Biomedical subjects

Cui-Fen Huang

Publications and source records attributed to Cui-Fen Huang.

At least 19 recordsLinked to original sources

PubMed2006

[Stress proteins of Bifidobacterium longum NCC2705].

Stress proteins of Bifidobacterium longum strain NCC2705 were identified and characterized during stationary phase. According to the proteomic map of L. lactics IL1403 and theoretical Mr/pl of stress proteins in B. longum NCC2705 genome annotation, spots of stress proteins in gels were localized, and proteins were identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and/or ESI-MS/MS mass spectrometry. For protein identification by peptide mass fingerprinting, peptide masses were searched against database of B. longum NCC2705 by Mascot licensed in-house. 44 spots representing 8 protein entries have been identified, these proteins were hydrophilic proteins and predicted acid proteins. Spot and protein analysis revealed that post-translational modifications might be common in these proteins. Except for DnaJ, the stress proteins were encoded by genes with CAI value above 0.5, and represented a large proportion of the most abundant proteins. Moreover, the results of scavenging effects on free radicals in vitro showed that B. longum NCC2705 can inhibit fatty acid oxidation and scavenge DPPH, but they scavenge weakly active oxygen free radicals. We identified a key protein that can reverse oxidative damage to proteins and lipids: alkyl hydroperoxide reductase (ahpC, BL0615)synthesized under our experimental conditions.

Bacterial Proteins↗

PubMed2005

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T.

AIM: To screen the immunogenic membrane proteins of Shigella flexneri 2a 2457T. METHODS: The routine two-dimensional polyacrylamide gel electrophoresis (2-DE) and Western blotting were combined to screen immunogenic proteins of S. flexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was performed to determine the molecular weight of peptides. Electrospray ionization (ESI-MS/MS) was performed to determine the sequences of the interesting peptides. RESULTS: A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein (YaeT) was detected, which might be a candidate target of vaccine. CONCLUSION: Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.

Animals↗

PubMed2005

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli.

AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri 2a T32 against enterotoxigenic E.coli (ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LTB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.

Animals↗

PubMed2005

[Screen in vivo induced gene of Mycobacterium tuberculosis by IVIAT].

In order to search new candidates of pharmaceutical target, in vivo induced antigen technology (IVIAT) was used to screen in vivo induced (ivi) genes of Mycobacterium tuberculosis (M. TB). Genomic expression library of M. TB was first constructed with an inducible plasmid pKK223-8; the titer of the library was 1.02 x 10(5) CFU. Sera from ten tuberculosis patients were pooled and absorbed against in vitro-grown M. TB and Escherichia coli, and used to probe the genomic expression library. 16 positive clones were identified by immunological screen, including 22 ORF: two encoding lipid metabolism proteins, five information pathways proteins, two PE/PPE proteins, six intermediary metabolism and respiration proteins, one cell wall and cell processes protein, four conserved hypothetical proteins and two conserved hypothetical proteins with an orthologue in Mycobacterium bovis. Parts of these genes can be used as candidates of pharmaceutical target because they may be relate with virulence.

Antigens, Bacterial↗

PubMed2005

[SMAD3 inhibits the expression of MMP-2 in mouse embryonic fibroblasts].

SMAD3 is one of the receptor-activated SMADs which are important in TGF-beta signal transduction. Smad3-null mice show accelerated cutaneous wound healing compared with wild-type mice. In this work, we investigated the functions and the mechanism of Smad3-mediated TGF-beta signal on matrix metaloproteinase-2 (MMP-2) in mouse fibroblast. We found that MMP-2 at wound bed was expressed earlier in Smad3-null mice than that in wild type and heterozygotes. In the sera of wounding mice, the activity of MMP-2 was also remarkably higher in Smad3-null mice than that in the other two. The embryonic fibroblasts were separated from Smad3 knockout mice to test the function of Smad3 in modulating the expression of MMP-2. The results showed that the expression and activity of MMP-2 in Smad3-null fibroblasts were higher than those in wild type cells. TGF-beta1 could increase the MMP-2 activities in both Smad3 mutant and wild type fibroblasts. The expression and activity of MMP-2 were inhibited by over expression of SMAD3 in Smad3-null fibroblasts, while the expression and activity of MMP-2 were increased by over expression of anti-sense Smad3 in wild type cells. All these results showed that SMAD3 inhibited the expression of MMP-2 in mouse embryonic fibroblasts.

Animals↗

PubMed2005

[Development of a new recombineering system by gap repair].

Using lambda phage Red recombinase mediated in vivo homologous recombination system, a 6.7 kb lambda PL operon sequence including the Red encoding genes was subcloned into pBR322 by gap repair technique, and generated a pBR322-Red recombinant plasmid that can provide the Red recombination function and can be transferred into many kinds of bacteria. To confirm the recombination functions of pBR322-Red, a single-stranded 70-bases oligo was introduced into W3110 by electroporation to create a single base T-->G mutation in galK gene on the bacterial chromosome. The result demonstrated that a new lambda Red-mediated recombineering system based on pBR322-Red was successfully established.

Bacteriophage lambda↗

PubMed2005

[Effect of PC-1 gene expression on migration ability of prostate cancer cells].

BACKGROUND & OBJECTIVE: PC-1 gene is highly expressed in C4-2 cell line, an androgen-independent and aggressive prostate cancer cell line, but lowly expressed in LNCaP cell line, an androgen-dependent prostate cancer cell line. This study was to assess the contribution of PC-1 gene to migration of prostate cancer cells. METHODS: LNCaP cell line with stable expression of PC-1 gene and C4-2 cell line with antisense nucleotide-down-regulated PC-1 were established. The migration abilities of LNCaP and C4-2 cells were tested using an in vitro transwell invasion assay. RESULTS: Migration cell count of LNCaP cells with up-regulation of PC-1 gene was similar to that of control LNCaP cells(P>0.05); migration cell count was significantly lower in C4-2 cells with down-regulation of endogenous PC-1 gene than in control C4-2 cells (P<0.05). CONCLUSION: PC-1 may be involved in the invasion of prostate cancer cells.

Cell Movement↗

PubMed2005

[Construction of recombinant plasmid using Neo/E Technology].

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.

Bacteriophage lambda↗

PubMed2004

[Expression of XBP-1 in breast cancer cell lines and its role in ERalpha signaling].

Estrogen receptor (ERalpha) plays an important role in the development and progression of breast cancer. Several recent studies have demonstrated that expression of human X-box binding protein 1 (XBP-1) is associated with ERalpha status in breast tumors and overexpressed in a subset of breast tumors. XBP-1 has two splicing variants, which were designated as XBP-1S and XBP-1U, respectively. However, little is known about the expression pattern of XBP-1S and XBP-1U in breast cancer cells and about their roles in ERalpha signaling. In this study, the expression of two splicing forms of XBP-1 was detected in breast cancer cell lines with RT-PCR. Estrogen response element (ERE) -containing luciferase reporter assay was used to determine the effects of XBP-1S and XBP-1U on the transcription activity of ERalpha in MDA-MB-435 breast cancer cells. The result showed that both XBP-1S and XBP-1U enhanced the transcription activity of ERalpha in a hormone-independent and dose-dependent manner and the activity of of XBP-1S is higher than that of XBP-1U. Enhancement of ERE-containing luciferase reporter gene expression by XBP-1S and XBP-1U was dependent on ERalpha. These data suggest that XBP-1S and XBP-1U may play important roles in breast cancer growth and progression through ERalpha signaling.

Breast Neoplasms↗

PubMed2004

[Advance in researches on BRCA1].

BRCA1 is one of the most important breast cancer susceptibility genes. It plays key roles in DNA damage repair, cell cycle checkpoint regulation, gene transcription chromatin stability, and cell proliferation. In this paper, advance in basic researches on BRCA1 is reviewed, and the role of BRCA1 in cancer development and progression is discussed, that may facilitate potential clinical application of BRCA1.

Animals↗

PubMed2004

[Research on the structure of the PSA promoter and the mechanisms of its expression regulation].

Prostate-specific antigen (PSA) as a prostate cancer marker is a chymotrypsin-like serine protease which is expressed primarily by both normal prostate epithelium and the vast majority of prostate cancers. PSA expression is tightly regulated by androgen through the activation of androgen receptor. However, in the absence of androgens, PSA gene expression can become elevated. This suggests that either the AR can be activated in the absence of androgen to elevate PSA gene expression or that another transcription factor acting on the PSA promoter is stimulated. This article reviews the research on the structure of the PSA promoter and enhancer and the mechanisms of the PSA expression.

Androgens↗

PubMed2004

[XBP-1 interacts with estrogen receptor alpha (ERalpha)].

Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.

Breast Neoplasms↗

PubMed2003

[Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Shigella flexneri 2a].

Enterotoxigenic Escherichia coli (ETEC) causes watery dehydrating diarrhea in infants in developing countries, and is the most common cause of travelers diarrhea. It has been known that the colonazition factor antigens (CFAs) and enterotoxins are important virulence factors of ETEC, and these two kinds of proteins should be included in any effective vaccine against ETEC. In this study, a host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express CS3 antigens and the fusion LT-B/ST enterotoxins of Escherichia coli. Both of these antigens were expressed steadily in the S. flexneri vector without any antibiotic markers. Antibodies against CS3, LT, ST and LPS of Shigella were detected in sera of mice that were immunized with recombinant bacteria either oragastrically (o.g.) or intranasally (i.n.). SIgA against CS3 and enterotoxins were detected simultaneously in feces of mice. This work is helpful for constructing multivalent recombinant vaccine for prevention of bacterial diarrhea.

Animals↗

PubMed2003

[Mapping of FHL2 transcription activation domain].

FHL2, a member of LIM-only protein family, plays an important role in transcription regulation, apoptosis, cancer development and progression. In this study, a mammalian transcription activation system was constructed by using DNA binding domain(DBD) of GAL4 and luciferase reporter gene with DBD binding sequence, and used for mapping of FHL2 transcription activation domain. First, the coding sequence of GAL4-DBD was inserted into expression vector pcDNA3, generating the pDBD recombinant plasmid, then the wild-type FHL2 and its mutants were fused in-frame with GAL4-DBD, resulting in expression vectors for wild-type FHL2 and its mutants. All of the recombinant plasmids were transfected into 293T cells. Western blot assay showed that all of the fusion proteins were expressed. The analysis of FHL2 transcription activation properties by using the GAL4-luciferase reporter gene indicated that wild-type FHL2 had activation activity in both 293T and MCF-7 cells. The deletion of the half LIM domain at the N-terminus severely impaired the capacity of FHL2 to stimulate transcription. The mutant lacking the LIM domain at the C-terminus was totally inactive, while the deletion of two LIM domains at the C-terminus partially recovered its ability to stimulate transcription. The deletion of the second LIM domain at C-terminus did not alter the activation capacity of FHL2. These results suggest that the last LIM domain at the C-terminus of FHL2 is critical for its transcription activation function, the second LIM domain at the C-terminus may be a negative regulation region, but this negative regulation depends on the last LIM domain. Mapping of transcription activation domain of FHL2 lays solid basis for further study on various FHL2 functions.

Binding Sites↗

PubMed2003

[Establishment of keratinocyte-specific Cre recombinase transgenic mice].

A keratinocyte-specific Cre transgenic construct (pK5-Cre) containing the keratin 5 promoter, Cre recombinase gene and polyA of human growth hormone gene was generated. The 4.2 kb DNA fragment of K5-Cre-hGH was introduced into 720 fertilized zygotes by microinjection. 695 injected eggs were implanted into the oviduct of 29 female mice respectively, from which 48 off-spring were obtained. Twelve mice carrying the transgene were identified by genotyping, and the integration efficiency is 25%. The K5-Cre transgenic mice were crossed with Smad4 conditional gene targeting mice to check the tissue-specific expression of the Cre recombinase and the Cre mediated recombination in multiple tissues. The results showed that the Cre recombinase was expressed in skin tissue only and successfully mediated the recombination between the loxP sites in vivo.

Animals↗

PubMed2003

[XBP-1 enhances the transcriptional activity of estrogen receptor alpha].

The estrogen receptor (ERalpha) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that human X-box binding protein 1 (XBP-1) mRNA expression is associated with ERalpha status in breast tumors. More recently, two forms of XBP-1 were identified due to their unique splicing. The two splicing variants of XBP-1 were designated XBP-1S and XBP-1U, respectively. In this study, the coding sequences of XBP-1S and XBP-1U were cloned respectively into the expression vector pcDNA3 harboring FLAG epitope, generating the recombinant plasmids pcDNA3-FLAG-XBP-1S and pcDNA3-FLAG-XBP-1U. Western blot analysis showed that both XBP-1S and XBP-1U were expressed in mammalian cells. To determine the effects of XBP-1S and XBP-1U on the transcriptional activity of ERalpha, MDA-MB-231 breast cancer cells were cotransfected with the expression vectors for ERalpha and either pcDNA3-FLAG-XBP-1S or pcDNA3-FLAG-XBP-1U. The results indicated that XBP-1S and XBP-1U enhanced ERalpha-mediated transcriptional activities in a hormone-independent manner. GST pull-down assay showed that both XBP-1S and XBP-1U interacted with ERalpha. These data suggest that XBP-1S and XBP-1U may play an important role in breast cancer growth and progression through ERalpha signaling.

Alternative Splicing↗

PubMed2003

[Co-expression of CFA/I and CS6 of Enterotoxigenic Escherichia coli ( ETEC ) in Shigella flexneri 2a T32 Derivative Strain FWL01].

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS6 (the common antigen in the CFA/IV fimbrial antigens ) are two of the most prevalent fimbriae found in clinical isolates but are never expressed by the same wild-type strains. In this study, CFA/I and CS6 of ETEC were co-expressed in Shigella flexneri 2a T32 derivative strain FWL01 by using a host-plasmid lethal balancing system based on asd gene. The results indicate that the recombinant plasmid carrying CFA/I and CS6 could be stably integrated in FWL01. Expression of the two antigens did not interfere the host growth. The results of immunofluorescence analysis showed that CFA/I and CS6 were localized on the surface of the strain FWL01. In Balb/c mice orally immunized with the recombinant strain, the immune responses against CFA/I and CS6 were observed. Those observations show the feasibility of a multivalent vaccine expressing different fimbrial antigens in attenuated Shigella flexneri.

Animals↗

PubMed2003

[Recombineering and its application].

Driven by the need of functional genomics, a homologous recombination-based, highly efficient genetic engineering system that termed "recombineering" has recently been developed. Recombineering has been defined as a genetic engineering with phage-encoded recombination function that utilizes short homologies, a convenient term to describe homologous-dependent, recombination-mediated, genetic engineering. The bacteriophage lambda Red recombination system has critical differences from standard E. coli RecA-dependent recombination pathway. The phage systems have unique advantage in that they can catalyze efficient recombination with very short regions of sequence homology (< 50 bp). Recombineering does not require construction of plasmid or phage DNA intermediates containing the appropriately pre-engineered homology segment. All that is required in vitro is the synthesis of standard oligonucleotides or construction of PCR products that provide the homology. Importantly, they function even in the absence of RecA. These approaches do not rely on the presence of suitable restriction site, and can be used to insert, delete, clone or substitute genomic DNA sequences at any desired position on a target molecule in Escherichia Coli. Recombineering also facilitates many kinds of genomic experiments difficult to be carried out. In this article, the bacteriophage lambda Red recombinase system, the progression and applications of this powerful new technique are reviewed according to the data published recently.

Bacteriophage lambda↗