Development of a Competitive Double Antibody Radioimmunoassay for Rat Metallothionein

Gasull, Teresa; Dv, Rebollo; Romero, Beatriz; Hidalgo, Juan

doi:10.1080/15321819308019851

Cited by 67 publications

(37 citation statements)

References 20 publications

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“…With one of the hemispheres, serial sagittal sections (20 m in thickness) were obtained with a cryostat and mounted on slides coated with poly(L)lysine, where an in situ hybridization analysis of the MT-I and MT-III isoforms was carried out as previously described (11). With the other hemisphere, the cerebellum, hippocampus, medulla ϩ pons, and remaining brain were dissected, homogenized, and centrifuged, and MT-I ϩ II protein levels were measured by radioimmunoassay (24,30).…”

Section: In Situ Hybridization and Radioimmunoassaymentioning

confidence: 99%

Metallothioneins Are Upregulated in Symptomatic Mice with Astrocyte-Targeted Expression of Tumor Necrosis Factor-α

Carrasco

Giralt

Penkowa

et al. 2000

Experimental Neurology

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Transgenic mice expressing TNF-␣ under the regulatory control of the GFAP gene promoter (GFAP-TNF␣ mice) exhibit a unique, late-onset chronic-progressive neurological disorder with meningoencephalomyelitis, neurodegeneration, and demyelination with paralysis. Here we show that the metallothionein-I ؉ II (MT-I ؉ II) isoforms were dramatically upregulated in the brain of symptomatic but not presymptomatic GFAP-TNF␣ mice despite TNF-␣ expression being present in both cases. In situ hybridization analysis for MT-I RNA and radioimmunoassay results for MT-I ؉ II protein revealed that the induction was observed in the cerebellum but not in other brain areas. Increased MT-I RNA levels occurred in the Purkinje and granular neuronal layers of the cerebellum but also in the molecular layer. Reactive astrocytes, activated rod-like microglia, and macrophages, but not the infiltrating lymphocytes, were identified as the cellular sources of the MT-I ؉ II proteins. In situ hybridization for MT-III RNA revealed a modest increase in the white matter of the cerebellum, which was confirmed by immunocytochemistry. MT-III immunoreactivity was present in cells which were mainly round or amoeboid monocytes/macrophages. The pattern of expression of the different MT isoforms in the GFAP-TNF␣ mice differed substantially from that described previously in GFAP-IL6 mice, demonstrating unique effects associated with the expression of each cytokine. The results suggest that the MT expression in the CNS reflects the inflammatory response and associated damage rather than a direct role of the TNF-␣ in their regulation and support a major role of these proteins during CNS injury.

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Section: In Situ Hybridization and Radioimmunoassaymentioning

confidence: 99%

Metallothioneins Are Upregulated in Symptomatic Mice with Astrocyte-Targeted Expression of Tumor Necrosis Factor-α

Carrasco

Giralt

Penkowa

et al. 2000

Experimental Neurology

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“…To determine metallothionein quantities in adipocytes, cytoplasmic proteins from human adipocytes were prepared in Tris-HCl (20 mmol/l, pH 8) buffer. Metallothionein measurement was realized by a radioimmunoassay (17). GyK activity and glycerol incorporation into triglycerides were assessed according to Noel et al (18) and Guan et al (12), respectively.…”

Section: Methodsmentioning

confidence: 99%

The Transcriptional Coactivator Peroxisome Proliferator–Activated Receptor (PPAR)γ Coactivator-1α and the Nuclear Receptor PPARα Control the Expression of Glycerol Kinase and Metabolism Genes Independently of PPARγ Activation in Human White Adipocytes

et al. 2007

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OBJECTIVE—The purpose of this work was to determine the pattern of genes regulated by peroxisome proliferator–activated receptor (PPAR) γ coactivator 1α (PGC-1α) in human adipocytes and the involvement of PPARα and PPARγ in PGC-1α transcriptional action. RESEARCH DESIGN AND METHODS—Primary cultures of human adipocytes were transduced with a PGC-1α adenovirus and treated with PPARγ and PPARα agonists. Variation in gene expression was assessed using pangenomic microarrays and quantitative RT-PCR. To investigate glycerol kinase (GyK), a target of PGC-1α, we measured enzymatic activity and glycerol incorporation into triglycerides. In vivo studies were performed on wild-type and PPARα−/− mice. The GyK promoter was studied using chromatin immunoprecipitation and promoter reporter gene assays. RESULTS—Among the large number of genes regulated by PGC-1α independently of PPARγ, new targets involved in metabolism included the gene encoding GyK. The induction of GyK by PGC-1α was observed at the levels of mRNA, enzymatic activity, and glycerol incorporation into triglycerides. PPARα was also upregulated by PGC-1α. Its activation led to an increase in GyK expression and activity. PPARα was shown to bind and activate the GyK promoter. Experiments in mice confirmed the role of PGC-1α and PPARα in the regulation of GyK in vivo. CONCLUSIONS—This work uncovers novel pathways regulated by PGC-1α and reveals that PPARα controls gene expression in human white adipocytes. The induction of GyK by PGC-1α and PPARα may promote a futile cycle of triglyceride hydrolysis and fatty acid reesterification.

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“…Confluent astrocytes cultures were maintained in a total of 500 l of culture medium and treated with either 10 units/ml IL-1␣ (Chemicon catalog number IL001), 10 M ZnSO 4 , or 10 units/ml IL-1␣ ϩ 10 M ZnSO 4 combined for the allotted time period, and media were collected, centrifuged at 13,000 ϫ g (5 min), and stored at Ϫ20°C. MT-I/-II was measured by radioimmunoassay as described previously (16).…”

Section: Methodsmentioning

confidence: 99%

“…Cortical Astrocytes-To test the hypothesis that astrocytes are capable of releasing MT-I/-II, primary cortical astrocytes were maintained in vitro, and the presence of MT-I/-II in the culture media was assessed using an MT-I/-II-specific radioimmunoassay (RIA) that is capable of detecting picogram amounts (16,17). In confluent astrocyte cultures, MT-I/-II was detected in the medium at a range of 15.6 Ϯ 0.49 pg of MT/g of total protein (average over 12 different experiments).…”

Section: Detection Of Extracellular Mt-i/-ii Released From Culturedmentioning

confidence: 99%

Redefining the Role of Metallothionein within the Injured Brain

Chung

Penkowa

Dittmann

et al. 2008

Journal of Biological Chemistry

130

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A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

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Development of a Competitive Double Antibody Radioimmunoassay for Rat Metallothionein

Cited by 67 publications

References 20 publications

Metallothioneins Are Upregulated in Symptomatic Mice with Astrocyte-Targeted Expression of Tumor Necrosis Factor-α

Metallothioneins Are Upregulated in Symptomatic Mice with Astrocyte-Targeted Expression of Tumor Necrosis Factor-α

The Transcriptional Coactivator Peroxisome Proliferator–Activated Receptor (PPAR)γ Coactivator-1α and the Nuclear Receptor PPARα Control the Expression of Glycerol Kinase and Metabolism Genes Independently of PPARγ Activation in Human White Adipocytes

Redefining the Role of Metallothionein within the Injured Brain

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