Biomedical subjects

Junjie Chen

Publications and source records attributed to Junjie Chen.

65 records · Page 4Linked to original sources

PubMed2003

[Recombinant human brain myelin basic protein and its antibody].

We constructed the expression vector by inserting 21.5 KDa MBP human brain full-length cDNA coding sequence digested with restriction enzyme EcoR I and Sal I into downstream of pGEX-5T expression vector. The recombinant vector p5TMP was transformed into E. coli and the positive clonies were selected and incubated in LB medium induced by IPTG (isopropyl- -D-thiogalactoside). A new polypeptide band with apparent molecular weight 42 KDa was detected in transformed cell lysates by SDS-PAGE. Western blotting analysis confirmed that this fusion protein reacted specifically with antibodies to MBP, the expression level of MBP was about 414.6 mg/L medium estimated by immuno-dot blot, ELISA and absorbance scanning. Newzealand rabbits were immunized by subcutaneous injection of the purified recombinant MBP. The titer was obtained at 1:16 after 5 injections. The specificity of the antibody to MBP was confirmed by immuno-blot and Western blotting.

Animals↗

PubMed2003

[Effects of brain-derived neurotrophic factor on H2O2-induced apoptosis of human arterial smooth muscle cell].

OBJECTIVE: To investigate the protective effects of brain-derived neurotrophic factor (BDNF) on hydrogen peroxide (H2O2) induced apoptosis of human arterial smooth muscle cells (SMC). METHODS: The hBDNF cDNA was transfected into the SMC mediated by Lipofect- AMINE, and ectopic expression in SMC was driven by the regulatory elements of I type collagen gene(COLIA1). The cell proliferation was measured with a colorimetric assay based on the cleavage of the tetrazolium salt MTT. Microscopic analysis of cell apoptosis was performed with fluorescent stain of acridine orange (AO). Apoptosis rate of SMC was tested by flow cytometry (FCM). DNA pattern was examined by agarose gel electrophoresis. RESULTS: Exposure of growing SMC to 100-800 mumol/L H2O2 for 24 h remarkably suppressed the cell proliferation. After treatment of the cells with 200 mumol/L H2O2 for 24 h, morphological and biochemical changes of classic apoptosis such as condensation of cytoplasm, fragmentation of the nuclear chromatin, DNA "ladder" pattern after electrophoresis were observed. Yet, the above-mentioned apoptosis changes were not observed and the apoptosis rate was reduced obviously in the BDNF-transfected SMC treated with the same concentration of H2O2. CONCLUSION: H2O2 can induce apoptosis of SMC, but BDNF inhibits H2O2-induced SMC apoptosis markedly and has strong anti-apoptotic activity.

Apoptosis↗

PubMed2002

Synthesis and cytotoxic activity of N-(2-diethylamino)ethylcarboxamide and other derivatives of 10H-quindoline.

A series of mono- and dimeric N-methylquindoline carboxamides were prepared by Friedlander condensation between methyl 2-amino-3-formyl benzoate and 3-acetoxy-1-acetylindoles, followed by exhaustive methylation with methyl iodide to give N-methylquindoline esters. Direct amination of these, or hydrolysis to the acids and amine coupling via intermediate imidazolides gave the desired carboxamides. The compounds were evaluated in a panel of cell lines in culture. The monomeric compounds showed similar structure-activity relationships to the known indeno[1,2-b]quinolines, with a 4-methyl group increasing potency several-fold. Bis analogues linked through the carboxamide were more cytotoxic than the corresponding monomers in the human leukemia lines, but N-N linked dimers were generally less potent, except for a tetracationic derivative. The most potent monomeric analogue showed moderate growth delay (ca. 5 days) against sub-cutaneously implanted colon 38 tumors in mice.

Alkaloids↗

PubMed2002

DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1.

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.

Animals↗

PubMed2002

A DNA damage-regulated BRCT-containing protein, TopBP1, is required for cell survival.

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.

Amino Acid Motifs↗

PubMed2002

[Biology and pathobiology of death receptors in liver].

Death receptors induce apoptosis by intracellular signaling transmission and relate to body growth, development, disease and death. This paper reviews the structural character of death receptors, and the mechanisms by which apoptosis is induced. The biology and pathobiology of these death receptors in liver are discussed as well.

Apoptosis↗

PubMed2002

[Study on expression of beta-NGF in myoblasts transfected by PSVCEP NGF-CAT with Lipofect AMINE].

OBJECTIVE: To gain an understanding of the expression of beta-NGF in myoblasts transfected by PSVCEP NGF-CAT and the transfecting efficiency of Lipofect AMINE. METHODS: PSVCEP NGF-CAT was delivered into cultured myoblasts by Lipofect AMINE. Expression of beta-NGF in the transfected myoblasts was detected by immunocytochemistry. RESULTS: Lipofect AMINE was evidently engulfed by myoblasts at the 6th hour; about 40% myoblasts could be detected with the expression of beta-NGF by immunocytochemistry at the 48th hour. CONCLUSION: PSVCEP NGF-CAT can express beta-NGF in cultured myoblasts, and Lipofect AMINE is a convenient vector for gene transfection with simplicity and efficacy.

Animals↗

PubMed2002

[Expression of human nerve growth factor gene in E. coli].

OBJECTIVE: To investigate the structure and function of human nerve growth factor (beta-NGF) and the gene encoding beta-NGF. METHODS: A pair of specific primers (29 mer) for the sequence encoding human beta-NGF was designed and synthesized. A 380 bp fragment was amplified from human blood genomic DNA by polymerase chain reaction, and cloned into pGEM-T Easy vector. The identified insert fragment from the recombinant pGEM-T-NGF was directionally ligated with linearized pGEX-5T with the compatible termini. E. coli JM 109 was transformed with the expression recombinant p5TNGF and induced by IPTG. RESULTS: The cloned DNA fragment was identified as the full-length sequence encoding human beta-NGF by restriction analysis and DNA sequencing. SDS-PAGE and Western blot revealed the cloned NGF gene expressed as a fusion protein (40.5 x 10(3) u) in the cells transformed by p5TNGF. The soluble fusion protein was determined to be 503.2 mg/L, accounting for 6.8% of the total soluble protein (7.4 g/L) of bacterial cells. This fusion protein was found to have antigenic activities of NGF. CONCLUSION: The clone containing the full-length sequence encoding human beta-NGF is obtained and successfully expressed in E. coli to be of use for studying the biological functions of human beta-NGF gene.

Base Sequence↗

PubMed2002

[The cloning and sequencing of H-2Kk gene cDNA of 615 mice].

OBJECTIVE: The purposes of this study were to clone and sequence the major histocompatibility complex type I (MHC I) molecular antigen recognizing gene (H-2Kk) of 615 mice, and to provide the functional gene for transgenic therapy. METHODS: The 1.4 kb full-length fragment of H-2Kk gene complementary DNA (cDNA) was amplified from the total RNA of 615 mouse liver by using reverse transcription polymerase chain reaction (RT-PCR). The cDNA was inserted into PGEM3Zf(+) vector directionally, and the competent E. coli JM109 was transformed with the ligated product. The recombinant PGEM3Zf(+)-H-2Kk cDNA plasmid was obtained using restricted enzyme analysis of the transfectants. The complete sequence of 615 mouse H-2Kk cDNA was determined by using Sanger's method. RESULTS: The sequences of 615 mouse H-2Kk cDNA were 99% similar with those of H-2Kk cDNA which were reported by other researchers, and the sequences encoding antigen recognizing regions (ARS) were identical with each other. CONCLUSION: The authors cloned the MHC I molecular antigen recognizing gene (H-2Kk) of 615 mice successfully and got the functional gene of MHC I.

Animals↗

PubMed2001

The LIM domain protein LMO4 interacts with the cofactor CtIP and the tumor suppressor BRCA1 and inhibits BRCA1 activity.

LMO4 belongs to the LIM-only (LMO) group of transcriptional regulators that appear to function as molecular adaptors for protein-protein interactions. Expression of the LMO4 gene is developmentally regulated in the mammary gland and is up-regulated in primary breast cancers. Using LMO4 in a yeast two-hybrid screen, we have identified the cofactor CtIP as an LMO4-binding protein. Interaction with CtIP appeared to be specific for the LMO subclass of LIM domain proteins and could be mediated by a single LIM motif of LMO4. We further identified the breast tumor suppressor BRCA1 as an LMO4-associated protein. The C-terminal BRCT domains of BRCA1, previously shown to bind CtIP, also mediated interaction with LMO4. Tumor-associated mutations within the BRCT repeats that abolish interaction between BRCA1 and CtIP had no effect on the association of BRCA1 with LMO4. A stable complex comprising LMO4, BRCA1, and CtIP was demonstrated in vivo. The LIM domain binding-protein Ldb1 also participated in this multiprotein complex. In functional assays, LMO4 was shown to repress BRCA1-mediated transcriptional activation in both yeast and mammalian cells. These findings reveal a novel complex between BRCA1, LMO4, and CtIP and indicate a role for LMO4 as a repressor of BRCA1 activity in breast tissue.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

Claspin, a regulator of Chk1 in DNA replication stress pathway.

Regulation of the vertebrate checkpoint kinase Chk1 involves several protein complexes including the recently identified protein Claspin. Claspin associates with Chk1 upon replication stress and DNA damage and is required for Chk1 activation in both Xenopus and human systems. More importantly, Claspin is involved in regulation of cell cycle checkpoints. Here, we discuss the emerging roles of Claspin in the Chk1 pathway and its functions in checkpoint control.

Adaptor Proteins, Signal Transducing↗