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Zheng-Ping Xu

Publications and source records attributed to Zheng-Ping Xu.

12 recordsLinked to original sources
PubMed2006

Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines.

AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines. METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

Breast Neoplasms↗
PubMed2004

ATM and ATR: sensing DNA damage.

Cellular response to genotoxic stress is a very complex process, and it usually starts with the "sensing" or "detection" of the DNA damage, followed by a series of events that include signal transduction and activation of transcription factors. The activated transcription factors induce expressions of many genes which are involved in cellular functions such as DNA repair, cell cycle arrest, and cell death. There have been extensive studies from multiple disciplines exploring the mechanisms of cellular genotoxic responses, which have resulted in the identification of many cellular components involved in this process, including the mitogen-activated protein kinases (MAPKs) cascade. Although the initial activation of protein kinase cascade is not fully understood, human protein kinases ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) are emerging as potential sensors of DNA damage. Current progresses in ATM/ATR research and related signaling pathways are discussed in this review, in an effort to facilitate a better understanding of genotoxic stress response.

Animals↗
PubMed2003

Bacterial expression, purification, and in vitro N-myristoylation of fusion hepatitis B virus preS1 with the native-type N-terminus.

Very low-level expression of hepatitis B virus (HBV) preS1 with the native-type N-terminus hampered the biochemical and functional studies on its myristoylation. In the present study, the fusion HBV preS1 with the native-type N-terminus and a His6-Tag fused to C-terminus (HBV preS1-HT) was highly expressed in Escherichia coli. This was due to an introduced mutation of the rare codon GGA found in the HBV preS1 to the codon preferred by E. coli, GGU. The protein was rapidly purified from bacterial lysate by Ni-IDA affinity chromatography. The experimental assays using 3H-labeled substrate demonstrate that the purified HBV preS1-HT can be effectively N-myristoylated by recombinant human protein N-myristoyltransferase (NMT) in vitro.

Acyltransferases↗
PubMed2003

[An in vitro nuclear run-on assay for nuclear function of angiogenin].

A sensitive and quantitative in vitro analysis method was established to detect the nascent RNAs stimulated by angiogenin using the nuclei isolated from human umbilical vein endothelial cells (HUVE). Angiogenin was mixed with nuclei in the reaction buffer, then transcription was initiated by adding the NTPs mixture. The RNA products were measured quantitatively by [alpha-(32)P]CTP incorporation with a liquid scintillation counter, either after removing free isotope by using spin column, or cutting the electrophoresis lane and counting after autoradiography. It was found that the optimum reacting temperature was 30 degrees, the most suitable reaction time was 30 min for this system, and the transcription enhancement activity of angiogenin was dose-dependent with the feasible concentration being 1 mg/L. Higher concentration of angiogenin degraded the RNA products in the system, suggesting that there is a mechanism to control the entry and accumulation of angiogenin in the target cells, which ensured angiogenin to play its role properly in the cells. Based on the evidence that angiogenin bound to DNA in nucleolus and enhanced RNA transcription, it was proposed that angiogenin might act as a trans-acting factor in nucleus to regulate RNA transcription, and inhibition of angiogenin-stimulated RNA transcription might be a promising target for screening anti-angiogenesis inhibitor.

Cell Line↗
PubMed2002

Functional and structural characterization of the human gene BHLHB5, encoding a basic helix-loop-helix transcription factor.

The genes encoding basic helix-loop-helix (bHLH) transcription factors have been implicated in many aspects of neural development, including cell growth, differentiation, and cell migration. Using both genomic and cDNA mouse and human clones encoding a neural-specific bHLH protein, human BHLHB5 was cloned and mapped to a region on chromosome 8q13 that segregates with Duane syndrome. Genomic sequence analysis of human BHLHB5 and mouse Bhlhb5 revealed that they contain a single exon encoding 381- and 355-amino-acid bHLH proteins, respectively. Multiple amino acid sequence alignments of the Bhlhb5 family members revealed several conserved motifs and an identical 147-amino-acid carboxy-terminal region that contains a 60-amino-acid bHLH domain. A 27-bp trinucleotide repeat (CAG)(9) encoding polyserine was found in human BHLHB5, but only one CAG was found at the corresponding position in the mouse Bhlhb5 and hamster BETA3 genes. Northern blot analysis of human BHLHB5 revealed brain-specific expression with the highest abundance in the cerebellum. Mouse Bhlhb5 can strongly repress a human PAX6 promoter.

Amino Acid Sequence↗
PubMed2002

Transketolase haploinsufficiency reduces adipose tissue and female fertility in mice.

Transketolase (TKT) is a ubiquitous enzyme used in multiple metabolic pathways. We show here by gene targeting that TKT-null mouse embryos are not viable and that disruption of one TKT allele can cause growth retardation ( approximately 35%) and preferential reduction of adipose tissue ( approximately 77%). Other TKT(+/-) tissues had moderate ( approximately 33%; liver, gonads) or relatively little ( approximately 7 to 18%; eye, kidney, heart, brain) reductions in mass. These mice expressed a normal level of growth hormone and reduced leptin levels. No phenotype was observed in the TKT(+/-) cornea, where TKT is especially abundant in wild-type mice. The small female TKT(+/-) mice mated infrequently and had few progeny (with a male/female ratio of 1.4:1) when pregnant. Thus, TKT in normal mice appears to be carefully balanced at a threshold level for well-being. Our data suggest that TKT deficiency may have clinical significance in humans and raise the possibility that obesity may be treated by partial inhibition of TKT in adipose tissue.

Adipose Tissue↗
PubMed1999

Expression Purification and Activity ofhuman Myristoyl-CoA:N-myristoyltransferase.

A gene encodinghuman myristoyl-CoA: protein N-myristoyltransferase (hNMT) from a brain cDNA libraryhas been obtained by PCR amplification and DNA sequencing. Then the mature-type and His(6)-fusion-type expression plasmids (pMF-hNMT(3) and pMFHT-hNMT(2)) containing the hNMT gene under control of T7 promoter have been constructed and transformed into E. coli BL21(DE3). SDS-PAGE analysis showed that the recombinant hNMT products expressed at 37 degrees were almost unsoluble but most of the His(6)-hNMT product expressed at lower temperature (22 degrees ) was soluble and its yield was about 7% of the total soluble cellular proteins. By immobilized metal (Ni(2+)) chelation affinity chromatography up to 80% His(6)-hNMT was purified by one step from bacterial lysate. The labelling experiment in vitro domonstrated that the expressed and purified His(6)-hNMT had an obvious catalytic activity to transferring myristoyl group.

Journal Article↗
PubMed1998

Studies on Structure and Function of the Myristoyltransferase Inhibitor Peptide Displayed on Phage Surface.

Site-directed mutagenesis was used to identify the critical residues and functional sequences of a potent NMT inhibitor peptide displayed on phage surface. W2A, H6N and H6R largely lowered the inhibitory activities of the mutated phages, and the activity of P3A and V4V5 right curved arrow A4A5 was also slightly reduced. However, V4V5 right curved arrow W4W5, H12N, H14N, C9S, C15S, deltaC15, deltaH14C15 and deltaC9--C15 had no obvious effect on the inhibition. These results suggest that W2, P3 and H6 are important in the peptide structure and/or the interaction with NMT, that amino acids with larger side chain are the more favorable residues between P3 and H6, and also that the sequences between C9 and C15 have no clear contribution to the inhibitory activity. In summary, the functional prptide directly related to the role of inhibition has been located between the N terminal T1 and A8 of the inhibitor peptide.

Journal Article↗
PubMed1998

C-terminal His-tag Fusion Expression and Purification of Truncated cAMP-dependent Protein Kinase.

The truncated mCalpha lacking 3'-coding sequence of 96 base paires was fused with a His-tag (mCalpha4H) and a C-terminal fusion expression plasmid pZP mCalpha4H was constructed. With the induction of IPTG, the Expressed mCalpha4H was up to about 20% of the total bacterial proteins in E. coli BL21 (DE3). Using immobilized metal (Ni(2+)) chelation affinity chromatography, the target protein mCalpha4H was purified from crude lysates and inclusion bodies respectively. The results of in vitro and in vivo myristoylation assay showed that the purifed mCalpha4H is a substrate of NMT as the mCalpha.

Journal Article↗
PubMed1998

Selection of Myristoyltransferase Inhibitor Phages from Phage Display Random Peptide Library.

Through screening library with either immobilized or coated enzymes, we selected disease-related myristoyltransferase inhibitor phages from phage display random peptide library. After high-affinity bound phages were obtained, they were subjected to in vitro NMT inhibition assay to identify inhibitor phages. The results of DNA sequencing, peptide sequence deducing and sequence aligning of the 16 inhibitor phages suggested that the sequences of inhibitor peptides obtained by the two separate screening methods appeared overlapping, and included a consensus motif PX(0-3)H/R or H/RX(0-3)R, in which X represented a non-specific amino acid.

Journal Article↗
PubMed1997

His(6) Fusion Expression of Myristoyl-CoA: Protein N-myristoyltransferase in E. coli and its Purification.

Saccharomyces cerevisiae Myristoyl-CoA: protein N-myristoyltransferase (NMT) gene was cloned into a His(6)-fusion expression vector pMFHT. After transforming into E. coli BL21 (DE3), His(6)-NMT was induced to express at 37 degrees by IPTG. SDS-PAGE analysis showed an induced expression product band of about 54 kD which constituted about 10% of the total bacterial proteins. The analysis of product solubility revealed that His(6)-NMT was predominantly soluble. On the basis of these results, His(6)-NMT was purified in one-step to 95% of purity from bacterial lysates using immobilized metal (Ni(2+)) chelation affinity chromatography. The in vitro labelling experiment demonstrated that His(6)-NMT had an activity similar to that of mature NMT, suggesting that the His(6)-tag did not affect the enzyme activity. His(6)-tag in the N-terminal of NMT makes it be possible to immobilized simply on Ni(2+)-IDA Sepharose 6B resin, which can be used to screen the peptide inhibitors of NMT from Phage Display random peptide library.

Journal Article↗