Epac2a-null mice exhibit obesity-prone nature more susceptible to leptin resistance.

BACKGROUND: The exchange protein directly activated by cAMP (Epac), which is primarily involved in cAMP signaling, has been known to be essential for controlling body energy metabolism. Epac has two isoforms: Epac1 and Epac2. The function of Epac1 on obesity was unveiled using Epac1 knockout (KO) mice. However, the role of Epac2 in obesity remains unclear. METHODS: To evaluate the role of Epac2 in obesity, we used Epac2a KO mice, which is dominantly expressed in neurons and endocrine tissues. Physiological factors related to obesity were analyzed: body weight, fat mass, food intake, plasma leptin and adiponectin levels, energy expenditure, glucose tolerance, and insulin and leptin resistance. To determine the mechanism of Epac2a, mice received exogenous leptin and then hypothalamic leptin signaling was analyzed. RESULTS: Epac2a KO mice appeared to have normal glucose tolerance and insulin sensitivity until 12 weeks of age, but an early onset increase of plasma leptin levels and decrease of plasma adiponectin levels compared with wild-type mice. Acute leptin injection revealed impaired hypothalamic leptin signaling in KO mice. Consistently, KO mice fed a high-fat diet (HFD) were significantly obese, presenting greater food intake and lower energy expenditure. HFD-fed KO mice were also characterized by greater impairment of hypothalamic leptin signaling and by weaker leptin-induced decrease in food consumption compared with HFD-fed wild-type mice. In wild-type mice, acute exogenous leptin injection or chronic HFD feeding tended to induce hypothalamic Epac2a expression. CONCLUSIONS: Considering that HFD is an inducer of hypothalamic leptin resistance and that Epac2a functions in pancreatic beta cells during demands of greater work load, hypothalamic Epac2a may have a role in facilitating leptin signaling, at least in response to higher metabolic demands. Thus, our data indicate that Epac2a is critical for preventing obesity and thus Epac2a activators may be used to manage obesity and obesity-mediated metabolic disorders.

Characterization of microRNAs and their target genes associated with transcriptomic changes in gamma-irradiated Arabidopsis.

MicroRNAs (miRNAs) regulate gene expression in response to biotic and abiotic stress in plants. We investigated gamma-ray-responsive miRNAs in Arabidopsis wild-type and cmt3-11t mutant plants using miRNA microarray analysis. miRNA expression was differentiated between the wild-type and cmt3-11t mutants. miR164a, miR169d, miR169h, miR172b*, and miR403 were identified as repressible in the wild-type and/or cmt3-11t mutant in response to gamma irradiation, while miR827, miR840, and miR850 were strongly inducible. These eight miRNA genes contain UV-B-responsive cis-elements, including G-box, I-box core, ARE, and/or MBS in the putative promoter regions. Moreover, Box 4, MBS, TCA-element, and Unnamed_4, as well as CAAT- and TATA-box, were identified in these eight miRNA genes. However, a positive correlation between the transcriptions of miRNAs and their putative target genes was only observed between miR169d and At1g30560 in the wild-type, and between miR827 and At1g70700 in the cmt3-11t mutant. Quantitative RT-PCR analysis confirmed that the transcription of miR164a, miR169d, miR169h, miR172b*, miR403, and miR827 differed after gamma irradiation depending on the genotype (wild-type, cmt3-11t, drm2, drd1-6, and ddm1-2) and developmental stage (14 or 28 days after sowing). In contrast, high transcriptional induction of miR840 and miR850 was observed in these six genotypes regardless of the developmental stage. Although the actual target genes and functions of miR840 and miR850 remain to be determined, our results indicate that these two miRNAs may be strongly induced and reproducible genetic markers in Arabidopsis plants exposed to gamma rays.

Fibroblast growth factor 21 analogue LY2405319 lowers blood glucose in streptozotocin-induced insulin-deficient diabetic mice by restoring brown adipose tissue function.

AIM: To investigate the effects of LY2405319, an analogue of fibroblast growth factor 21 (FGF21), on glucose homeostasis in streptozotocin (STZ)-induced insulin-deficient mice (STZ mice). METHODS: Nine-week-old male C57BL/6J mice were administered a single intraperitoneal injection of STZ (150 mg/kg). One week later, after confirmation of hyperglycaemia, saline or LY2405319 (5 mg/kg) was injected subcutaneously daily for 4 weeks. Changes in glucose homeostasis, energy metabolism and brown adipose tissue (BAT) function were assessed. RESULTS: The STZ mice had elevated blood glucose and reduced plasma FGF21 levels, impaired glucose uptake in the BAT, and BAT mitochondria with absent or swollen cristae and fewer lipid vacuoles. LY2405319 significantly reduced blood glucose levels and this was associated with increased BAT glucose uptake and changes in gene expression and morphology, indicating improved mitochondrial lipid metabolism in the BAT. Importantly, the ability of LY2405319 to lower blood glucose in STZ mice was compromised after removing interscapular BAT. CONCLUSIONS: Our results show that LY2405319 reduces blood glucose levels in insulin-deficient diabetes by improving BAT metabolism. Additional studies investigating the therapeutic potential of FGF21 for the treatment of type 1 diabetes are warranted.

cAMP response element binding protein H mediates fenofibrate-induced suppression of hepatic lipogenesis.

AIMS/HYPOTHESIS: Fenofibrate is a drug used to treat hyperlipidaemia that works by inhibiting hepatic triacylglycerol synthesis. Sterol regulatory element binding protein-1c (SREBP-1c) is a major regulator of the expression of genes involved in hepatic triacylglycerol synthesis. In addition, endoplasmic reticulum (ER)-bound transcription factor families are involved in the control of various metabolic pathways. Here, we show a novel function for an ER-bound transcription factor, cAMP response element binding protein H (CREBH), in fenofibrate-mediated inhibition of hepatic lipogenesis. METHODS: The effects of fenofibrate and adenovirus-mediated Crebh (also known as Creb313) overexpression (Ad-Crebh) on hepatic SREBP-1c production and lipogenesis in vitro and in vivo were investigated. We also examined whether downregulation of endogenous hepatic Crebh by small interfering (si)RNA restores the fenofibrate effect on hepatic lipogenesis and SREBP-1c production. Finally, we examined the mechanism by which CREBH inhibits hepatic SREBP-1c production. RESULTS: Fasting and fenofibrate treatment induced CREBH production and decreased SREBP-1c levels. Indeed, Ad-Crebh inhibited insulin- and liver X receptor agonist TO901317-induced Srebp-1c (also known as Srebf1) mRNA expression in cultured hepatocytes. Moreover, increased production of CREBH in the liver of mice following tail-vein injection of Ad-Crebh inhibited high-fat diet-induced hepatic steatosis through inhibition of Srebp-1c expression. The inhibition of endogenous Crebh expression by siRNA restored fenofibrate-induced suppression of Srebp-1c expression and hepatic lipid accumulation both in vitro and in vivo. CONCLUSIONS/INTERPRETATION: These results show that fenofibrate decreases hepatic lipid synthesis through induction of CREBH. This study suggests CREBH as a novel negative regulator of SREBP-1c production and hepatic lipogenesis.

Magnetic and structural studies of the multifunctional material SrFe(0.75)Mo(0.25)O(3-δ).

SrFe0.75Mo0.25O3-δ has been recently discovered as an extremely efficient electrode for intermediate temperature solid oxide fuel cells (IT-SOFCs). We have performed structural and magnetic studies to fully characterize this multifunctional material. We have observed by powder neutron diffraction (PND) and transmission electron microscopy (TEM) that its crystal symmetry is better explained with a tetragonal symmetry (I4/mcm space group) than with the previously reported orthorhombic symmetry (Pnma space group). The temperature dependent magnetic properties indicate an exceptionally high magnetic ordering temperature (TN ∼ 750 K), well above room temperature. The ordered magnetic structure at low temperature was determined by PND to be an antiferromagnetic coupling of the Fe cations. Mössbauer spectroscopy corroborated the PND results. A detailed study, with X-ray absorption spectroscopy (XAS), in agreement with the Mössbauer results, confirmed the formal oxidation states of the cations to be mixed valence Fe(3+/4+) and Mo(6+).

Redox compartmentalization and cellular stress.

Mammalian cells are highly organized to optimize function. For instance, oxidative energy-producing processes in mitochondria are sequestered away from plasma membrane redox signalling complexes and also from nuclear DNA, which is subject to oxidant-induced mutation. Proteins are unique among macromolecules in having reversible oxidizable elements, 'sulphur switches', which support dynamic regulation of structure and function. Accumulating evidence shows that redox signalling and control systems are maintained under kinetically limited steady states, which are highly displaced from redox equilibrium and distinct among organelles. Mitochondria are most reducing and susceptible to oxidation under stressed conditions, while nuclei are also reducing but relatively resistant to oxidation. Within compartments, the glutathione and thioredoxin systems serve parallel and non-redundant functions to maintain the dynamic redox balance of subsets of protein cysteines, which function in redox signalling and control. This organization allows cells to be poised to respond to cell stress but also creates sites of vulnerability. Importantly, disruption of redox organization is a common basis for disease. Research tools are becoming available to elucidate details of subcellular redox organization, and this development highlights an opportunity for a new generation of targeted antioxidants to enhance and restore redox signalling and control in disease prevention.

Spontaneous improvement of persistent ulceration after carotid artery stenting.

BACKGROUND AND PURPOSE: Because carotid plaque ulceration is associated with an increased risk of cerebral embolism, residual carotid plaque ulceration directly around a stent (persistent ulceration) after carotid angioplasty and stent placement (CAS) could still be a risk factor for a stroke. The purpose of this study is to understand the morphologic and clinical prognosis of persistent ulceration. PATIENTS AND TECHNIQUES: CAS was attempted on 91 consecutive stenotic lesions (80 patients). Of these, 54 lesions (48 patients) had ulceration before CAS. Angiograms were evaluated immediately after the procedure. Persistent ulceration was found in 34 lesions (30 patients). The mean depth and length of persistent ulcers were 2.1 mm (range, 1-4.7 mm) and 8.9 mm (range, 1.5-22 mm), respectively. All patients with persistent ulceration were followed with antiplatelet therapy. RESULTS: No ischemic event due to the lesions occurred during the mean follow-up period of 25.5 months (range, 3-48 months). Angiography on 25 lesions (21 patients) at a mean of 5.8 months (range, 1-21 months) after CAS showed that persistent ulceration disappeared in 12 lesions (48%), improved in 11 lesions (44%), and remained unchanged in 2 lesions (8%). Nine lesions (36%) showed restenosis, which were < or =30% and did not require any additional intervention. New ischemic lesions were not detected in any of the 14 patients (17 lesions) who underwent follow-up MR imaging at a mean of 9 months (range, 1-32 months) after CAS. CONCLUSION: We conclude that persistent ulceration after CAS improves spontaneously and is not a risk factor for cerebral embolism.

Direct carotid puncture for the endovascular treatment of anterior circulation aneurysms.

We report the usefulness of Guglielmi detachable coil (GDC) embolization by direct carotid puncture for anterior circulation aneurysms. For all 27 patients, GDC embolization by direct carotid puncture was safely performed by using a 5F sheath introducer 5 cm long and a Tracker-38 catheter. Neurologic deficits and hemorrhage were not found in any patient during the follow-up period. If the transfemoral approach cannot be applied, GDC embolization should be considered as an alternative method.

Further evaluation and validation of a commercially available competitive ELISA (cELISA) for the detection of antibodies specific to equine arteritis virus (EAV).

The purpose of this study was to further evaluate and validate two commercially available equine arteritis virus (EAV) competitive ELISAs (original and enhanced cELISAs) using archived equine sera from experimentally inoculated animals and field sera submitted for laboratory diagnosis. First, the original and subsequently enhanced cELISAs were compared with the virus neutralisation test (VNT) using a panel of archived serum samples from experimentally inoculated animals. Then, the enhanced cELISA was compared with the VNT using a large panel of archived serum samples. The total number of equine sera tested was 3255, which included sera against 25 different EAV strains. The study confirmed that the enhanced cELISA was more sensitive than the original cELISA. Based on testing sera from experimentally inoculated animals and field sera, the enhanced cELISA had an estimated sensitivity (98.9 percent and 99.6 percent, respectively) and specificity (98.3 percent and 98.7 percent, respectively). The currently marketed enhanced VMRD EAV antibody cELISA test kit (VMRD Inc., Pullman, Washington, USA) has high sensitivity and specificity relative to the VNT. Based on the findings of this study, the authors would propose that the enhanced cELISA should be considered as an alternative approved method to the VNT for the detection of antibodies to EAV.

In vitro inhibition of human glioblastoma cell line invasiveness by antisense uPA receptor.

The cell surface urokinase-type plasminogen activator receptor (uPAR) has been shown to be a key molecule in regulating plasminogen-mediated extracellular proteolysis. To investigate the role of uPAR in invasion of brain tumors, human glioblastoma cell line SNB19 was stably transfected with a vector capable of expressing an antisense transcript complementary to the 300 base pair of the 5' end of the uPAR mRNA. Parental and stably transfected (vector, sense, and antisense) cell lines were analysed for uPAR mRNA transcript by Northern blot analysis, and receptor protein levels were measured by radioreceptor assays and Western blotting. Significant reduction of uPAR sites was observed in the antisense transfected cell lines. The levels of uPAR mRNA were significantly decreased in antisense clones compared to control, vector and sense clones. The invasive potential of the cell lines in vitro was measured by Matrigel invasion assay and migration of cells from spheroids to monolayers. The antisense transfected cells showed a markedly lower level of invasion and migration than the controls. The antisense clones were more adhesive to the ECM components compared to parental, vector and sense clones. All transfected (vector, sense and antisense) clones and parental cells produced similar levels of uPA activity without any significant difference however, MMP-2 activity was decreased in antisense clones compared to controls. These results demonstrate that uPAR expression is critical for the invasiveness of human gliomas and down regulation of uPAR expression may be a feasible approach to decrease invasiveness.

Anti-proliferative and pro-apoptotic effects of herbal medicine on hepatic stellate cell.

Hepatic stellate cells (HSC) play a central role in hepatic fibrosis and compounds that promote apoptosis in HSC may have anti-fibrotic potentials. Herbal medicine has long been used in chronic liver disease but there is little scientific evidence for their actions. The present study investigated the effects of 14 commonly used herbs on cellular proliferation and apoptosis of a rat hepatic stellate cell line, HSC-T6 and the underlying mechanism of herb-induced apoptosis. HSC-T6 cell were incubated with herbal extracts and their proliferation was assessed by colorimetric assay. Apoptosis was measured and confirmed by flow cytometry, terminal transferase uridyl nick end labeling (TUNEL) assay and morphological features in hematoxylin and eosin staining. Apoptotic pathways involving Fas receptor and Bcl-2 family were investigated by Western blot. Five herbs, namely Angelica sinensis (AS), Carthamus tinctorius (CT), Ligusticum chuanxiong (LC), Salvia miltiorrhiza (SM) and Stephania tetrandra (ST) demonstrated both anti-proliferative and pro-apoptotic activities in HSC-T6. The highest potency was detected in SM and ST with 51.63 and 44.52% of HSC-T6 showing apoptotic changes, respectively. This was associated with upregulation of Fas and Bax and down-regulation of Bcl-xL in HSC. Fas ligand and Bcl2 expressions remained unchanged. The potential anti-fibrotic effect of herbal medicine warrants further evaluation.

Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases.

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

Role of extracellular matrix proteins in regulation of human glioma cell invasion in vitro.

Primary brain tumors lack the metastatic behavior that is in part believed to be promoted by the extracellular matrix (ECM) components of the basement membrane. This study was intended to examine the influence of the ECM components present in the basement membrane that may act as natural barriers to tumor cell invasion. We examined the effect of type I and type IV collagens, fibronectin, laminin, and hyaluronic acid on the migration and invasion of four established glioblastoma cell lines, SNB19, U251, UWR1, and UWR2. Lower concentrations of all the ECM components induced the migration and invasion of all the cell lines. However, in the case of SNB19, laminin inhibited both migration and invasion in a concentration-dependent manner. We have also examined the influence of individual ECM components on the migration of cells from a spheroid to a monolayer on ECM component-coated coverslips. Consistent with the invasion studies using the modified Boyden chamber assays, lower concentrations of ECM components induced the migration of cells from spheroids to monolayer. Again, laminin inhibited the migration of cells from SNB19 spheroids. These results indicate that ECM components induce the invasion of glioma cells, apart from components like laminin, which may act as natural inhibitors.

Inhibition of in vivo tumorigenicity and invasiveness of a human glioblastoma cell line transfected with antisense uPAR vectors.

Our previous studies showed that glioblastomas express increased urokinase-type plasminogen activator receptors (uPARs) in comparison to low-grade gliomas (Yamamoto et al., Cancer Res., 54, 5016-5020, 1994). To explore whether downregulation of uPAR inhibits tumor formation and invasiveness, a human glioblastoma cell line was transfected with a cDNA construct corresponding to 300 bp of the human uPAR's 5' end in an antisense orientation, resulting in a reduced number of uPA receptors. Co-culture studies with tumor spheroids and fetal rat brain aggregates showed that antisense SNB19-AS1 cells expressing reduced uPAR failed to invade fetal rat brain aggregates. Intracerebral injection of SNB19-AS1 stable transfectants failed to form tumors and were negative for uPAR expression in nude mice. Thus uPAR appears in this model to be essential for tumorigenicity and invasion of glioblastomas in vivo.

Cisplatin but not BCNU inhibits urokinase-type plasminogen activator levels in human glioblastoma cell lines in vitro.

Glioblastomas extensively invade the surrounding normal brain tissue, with a concomitant expression of various proteolytic enzymes, in particular urokinase-type plasminogen activator (uPA). In this study we used cis-diamminedichloroplatinum (cisplatin) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), commonly used anti-cancer drugs for the treatment of glioblastomas, to study the expression of uPA in three human glioblastoma cell lines in vitro. Cells were treated with 25 microM cisplatin and 50 microM BCNU, and uPA levels were estimated by fibrin zymography during a 72-h time course. Treatment of glioblastoma cells with cisplatin resulted in significantly decreased levels of uPA in serum-free conditioned medium and cell extracts, compared to BCNU-treated and untreated cell lines. Quantitative levels of uPA enzyme activity assessed by scanning laser densitometry and uPA protein by ELISA using antibody against uPA showed decreased levels of uPA in cisplatin-treated glioma cell lines relative to BCNU and untreated cell lines. Our results suggest that anti-tumor compound, cisplatin, may exert its anti-neoplastic effects by inhibiting uPA in malignant glioblastomas.

Effect of cisplatin and BCNU on MMP-2 levels in human glioblastoma cell lines in vitro.

Matrix metalloproteinases (MMPs) play an important role in various physiological and pathological conditions such as tissue remodeling, and cancer cell invasion and metastasis. The aim of this study was to determine the effect of the antitumor compounds cis-dichlorodiammine platinum (ii) (cisplatin) and 1, 3 bis (2-chloroethyl)-1-nitrosourea (BCNU) on 72-kDa type IV collagenase activity (MMP-2) in human gliomas. Human glioblastoma cell lines were treated with cisplatin (25 microM), and BCNU (50 microM), and the levels of MMP-2 were estimated in serum-free conditioned medium and in cell extracts at different time intervals. Gelatin zymography revealed increased levels of MMP-2 in serum-free conditioned medium and in cell extracts of untreated glioblastoma cell cultures during a 72-h period. In contrast, MMP-2 levels were significantly decreased in cisplatin-treated cells both in conditioned medium and cell extracts. However, no significant changes of MMP-2 levels were noted in BCNU-treated cells. Quantitative analysis of MMP-2 enzyme activity by densitometry and amount of MMP-2 protein by ELISA showed significantly decreased levels of MMP-2 in cisplatin-treated cells compared to BCNU and untreated glioblastoma cells. The results indicate that decreased levels of MMP-2 might represent an additional mechanism by which cisplatin provides its antineoplastic effects.

In vitro system to study role of blood flow on nitric oxide production and cell signaling in endothelial cells.

Through the approaches illustrated in this article we have demonstrated how molecular signaling events can be elucidated in cells responding to physiological forces. With the recent findings that endothelial form of nitric oxide synthase is associated with proteins such as caveolin and the availability of these cDNA constructs, this methodology allows a possible avenue to determine the physiological significance of such associations and the regulation of NO formation in response to shear stress.

Invasive pattern of lac-Z-transfected human glioblastoma cells in nude mice brain.

Primary, malignant brain tumors show an extensive infiltrative invasion into surrounding normal brain. At present, little information is available regarding the local invasive behavior of human brain tumors and until now no animal model suitable to mimic human gliomas has been reported. To identify the infiltrative behavior of an established glioblastoma cell line (SNB19), we achieved a stable transfection of the SNB19 cell line with beta-galactosidase (lac-Z) plasmid. The stable beta-galactosidase-expressing cells were then injected intracerebrally into nude mice in an attempt to follow its pattern of spread. The mice were sacrificed at 3, 4, and 6 weeks postinjection. We could detect tumor formation in all of the animals, and the tumor size increased gradually over the 6 week time period. Three weeks after injection, tumor cells showed characteristic infiltrative invasion along the corpus callosum. We also observed tumor-cell invasion into the anterior commissure in some animals, and each tumor cell could be identified by lac-Z expression as visualized by its blue color. Further invasion was identified at 4 and 6 weeks postinjection. Our results suggest that this model could be used to study the molecular mechanisms involved in the invasion of gliomas so that appropriate therapeutic intervention strategies could be designed.

Isochromosome 11q in a case of acute myelomonocytic leukemia FAB type M4.

The clinical and cytogenetic findings in a 42-year-old female with acute myelomonocytic leukemia (AMMoL) were reported. At diagnosis, cytogenetic studies of bone marrow cells revealed the karyotype 45; XX, +8, -11, -17, -18, +i (11q). To our knowledge, this is the second case of AMMoL with i(11q) in the literature.