Biomedical subjects

D Chen

Publications and source records attributed to D Chen.

At least 91 records · Page 5Linked to original sources

PubMed2001

Microminiaturized immunoassays using quantum dots as fluorescent label by laser confocal scanning fluorescence detection.

An immunoassay readout method based on fluorescent imaging analysis with laser confocal scanning is described. The ZnS-coated CdSe quantum dots (ZnS/CdSe QDs) were linked to a detection antibody. Immunoassay was carried out on a glass chip using a sandwich assay approach, where antibody covalently bound to a glass chip was allowed to capture antigen specially. Afterwards, the detection antibody labeled with QD was allowed to bind selectively to the captured antigen. The fluorescent signals of the sandwich conjugate were detected by a laser confocal scanner. A diode laser was used to excite efficiently the fluorescent signals while bovine serum albumin was used to eliminate nonspecific binding sites. The detection limit of this approach was up to 10(-9) M under current experimental conditions. The specificity of the QDs-labeled immunoglobulin (IgG) was tested by an experiment using goat IgG and human IgG samples. The result was consistent with the binding specificity in a sandwich-type assay. The potential of this method to function as a simple and efficient readout strategy for immunoassay in biochip is discussed.

Animals↗

PubMed2001

Transferrin receptor-mediated gene transfer to the corneal endothelium.

BACKGROUND: The application of gene therapy to prevent allograft rejection requires the development of noninflammatory vectors. We have therefore investigated the use of a nonviral system, transferrin-mediated lipofection, to transfer genes into the cornea with the aim of preventing corneal graft rejection. METHODS: Rabbit and human corneas were cultured ex vivo and transfected with either lipofection alone or in conjunction with transferrin. The efficiency of transfection, localization, and kinetics of marker gene expression were determined. Strategies to increase gene expression, using chloroquine and EDTA, were investigated. In addition to a marker gene, a gene construct encoding viral interleukin 10 (vIL-10) was transfected and its functional effects were examined in vitro. RESULTS: Transferrin, liposome, and DNA were demonstrated to interact with each other, forming a complex. This complex was found to deliver genes selectively to the endothelium of corneas resulting in gene expression. Treatment of corneas with chloroquine and EDTA increased the transfection efficiency eight-fold and threefold, respectively. We also demonstrated that constructs encoding vIL-10 could be delivered to the endothelium. Secreted vIL-10 was shown to be functionally active by inhibition of a mixed lymphocyte reaction. CONCLUSIONS: Our data indicate that transferrin-mediated lipofection is a comparatively efficient nonviral method for delivering genes to the corneal endothelium. Its potential for use in preventing graft rejection is shown by the ability of this system to induce vIL-10 expression at secreted levels high enough to be functional.

Animals↗

PubMed2001

Multimers of the bluetongue virus nonstructural protein, NS2, possess nucleotidyl phosphatase activity: similarities between NS2 and rotavirus NSP2.

The nonstructural protein, NS2, of bluetongue virus is a nonspecific single- stranded RNA-binding protein that forms large homomultimers and accumulates in viral inclusion bodies of infected cells. NS2 shares these features with the nonstructural protein, NSP2, of rotavirus, which like BTV is a member of the family Reoviridae. Recently, NSP2 was shown to have an NTPase activity and an autokinase activity that catalyzed its phosphorylation in vitro. To examine NS2 for similar enzymatic activities, the protein was expressed in bacteria with a C-terminal His-tag and purified to homogeneity. Recombinant (r)NS2 possessed nonspecific RNA-binding activity and formed 8-10S homomultimers of the same approximate size as rNSP2 homomultimers. Notably, enzymatic assays performed with rNS2 showed that the protein hydrolyzed the alpha, beta, and gamma phosphodiester bonds of all four NTPs. Therefore, rNS2 possesses a nucleotidyl phosphatase activity instead of the NTPase activity of NSP2, which only hydrolyzes the gamma phosphodiester bonds of NTPs. NS2 did not exhibit any autokinase activity in vitro, unlike NSP2. However, both NS2 and NSP2 were phosphorylated in vitro by cellular kinases. Although the nature of the enzymatic activities differs significantly, the fact that both NS2 and NSP2 hydrolyze NTPs, undergo phosphorylation, bind RNA, and assemble into multimers consisting of 6 +/- 2 subunits suggests that they are functional homologs.

Adenosine Triphosphate↗

PubMed2001

Palladium film decoupler for amperometric detection in electrophoresis chips.

Demonstrated in this article is that a palladium metal film can be applied to decouple the electric circuitry of electrochemical detection from that of the electrophoretic separation in an electrophoresis chip. The Pd solid-state field decoupler, as well as the working electrodes, is thermally evaporated onto the plastic chip and oriented vertically across the separation channel. After the sample zones flow over the Pd decoupler, their electrochemical response is measured at working electrodes in the downstream pathway. Because the electrodes are on the separation channel, the electrode channel alignment is no longer a problem. For a separation channel of roughly 200 microm in width and 75 microm in depth in 10 mM phosphate (pH 5.1), the noise level at the working electrode is < 15 pA at an electric field of 570 V/cm.

Journal Article↗

PubMed2001

[Molecular cloning of the tumor associated antigen recognized by monoclonal antibody 3H11].

OBJECTIVE: Monoclonal antibody (McAb) 3H11 can specifically bind to different cancer cells from different tissues. Meanwhile, McAb 3H11 labeled with radioactive isotopes has been successfully applied to detect primary cancer and metastatic cancer in clinical imaging. Molecular cloning of the antigen recognized by McAb 3H11 will be significant for understanding tumor development. METHODS: Based on the fragment of the McAb 3H11 antigen cloned by Immunoscreening MGC803 cDNA expression library, the whole length of the cDNA molecule was gotten by RACE and nested PCR. RESULTS: Sequence data of the cDNA molecule indicates that there was no homologous gene in GenBank. Northern blot experiments showed that mRNA of McAb 3H11 antigen extensively distributes in different cancer cells and tissues but not in corresponding normal tissues. Moreover in producing antibodies of the antigen expressed prokaryotically, it was found that the immunogenicity of the antigen was quite low in mammalian. CONCLUSION: The antigen recognized by McAb 3H11 acts as a regulator in embryo cells and regains expression in tumor cells; and it may be associated with low differentiation and high proliferation.

Animals↗

PubMed2001

A DNA vaccine encoding a peptide mimic of Streptococcus pneumoniae serotype 4 capsular polysaccharide induces specific anti-carbohydrate antibodies in Balb/c mice.

Streptococcus pneumoniae is a leading cause of morbidity and mortality in both the developing and developed world. The T-independent nature of the current polysaccharide vaccine renders it ineffective in elderly adults and children <2 years of age. Although the recently licensed conjugate vaccines are capable of producing T-cell-dependent immunity, they also have their limitations, namely a lack of response in certain populations. Our laboratory has focused on a different approach, DNA vaccination. We have defined a peptide sequence (pep4) that mimics the S. pneumoniae serotype 4 capsular polysaccharide (PPS4) using a monoclonal antibody to PPS4 (mAb4) and phage display library. Pep4 was synthesized, complexed to proteosomes and used to immunize mice. We have shown by ELISA that this peptide mimic is capable of eliciting an anti-PPS4 immune response significantly higher than in negative control mice (P<0.05). A PPS4-DNA vaccine was made by cloning an oligodeoxynucleotide encoding pep4 into the HBcAg vector. Following epidermal immunization with the PPS4-DNA vaccine, mice produced an anti-PPS4 antibody response significantly higher than in mice immunized with an impertinent DNA vector (P<0.05). Our results demonstrate the feasibility of peptide mimicry in DNA vaccine development.

Amino Acid Sequence↗

PubMed2001

Gaf-1, a gamma -SNAP-binding protein associated with the mitochondria.

The role of alpha/beta-SNAP (Soluble NSF Attachment Protein) in vesicular trafficking is well established; however, the function of the ubiquitously expressed gamma-SNAP remains unclear. To further characterize the cellular role of this enigmatic protein, a two-hybrid screen was used to identify new, gamma-SNAP-binding proteins and to uncover potentially novel functions for gamma-SNAP. One such SNAP-binding protein, termed Gaf-1 (gamma-SNAP associate factor-1) specifically binds gamma- but not alpha-SNAP. The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically associated with gamma-SNAP in cellular extracts. This binding is distinct from other SNAP interactions since no alpha-SNAP or NSF coprecipitated with Gaf-1. Subcellular fractionation and immunofluorescence analysis show that Gaf-1 is peripherally associated with the outer mitochondrial membrane. Only a fraction of gamma-SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions. GFP-gamma-SNAP and the C-terminal domain of Gaf-1 both show a reticular distribution in HEK-293 cells. This reticular structure colocalizes with Gaf-1 and mitochondria as well as with microtubules but not with other cytoskeletal elements. These data identify a class of gamma-SNAP interactions that is distinct from other members of the SNAP family and point to a potential role for gamma-SNAP in mitochondrial dynamics.

Adaptor Proteins, Signal Transducing↗

PubMed2001

[Prediction of prognosis of patients with multiple organ dysfunction syndrome by sepsis-related organ failure assessment].

OBJECTIVE: To describe the clinical properties of multiple organ dysfunction syndromB (MODS) with sepsis-related organ failure assessment (SOFA). METHODS: A total of 366 MODS patients admitted to the 5 participating ICUs from 1990 to 1996 were included in this study. SOFA score and demographic data were retrospectively evaluated. RESULTS: Total maximum SOFA score, delta SOFA and admission total SOFA score exhibited a good correlation with hospital mortality rate, with the area under the ROC curve of 0.819, 0.750 and 0.616, respectively. The SOFA score for patients without organ failure was 3.7 +/- 1.2, with a mortality rate of 21.7%, and the SOFA score for patients with failure of all 6 organs was 20.2 +/- 1.4, with a mortality rate of 77.8%. A maximum score was firstly reached for respiratory system (1.41 days after admission), and last for cardiovascular system (4.89 days). Logistic regression model revealed that the central nervous system was associated with the highest relative contribution (odds ratio 1.75) to hospital outcome, followed by renal (OR 1.42), cardiovascular (OR 1.38), coagulation (OR 1.34) and respiratory (OR 1.17) systems. No independent contribution was found for the hepatic score (OR 0.99). CONCLUSION: Total maximum SOFA score and delta SOFA can be used to quantify the progress of MODS during ICU stay.

Adult↗

PubMed2001

Identification of lysine 346 as a functionally important residue for pyridoxal 5'-phosphate binding and catalysis in lysine 2, 3-aminomutase from Bacillus subtilis.

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains pyridoxal 5'-phosphate (PLP) and a [4Fe-4S] center and requires S-adenosylmethionine (SAM) for activity. The hydrogen transfer is mediated by the 5'-deoxyadenosyl radical generated in a reaction of the iron-sulfur cluster with SAM. PLP facilitates the radical rearrangement by forming a lysine-PLP aldimine, in which the imine group participates in the isomerization mechanism. We here report the identification of lysine 346 as important for PLP binding and catalysis. Reduction of LAM with NaBH(4) rapidly inactivated the enzyme with concomitant UV/visible spectrum changes characteristic of reduction of an aldimine formed between PLP and lysine. Following reduction with NaBH(4) and proteolysis with trypsin, a single phosphopyridoxyl peptide of 36 amino acid residues was identified by reverse-phase liquid chromatography/mass spectrometry (LC/MS). The purified phosphopyridoxyl peptide exhibited an absorption band at 325 nm, and its identity was further confirmed by tandem mass spectrometry (MS/MS) sequencing. The bound PLP is linked to lysine 346 in a PGGGGK (PLP) structure. The sequence of this binding motif is conserved in LAMs from Bacillus and Clostridium and other homologous proteins but is distinct from the PLP-binding motifs found in other PLP enzymes. The function of lysine 346 was further studied by site-directed mutagenesis. The purified K346Q mutant was inactive, and its content of PLP was only approximately 15% of that of the wild-type enzyme. The data indicate that the formation of the aldimine linkage between lysine 346 and PLP is important for LAM catalysis. Sequences similar to the PLP-binding motifs in other enzymes were also present in LAM. However, lysine residues within these motifs neither are the PLP-binding sites in LAM nor are directly involved in LAM catalysis. This study represents the first comprehensive investigation of PLP binding in a SAM-dependent iron-sulfur enzyme.

Bacillus subtilis↗

PubMed2001

Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11.

Monoclonal antibody (MAb) 3H11 can bind specifically to different cancer cells from different tissues. MAb 3H11 labeled with radioactive isotopes has been used clinically to detect primary cancer and metastatic cancer. Molecular cloning of the antigen recognized by MAb 3H11 is important in studying tumor occurrence and in developing new biotherapy for cancer. Using MAb 3H11, we screened cDNA library made from the human gastric cancer cell line MGC 803, which reacts with MAb 3H11, and isolated one positive clone specifically recognized by the antibody. The insert cDNA fragment was 0.5 kb. After recombining with glutathione-S-transferase expression vector pGEX-4T, the cDNA fragment could be expressed into a fusion protein that specifically reacted with MAb 3H11. Moreover, the fusion protein could competitively inhibit MAb 3H11 binding to MGC 803 cells. Based on the nucleotide sequence of the cDNA fragment, the full length of the cDNA (2156 bp) was obtained by Rapid-Amplification-cDNA-End (RACE) and nested PCR. Its reading frame was 1767 bp encoding a protein of 589 amino acids. Sequence analysis indicated that there is no highly homologous gene in the GenBank. Northern blot and RT-PCR showed that the mRNA of MAb 3H11 antigen was extensively distributed in embryonic tissue and in different cancerous tissues, but not in corresponding normal tissues. Moreover, in producing antibodies to the antigen expressed prokaryotically, we found that the immunogenicity of the antigen was low in mammalian. Thus we believe that this novel antigen acts as an expression regulator in embryo cells and regains expression in tumor cells. In addition, this antigen is characterized by low differentiation and high proliferation. Molecular function of the antigen needs to be investigated.

Amino Acid Sequence↗

PubMed2001

Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities.

Nuclear receptors (NRs) activate gene transcription by binding to specific enhancer elements and recruiting coactivators of the p160 family to promoters of target genes. The p160 coactivators in turn enhance transcription by recruiting secondary coactivators, including histone acetyltransferases such as CREB-binding protein (CBP) and p300/CBP-associated factor (p/CAF), as well as the recently identified protein methyltransferase, coactivator-associated arginine methyltransferase 1 (CARM1). In the current study, protein arginine methyltransferase 1 (PRMT1), another arginine-specific protein methyltransferase that shares a region of high homology with CARM1, was also found to act as a coactivator for NRs. PRMT1, like CARM1, bound to the C-terminal AD2 activation domain of p160 coactivators and thereby enhanced the activity of NRs in transient transfection assays. The shape of the graphs of reporter gene activity versus the amounts of CARM1 or PRMT1 expression vector indicated a cooperative relationship between coactivator concentration and activity. Moreover, CARM1 and PRMT1 acted in a synergistic manner to enhance reporter gene activation by both hormone-dependent and orphan NRs. The synergy was most evident at low levels of transfected NR expression vectors, where activation of reporter genes was almost completely dependent on the presence of NR and all three exogenously supplied coactivators, i.e. GRIP1, CARM1, and PRMT1. In contrast, with the higher levels of NR expression vectors typically used in transient transfection assays, NR activity was much less dependent on the combination of coactivators, suggesting that target gene activation occurs by different mechanisms at high versus low cellular concentrations of NR. Because multiple coactivators are presumably required to mediate transcriptional activation of native genes in vivo, the low-NR conditions may provide a more physiologically relevant assay for coactivator function.

Animals↗

PubMed2001

Heat shock protein 70 moderately enhances peptide binding and transport by the transporter associated with antigen processing.

Hsp70 molecules are capable of binding antigenic peptides and eliciting CTL responses to the bound peptide. However, the precise mechanism for the induction of CTL has not been determined. One possibility is that hsp molecules can directly shuttle peptides in the MHC class I antigen processing and presentation pathway, as previously postulated. Here, we have addressed this issue by testing the effect of purified hsp70 molecules on peptide binding and transport by the transporter associated with antigen processing (TAP). Our results indicate that purified hsp70 molecules moderately enhance TAP function. In addition, we detect a physical association between hsp70 molecules and TAP, as well as the homologous drug transporter P-glycoprotein. We conclude that while hsp70 molecules may not be directly involved in the delivery of peptide to TAP, they may play an important role in TAP transport by binding to TAP and promoting its function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Removal of the circumsporozoite protein (CSP) glycosylphosphatidylinositol signal sequence from a CSP DNA vaccine enhances induction of CSP-specific Th2 type immune responses and improvesprotection against malaria infection.

The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence had a profound effect on induction and efficacy of CSP-specific immune response after genetic immunization of BALB/c mice with a gene gun. The CSP produced from the plasmid lacking the GPI anchor signal sequence (CSP-A) was secreted and soluble, but that produced by the CSP+A plasmid was not. The CSP-A plasmid induced a highly polarized Th2 type response, in which the CSP-specific IgG antibody titer was three- to fourfold higher, and the protective effect was significantly greater than that induced by the CSP+A plasmid. Thus, these two physical forms of CSP induced quantitatively and qualitatively different immune responses that also differed in protective efficacy. Engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines.

Animals↗

PubMed2001

Vaccination with allogeneic dendritic cells fused to carcinoma cells induces antitumor immunity in MUC1 transgenic mice.

Fusions of autologous tumor cells with allogeneic dendritic cells (DC) represent an approach for the induction of antitumor immunity. In the present studies, we investigated the antitumor effects of vaccinating MUC1-transgenic (MUC1.Tg) mice with MC38/MUC1 carcinoma cells fused to allogeneic DC from BALB/c mice (allo-DC, H-2(d)) or syngeneic DC from C57BL/6 mice (syn-DC, H-2(b)). Both allo and syn fusion cells (FC/MUC1) expressed MHC class II, costimulatory molecules, and the MUC1 antigen. Allo-FC/MUC1 exhibited dual expression of MHC class I haplotypes (H-2(d)/H-2(b))and MUC1 antigen. By contrast, only H-2(b) and MUC1 antigen were expressed by syn-FC/MUC1. CTLs from MUC1.Tg mice immunized with allo- or syn-FC/MUC1 fusion cells lysed MC38/MUC1 targets. Moreover, immunization with allo- or syn-FC/MUC1 was effective in eliminating established MUC1-positive pulmonary metastases in MUC1.Tg mice. These results indicate that immunization of MUC1.Tg mice with syn- or allo-FC/MUC1 is effective in reversing immunologic unresponsiveness to MUC1 antigen and inducing immunity against MUC1-positive tumors. The findings in the present study have broader clinical implications for fusion cell vaccines.

Animals↗

PubMed2001

Cloning, expression, purification, and characterization of rat MMP-12.

Macrophage metalloelastase (MMP-12) is implicated in the pathology of many diseases such as emphysema, aortic lesions and cancer. Recently, MMP-12 was cloned and purified from mouse and human macrophages. We report here the expression of the full-length and catalytic domain of rat MMP-12 in Escherichia coli and characterization of the purified enzyme. Inclusion bodies of expressed rat MMP-12 catalytic domain were denatured and refolded using a new method, and then affinity purified to near homogeneity with zinc-chelating Sepharose. The purified rat MMP-12 catalytic domain was highly active in digesting substrates, having a K(m) of 12 microM and optimal pH of 7.5--8.5. During investigation of natural substrate specificity, we found that rat MMP-12 catalytic domain was able to completely degrade collagen-V, partially degrade collagen-I, but it was unable to digest collagen-IV. The enzyme could also degrade osteonectin, vitronectin, and fibronectin, but not laminin and albumin. The catalytic properties and natural substrate specificity of rat MMP-12 catalytic domain differed from those of human MMP-12 catalytic domain.

Animals↗

PubMed2001

A modified method for preparation of adriamycin carried by magnetic albumin microspheres.

The targeting of antineoplastic agents to restricted anatomic sites and specific target cells have been challenged clinicians all the time in cancer chemotherapy, which resulted in recent efforts to focus the effects of existing antitumor agents and treatments on tumor cells and spare their effects on normal cells. The drug-carrier complex, adriamycin carried by magnetic albumin microspheres (ADM-MAM) was prepared by using our discovered new and modified method. The physical feature of the prepared drug-carrier microspheres was much better than by the traditional method in comparison. The successful preparation of the drug-carrier complex, ADM-MAM, is one of the key steps for our later further researches in the targeted chemotherapy.

Antineoplastic Agents↗

PubMed2001

The influence of phorbol ester on the effect of tamoxifen in breast cancer cells.

To investigate the correlation between the activity of kinases in the growth factor signal transduction pathway and the development of resistance of breast cancer to tamoxifen, reporter gene regulated by the regulating fragment of CCD1 was transfected into the MCF-7 cells, and the influence of tamoxifen on the reporter gene expression was examined under different conditions of TPA treatment. Our results showed that the reporter gene expression was inhibited by tamoxifen and promoted by TPA. Furthermore, tamoxifen exerts an agonist effect on the reporter gene expression when the cells was treated by TPA previously for 12 h. It is concluded that TPA could induce estrogen-like effect of tamoxifen on estrogen receptor positive breast cancer cells and it may be one of the mechanisms responsible for the development of tamoxifen resistance.

Antineoplastic Agents, Hormonal↗

PubMed2001

The effect of hydroxyapatite ultrofine powder on the immunity function of tumor-bearing mice.

The inhibitory effect of hydroxyapatite ultrofine powder (HAUFP) on tumor and the effect on the immunity function of body were investigated. The levels of IL-2 in the spleen cells and serum TNF levels in the tumor-bearing mice at the 7th day and 14th after peritoneal injection of HAUFP were detected by using the methods of colorimetric analysis of MTT and crystal purple decoration, respectively. The disappearance of the ascites of the mice was observed. The results showed that the levels of IL-2 and TNF in the tumor-bearing mice were higher obviously in the drug-treated group than in the control group (P < 0.01), the ascites growth was inhibited. It was suggested that HAUFP could increase the levels of IL-2 and TNF of the tumor-bearing mice and improve the immune function of body.

Animals↗