Objective To screen the proteins associated with four-and-a-half LIM domains 3 (FHL3) 3’ untranslated region (3’UTR) in glioma cells.
Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2 (PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3’UTR. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immuno-precipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1 (PTBP1), a binding protein identified by LC-MS/MS.
Results PCBP2 could bind to FHL3 mRNA 3’UTR-A and inhibited the expression of FHL3 in T98G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3’UTR and interacted with PCBP2 protein.
Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3’UTR through forming a protein complex.

OBJECTIVETo study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.

METHODSSeries of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.

RESULTSEach fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.

CONCLUSIONDok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Neurites ; drug effects ; physiology ; Neurotrophin 3 ; pharmacology ; PC12 Cells ; Rats ; Receptor, trkC ; metabolism ; Transfection

OBJECTIVETo study the interaction between ShcD and TrkC and to reveal the molecular mechanism of the downstream signal transduction of TrkC.

METHODSYeast two-hybrid assay was used. The intracellular domains of TrkC and TrkC mutants were cloned into pAS2-1, and ShcD and its four domains (CH2, PTB, CH1, and SH2 domains) were cloned into pACT2 vector respectively. The constructs were separately cotransformed into yeast. beta-galactosidase activity was measured to detect their interactions. TrkC was cloned into pmRFP (carrying red fluorescent protein), and ShcD was cloned into pEGFP (carrying green fluorescent protein). pmRFP-TrkC and pEGFP-ShcD were co-transfected into 293T cells, and then the cells were fixed and subjected to confocal analysis to study their subcellular localization.

RESULTSShcD interacted with TrkC but not with kinase dead mutant TrkCM1(K572A). Both PTB and SH2 domains were capable of binding to TrkC, and PTB domain bound NPQY motif of TrkC. ShcD colocalized with TrkC throughout the cytoplasm and in the plasma membrane in 293T cells.

CONCLUSIONShcD binds to TrkC in a kinase-activity-dependent manner through its PTB and SH2 domains.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Binding Sites ; Cells, Cultured ; Genetic Vectors ; Humans ; Plasmids ; genetics ; Protein Binding ; Receptor, trkC ; genetics ; metabolism ; Shc Signaling Adaptor Proteins ; genetics ; metabolism ; Transfection ; Transformation, Bacterial ; Two-Hybrid System Techniques ; src Homology Domains ; genetics

OBJECTIVETo explore the influences of the restoration of neural adhesion molecule NECL1 on the morphology, migration, and invasion of NECL1-deficient glioma cell lines.

METHODSScratch and Transwell assays were used to observe the cell migration and invasion, the activities of extracellular metalloproteinases were measured, and the cell morphology was observed. Astrocytes marker glial fibrillary acidic protein was detected by Western blot after the restoration of NECL1 in glioma U251 cell line.

RESULTSIn NECL1-deficient U251 glioma cell lines, migration and invasion were inhibited. The U251 cells was differentiated potentially to astrocytes, and glial fibrillary acidic protein was up-regulated after the restoration of the NECL1 expression.

CONCLUSIONAs a potential tumor repressor, the neural adhesion molecule NECL1 can inhibit the migration and invasion of glioma cell and induces its differentiation.

Brain Neoplasms ; metabolism ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Movement ; Glioma ; metabolism ; pathology ; Humans ; Neoplasm Invasiveness ; Neural Cell Adhesion Molecules ; metabolism

OBJECTIVETo screen the asymmetric dimethyl arginines (ADMA)-containing proteins which could combine with protein arginine methyltransferase 1 (PRMT1).

METHODSWestern blot was adopted to identify the expression of PRMT1 and the proteins with ADMA in glioma cell lines and normal brain tissues, and then to detect the changes of ADMA level after knock-down of PRMT1 with RNAi transfection in U87MG cells. Co-Immunoprecipitation (Co-IP), western blot, and sliver staining were employed to screen the candidate binding proteins of PRMT1. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the binding proteins of PRMT1.

RESULTSThe expression of PRMT1 and some levels of ADMA were higher in glioma cell lines than in normal brain tissues. After knocking down PRMT1, some ADMA levels were found declined. After screening the binding proteins of PRMT1 with Co-IP and LC-MS/MS, 26 candidate binding proteins were identified. Among them, 6 candidate proteins had higher ions scores (> 38) and bioinformation analysis predicted that SEC23-IP, ANKHD1-EIF4EBP3 protein, and 1-phosphatidylinositol-3-phosphate 5-kinase isoform 2 had possible methylated aginine sites.

CONCLUSIONSThe high expression of PRMT1 in glioma may induce the change of ADMA levels. Altogether 26 candidate proteins were identified, which contain ADMA and specifically bind with PRMT1.

Arginine ; analogs & derivatives ; analysis ; Cell Line, Tumor ; Chromatography, Liquid ; Glioma ; chemistry ; Humans ; Immunoprecipitation ; Protein-Arginine N-Methyltransferases ; analysis ; physiology ; Repressor Proteins ; analysis ; physiology ; Substrate Specificity ; Tandem Mass Spectrometry

OBJECTIVETo express and purify the recombinant human pigment epithelium-derived factor (PEDF) which inhibits the proliferation of the endothelium cells from blood vessel in E.coli.

METHODSPEDF gene was inserted into the prokaryotic expression vector pGEX-4T-2. The recombinant protein PEDF was expressed in E.coli BL-21, and purified by the GST Sepharose 4B affinity column. The recombinant human PEDF protein was identified by Western blot and mass spectrum. The biological activity of the recombinant human PEDF protein was measured by using MTT.

RESULTSThe 46 kDa recombinant human PEDF protein was obtained. It significantly inhibited the proliferation of the human umbilical vein cell line HUVEC.

CONCLUSIONThe recombinant human PEDF with anti-angiogenesis activity protein may be successfully purify through prokaryotic expression.

Angiogenesis Inhibitors ; pharmacology ; Apoptosis ; drug effects ; Base Sequence ; Cell Division ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Endothelium, Vascular ; cytology ; Eye Proteins ; pharmacology ; Humans ; Molecular Sequence Data ; Nerve Growth Factors ; pharmacology ; Prokaryotic Cells ; metabolism ; Recombinant Proteins ; pharmacology ; Serpins ; pharmacology ; Umbilical Veins ; cytology

OBJECTIVETo screen and identify the possible existence of natural antisense transcript (NAT) within the mouse neocortex.

METHODSSixty-three cerebral cortex layer-specific genes were screened by bioinformatics prediction in mice, among which 31 mice with potential NATs were screened. NAT was identified using reverse transcription polymerase chain reaction (RT-PCR) and then cloned in pGEM-T Vector System for sequencing.

RESULTSAmong 31 genes predicted using bioinformatics, 8 were proved to be NAT positive by RT-PCR.

CONCLUSIONSNATs exist in the mouse neocortex tissue during the development of cerebral cortex. NATs may influence mouse cortical development by regulating the related coding genes.

Animals ; Cell Line ; Cerebral Cortex ; Mice ; Molecular Sequence Data ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics

OBJECTIVETo investigate the molecular mechanism of nectin-like molecule 1 (NECL1) inhibiting the migration and invasion of U251 glioma cells.

METHODSWe infected U251 glioma cells with adeno-nectin-like molecule 1 (Ad-NECL1) or empty adenovirus (Ad). Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells. DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines. The differential expression of osteopontin (OPN), a gene related to migration and invasion, was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry.

RESULTSThe restoration of NECL1 inhibited migration of U251 cells significantly (P<0.05). Altogether 195 genes were found differentially expressed by microarray, in which 175 were up-regulated and 20 down-regulated, including 9 extracellular matrix proteins involved in the migration of cells. Both mRNA and protein expressions of OPN, the most markedly reduced extracellular matrix protein, were found decreased in U251 cells after restoration of NECL1. Immunohistochemical assay also detected an increase of OPN in glioma tissues, related with the progressing of malignant grade.

CONCLUSIONA link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.

Brain Neoplasms ; metabolism ; pathology ; Cell Adhesion Molecules ; physiology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; physiology ; Glioma ; metabolism ; pathology ; Humans ; Nectins ; Neoplasm Invasiveness ; physiopathology ; Neoplasm Metastasis ; physiopathology ; Osteopontin ; genetics

OBJECTIVETo assess the expression level of D-Tyr-tRNA(Tyr) deacylase (DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease (AD).

METHODSAltogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice. Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice. The behavior of the mice was tested by using a Step-through Test System.

RESULTSBoth mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice (P<0.05). In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice.

CONCLUSIONSDTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.

Alzheimer Disease ; enzymology ; Aminoacyltransferases ; metabolism ; Animals ; Base Sequence ; DNA Primers ; Disease Models, Animal ; Mice ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo get the cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain.

METHODSUsing the SARS-CoV PUMC2 strain genomic RNA as the template, the cDNA fragments were amplified by RT-PCR, the PCR products were further purified and ligated into the pGEM-T vector, and all the clones obtained were sequenced.

RESULTSThe cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain were obtained.

CONCLUSIONSThese cDNAs can be provided for the function study of SARS-CoV proteins and the construction of full-length infectious cDNA clone of SARS-CoV.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; genetics ; Genome, Viral ; Molecular Sequence Data ; Nucleic Acid Amplification Techniques ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; isolation & purification ; Sequence Analysis, DNA ; Viral Proteins ; genetics