59 research outputs found
Detection of Peptide-Based Nanoparticles in Blood Plasma by ELISA
Aims The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. Materials & Methods A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. Results The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. Conclusions We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions
Ceramide Analog [\u3csup\u3e18\u3c/sup\u3eF]F-HPA-12 Detects Sphingolipid Disbalance in the Brain of Alzheimer’s Disease Transgenic Mice by Functioning as a Metabolic Probe
The metabolism of ceramides is deregulated in the brain of Alzheimer’s disease (AD) patients and is associated with apolipoprotein (APO) APOE4 and amyloid-β pathology. However, how the ceramide metabolism changes over time in AD, in vivo, remains unknown. Distribution and metabolism of [18F]F-HPA-12, a radio-fluorinated version of the ceramide analog N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide, was investigated in the brain of AD transgenic mouse models (FAD) on an APOE4 or APOE3 genetic background, by positron emission tomography and by gamma counter. We found that FAD mice displayed a higher uptake of [18F]F-HPA-12 in the brain, independently from the APOE4 or APOE3 genetic background. FAD mice could be distinguished from littermate control animals with a sensitivity of 85.7% and a specificity of 87.5%, by gamma counter measurements. Metabolic analysis of [18F]F-HPA-12 in the brain suggested that the tracer is degraded less efficiently in the FAD mice. Furthermore, the radioactive signal registered in the hippocampus correlated with an increase of Cer d18:1/20:2 levels measured in the same brain region by mass spectrometry. Our data gives additional proof that ceramide metabolism is different in FAD mice compared to controls. Ceramide analogs like HPA-12 may function as metabolic probes to study ceramide disbalance in the brain
IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients
Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies are pathogenic. publisher: Elsevier articletitle: IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients journaltitle: Journal of Autoimmunity articlelink: http://dx.doi.org/10.1016/j.jaut.2016.11.005 content_type: article copyright: © 2016 The Authors. Published by Elsevier Ltd. ispartof: Journal of Autoimmunity vol:77 pages:104-115 ispartof: location:England status: publishe
CERT\u3csub\u3eL\u3c/sub\u3e Reduces C16 Ceramide, Amyloid-β Levels, and Inflammation in a Model of Alzheimer’s Disease
BACKGROUND: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer\u27s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain.
METHODS: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay.
RESULTS: Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype.
CONCLUSION: Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases
Ceramide analog [18F]F-HPA-12 detects sphingolipid disbalance in the brain of Alzheimer’s disease transgenic mice by functioning as a metabolic probe
Ceramide analog [18F]F-HPA-12 detects sphingolipid disbalance in the brain of Alzheimer’s disease transgenic mice by functioning as a metabolic probe
The metabolism of ceramides is deregulated in the brain of Alzheimer's disease (AD) patients and is associated with apolipoprotein (APO) APOE4 and amyloid-β pathology. However, how the ceramide metabolism changes over time in AD, in vivo, remains unknown. Distribution and metabolism of [18F]F-HPA-12, a radio-fluorinated version of the ceramide analog N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide, was investigated in the brain of AD transgenic mouse models (FAD) on an APOE4 or APOE3 genetic background, by positron emission tomography and by gamma counter. We found that FAD mice displayed a higher uptake of [18F]F-HPA-12 in the brain, independently from the APOE4 or APOE3 genetic background. FAD mice could be distinguished from littermate control animals with a sensitivity of 85.7% and a specificity of 87.5%, by gamma counter measurements. Metabolic analysis of [18F]F-HPA-12 in the brain suggested that the tracer is degraded less efficiently in the FAD mice. Furthermore, the radioactive signal registered in the hippocampus correlated with an increase of Cer d18:1/20:2 levels measured in the same brain region by mass spectrometry. Our data gives additional proof that ceramide metabolism is different in FAD mice compared to controls. Ceramide analogs like HPA-12 may function as metabolic probes to study ceramide disbalance in the brain
The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria
Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, 14C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (PÂ =Â 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation
Paradoxical Increase in TAG and DAG Content Parallel the Insulin Sensitizing Effect of Unilateral DGAT1 Overexpression in Rat Skeletal Muscle
BACKGROUND: The involvement of muscle triacylglycerol (TAG) storage in the onset of insulin resistance is questioned and the attention has shifted towards inhibition of insulin signalling by the lipid intermediate diacylglycerol (DAG). The enzyme 1,2-acylCoA:diacylglyceroltransferase-1 (DGAT1) esterifies a fatty acyl-CoA on DAG to form TAG. Therefore, the aim of the present study was to investigate if unilateral overexpression of DGAT1 in adult rat Tibialis anterior (TA) muscle will increase conversion of the lipid intermediate DAG into TAG, thereby improving muscle insulin sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: The DGAT1 gene construct was injected in the left TA muscle of male rats on chow or high-fat (45% kcal) diet for three weeks, followed by application of one 800 V/cm and four 80 V/cm pulses, using the contralateral leg as sham-electroporated control. Seven days after electroporation, muscle specific insulin sensitivity was assessed with a hyperinsulinemic euglycemic clamp using 2-deoxy-[3H]glucose. Here, we provide evidence that unilateral overexpression of DGAT1 in TA muscle of male rats is associated with an increased rather than decreased DAG content. Strikingly, this increase in DAG content was accompanied by improved muscle insulin sensitivity. Interestingly, markers of muscle lipolysis and mitochondrial function were also increased in DGAT1 overexpressing muscle. CONCLUSIONS/SIGNIFICANCE: We conclude that unilateral DGAT1 overexpression can rescue insulin sensitivity, possibly by increasing DAG and TAG turnover in skeletal muscle. In case of a proper balance between the supply and oxidation of fatty acids in skeletal muscle, the lipid intermediate DAG may not exert harmful effects on insulin signalling
CERTL reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease
Background: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. Methods: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. Results: Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. Conclusion: Our results demonstrate a crucial role of CERTL in r
Quasi-continuous parallel online scattered light, fluorescence and dissolved oxygen tension measurement combined with monitoring of the oxygen transfer rate in each well of a shaken microtiter plate
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