ABSTRACT
BACKGROUND: The Ras/Raf/ERK1/2 signaling pathway controls many cellular responses such as cell proliferation, migration, differentiation, and death. In the nervous system, emerging evidence also points to a death-promoting role for ERK1/2 in both in vitro and in vivo models of neuronal death. To further investigate how Ras/Raf/ERK1/2 up-regulation may lead to the development of spinal cord injury, we developed a cellular model of Raf/ERK up-regulation by overexpressing c-Raf in cultured spinal cord neurons (SCNs) and dorsal root ganglions (DRGs). METHODS: DRGs and SCNs were prepared from C57BL/6J mouse pups. DRGs or SCNs were infected with Ad-Raf-1 or Ad-Null adenovirus alone. Cell adhesion assay and cell migration assay were investigated, DiI labeling was employed to examine the effect of the up-regulation of Ras/Raf/ERK1/2 signaling on the dendritic formation of spinal neurons. We used the TO-PRO-3 staining to examine the apoptotic effect of c-Raf on DRGs or SCNs. The effect on the synapse formation of neurons was measured by using immunofluorescence. RESULTS: We found that Raf/ERK up-regulation stimulates the migration of both SCNs and DRGs, and impairs the formation of excitatory synapses in SCNs. In addition, we found that Raf/ERK up-regulation inhibits the development of mature dendritic spines in SCNs. Investigating the possible mechanisms through which Raf/ERK up-regulation affects the excitatory synapse formation and dendritic spine development, we discovered that Raf/ERK up-regulation suppresses the development and maturation of SCNs. CONCLUSION: The up-regulation of the Raf/ERK signaling pathway may contribute to the pathogenesis of spinal cord injury through both its impairment of the SCN development and causing neural circuit imbalances.
Subject(s)
Dendritic Spines/physiology , Neurogenesis/physiology , Signal Transduction/physiology , Spinal Cord/cytology , Synapses/physiology , raf Kinases/metabolism , ras Proteins/metabolism , Animals , Cell Movement/physiology , Dendritic Spines/metabolism , Female , Ganglia, Spinal/cytology , MAP Kinase Signaling System/physiology , Mice , Neurogenesis/genetics , Neurons/cytology , Pregnancy , Signal Transduction/genetics , Synapses/metabolism , Up-Regulation , raf Kinases/genetics , ras Proteins/geneticsABSTRACT
<p><b>OBJECTIVE</b>To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus, and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS.</p><p><b>METHODS</b>SARS-associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT-nested-PCR) and cloned into pBAD/Thio-TOPO prokaryotic expression vector. The recombinant N fusion protein was expressed and purified, and its antigenicity and specificity was analyzed by Western Blot, to establish the recombinant N protein-based ELISA for detection of IgG antibodies to SARS-associated coronavirus, and SARS-associated coronavirus lysates-based ELISA was compared parallelly.</p><p><b>RESULTS</b>The recombinant expression vector produced high level of the N fusion protein after induction, and that protein was purified successfully by affinity chromatography and displayed higher antigenicity and specificity as compared with whole virus lysates.</p><p><b>CONCLUSION</b>The recombinant SARS-associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new, sensitive, and specific ELISA for SARS diagnosis.</p>
Subject(s)
Humans , Antibodies, Viral , Blood , Blotting, Western , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Gene Expression , Genome, Viral , Immunoglobulin G , Blood , Nucleocapsid Proteins , Genetics , Allergy and Immunology , Metabolism , RNA, Viral , Genetics , Recombinant Proteins , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus , Genetics , Allergy and Immunology , Metabolism , Severe Acute Respiratory Syndrome , Blood , Diagnosis , VirologyABSTRACT
<p><b>OBJECTIVE</b>Angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor of severe acute respiratory syndrome coronavirus (SARS-CoV), so its gene was cloned and eukaryotic expressed for further insight into mechanisms in SARS-CoV entry and pathogenesis, as well as development of a safe and reliable neutralization assay for SARS-CoV.</p><p><b>METHODS</b>Total RNA was extracted from right atrial tissue of a patient with right heart failure resected during a valvular replacement surgery by Trizol one-step method, and the full-length ACE-2 encoding gene was acquired by RT-nested-PCR. The ACE-2 encoding gene was then cloned into pcDNA4/HisMax-TOPO eukaryotic expression vector to construct the recombinant plasmid pcDNA4/ ACE-2, which was then transfected into 293 T cell and ACE-2 eukaryotic transient expression was detected by Western Blot. Syncytia inhibition assay was established to detect SARS-CoV neutralizing antibody, and compared parallelly with SARS pseudovirus neutralization assay.</p><p><b>RESULTS</b>The recombinant plasmid pcDNA4/ ACE-2 could express ACE-2 protein in eukaryotic cells and induce cell-cell fusion between S protein- and ACE2-expressing cells. This cell-cell fusion assay could be used to detect SARS-CoV neutralizing antibody.</p><p><b>CONCLUSION</b>SARS-CoV receptor ACE-2 gene was successfully cloned and eukaryotic expressed, and used to establish syncytia inhibition assay for SARS-CoV neutralizing antibody assay.</p>
