Interaction of cadmium with actin microfilaments in vitro

Dı́az-Barriga, Fernando; Carrizales, Leticia; Yáñez, Leticia; Hernández, José Manuel; Robles, M.C. Domínguez; Palmer, E; Saborío, José L.

doi:10.1016/0887-2333(89)90034-9

Cited by 35 publications

(16 citation statements)

References 49 publications

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“…The appearance of a single protein band of 75kDa in the nuclear and cytoplasmic fractions using the anti-peptide EhDNApol A antibody indicates that a population of EhDNApolA is translocated from the cytoplasm into the nucleus (Figure 7B, lanes 1 and 2). The same patter is observed with the anti-actin antibody, as actin is a protein with cytoplasmic and nuclear localization [30]. Because nuclear fractions are often contaminated with cytosolic fractions, we used the identification of C/EBPβ, as a control of the nuclear extract purification protocol.…”

Section: Resultsmentioning

confidence: 68%

“…Protein extracts were separated using a 15% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membranes were incubated with a 1 to 2000 dilution of the purified immune sera and an anti-actin antibody [30] in 1% nonfat dry milk, 0.05% Tween-20 in PBS 7.4 for 2 hours. The reactivity was detected using peroxidase conjugated secondary antibodies (1 to 2000 dilution) with the ECL Plus detection kit (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

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A Nuclear Family A DNA Polymerase from Entamoeba histolytica Bypasses Thymine Glycol

2010

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BackgroundEukaryotic family A DNA polymerases are involved in mitochondrial DNA replication or translesion DNA synthesis. Here, we present evidence that the sole family A DNA polymerase from the parasite protozoan E. histolytica (EhDNApolA) localizes to the nucleus and that its biochemical properties indicate that this DNA polymerase may be involved in translesion DNA synthesis.Methodology and ResultsEhDNApolA is the sole family A DNA polymerase in E. histolytica. An in silico analysis places family A DNA polymerases from the genus Entamoeba in a separate branch of a family A DNA polymerases phylogenetic tree. Biochemical studies of a purified recombinant EhDNApolA demonstrated that this polymerase is active in primer elongation, is poorly processive, displays moderate strand displacement, and does not contain 3′–5′ exonuclease or editing activity. Importantly, EhDNApolA bypasses thymine glycol lesions with high fidelity, and confocal microscopy demonstrates that this polymerase is translocated into the nucleus. These data suggest a putative role of EhDNApolA in translesion DNA synthesis in E. histolytica.ConclusionThis is the first report of the biochemical characterization of a DNA polymerase from E. histolytica. EhDNApolA is a family A DNA polymerase that is grouped into a new subfamily of DNA polymerases with translesion DNA synthesis capabilities similar to DNA polymerases from subfamily ν.

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Section: Resultsmentioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

A Nuclear Family A DNA Polymerase from Entamoeba histolytica Bypasses Thymine Glycol

2010

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“…Antibodies directed to a1-, a2-and b-dystrobrevin (bCT-FP) [23] and Dp71 (2166) [24] were provided by Dr. Dereck Blake. Antibody antiactin was supplied by Dr. Manuel Hernández [25]. The polyclonal antibodies against Dp71 (DPC20, used for the immunoprecipitation assays) and a1-syntrophin (N-19, used for Western blotting assays) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).…”

Section: Antibodiesmentioning

confidence: 99%

Dystrophin Dp71 is Critical for Stability of the DAPs in the Nucleus of PC12 Cells

et al. 2009

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We have adopted the PC12 cell line as in vitro cell model for studying Dp71 function in neuronal cells. These cells express a cytoplasmic (Dp71f) and a nuclear (Dp71d) isoform of Dp71 as well as various dystrophin-associated proteins (DAPs). In this study, we revealed by confocal microscopy analysis and Western blotting evaluation of cell fractions the presence of different DAPs (beta-dystroglycan, beta-dystrobrevin, epsilon-sarcoglycan and gamma1-syntrophin) in the nucleus of PC12 cells. Furthermore, we established by immunoprecipitation assays that Dp71d and the DAPs form a dystrophin-associated protein complex (DAPC) in the nucleus. Interestingly, depletion of Dp71 by antisense treatment (antisense-Dp71 cells) provoked a drastic reduction of nuclear DAPs, which indicates that Dp71d is critical for DAPs stability within the nucleus. Although Up71, the utrophin gene product homologous to Dp71, exhibited increased expression in the antisense-Dp71 cells, its scarce nuclear levels makes unlikely that could compensate for Dp71 nuclear deficiency.

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“…The protein pellets were resuspended in 1% sodium dodecyl sulfate (SDS) and Western analyses were performed using 100 g of protein per lane with the TH monoclonal antibody described above. Membranes were stripped and incubated with a monoclonal antibody against actin (Diaz-Barriga et al, 1989). Presence of proteins was revealed using the enhanced chemoluminescence (ECL TM ) detection system (Amersham, Arlington Heights, IL) as previously described (Segovia et al, 1998b;Trejo et al, 1999a).…”

Section: Western Blot Analysismentioning

confidence: 99%

Primary astrocytes retrovirally transduced with a tyrosine hydroxylase transgene driven by a glial-specific promoter elicit behavioral recovery in experimental Parkinsonism

Cortez

Trejo

Vergara

et al. 2000

Journal of Neuroscience Research

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We used a retroviral-mediated gene transfer system to transduce primary rat astrocytes with a transgene in which the activity of a tyrosine hydroxylase (TH) cDNA is under the transcriptional control of a human promoter of the glial fibrillary acidic protein (GFAP). The engineered cells were tested for their therapeutic efficacy in a rodent model of Parkinson's disease (PD). The method is based both on the properties of astrocytes, as well as on those of the promoter. Astrocytes are an integral part of the neural tissue, have a long life span, are more resistant to oxidative stress than neurons, and possess an efficient secretory system. The GFAP promoter is active throughout postnatal life, and its activity is up-regulated by many insults to the brain, including PD. Transduced astrocytes were implanted into the striata of rats lesioned with 6-hydroxydopamine (6-OHDA), and the efficacy of grafted cells tested. Implanted astrocytes induced a significant reduction in the turning behavior that occurs in response to apomorphine for at least 4 weeks after grafting, and transgenic mRNA and protein could be detected in implanted brains. These results indicate that the gFa2-TH construct can be readily adapted to be used with a retroviral gene transfer system to obtain nontumorigenic cells that sustain a sufficient level of transgene activity to enable therapeutic effectiveness for prolonged periods. These results further endorse the use of astrocytes for gene therapy in the central nervous system.

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Interaction of cadmium with actin microfilaments in vitro

Cited by 35 publications

References 49 publications

A Nuclear Family A DNA Polymerase from Entamoeba histolytica Bypasses Thymine Glycol

A Nuclear Family A DNA Polymerase from Entamoeba histolytica Bypasses Thymine Glycol

Dystrophin Dp71 is Critical for Stability of the DAPs in the Nucleus of PC12 Cells

Primary astrocytes retrovirally transduced with a tyrosine hydroxylase transgene driven by a glial-specific promoter elicit behavioral recovery in experimental Parkinsonism

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