Controllable DNA Condensation-Release Induced by Simple Azaheterocyclic-Based Metal Complexes

The condensation of DNA is essential for biological processes such as DNA transcription and replication, and its study receives additional impetus from an interest in gene therapy. Although many efficacious condensing agents have been discovered and investigated, little is known about the conversation of condensation-release under suitable conditions. A novel class of DNA condensing agents based on small azaheterocyclic metal-binding molecules has been discovered and described. Both linear and plasmid DNA can be condensed to nanoparticles by the title compounds with 50 °C incubation, especially in the presence of divalent metal ions. Importantly, this condensation may be released to original forms with little or no damage to the DNA under incubation at physiological temperatures. These changes in DNA morphology over time have been analyzed by gel electrophoresis, circular dichroism (CD), and atomic force microscopy (AFM). The present work might help to develop strategies for the design and synthesis of controllable condensing agents, which may also be applied to control gene expression and delivery

Table_1_mTOR Signaling Pathway Regulates the Release of Proinflammatory Molecule CCL5 Implicated in the Pathogenesis of Autism Spectrum Disorder.xlsx

Autism spectrum disorder (ASD) is a complex pervasive neurodevelopmental disorder and neuroinflammation may contribute to the pathogenesis of ASD. However, the exact mechanisms of abnormal release of proinflammatory mediators in ASD remain poorly understood. This study reports elevated plasma levels of the proinflammatory chemokine (C-C motif) ligand 5 (CCL5) in children with ASD, suggesting an aberrant inflammatory response appearing in the development of ASD. Mining of the expression data of brain or blood tissue from individuals with ASD reveals that mTOR signaling is aberrantly activated in ASD patients. Our in vitro study shows that suppression of mTOR reduces the gene expression and release of CCL5 from human microglia, supporting that CCL5 expression is regulated by mTOR activity. Furthermore, bacterial lipopolysaccharide (LPS)-induced CCL5 expression can be counteracted by siRNA against NF-κB, suggests a determining role of NF-κB in upregulating CCL5 expression. However, a direct regulatory relationship between the NF-κB element and the mTOR signaling pathway was not observed in rapamycin-treated cells. Our results show that the phosphorylated CREB can be induced to suppress CCL5 expression by outcompeting NF-κB in binding to CREB-binding protein (CREBBP) once the mTOR signaling pathway is inhibited. We propose that the activation of mTOR signaling in ASD may induce the suppression of phosphorylation of CREB, which in turn results in the increased binding of CREBBP to NF-κB, a competitor of phosphorylated CREB to drive expression of CCL5. Our study sheds new light on the inflammatory mechanisms of ASD and paves the way for the development of therapeutic strategy for ASD.</p

Image_1_mTOR Signaling Pathway Regulates the Release of Proinflammatory Molecule CCL5 Implicated in the Pathogenesis of Autism Spectrum Disorder.tif

Autism spectrum disorder (ASD) is a complex pervasive neurodevelopmental disorder and neuroinflammation may contribute to the pathogenesis of ASD. However, the exact mechanisms of abnormal release of proinflammatory mediators in ASD remain poorly understood. This study reports elevated plasma levels of the proinflammatory chemokine (C-C motif) ligand 5 (CCL5) in children with ASD, suggesting an aberrant inflammatory response appearing in the development of ASD. Mining of the expression data of brain or blood tissue from individuals with ASD reveals that mTOR signaling is aberrantly activated in ASD patients. Our in vitro study shows that suppression of mTOR reduces the gene expression and release of CCL5 from human microglia, supporting that CCL5 expression is regulated by mTOR activity. Furthermore, bacterial lipopolysaccharide (LPS)-induced CCL5 expression can be counteracted by siRNA against NF-κB, suggests a determining role of NF-κB in upregulating CCL5 expression. However, a direct regulatory relationship between the NF-κB element and the mTOR signaling pathway was not observed in rapamycin-treated cells. Our results show that the phosphorylated CREB can be induced to suppress CCL5 expression by outcompeting NF-κB in binding to CREB-binding protein (CREBBP) once the mTOR signaling pathway is inhibited. We propose that the activation of mTOR signaling in ASD may induce the suppression of phosphorylation of CREB, which in turn results in the increased binding of CREBBP to NF-κB, a competitor of phosphorylated CREB to drive expression of CCL5. Our study sheds new light on the inflammatory mechanisms of ASD and paves the way for the development of therapeutic strategy for ASD.</p

Image_2_mTOR Signaling Pathway Regulates the Release of Proinflammatory Molecule CCL5 Implicated in the Pathogenesis of Autism Spectrum Disorder.tif

Autism spectrum disorder (ASD) is a complex pervasive neurodevelopmental disorder and neuroinflammation may contribute to the pathogenesis of ASD. However, the exact mechanisms of abnormal release of proinflammatory mediators in ASD remain poorly understood. This study reports elevated plasma levels of the proinflammatory chemokine (C-C motif) ligand 5 (CCL5) in children with ASD, suggesting an aberrant inflammatory response appearing in the development of ASD. Mining of the expression data of brain or blood tissue from individuals with ASD reveals that mTOR signaling is aberrantly activated in ASD patients. Our in vitro study shows that suppression of mTOR reduces the gene expression and release of CCL5 from human microglia, supporting that CCL5 expression is regulated by mTOR activity. Furthermore, bacterial lipopolysaccharide (LPS)-induced CCL5 expression can be counteracted by siRNA against NF-κB, suggests a determining role of NF-κB in upregulating CCL5 expression. However, a direct regulatory relationship between the NF-κB element and the mTOR signaling pathway was not observed in rapamycin-treated cells. Our results show that the phosphorylated CREB can be induced to suppress CCL5 expression by outcompeting NF-κB in binding to CREB-binding protein (CREBBP) once the mTOR signaling pathway is inhibited. We propose that the activation of mTOR signaling in ASD may induce the suppression of phosphorylation of CREB, which in turn results in the increased binding of CREBBP to NF-κB, a competitor of phosphorylated CREB to drive expression of CCL5. Our study sheds new light on the inflammatory mechanisms of ASD and paves the way for the development of therapeutic strategy for ASD.</p

Subcellular distribution of copine1 and its calcium-dependent association with membrane

NTM and GTM cells either untreated or treated with A23187 were fractionated into a total membrane fraction (Mem) and a whole cell lysate (Cyt) as described in the Methods section. Following SDS–PAGE and western blot, copine1 was detected using a mouse monoclonal copine1 antibody. Equal amounts of protein were loaded in each lane, and the protein content was determined using the Bradford method (Bio-Rad) and BSA as the standard. For the A23187-treated cells, Ca and A23187 were incubated with TM cells for 30 min before cell collection. NTM: normal TM cells; GTM: glaucomatous TM cells. Data from the three experiments were averaged and graphed with error bars representing standard errors.<p><b>Copyright information:</b></p><p>Taken from "Upregulation of Copine1 in trabecular meshwork cells of POAG patients: a membrane proteomics approach"</p><p></p><p>Molecular Vision 2008;14():1028-1036.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2408776.</p><p></p

Confirmation of mRNA expression in trabecular meshwork cells by real-time polymerase chain reaction using a relative quantification protocol

mRNA expression in untreated and A23187-treated TM cells was examined using real-time PCR. The comparative C method was employed, and the change in gene expression is expressed as fold change in relation to every control. For the A23187-treated cells, Ca and A23187 were incubated with TM cells for 30 min before cell collection. NTM: normal TM cells; GTM: glaucomatous TM cells. Data are means±SEM of four independent experiments. The asterisk indicates a p<p><b>Copyright information:</b></p><p>Taken from "Upregulation of Copine1 in trabecular meshwork cells of POAG patients: a membrane proteomics approach"</p><p></p><p>Molecular Vision 2008;14():1028-1036.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2408776.</p><p></p

Immunohistochemical characterization of primary trabecular meshwork cell cultures

Immunohistochemical assays on the TM cells were positive for fibronectin (), laminin (), vimentin (), neuronal specific enolase () and negative for the endothelial cell marker, factor VIII (), which indicated that the primary cultures obtained from normal and POAG individuals were indeed TM cells.<p><b>Copyright information:</b></p><p>Taken from "Upregulation of Copine1 in trabecular meshwork cells of POAG patients: a membrane proteomics approach"</p><p></p><p>Molecular Vision 2008;14():1028-1036.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2408776.</p><p></p

Western blot analysis using monoclonal antibodies against copine1 and β-actin on the traditional one-dimensional SDS–PAGE of trabecular meshwork cell total proteins

Expressions of untreated and A23187-treated copine1 were analyzed using the western blot method. The expression of β-actin was used as a control. The expression levels were quantitated by measuring band intensities and were expressed as fold induction of that in controls. NTM: normal TM cells; GTM: glaucomatous TM cells. Data are means±SEM of three independent experiments. The asterisk indicates a p<p><b>Copyright information:</b></p><p>Taken from "Upregulation of Copine1 in trabecular meshwork cells of POAG patients: a membrane proteomics approach"</p><p></p><p>Molecular Vision 2008;14():1028-1036.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2408776.</p><p></p

Two-dimensional electrophoresis comparison between NTM and GTM membrane protein extracts

Silver-stained 2D gels of membrane protein extracts from NTM () and GTM () are shown. Differentially abundant protein spots are highlighted in the red frames. Spot 1 was identified as copine1 (MW 58.6 kDa, pI 6.43). The expanded regions of differentially expressed copine1 were cut from the representative gels and pasted in the top left corner of the image. NTM: normal TM cells; GTM: glaucomatous TM cells.<p><b>Copyright information:</b></p><p>Taken from "Upregulation of Copine1 in trabecular meshwork cells of POAG patients: a membrane proteomics approach"</p><p></p><p>Molecular Vision 2008;14():1028-1036.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2408776.</p><p></p

Detection of the Pro370Leu mutation by direct polymerase chain reaction DNA sequencing in the GZ

1 pedigree. Representative chromatogram contains sequence from the noncoding DNA strand. The location of the mutation within and the nature of the nucleotide change (C->T ) were shown as a double peak in the heterozygous condition.<p><b>Copyright information:</b></p><p>Taken from "Pro370Leu gene mutation in a large Chinese family with juvenile-onset open angle glaucoma: correlation between genotype and phenotype"</p><p></p><p>Molecular Vision 2008;14():1533-1539.</p><p>Published online 22 Aug 2008</p><p>PMCID:PMC2518531.</p><p></p