A BODIPY-luminol chemiluminescent resonance energy-transfer (CRET) cassette for imaging of cellular superoxide.

Spectroscopic and in cellulo studies are here reported on the very first BODIPY-luminol chemiluminescent resonance energy-transfer (CRET) cassette where the luminol CL agent is covalently linked to the BODIPY energy-transfer acceptor in a molecular dyad. The efficiency of intramolecular CRET investigated for the BODIPY-luminol dyad was found to be 64% resulting in a dual emissive response. Successful in cellulo biochemiluminescence via CRET was achieved in PMA activated splenocytes.

Identification and Characterisation of a Novel Pathogenic Mutation in the Human Lipodystrophy Gene AGPAT2 : C48R: A Novel Mutation in AGPAT2.

Loss-of-function mutations in AGPAT2, encoding 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2), produce congenital generalised lipodystrophy (CGL). We screened the AGPAT2 gene in two siblings who presented with pseudoacromegaly, diabetes and severe dyslipidaemia and identified a novel mutation in AGPAT2 causing a single amino acid substitution, p.Cys48Arg. We subsequently investigated the molecular pathogenic mechanism linking both this mutation and the previously reported p.Leu228Pro mutation to clinical disease. Wild-type and mutant AGPAT2 were expressed in control and AGPAT2-deficient preadipocyte cell lines. mRNA and protein expression was determined, and the ability of each AGPAT2 species to rescue adipocyte differentiation in AGPAT2-deficient cells was assessed. Protein levels of both p.Cys48Arg and p.Leu228Pro AGPAT2 were significantly reduced compared with that of wild-type AGPAT2 despite equivalent mRNA levels. Stable expression of wild-type AGPAT2 partially rescued adipogenesis in AGPAT2 deficient preadipocytes, whereas stable expression of p.Cys48Arg or p.Leu228Pro AGPAT2 did not. In conclusion, unusually severe dyslipidaemia and pseudoacromegaloid overgrowth in patients with diabetes should alert physicians to the possibility of lipodystrophy. Both the previously unreported pathogenic p.Cys48Arg mutation in AGPAT2, and the known p.Leu228Pro mutation result in decreased AGPAT2 protein expression in developing adipocytes. It is most likely that the CGL seen in homozygous carriers of these mutations is largely accounted for by loss of protein expression.

Investigating the involvement of the ATF6α pathway of the unfolded protein response in adipogenesis.

The unfolded protein response (UPR) is activated by endoplasmic reticulum stress resulting from an accumulation of unfolded or mis-folded proteins. The UPR is divided into three arms, involving the activation of ATF-6, PERK and IRE-1, that together act to restrict new protein synthesis and increase the production of chaperones. Recent studies have implicated the PERK and IRE-1 components of the UPR in adipocyte differentiation. In this study, we investigate the importance of ATF6α during adipogenesis using stable knockdown of this protein in the model adipogenic cell line, C3H10T1/2. Reduction of ATF6α expression by >70% resulted in impaired expression of key adipogenic genes and reduced lipid accumulation following the induction of adipogenesis. In contrast, loss of ATF6α did not impair the ability of cells to undergo osteogenic differentiation. Overall, our data indicate that all three arms of the UPR, including ATF6α, must be intact to permit adipogenesis to occur.

Demonstration of a high-field short-period superconducting helical undulator suitable for future TeV-scale linear collider positron sources.

The first demonstration of a full-scale working undulator module suitable for future TeV-scale positron-electron linear collider positron sources is presented. Generating sufficient positrons is an important challenge for these colliders, and using polarized e(+) would enhance the machine's capabilities. In an undulator-based source polarized positrons are generated in a metallic target via pair production initiated by circularly polarized photons produced in a helical undulator. We show how the undulator design is developed by considering impedance effects on the electron beam, modeling and constructing short prototypes before the successful fabrication, and testing of a final module.

Influence of the electron-cation interaction on electron mobility in dye-sensitized ZnO and TiO2 nanocrystals: a study using ultrafast terahertz spectroscopy.

Charge transport and recombination in nanostructured semiconductors are poorly understood key processes in dye-sensitized solar cells. We have employed time-resolved spectroscopies in the terahertz and visible spectral regions supplemented with Monte Carlo simulations to obtain unique information on these processes. Our results show that charge transport in the active solar cell material can be very different from that in nonsensitized semiconductors, due to strong electrostatic interaction between injected electrons and dye cations at the surface of the semiconductor nanoparticle. For ZnO, this leads to formation of an electron-cation complex which causes fast charge recombination and dramatically decreases the electron mobility even after the dissociation of the complex. Sensitized TiO2 does not suffer from this problem due to its high permittivity efficiently screening the charges.

The NR4A family of orphan nuclear receptors are not required for adipogenesis.

Nuclear hormone receptors of the NR4A subfamily are rapidly induced during the early stages of adipogenesis, leading to the speculation that they may have important roles in this process. One of the three subfamily members, Nur77 has also been shown to play key roles in energy expenditure and lipolysis in skeletal muscle and in the control of hepatic gluconeogenesis. We, therefore, examined the role of NR4A factors in adipogenesis using the well-characterized 3T3-L1 preadipocyte model. Inhibition of Nur77 expression using siRNA did not affect induction of adipogenic genes, nor the accumulation of lipid. To inhibit the activity of all the three NR4A family members, we generated preadipocytes stably expressing a well-characterized dominant-negative Nur77 (DN-Nur77), known to block the function of the other NR4A factors, Nurr1 and Nor1, as well as Nur77. While the increased NR4A activity observed following adipogenic induction was completely abolished in these cells, DN-Nur77 expression did not affect the expression of genes characteristic of terminally differentiated adipocytes and had no impact on lipid accumulation in these cells. Thus, while members of the NR4A subfamily of nuclear receptors may have important metabolic roles in skeletal muscle and liver, we demonstrate that they are dispensable for normal adipocyte development.

Effect of apolipoprotein E deficiency on reactive sprouting in the dentate gyrus of the hippocampus following entorhinal cortex lesion: role of the astroglial response.

This study investigated the effect of apolipoporotein E (apoE) deficiency on hippocampal reactive sprouting responses of the septohippocampal cholinergic (SHC) and commissural/associational fibers (C/A) following an electrolytic lesion of the entorhinal cortex (ECL), using apoE knockout (apoEKO) and age-matched control wild-type mice. Based on recent evidence suggesting that apoE plays a role in the modulation of glial inflammation, we also tested the hypothesis that the pattern of the astroglial response to ECL might be related to the defective reinnervation previously reported in apoEKO mice. Consistent with our hypothesis, we report a differential pattern of astroglial response that concurred with impairments in the sprouting of the SHC and corresponding synaptic replacement in apoEKO mice at 14 and 30 days post-lesion (DPL), a time range covering the onset of axonal/terminal sprouting to synaptogenesis. We also report a limited sprouting of the C/A fiber system in apoEKO relative to control mice at 30 DPL, a period of active dendritic remodeling. The results of the present study confirm and extend previous findings that apoEKO mice display impaired regenerative capacity in response to ECL and argue that in addition to the effect of apoE on lipid trafficking, apoE may also influence the astroglial response to damage, and that both of these effects account for the defective reinnervation observed in apoEKO mice.

Mice lacking pro-opiomelanocortin are sensitive to high-fat feeding but respond normally to the acute anorectic effects of peptide-YY(3-36).

Inactivating mutations of the pro-opiomelanocortin (POMC) gene in both mice and humans leads to hyperphagia and obesity. To further examine the mechanisms whereby POMC-deficiency leads to disordered energy homeostasis, we have generated mice lacking all POMC-derived peptides. Consistent with a previously reported model, Pomc(-/-) mice were obese and hyperphagic. They also showed reduced resting oxygen consumption associated with lowered serum levels of thyroxine. Hypothalami from Pomc(-/-) mice showed markedly increased expression of melanin-concentrating hormone mRNA in the lateral hypothalamus, but expression of neuropeptide Y mRNA in the arcuate nucleus was not altered. Provision of a 45% fat diet increased energy intake and body weight in both Pomc(-/-) and Pomc(+/-) mice. The effects of leptin on food intake and body weight were blunted in obese Pomc(-/-) mice whereas nonobese Pomc(-/-) mice were sensitive to leptin. Surprisingly, we found that Pomc(-/-) mice maintained their acute anorectic response to peptide-YY(3-36) (PYY(3-36)). However, 7 days of PYY(3-36) administration had no effect on cumulative food intake or body weight in wild-type or Pomc(-/-) mice. Thus, POMC peptides seem to be necessary for the normal response of energy balance to high-fat feeding, but not for the acute anorectic effect of PYY(3-36) or full effects of leptin on feeding. The finding that the loss of only one copy of the Pomc gene is sufficient to render mice susceptible to the effects of high fat feeding emphasizes the potential importance of this locus as a site for gene-environment interactions predisposing to obesity.

The cholesterol-lowering drug probucol increases apolipoprotein E production in the hippocampus of aged rats: implications for Alzheimer's disease.

Several recent epidemiological studies have proposed that cholesterol-lowering drug Statin may provide protection against Alzheimer's disease (AD). Probucol is a non-Statin cholesterol-lowering drug and a potent inducer of apolipoprotein E (apoE) production in peripheral circulation. A recent clinical study using Probucol in elderly AD subjects revealed a concomitant stabilisation of cognitive symptoms and significant increases in apoE levels in the cerebral spinal fluid in these patients. To gain insight into the mechanisms underlying these effects, we treated a cohort of aged male rats (26-month-old) with oral dose of Probucol for 30 days. Specifically, we examined the effects of Probucol on apoE production and its receptors (low density lipoprotein receptor [LDLr] and low density lipoprotein receptor-related protein [LRP]), astroglial marker of cell damage (glial fibrillary acidic protein [GFAP]), markers of neuronal synaptic plasticity and integrity (synaptosomal associated protein of 25 kDa [SNAP-25] and synaptophysin) as well as cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase [HMGCoAr]) in the hippocampus. We report that Probucol induces the production of apoE and one of its main receptors, LRP, increases HMGCoAr (rate-limiting enzyme in cholesterol synthesis), substantially attenuates age-related increases in glial activation, and induces production of synaptic marker SNAP-25, a molecule commonly associated with synaptogenesis and dendritic remodeling. These findings suggest that Probucol could promote neural and synaptic plasticity to counteract the synaptic deterioration associated with brain aging through an apoE/LRP-mediated system. Consistent with the beneficial effects of other cholesterol-lowering drugs such as the Statin, Probucol could also offers additional benefits based on apoE neurobiology.

Apolipoprotein E knockout mice display procedural deficits in the Morris water maze: analysis of learning strategies in three versions of the task.

Apolipoprotein E knockout (apoEKO) mice have been shown to be impaired in the spatial Morris water maze (MWM). However, several groups failed to replicate this finding. One reason for this inconsistency may stem from variations in the experimental protocols and environment between laboratories. In the present study, we have tested if age and variations in protocol implementation that specifically affect salience of the visual extramaze cues influence performance and navigational strategies in the MWM. We tested three- and 12-month-old apoEKO and wild type mice in three versions of the MWM differing on the availability of visual extramaze cues: (1) salient cues, (2) diffuse cues, and (3) absence of cues. Our results show that the presence of salient cues enhances acquisition performance of wild type, but not apoEKO mice in the MWM. This effect was restricted to the acquisition phase since apoEKO mice reached a level of performance that was comparable to that of controls toward the end of the task. No significant differences were detected between apoEKO and controls in either the diffuse cues or absence of cues paradigms. Thigmotaxic tendencies were observed in apoEKO mice and correlated high latency scores. Thigmotaxis may have interfered with the initial ability to engage in a proficient navigational strategy. These findings suggest that, in contrast to what has been proposed in the past, apoEKO mice appear not to be impaired in spatial memory per se but are deficient in a procedural component of the MWM. Furthermore, the procedural deficit and corresponding thigmotaxic tendencies of apoEKO mice appeared to increase with age. Taken together, these findings confirm our hypothesis that age and variations in experimental protocols can influence MWM performances.

Central 5-hydroxytryptamine-2A receptor expression in transgenic mice bearing a glucocorticoid receptor antisense.

Transgenic mice bearing a transgene coding for a glucocorticoid receptor antisense mRNA that partially blocks glucocorticoid receptor expression were used to investigate the long-term effect of hypothalamic-pituitary-adrenal dysfunction on brain 5-hydroxytryptamine-2A (5-HT2A) receptor expression. The brain 5-HT2A receptor mRNA levels in transgenic mice were measured by in situ hybridization and compared to those in control mice. We also studied the effect of a 3-week treatment with fluoxetine on brain 5-HT2A receptor expression in the transgenic mice. No difference in 5-HT2A mRNA levels was observed between transgenic and control mice in cortical or striatal regions, and fluoxetine treatment was without effect. No difference in hypothalamic 5-HT2A mRNA levels was observed between transgenic and control mice, while fluoxetine treatment increased these levels in both transgenic as well as in the hypothalamic ventromedial and paraventricular nuclei of control mice. 5-HT2A receptor mRNA levels were similar in hippocampal CA1 and CA2 subregions of control and transgenic, but were lower in the CA3 and CA4 subregions of transgenic mice. Fluoxetine had no effect on 5-HT2A mRNA levels of transgenic mice but reduced control mouse 5-HT2A receptor mRNA levels in the CA3 subregion. These results suggest that impaired glucocorticoid receptor function can affect hippocampal 5-HT2A receptor expression in transgenic mice and that this is not corrected by fluoxetine treatment.

Dopaminergic activity in transgenic mice underexpressing glucocorticoid receptors: effect of antidepressants.

Transgenic mice bearing a transgene coding for a glucocorticoid receptor antisense mRNA, which partially blocks glucocorticoid receptor expression, were used to investigate the long-term effect of hypothalamic-pituitary-adrenal axis dysfunction on brain dopamine transmission. Compared to control mice, the transgenic animals showed increased amphetamine-induced locomotor activity and increased concentrations of striatal dopamine and its metabolites dihydroxyphenylacetic acid and homovanillic acid. Binding of [3H]SCH 23390 and [3H]spiperone to, respectively, D1 and D2 dopamine receptors was increased in transgenic mice. In contrast, autoradiography of striatal [3H]GBR 12935 binding to the dopamine transporter was decreased and the mRNA levels of this transporter, measured by in situ hybridization, remained unchanged in the substantia nigra pars compacta. The effect of chronic treatment for two weeks with amitriptyline or fluoxetine was compared in control and transgenic mice. No significant changes were observed in control mice following antidepressant treatment, whereas in transgenic mice both antidepressants reduced striatal [3H]SCH 23390 and [3H]raclopride specific binding to D1 and D2 receptors. Amitriptyline, but not fluoxetine, increased striatal [3H]GBR 12935 binding to the dopamine transporter, whereas its mRNA level in the substantia nigra pars compacta was decreased in fluoxetine, compared to vehicle- or amitriptyline-treated transgenic mice. From these results we suggest that hyperactive dopaminergic activity of the nigrostriatal pathway controls motor activity in the transgenic mice. Furthermore, antidepressant treatment corrected the increased striatal D1 and D2 receptors and decreased dopamine transporter levels in the transgenic mice.

Antidepressants preferentially enhance habituation to novelty in the olfactory bulbectomized rat.

RATIONALE: The mechanisms whereby antidepressant drugs exert their therapeutic effects remain unknown. Responses to stressful stimuli are currently thought to contribute to the onset and course of affective disorders. It has been postulated that antidepressants might act by ameliorating response patterns to challenging life events, such as processes of reactivity and/or habituation. OBJECTIVE: Using the olfactory bulbectomy (OBX) rat model, this study examined the effects of various antidepressants on measures of reactivity and habituation in behavioral tests assessing responses to novel stimuli. METHODS: Sham-operated and OBX rats received 21 daily injections of fluoxetine (10 mg/kg), amitriptyline (10 mg/kg), desipramine (10 mg/kg), buspirone (3 mg/kg), or vehicle. Forty-eight hours after the last injection, animals were tested in the open field, elevated plus maze, and startle apparatus. For each test, time series data were collected and fit with exponential random effects models, in which estimated parameters assessed behavioral reactivity and habituation. RESULTS: Relative to sham controls, OBX rats displayed increased total locomotor activity in the open field and exhibited increased open arm behavior in the elevated plus maze. Through comparison with zinc sulfate-treated anosmic controls, these OBX-induced increases were attributed to both an augmentation of initial reactivity due to anosmia and an attenuation of the average rate of habituation. Chronic antidepressant treatment did not reduce the anosmia-related initial reactivity levels of OBX rats to that of sham controls. Rather, the antidepressants evoked their restorative effects by increasing the rate of habituation. CONCLUSIONS: These findings suggest that antidepressants restore normal responding by permitting more effective adaptation to novel stimuli.

Insulin and insulin-like growth factor-I induce vascular endothelial growth factor mRNA expression via different signaling pathways.

In this study we have investigated the molecular mechanisms of insulin and insulin-like growth factor-I (IGF-I) action on vascular endothelial growth factor (VEGF) gene expression. Treatment with insulin or IGF-I for 4 h increased the abundance of VEGF mRNA in NIH3T3 fibroblasts expressing either the human insulin receptor (NIH-IR) or the human IGF-I receptor (NIH-IGFR) by 6- and 8-fold, respectively. The same elevated levels of mRNA were maintained after 24 h of stimulation with insulin, whereas IGF-I treatment further increased VEGF mRNA expression to 12-fold after 24 h. Pre-incubation with the phosphatidylinositol 3-kinase inhibitor wortmannin abolished the effect of insulin on VEGF mRNA expression in NIH-IR cells but did not modify the IGF-I-induced VEGF mRNA expression in NIH-IGFR cells. Blocking mitogen-activated protein kinase activation with the MEK inhibitor PD98059 abolished the effect of IGF-I on VEGF mRNA expression in NIH-IGFR cells but had no effect on insulin-induced VEGF mRNA expression in NIH-IR cells. Expression of a constitutively active PKB in NIH-IR cells induced the expression of VEGF mRNA, which was not further modified by insulin treatment. We conclude that VEGF induction by insulin and IGF-I occurs via different signaling pathways, the former involving phosphatidylinositol 3-kinase/protein kinase B and the latter involving MEK/mitogen-activated protein kinase.

Selective mu and delta, but not kappa, opiate receptor antagonists inhibit the habituation of novelty-induced hypoalgesia in the rat.

RATIONALE: There is now extensive evidence demonstrating that exposure to novel stimuli induces hypoalgesia and that this effect habituates over repeated exposure to the stimuli. Moreover, it has been shown that administration of the nonselective opiate receptor antagonist naloxone can attenuate the rate of habituation of novelty-induced hypoalgesia. OBJECTIVES: The present experiments were conducted to determine the relative influence of different opiate receptor subtypes in the attenuation of the habituation of novelty-induced hypoalgesia. METHODS: In experiments 1-3, different groups of male, Wistar rats (275-300 g) were administered vehicle, 0. 5, 1.0 or 2.0-nmol doses of the mu-selective antagonist Cys(2)-Tyr(3)-Orn(5)-Pen(7)-amide (CTOP), the delta-receptor selective antagonist naltrindole, or the kappa-selective antagonist nor-binaltorphimine (nor-BNI). In experiment 4, animals were administered vehicle, 5, 25 or 75-nmol doses of nor-BNI. All injections were delivered to the right lateral ventricle 30 min prior to exposure to a novel hot-plate apparatus (48.5 degrees C), once a day for eight consecutive days. RESULTS: Paw-lick latencies in vehicle-treated animals were long during the initial exposures and declined over repeated tests, suggesting the habituation of novelty-induced hypoalgesia. The rate of habituation was significantly attenuated by administration of 1.0-nmol and 2.0-nmol doses of CTOP, by a 2.0-nmol dose of naltrindole, but was unaffected by all doses of nor-BNI. CONCLUSIONS: These results support the involvement of the mu and delta, but not the kappa, opiate receptor subtypes in the habituation of novelty-induced hypoalgesia.

Cholinergic systems and long-term potentiation in memory-impaired apolipoprotein E-deficient mice.

Impairments in cholinergic neurotransmitter systems of the basal forebrain are a hallmark of Alzheimer's disease pathophysiology. The presence of the epsilon4 allele of apolipoprotein E was recently implicated as a major risk factor in both familial and sporadic Alzheimer's disease. The present study examined the integrity of cholinergic and non-cholinergic systems in apolipoprotein E-deficient, memory-impaired mice. Choline acetyltransferase activity, hippocampal acetylcholine release, nicotinic and muscarinic (M1 and M2) receptor binding sites and acetylcholinesterase cell or terminal density showed no signs of alteration in either three-month or 9.5-month-old apolipoprotein E-deficient mice compared to controls. In contrast, long-term potentiation was found to be markedly reduced in these mice, but increases in the strength of stimulation induced the same level of long-term potentiation as that observed in controls. These alterations did not appear to be the consequence of modifications in the binding properties of glutamatergic receptors (N-methyl-D-aspartate and [RS]-alpha-amino-3-hydroxy-5-methylisoxazole propionic acid) but from defective regulation of the (RS)-alpha-amino-3-hydroxy-5-methylisoxazole propionic acid receptor by phospholipase A2 activity. These results support the notion that apolipoprotein E plays a fundamental role in neuronal plasticity, which could in turn affect cognitive performance through imbalances in extra- and intracellular lipid homeostasis.

Lactation-induced reduction in rats' acoustic startle is associated with changes in noradrenergic neurotransmission.

The acoustic startle response (ASR) with or without fear conditioning was compared between cycling (CYC) and lactating (LACT) female rats. ASR sensitivity to changes in endogenous noradrenergic (NA) release was examined using the alpha-2 NA receptor drugs yohimbine and clonidine. Groups of CYC and LACT females were also tested in the open field. ASR was reduced in all LACT, compared with that in CYC females. Both groups exhibited a robust response to fear conditioning and unpotentiated ASR subsequent to conditioning was increased in LACT females. The lowest dose of yohimbine significantly increased ASR in LACT females, but not in CYC females. Clonidine reduced ASR in both groups of females, with a greater potency in CYC females. In the open field, LACT females displayed a shorter latency to emerge, less freezing behavior, and more entries into the field than did CYC females. The authors concluded that (a) LACT females are less anxious in a novel environment and that decreased anxiety can be efficiently counteracted by fear conditioning, and (b) changes in NA neurotransmission contribute to lactation-induced modifications in ASR.

Control of glycogen synthesis in cultured human muscle cells.

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the alpha isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70(S6k) activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase.