Biomedical subjects

Cui-Fen Huang

Publications and source records attributed to Cui-Fen Huang.

35 records · Page 2Linked to original sources

PubMed2003

[Isolation and characterization of a BRCA1-interacting protein].

BRCA1 (breast cancer susceptibility gene-1) plays important roles in DNA damage repair, cell checkpoint regulation, gene transcription, chromosome stability, and apoptosis. At the C-terminus of BRCA1 is the activation domain with a number of acidic amino acid residues that includes two tandem repeats of BRCT(BRCT1 and BRCT2). In this study, to identify proteins that interact with the BRCT2 domain of BRCA1, the standard yeast two-hybird screen was performed. FHL2 was isolated from a human ovary library, with the BRCT2 domain of BRCA1 as bait. Furthermore, the specific interaction of FHL2 with the BRCT2 domain of BRCA1, but not with the BRCT1 domain of BRCA1 and the BRCT domain of Rap1, was verified by yeast mating. To confirm the interaction between BRCA1 and FHL2 in vitro, the GST pull-down assay was performed, the coding sequences of BRCT1 and BRCT2 domains were fused in-frame with the coding region of GST in the pGEX-2T vector, generating the pGST-BRCT1 and pGST-BRCT2 recombinant plasmids the fusion proteins GST-BRCT1 and GST-BRCT2 were expressed in E. coli DH5 alpha. The purified fusion proteins were obtained by GST-Sepharose 4B affinity chromatography. The purified fusion proteins were incubated with in vitro translated 35S-methinine-labeled FHL2. Consistent with the two-hybird results, FHL2 could specifically bind to the BRCT2 domain, but not BRCT1 in vitro. To further assess the binding specificity of FHL2 to the BRCT2 domain of BRCA1 in vivo, pFLAG-FHL2 and pHABRCT1/pHA-BRCT2 recombinant plasmids were cotransfected into 293T cells. Then the coimmunoprecipitation assay were performed. The results also showed that FHL2 specifically interacted with the BRCT2 domain in vivo. Furthermore, the coimmunoprecipitation assay demonstrated that FHL2 could interact with endogenous BRCA1 in vivo. These findings lay solid foundations for study on the function of BRCA1 and FHL2 in cancer development and progression.

BRCA1 Protein↗

PubMed2002

Ectopic expression of a COOH-terminal fragment of the human telomerase reverse transcriptase leads to telomere dysfunction and reduction of growth and tumorigenicity in HeLa cells.

The COOH-terminus of telomerase reverse transcriptase (hTERT) has been shown to participatein the nuclear translocation of TERT. Here, we constructed plasmids expressing the COOH-terminal M(r) 27,000 polypeptide of hTERT (hTERTC27) withthe telomerase RNA-binding domains and the reverse transcriptase domains deleted. We showed that ectopic overexpression of this polypeptide caused a defect in telomere maintenance in hTERT-positive HeLa cells, which led to senescence-like growth arrest and apoptosis. The hTERTC27 appears to work by inducing telomere dysfunction, exemplified by significantly increased anaphase chromosome end-to-end fusion events in transfected cells. Significantly, it had no effect on the cellular telomerase enzymatic activity or telomere length. The in vivo effect was further demonstrated as HeLa cells stably expressing hTERTC27 have significantly lower growth rate and reduced tumorigenicity in nude mice xenografts. Results from this study revealed an important function for the COOH terminus of hTERT in maintaining the integrity of telomere structure and chromosome ends, as well as in cell senescence and apoptosis. Furthermore, hTERTC27 provides a new strategy for cancer therapy by inducing telomere dysfunction in cancer cells without affecting the telomerase enzymatic activity.

Animals↗

PubMed2002

Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Salmonella typhimurium.

LT and ST are the main enterotoxins of enterotoxigenic Escherichia coli (ETEC) found in clinical isolates, and CS3 (the common antigen in the CFA/II family of fimbrial antigens) is one of the most prevalent antigens of colonization factors. The genetic determinants encoding CS3 and LT-B/ST fusion toxin were manipulated so that these important antigens are expressed simultaneously in attenuated Salmonella typhimurium oral vaccine strain X4072. These antigens produced by X4072 (pXZL88) could be recognized with monospecific CS3, LT or ST antibodies respectively. The specific antibodies against CS3, LT and ST could be detected. In the sera of immunized mice via oral route with the live bacteria. Significantly, the antibody to ST was able to neutralize the biological activity of native ST. This prototype construct may be proved to be useful in investigating the live vector approach to immunoprophylaxis of ETEC diarrhea disease.

Animals↗

PubMed2002

[Generation and characterization of chondrocyte specific Cre transgenic mice].

A chondrocyte specific transgenic construct (pcol2A1-Cre) containing the cartilage specific type II collagen A1 promoter, the Cre recombinase gene and the polyA of human growth factor gene was generated. The 9.3 kb DNA fragments were recovered from Not I digested fragments and microinjected into 323 fertilized eggs. The injected eggs were implanted into the oviduct of 14 female mice. In the 52 offsprings, there were 10 mice carring the transgene identified by PCR, and the efficiency was 19.2%. The col2A1-Cre transgenic mice were crossed with a conditional gene targeting mice to check the Cre mediated recombination in multiple tissues. The results of the PCR analysis suggested that the Cre recombinase was expressed only in cartilaginous tissue and could mediate the recombination between the LoxP sites in vivo. The result was further confirmed by Southern-blot.

Animals↗

PubMed2002

A transgenic mouse that targets the expression of Cre recombinase in pancreatic tissue.

The transgenic mice that express Cre recombinase in a tissue specific manner is a powerful tool in generating the conditional gene knockout mice. The rat insulin promoter was cloned target the expression of Cre in pancreatic tissue. The Cre gene was modified by adding the nuclear localization signal and the sequence for initiation by eukaryotic ribosomes at 5' terminal of the Cre gene. Cre gene was linked to the intron of human growth factor gene. This construct was introduced into the mouse eggs using microinjection. Seven mice were identified as founders carrying the Cre gene by PCR. The results of RT-PCR showed that the transgenic mouse from one founder could transcribe the foreign gene in pancreas. The Southern blot analysis indicated that the Cre recombinase expressed in pancreas of the transgenic mouse was functional.

Animals↗

PubMed2002

[Analysis of the proteomic changes of the serum of the Smad3 targeted deficient mice].

We have studied the proteomic changes of the serum of the Smad3 targeted deficient mice using 2-DE and PMF approaches. 7 proteins expressed at different level in wild type mice and the Smad3 deficient mice were identified. These results would benefit the research on diagnosis and therapy of osteoarthritis and provided clues to studying the important function of Smad3 mediated TGF-beta signals during the skeletal development.

Animals↗

PubMed2002

[Studies of mutations in BRCA1 transactivation domain by visualization of chromatin structure].

Mutations in breast cancer susceptibility gene 1(BRCA1) account for approximately 40%-50% of familial breast cancer cases and for more than 80% of inherited breast and ovarian cancer cases. Many cancer-predisposing mutations are located in the C-terminal region that functions as a transcriptional activation domain, but most of the mutations in the transactivation domain identified to date cannot be readily distinguished as either disease-associated mutations or benign polymorphisms. Because chromatin structure regulation is an early event in gene transcription control, the chromatin unfolding activities of different transactivation domain variants were compared with that of the wild-type transactivation domain by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To do this, different constructs of the transactivation domain were selected as follows: (a) the wild-type transactivation domain; (b) two polymorphisms (S1613G and M1652I); and (c) four cancer-predisposing mutations (A1708E, M1775R, W1837R and Y1853 term). All of the constructs were made by fusing in frame with lac repressor. Western blot analysis indicated that all of the fusion proteins were expressed in A03 1 cells, in which multiple copies of the lac operator were integrated to produce a heterochromatic region of the genome. The chromatin unfolding assay showed that, like the wild-type transactivation domain, two variants that represent benign polymorphisms did not induce chromatin unfolding or only induced subtle change. Contrary to the behaviors of the wild type and two benign variants, four cancer-predisposing mutations in the transactivation domain superactivate the chromatin unfolding. The results suggest that the chromatin unfolding assay can aid in the characterization of deterious mutations in the C-terminal transactivation domain of BRCA1 and may provide more reliable presymptomatic risk assessment.

Animals↗

PubMed2002

[A novel system for characterization of the transcription activating proteins in mammalian cells].

A system used for detecting the transcriptional activating activity of the function-unknown gene products in mammalian cells was developed. Based on the plasmid pTet-Off and the eukaryotic expressing vector pCDNA3.1B(-)/myc-his, firstly, we constructed a set of recombinant plasmids namely pZHO1 (for cloning into the foreign gene fragment and as a negative control), pZHO2 (as a positive control). The system also includes the plasmids pTRE-luc (encoding the Firefly luciferase reporter gene) and pRL-TK (encoding Renilla luciferase gene as background control). To confirm the feasibility of the system, the plasmids pZHO1, pZHO2 and pZHO3 (encoding p53 transcriptional activating domain, containing 73 amino acids in its N terminal) was contransfected into such mammalian cells as C4-2, MCF-7, COS7 respectively, each with pTRE-luc and pRL-TK plasmids, the feasibility of the system was determined by comparing the relative activity of Firefly luciferase activity ratio of and Renilla in different transfecting panels. Our research result showed that the system we constructed can be used for detecting the transcriptional activating activity of the target protein molecules in mammalian cells.

Animals↗

PubMed2002

[Mapping of BRCT1 domain of BRCA1 with chromatin unfolding activity].

Breast cancer susceptibility gene 1(BRCA1) plays an important role in breast cancer development and progression. BRCA1 encodes a 1863-amino acid protein with two BRCA1 C-terminal (BRCT) domains at its C-terminus, BRCT1 and BRCT2. Many cancer-predisposing mutations are located in the BRCT domains, which have been shown to induce chromatin unfolding by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To map the important region of BRCT domain (amino acid residues 1642-1736), six deletion mutant constructs were made. The chromatin structure assay showed that amino acid residues 1691-1721 are involved in the induction of chromatin unfolding. To further localize the critical amino acid residues, ten alanine scanning mutant constructs were made. The chromatin structure assay demonstrated that the 1707IAGGK1711 region is critical for the chromatin unfolding activity. Based on the mapped important region, Blast analysis identified a novel homologous protein. Mapping of the BRCT1 domain may aid in the presymptomatic risk assessment and provide a valuable tool for further study on the BRCT1 structure and function.

BRCA1 Protein↗

PubMed2001

Expression of Colonization Factor Antigen I of Enterotoxigenic Escherichia coli in Shigella flexneri 2a T32.

A host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express colonization factor antigen I(CFA-I) of enterotoxigenic Escherichia coli.The results of Western blotting suggested that avirulant strain of S.flexneri Fwl01 expressed CFA-I steadily. Examination of negatively-stained preparation of cultures by electron microscopy showed that S.flexneri Fwl01 carrying the plasmid pZHY21 had thick pili on its surface. Antibodies against CFA-I were decteted in sera of mice immunized with recombinant bacteria either orogastrically (o.g.) or intranasally(i.n.); simultaneously sIgA against CFA-I was also decteted in the intestine. This work is helpful for constructing multivalent reombinant vaccine for prevention of bacterial diarrhea.

Journal Article↗

PubMed2000

Cloning and Secretion Expression of Heat-shock Protein 70 Gene of Helicobacter pylori.

Heat-shock protein 70 gene (hsp70)was obtained by PCR method from Helicobacter pylori chromosomal DNA. Sequencing analysis exhibited that the hsp70 gene isolated from Hp Y(2) was highly homologous with the gene encoded in Helicobacter pylori 26695 and J99, which had been sequenced for complete genome. The hsp70 gene was recombined in vitro with fusion secretion expression vector pMAL-p2 and was transformed into E.coli cells. The E.coli strains, containing hsp70 recombinant plasmid, expressed a 113 kD fusion protein which accounted for 19.4% of the total bacterial periplasm protein after the induction with IPTG for 5 h at 30 degrees. The expressed fusion protein could react specifically with anti-Helicobacter pylori rabbit IgG, as proved by Western blot method.

Journal Article↗

PubMed2000

Molecular Design and Bio-activity Analysis of a t-PA Derivative (t-PA S165W).

In order to obtain tissue-type plasminogen activator (t-PA) mutant with enhanced affinity for fibrin, redesigning t-PA strategies were proposed by computer molecular designing technology, and the re-designed t-PA derivative, t-PA S165W, was expressed in CHO cells. The bio-activities and affinity for fibrin of the expression product were tested. No apparent changes were observed between t-PA S165W and wild type t-PA. Therefore, single site mutation of the t-PA could not lead to enhanced affinity for fibrin of t-PA.

Journal Article↗

PubMed2000

Expression of Smad4 Gene in Pichia pastoris and Identification of the Protein.

Smad4 is a novel tumor suppressor gene which is mutated or deleted in about 50% of pancreatic carcinoma and 30% of colon cancer. SMAD4 was expressed in Pichia pastoris and was characterized. The molecular weight of the expression product was shown to be about 67 kD, and 13 amino acids in N terminal were determined and were identical with the putative sequence from Smad4 cDNA, and it was able to specifically react with the antibody against SMAD4.

Journal Article↗

PubMed1999

Simultneous Expression of CS3 Colonization Factor Antigen and Cholera Toxin B Subunit in Salmonella typhi.

A host-plasmid balancing system was established based on asd gene in an avirulent strain of Salmonella typhi to express enterotoxigenic E.coli surface antigen CS3 and V.cholerae toxin subunit B(CTB). The plasmid can be stably maintained in the host and can express CS3 and CTB in the host cell without any antibiotic selection, although expression level and growth characteristics of the recombinant strain expressing either CS3 or CTB are superior to that of the recombinant strain which expresses both of the antigens. Antibo-dies against CS3 and CTB can be detected in sera of mice immunized with recombinant bacteria either orally or subcutaneously, and mice immunized subcutaneously can be protected from challenging with virulent strain of Salmonella typhi. This work may be helpful in constructing multivalent recombinant vaccines for prevention of bacterial diarrhea.

Journal Article↗

PubMed1999

Cloning and Expression of alpha-Bungarotoxin Gene.

The genetic engineering methods to produce the snake neurotoxin obviously have potential advantages over the traditional methods of extracting neurotoxin from snakes. This work was to study the expression and the feasibility of scaled production of snake neurotoxin in the routine expression systems such as E.coli with the -bungarotoxin as an example. First, on the basis of the reported amino acid sequence of alpha-bungarotoxin, DNA sequence of -bungarotoxin was deduced and four partially complementary oligonucleotide fragments were designed. The coding region of -bungarotoxin was obtained by renaturing the DNA fragments, nick filling-in, ligation and PCR. The coding region of -bungarotoxin was cloned into plasmids pGEX-2T, named pDZ04, and transformed E.coli BL21 in order to study the expression of alpha-bungarotoxin gene. The results of SDS-PAGE analysis showed that the recombinant plasmid pDZ04 could express efficiently in BL21, and the fusion protein took up about 30%-40% of total bacterial protein. Finally, The biological activity of the recombinant alpha-bungarotoxin and the fusion protein was studied with the natural alpha-bungarotoxin purified from the snake venom as control, ELISA results showed that they had similar antigenicity.

Journal Article↗

PubMed1998

Screening of Cellulose Binding Motif (CBM) from Phage Peptides Library.

Bacteriophages capable of binding cellulose matrix were screened from a 15-mer phage peptides library. In the deduced amino acid sequences of the screened phages, a characteristic region containing a conserved aromatic residue [tyrosine (Y) or phenylalanine (F)] was found, which is similar to the normal cellulose binding domains found in fungal and bacterial cellulose catalysase. Dot-ELISA showed that the phage containing sequence as SWYL has higher affinity to cellulose fibre than other phages, such as those containing sequences as CWYGNC, CWYGEC and XSWYDXXSWFSX. Results indicated that SWYL maybe a good candidate for cellulose binding motif. This work laid basis for further study on cellulose binding motif.

Journal Article↗

PubMed1996

Studies on the Expression of Recombinant Human MCAF/GM-CSF Fusion Protein and Its Biological Activities.

With PCR technology and methods of DNA recombination in vitro, the fusion protein of GM-CSF with MCAF, the facto that has a directing effect on cells to a target, was generated by the construction of recombinant plasmids pMG 01, pMG 02 and pMG 03 with different nucleotide composition between its SD sequence and the initiation codon ATG under the control of lambdaP(R) P(L) promoter of vector pBV 220. Although no stable secondary structure exist in the translation initiation regions of all the recombinant plasmid constructed, the expression levels of the products with DH5 alpha (pMG 02) and DH5alpha (pMG 03) are much higher than that with DH5alpha (pMG01) which hardly expressed the product. The assay of Western blot indicated that the expressed product reacted with MCAF and GM-CSF antibodies, respectively. The assay of biological activities showed that the expressed product had apparent monocyte chemoattractant activity and supported the growth of the human GM-CSF-dependent cell line TF1, suggesting that the biological functions of MCAF and GM-CSF are compatible

Journal Article↗