, TNF-α, IL-6, IL-1β, BUN and Cre were increased in the I/R group compared to controls. Severe glomerular and tubular damage was apparent in the I/R group compared to controls. The level of salusin-β was decreased in the serum and kidney tissue of the I/R group compared to controls, whereas the level of salusin-α was decreased in the serum and increased in the kidney tissue. Salusin-α and salusin-β administration increased SOD and GSH-PX enzyme activation and decreased the levels of MDA, TNF-α, IL-6 and IL-1β compared to the I/R group. BUN and Cre levels were decreased in the I/R + salusin-α1 group and the level of Cre was decreased in I/R + salusin-β10 group compared to the I/R group. We demonstrated a protective effect of salusin-α and salusin-β against renal I/R damage. Changes in the levels of salusin-α and salusin-β in the I/R group suggest that these peptides may be associated with acute renal failure.     

Cytotoxic and cytogenetic effects of α-copaene on rat neuron and N2a neuroblastoma cell lines

Alpha-copaene (α-COP), a tricyclic sesquiterpene, is present in several essential oils of medicinal and aromatic plants and has antioxidant and antigenotoxic features. Its cytotoxic, cytogenetic and oxidative effects have not been investigated in neuron and N2a neuroblastoma (NB) cell cultures. Therefore, we aimed to describe in vitro: (i) cytotoxic properties by 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenlytetrazolium bromide test; (ii) antioxidant/oxidant activity by total antioxidant capacity (TAC) and total oxidative status (TOS) analysis; and (iii) genotoxic damage potential by single cell gel electrophoresis — of α-COP in healthy neuron and N2a-NB cell cultures for the first time. Significant (P < 0.05) decrease in cell proliferation were observed in cultured primary rat neurons starting with the concentration of 150 mg/L and in N2a-NB cells starting with 100 mg/L. In addition, 25 mg/L of α-COP treatment caused increase of TAC levels and α-COP treatments at higher doses led to increase of TOS levels in neuron N2a-NB cell cultures. Moreover, none of the tested concentrations of α-COP have shown a genotoxic effect on both cell lines. Our findings clearly demonstrate that α-COP exhibited mild cytotoxic effects on N2a-NB cell line. In conclusion, α-COP may have potential as an anticancer agent, which needs to be further studied.     

Radiolabeling and initial biological evaluation of [18F]KBM-1 for imaging RAR-α receptors in neuroblastoma

Retinoic acid receptor alpha (RAR-α) plays a significant role in a number of diseases, including neuroblastoma. Children diagnosed with high-risk neuroblastoma are treated 13-cis-retinoic acid, which reduces risk of cancer recurrence. Neuroblastoma cell death is mediated via RAR-α, and expression of RAR-α is upregulated after treatment. A molecular imaging probe that binds RAR-α will help clinicians to diagnose and stratify risk for patients with neuroblastoma, who could benefit from retinoid-based therapy. In this study, we report the radiolabeling, and initial in vivo evaluation of [¹⁸F]KBM-1, a novel RAR-α agonist. The radiochemical synthesis of [¹⁸F]KBM-1 was carried out through KHF₂ assisted substitution of [¹⁸F]^? from aryl-substituted pinacolatoesters-based retinoid precursor. In vitro cell uptake assay in human neuroblastoma cell line showed that the uptake of [¹⁸F]KBM-1 was significantly inhibited by all three blocking agents (KBM-1, ATRA, BD4) at all the selected incubation times. Standard biodistribution in mice bearing neuroblastoma tumors demonstrated increased tumor uptake from 5 min to 60 min post radiotracer injection and the uptake ratios for target to non-target (tumor: muscle) increased 2.2-fold to 3.7-fold from 30 min to 60 min post injection. Tumor uptake in subset of 30 min blocking group was 1.7-fold lower than unblocked. These results demonstrate the potential utility of [¹⁸F]KBM-1 as a RAR-α imaging agent.     

Myristoylation exerts direct and allosteric effects on Gα conformation and dynamics in solution

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen-deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.     

Central effects of TNFα on thermogenesis and fever in the rat

Intracerebroventricular (icv) injection of purified recombinant human tumour necrosis factor (TNF , 4–8g) in conscious rats, produced increases in colonic temperature (1.0°C) and resting oxygen consumption (VO₂, 14%) which were maximal after 80–90 minutes. Pretreatment with propranolol (10mg/kg s.c) significantly inhibited the rise in VO₂, and prevented the increase in body temperature. Icv injection of an antagonist to corticotropin releasing factor (-helical CRF 9-41, 25 g), which prevents the pyrogenic and thermogenic actions of interleukin-1, did not influence the effects of TNF on temperature or VO₂. Injection of a fragment of TNF (113–130 amino acid sequence) did not affect body temperature or VO₂. TNF injection (icv) significantly increased brown adipose tissue (BAT)in vitro mitochondrial GDP binding, and this effect was slightly inhibited, but not prevented, by surgical denervation of the tissue, and was unaffected by pretreatment with -helical CRF 9-41. These data indicate that TNF can stimulate thermogenesis by a direct central action. The effects are largely, but not totally, dependent on the sympathetic nervous system but, unlike the thermogenic actions of interleukin they do not require release of CRF.     

Nuclear prostaglandin receptors: role in pregnancy and parturition?

The key regulatory role of prostanoids [prostaglandins (PGs) and thromboxanes (TXs)] in the maintenance of pregnancy and initiation of parturition has been established. However, our understanding of how these events are fine-tuned by the recruitment of specific signaling pathways remains unclear. Whereas, initial thoughts were that PGs were lipophilic and would easily cross cell membranes without specific receptors or transport processes, it has since been realized that PG signaling occurs via specific cell surface G-protein coupled receptors (GPCRs) coupled to classical adenylate cyclase or inositol phosphate signaling pathways. Furthermore, specific PG transporters have been identified and cloned adding a further level of complexity to the regulation of paracrine action of these potent bioactive molecules. It is now apparent that PGs also activate nuclear receptors, opening the possibility of novel intracrine signaling mechanisms. The existence of intracrine signaling pathways is further supported by accumulating evidence linking the perinuclear localization of PG synthesizing enzymes with intracellular PG synthesis. This review will focus on the evidence for a role of nuclear actions of PGs in the regulation of pregnancy and parturition.