Biomedical subjects

Yin Gao

Publications and source records attributed to Yin Gao.

17 recordsLinked to original sources

PubMed2007

Identification of a new AT-rich-element binding factor PsATF1 and its combined effect with PsGBF on the activation of PsCHS1 promoter.

Chaltone synthase (CHS) is a key speed-limiting enzyme in the phenylpropanoid pathway which plays an important role in plant defense response against pathogens. In the PsCHS1 promoter, there is an AT-rich element (ATRE) which is required for the maximal elicitor-mediated activation. However, the transcription activator of the ATRE and its regulation mechanism in pea keep unclear. In this paper, a new ATRE-binding factor was isolated from an elicitor-induced pea cDNA expression library and was designated as PsATF1. Electrophoretic mobility shift assay (EMSA) indicated the ATRE-specific binding activity of PsATF1. Beta-galactosidase assays in yeast cells suggested that PsATF1 possessed transcription-activating activity because PsATF1 activated the expression of the reporter gene even without the GAL4 activation domain (AD). The current study also examined the co-activation effects of PsATF1 with another transcription factor PsGBF on ATRE or PsCHS1 promoter through a transient expression system. The present work reports that PsATF1 acts as a complete transcription activator and first indicates that there are combined effects of PsATF1 with PsGBF on the activation of PsCHS1 promoter. These results provide theoretical basis to the plant defense gene expression mechanism regulated by multiple activators.

AT Rich Sequence↗

PubMed2006

Identification of a bHLH-type G-box binding factor and its regulation activity with G-box and Box I elements of the PsCHS1 promoter.

With the use of in vivo recombination theory, the screening time of yeast one-hybrid system was decreased in the present study. A basic helix-loop-helix (bHLH) protein PsGBF was successfully obtained from a glutathione (GSH)-induced pea cDNA library using the G-box cis-element of the PsCHS1 promoter as a bait. Electrophoretic mobility shift assay (EMSA) and beta-galactosidase assay results suggested that PsGBF possesses both G-box-specific binding and transcription-activating activities. The specific interaction of PsGBF with G-box was further confirmed by in vivo transient expression assays in tobacco. The current study examined the combination effect of G-box with Box I elements in the interaction with PsGBF or OsMYC. The results indicated that PsGBF bound with the G-box, but not the Box I element. Moreover, this combination effect of G-box and Box I only associated with PsGBF but not with other bHLH-type proteins such as OsMYC.

Acyltransferases↗

PubMed2006

Retinoic acid-induced CD38 antigen promotes leukemia cells attachment and interferon-gamma/interleukin-1beta-dependent apoptosis of endothelial cells: implications in the etiology of retinoic acid syndrome.

All-trans retinoic acid (RA) treatment of patients with acute promyelocytic leukemia (APL) induces complete remission in more than 90% of the cases. Although RA therapy is well tolerated, about 25% of APL patients develop a potentially fatal condition called retinoic acid syndrome (RAS). Molecular mechanisms underlying the development of RAS pathogenesis, especially those that result in the damage of endothelial cells remain elusive. In the present study, we found that RA treatment induces the expression of interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta) in peripheral blast cells from APL patients. IFN-gamma and IL-1beta also exerted synergistic effect in driving human umbilical cord endothelial cells (HUVECs) and human lung microvascular endothelial cells (HLMVECs) into apoptosis. RA also upregulated the expression of CD38, an ectoenzyme responsible for the generation of the calcium messenger cyclic ADP-ribose. Importantly, RA-induced CD38 expression promoted strong attachment of leukemia cells to endothelial cells, and incubation of endothelial cells with either high concentration (100 ng/ml) of IFN-gamma alone or low concentration of IL-1beta and IFN-gamma (10 ng/ml, each) induced strong apoptotic responses as revealed by caspase-8 activation and DNA fragmentation. Our results suggest that these RA-induced events could contribute to the development of RAS pathogenesis in patients with APL.

ADP-ribosyl Cyclase 1↗

PubMed2006

N-linked glycosylation of CD38 is required for its structure stabilization but not for membrane localization.

CD38 is a type II transmembrane protein with 25% of its molecular mass consisting of glycosyl moieties. It has long been predicted that the carbohydrate moieties of glycoproteins play important roles in the physical function and structural stability of the proteins on cell surfaces. To determine the structural/functional significance of glycosylation of the human CD38, the four potential N-linked glycosylation sites asparagine residues, N100, N164, N209 and N219 were mutated. The mutant (CD38mu) and wild-type (CD38wt) were expressed separately in Escherichia coli, HeLa, and MCF-7 cells. SDS-polyacrylamide gel electrophoresis under reducing conditions and western blotting indicated that the molecular mass of the CD38wt is 45 kDa, and that of the CD38mu is 34 kDa in HeLa cells. Importantly, the CD38mu protein expressed in HeLa cells, showed the high molecular weight oligomers in addition to the 34 kDa monomeric form. Similarly, in E. coli, the CD38wt formed dimers and other oligomers besides the monomeric form. Moreover, MCF-7 cells stably transfected with CD38wt cDNA, also revealed the presence of cross-linked oligomers when treated with a N-linked glycosylation inhibitor tunicamycin (TM). These results suggested that the N-linked glycosylation of CD38 plays a crucial role in the structure stability by preventing the formation inter-molecular cross-links. In addition, immunostaining, enzyme activity (cyclase), and western blotting data revealed that the glycosylation of human CD38 protein is not required for its localization to the cell membrane.

ADP-ribosyl Cyclase 1↗

PubMed2006

Regulation of replication protein A functions in DNA mismatch repair by phosphorylation.

Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase delta-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR.

Base Pair Mismatch↗

PubMed2006

A novel bacterium Saprospira sp. strain PdY3 forms bundles and lyses cyanobacteria.

A helical filamentous cyanobactericidal bacterium was isolated from Dianchi Lake, a eutrophic freshwater lake in Kunming City of the Yunnan Province in China using a special solid medium. This species was designated strain PdY3. This bacterium was identified as a novel Saprospira sp. on the basis of its morphological characteristics and 16S rDNA sequence. Strain PdY3 showed apparent group behavior on the solid medium, forming orderly, bundle-like group structures. These bundles moved as groups. Individuals in a bundle responded to the bundle as a whole. PdY3 also showed group behavior and formed a three-dimensional reticular structure when co-cultured with Anabaena in liquid media. This helical bacterium lysed cyanobacteria through direct contact and its group behavior greatly accelerated the cyanobactericidal process. Our experiments showed that PdY3 caused lysis of 64% of Anabaena cells within 1 day and that its cyanobactericidal range was broad. These results underscore potential application of Saprospira on the control of blooms of cyanobacteria. PdY3 group behavior might allow a more efficient capture of bacterial prey.

Anabaena↗

PubMed2006

Y55 and D78 are crucial amino acid residues of a new IgE epitope on trichosanthin.

Trichosanthin (TCS) possesses many biological and pharmaceutical activities, but its strong immunogenicity limits its clinical application. To reduce the immunogenicity of TCS, we modified the reported method for the prediction of antigenic site and identified two crucial amino acid residues (Y55 and D78) for a new epitope. We mutated these two residues into glycine and serine, respectively, and obtained three mutants, Y55G, D78S, and Y55G/D78S. These mutants induced less amount of Ig and IgG antibodies in C57BL/6J mice than wild-type TCS (wTCS) (p<0.01) and almost lost the ability to induce IgE antibody production. The mutants stimulated fewer TCS-specific B cells in C57BL/6J mice than wTCS (p<0.01). Compared with wTCS, Y55G, D78S, and Y55G/D78S lost 26.9%, 17.9%, and 98.7% specific binding ability to anti-TCS monoclonal antibody TCS4E9, respectively. These mutants still retained RNA N-glycosidase activity. In conclusion, Y55 and D78 are two crucial amino acid residues of a new IgE epitope on TCS, and their mutation reduces the immunogenicity of TCS, but still retained the enzymatic activity.

Amino Acid Substitution↗

PubMed2005

Reconstitution of 5'-directed human mismatch repair in a purified system.

This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.

5' Flanking Region↗

PubMed2005

Enhanced green fluorescence protein tracks trichosanthin in human choriocarcinoma cells as a feasible and stable reporter.

Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) which can inhibit the growth of human choriocarcinoma (JAR) cells. There are no clear mechanisms to discover the interaction pathway and cytotoxicity of TCS in JAR cells. In this paper, we showed the distribution and transport of endogenously expressed TCS in JAR cells. Enhanced Green Fluorescence Protein (EGFP), fused with TCS, was applied as a reporter to track the behavior of TCS in JAR cells. Firstly, we investigated the expression stability of EGFP and physiological effects on JAR cells. A stable cell line expressing EGFP was created, which could reproduce and express EGFP even if transplanted into nude mice. Based on the proved stability and feasibility of EGFP in cultured cells and in vivo, the fusion gene of EGFP and TCS was constructed and transfected into JAR cells by liposome. The fluorescence microscopy showed that TCS-EGFP fusion gene was expressed in JAR cells in 24 to 48 hours and the fluorescence spread in cytoplasm mainly and in nucleus partially, which could trace the distribution and transport of TCS-EGFP in JAR cells. Most of fluorescent cells died after 48 hours for the cytotoxicity of expressed TCS-EGFP. These results first reported a stable expression and tracing method by EGFP in JAR cells, and provided theoretical basis to apply TCS in cancer therapy.

Animals↗

PubMed2005

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking.

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem-loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.

Arabidopsis↗

PubMed2005

Trichomislin, a novel ribosome-inactivating protein, induces apoptosis that involves mitochondria and caspase-3.

Trichomislin, a novel ribosome-inactivating protein, was cloned from the genome of Trichosanthes kirilowii Maxim. The gene was recombined to prokaryotic expression vector and the protein was purified by cation-exchange chromatography. The secondary structure of trichomislin was measured by circular-dichroism analysis and the ratios of alpha-helices and beta-sheets were calculated. Trichomislin could inhibit the synthesis of protein in rabbit reticulocyte lysate systems and its reaction mechanism was to inactivate ribosome as an rRNA N-glycosidase. Antitumor analyses indicated trichomislin induced the apoptosis and inhibited the growth of choriocarcinoma cells. Further investigation showed that trichomislin could bind to and enter choriocarcinoma cells, and then increase the caspase-3 activity in a time-dependent manner. At the same time, the concentration of cytochrome c in cytosol increased while that in mitochondria decreased. These results suggested that trichomislin induced apoptosis by releasing cytochrome c from mitochondria which then triggered the caspase family member activation.

Amino Acid Sequence↗

PubMed2005

Expression and purification of Huwentoxin-I in baculovirus system.

Huwentoxin-I (HWTX-I) is a novel neurotoxin isolated from the venom of Orinithoctonus huwena. Based on its biological activity, HWTX-I could be developed as a pain-killer for clinical purpose. Production of HWTX-I by the bacterium or yeast expression systems resulted in poor yields and the purified protein was proved to have lower biological activity than that of native one. So, for the first time, we introduced a new method to express HWTX-I gene in Sf9 cells using baculovirus expression system. Recombinant HWTX-I was recognized by Western blotting and then purified by nickel-chelating affinity chromatography under native conditions. Recombinant HWTX-I showed identical amino acid sequence as native form and exhibited similar effect on muscular transmission with that of native form. These results indicate that the baculovirus expression system and native purification strategy are viable ways to produce active HWTX-I.

Animals↗

PubMed2005

hMRE11 deficiency leads to microsatellite instability and defective DNA mismatch repair.

DNA mismatch repair (MMR) is essential in the surveillance of accurate transmission of genetic information, and defects in this pathway lead to microsatellite instability and hereditary nonpolyposis colorectal cancer (HNPCC). Our previous study raised the possibility that hMRE11 might be involved in MMR through physical interaction with hMLH1. Here, we show that hMRE11 deficiency leads to significant increase in MSI for both mono- and dinucleotide sequences. Furthermore, RNA-interference-mediated hMRE11-knockdown in HeLa cells results in MMR deficiency. Analysis of seven HNPCC-associated hMLH1 missense mutations located within the hMRE11-interacting domain shows that four mutations (L574P, K618T, R659P and A681T) cause near-complete disruption of the interaction between hMRE11 and hMLH1, and two mutations (Q542L and L582V) cause a 30% reduction of protein interaction. These findings indicate that hMRE11 represents a functional component of the MMR pathway and the disruption of hMLH1-hMRE11 interaction could be an alternative molecular explanation for hMLH1 mutations in a subset of HNPCC tumours.

Adaptor Proteins, Signal Transducing↗

PubMed2004

[Selection of surgical procedure for the treatment of idiopathic scoliosis].

OBJECTIVES: To discuss and evaluate the selection of surgical procedure for the treatment of idiopathic scoliosis according to the location and degree of the deformity. METHODS: 175 patients with idiopathic scoliosis underwent surgical treatment with correction and fusion. The patients were divided into four groups according to the location and degree of the deformity and four different procedures were used for each group. For each group, the blood loss, surgery time, correction rate, loss of correction at final follow up and complications were compared and analyzed. RESULTS: All patients underwent surgery safely and no neurological complication occurred. The correction rate was 81% for Group I, 86% for Group II, 68% for Group III and 72% for Group IV. All patients were followed up at least 2 years and the average time was 38 months (24 approximately 52). CONCLUSION: Proper selection of surgical procedure according to the location and degree of the scoliotic deformity, satisfactory results can be achieved in the treatment of idiopathic scoliosis.

Adolescent↗

PubMed2003

A novel antimicrobial protein isolated from potato (Solanum tuberosum) shares homology with an acid phosphatase.

The nucleotide and amino acids sequences for AP(1) will appear in the GenBank(R) and NCBI databases under accession number AY297449. A novel antimicrobial protein (AP(1)) was purified from leaves of the potato ( Solanum tuberosum, variety MS-42.3) with a procedure involving ammonium sulphate fractionation, molecular sieve chromatography with Sephacryl S-200 and hydrophobic chromatography with Butyl-Sepharose using a FPLC system. The inhibition spectrum investigation showed that AP(1) had good inhibition activity against five different strains of Ralstonia solanacearum from potato or other crops, and two fungal pathogens, Rhizoctonia solani and Alternaria solani from potato. The full-length cDNA encoding AP(1) has been successfully cloned by screening a cDNA expression library of potato with an anti-AP(1) antibody and RACE (rapid amplification of cDNA ends) PCR. Determination of the nucleotide sequences revealed the presence of an open reading frame encoding 343 amino acids. At the C-terminus of AP(1) there is an ATP-binding domain, and the N-terminus exhibits 58% identity with an/the acid phosphatase from Mesorhizobium loti. SDS/PAGE and Western blotting analysis suggested that the AP(1) gene can be successfully expressed in Escherichia coli and recognized by an antibody against AP(1). Also the expressed protein showed an inhibition activity the same as original AP(1) protein isolated from potato. We suggest that AP(1) most likely belongs to a new group of proteins with antimicrobial characteristics in vitro and functions in relation to phosphorylation and energy metabolism of plants.

Acid Phosphatase↗

PubMed2002

[Expression of cre gene in Escherichia coli and bioassay its expression product].

The Cre recombinase from bacteriophage P1 can recognize specific DNA sequences, cleave DNA at specific target sites, and then ligate it to the cleaved DNA of a second site. In this study, cre gene was cloned into the pGEM-T Easy vector via PCR procedure. Then the cre gene was inserted into an expression vector pET-29a and expressed in E. coli BL21 (DE3). A 38 kD soluble protein was expressed and named CRE. CRE was purified by DEAE-52 chromatography. Bioassay of the partially purified product showed that CRE can cleave the plasmid pGLGFP which contains two loxP site with the same direction.

Chromatography, DEAE-Cellulose↗

PubMed2002

[Progress in the study of the structure and function of Cre recombinase].

The Cre recombinase, an integrase from bacteriophage P1, catalyzes site-specific recombination between 34-bp repeats termed loxP sites, in the absence of any additional cofactors and energy. Mediated by Cre recombinase, specific DNA fragments can be excised, inversed or integrated depending on the orientation or position of loxP sites in vitro or in vivo. Because of its simplicity and high efficiency, Cre/loxP site-specific recombination system has been widely used in gene deletion and function identification, gene site-specific integration, gene trapping and chromosome engineering. It has been used as a useful tool for DNA recombination in transgenic yeast, plants, insects and mammals. Here progress in the study of the structure and function of Cre recombinase is discussed.

Integrases↗