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Biomedical subjects

G L Shen

Publications and source records attributed to G L Shen.

At least 19 recordsLinked to original sources
PubMed2001

Renewable amperometric immunosensor for Schistosoma japonium antibody assay.

A renewable amperometric immunosensor has been proposed for the determination of Schistosoma japonium antibody (SjAb) in rabbit serum. A paraffin-graphite-Schistosoma japonium antigen (SjAg) biocomposite, which needs no additional curing, was directly used to construct the immunosensors. The analytical sample containing the desired SjAb was mixed with SjAb labeled with horseradish peroxidase (HRP) to form the incubation solution for the competitive binding assay. Amperometry was used to determine the amount of HRP fixed on the sensor surface, which was related to the content of desired SjAb. Assay conditions were optimized, including the selection of substrate, the loading of SjAg in the biocomposite, the amount of labeled SjAb in the incubation solution, the incubation time, and the temperature. Using o-aminophenol (o-AP) as a substrate, amperometric detection at -200 mV (vs SCE) resulted in a pseudolinear detection range of about 0.36 to 14 microg/mL, with a detection limit of 0.36 microg/mL. Rabbit serum samples with varying infection degrees were analyzed, and the results demonstrated that the concentration that is detectable in this system meets the demands of clinical analyses. A new surface on the immunosensor for use in another competitive assay can be obtained by removing the original one and polishing the surface.

Animals↗
PubMed2001

Synthesis of glycosylated porphyrins as neutral ionophores for a berberine-sensitive electrode.

For preparing a berberine-sensitive electrode, 5,10,15,20-tetrakis[2-(2,3,4,6-tetraacetyl-beta-D-glucopyranosyl)-1-O- phenyl]porphyrin (T(o-glu)PPH2) was synthesized from the reaction of pyrrole with ortho-acetylglycosylated benzaldehyde by Lindsay's method. The electrode based on T(o-glu)PPH2 with an optimized membrane composition exhibits Nernstian response to berberine in the concentration range 2.4 x 10(-7)-5.0 x 10(-3) mol L-1, with a pH range from 3.9 to 10.2, and a fast response time of 30 s. The electrode shows fair selectivity towards berberine with respect to common co-existing species. T(o-glu)PPH2 shows better potentiometric response characteristics comparing to chloro[5,10,15,20-tetrakis[2-(2,3,4,6-tetraacetyl-beta-D- glucopyranosyl)-1-O-phenyl]-porphinato]-manganese (MnT(o-glu)PPCl) and better selectivity towards berberine than tetraphenylporphyrin (TPPH2). The effect of the composition of the electrode membrane has been studied and the experimental conditions optimized. The contents of berberine in pharmaceutical tablets were determined by direct potentiometry and the results agreed with values obtained by the pharmacopoeia method.

Berberine↗
PubMed2001

Renewable amperometric immunosensor for complement 3 assay based on the sol-gel technique.

A renewable amperometric immunosensor based on the sol-gel technique has been constructed by dispersing graphite, complement 3 (C3) antiserum, and sol-gel at low temperature. The prepared immunosensor is rigid, porous, and has a renewable external surface. A competitive binding assay has been used to determine C3 in human serum with the aid of C3 labeled with horseradish peroxidase. The enzyme-labeled antigen can readily diffuse toward the encapsulated antibody, which retains its binding properties. The experimental conditions for the assay with the biocomposite, including the loading of C3 antiserum in the biocomposite, the amount of labeled C3 in incubation solution, incubation time, and temperature, have been optimized. Using C3 labeled with horseradish peroxidase, and o-AP as the substrate, amperometric detection at -150 mV (relative to the SCE) results in a linear detection range of 1.17-35.1 microg mL(-1), with a detection limit of 0.56 microg mL(-1). Serum samples have been assayed and the results demonstrate the feasibility of the proposed immunosensor for clinical analysis. The surface of the immunosensor can be renewed simply by polishing to obtain a fresh immunocomposite ready to use in a new competitive assay.

Biosensing Techniques↗
PubMed2001

Picric acid sensitive optode based on a fluorescence carrier covalently bound to membrane.

An acryloyl group was attached to fluorescein through the phenol hydroxyl group to make it photopolymerizable with 2-hydroxypropyl methacrylate and covalently immobilized on an optode glass surface modified with gamma-(methacryloxy)propyltrimethoxysilane. The optode system with a plastic-clad fused silica bifurcated fiber optic bundle shows satisfactory analytical performance characteristics for determining picric acid in terms of selectivity and reproducibility with a linear range from 8.0 x 10(-7) to 4.0 x 10(-3) mol L-1. The quenching mechanism was investigated. The optode membrane can be applied to the indirect determination of cinchonine drug and the results obtained were satisfactory.

Fiber Optic Technology↗
PubMed2001

Fluorescence spectral study of interaction of water-soluble metal complexes of Schiff-base and DNA.

The fluorescence spectral characteristics and the interaction of several water-soluble metal complexes of Schiff-base with DNA are described. Among the complexes tested, Mn-Schiff-base bound to DNA showed a marked decrease in the fluorescence intensity with a blue shift of the excitation and emission peaks. Some hypochromism in the UV absorption spectra was also observed. KI quenching and competitive binding to DNA between Mn-Schiff-base and ethidium bromide (EB) were studied in connection with other experimental observations to show that the interactive model between Mn-Schiff-base and DNA is an intercalative one. The pH and salt effect on the fluorescence properties was also investigated. The linear relationship between F/F0 and the concentration of calf thymus DNA covers 3.0 x 10(-6)-2 x 10(-4) mol L-1, which can be utilized for determining traces of calf thymus DNA with a detection limit of 8.0 x 10(-7) mol L-1 in base pairs.

DNA↗
PubMed2000

Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning.

Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.

Amino Acid Sequence↗
PubMed2000

Cloning of a second dendritic cell-associated C-type lectin (dectin-2) and its alternatively spliced isoforms.

Using a subtractive cDNA cloning strategy, we isolated previously five novel genes that were expressed abundantly by the murine dendritic cell (DC) line XS52, but not by the J774 macrophage line. One of these genes encoded a unique, DC-associated C-type lectin, termed "dectin-1." Here we report the characterization of a second novel gene that was also expressed in a DC-specific manner. Clone 1B12 encoded a type II membrane-integrated polypeptide of 209 amino acids containing a single carbohydrate recognition domain motif in the COOH terminus. The expression pattern of this molecule, termed "dectin-2," was almost indistinguishable from that for dectin-1; that is, both were expressed abundantly at mRNA and protein levels by the XS52 DC line, but not by non-DC lines, and both were detected in spleen and thymus, as well as in skin resident DC (i.e. Langerhans cells). Interestingly, reverse transcriptase-polymerase chain reaction and immunoblotting revealed multiple bands of dectin-2 transcripts and proteins suggesting molecular heterogeneity. In fact, we isolated additional cDNA clones encoding two distinct, truncated dectin-2 isoforms. Genomic analyses indicated that a full-length dectin-2 (alpha isoform) is encoded by 6 exons, whereas truncated isoforms (beta and gamma) are produced by alternative splicing. We propose that dectin-2 and its isoforms, together with dectin-1, represent a unique subfamily of DC-associated C-type lectins.

Alternative Splicing↗
PubMed2000

A novel ethacrynic acid sensor based on a lanthanide porphyrin complex in a PVC matrix.

Lanthanide porphyrin complexes synthesized by a solid state method were used to prepare a novel ethacrynic acid (EA) sensor. The sensor, based on pentane-2,4-dionato(meso-tetraphenylporphinato)terbium [TbTPP(acac)] with an optimized membrane composition, exhibits a Nernstian response to EA- ion in the concentration range 7.4 x 10(-6)-1.0 x 10(-1) mol l-1 with a pH range from 3.2 to 6.8 and a fast response time of 30 s. The electrode shows improved selectivity towards EA- ion with respect to common co-existing ions compared with the previously reported EA sensor. As electroactive materials, lanthanide porphyrin complexes show better potentiometric response characteristics than copper porphyrin complexes. The effect of solvent mediators and lipophilic ion additives was studied and the experimental conditions were optimized. The electrode was applied to the determination of EA in human urine samples with satisfactory results.

Electrochemistry↗
PubMed2000

Bismetalloporphyrin-based ISE sensitive to fluoroborate.

Several mu-oxo-bismetalloporphyrin complexes were synthesized for preparing fluoroborate sensors. The electrode based on mu-oxo-bis[tetra(p-chlorophenyl)porphinatomanganese(III)] with an optimized membrane composition shows a potentiometric response towards fluoroborate ion over the concentration range from 4.3 x 10(-7) to 1.0 x 10(-1) mol l-1 with a Nernstian slope, a wide working pH range from 5.5 to 12.0 and a fast response time of 30 s. The electrode shows anti-Hofmeister selectivity towards BF4- with respect to common co-existing ions, which is an improvement over methods reported so far. As electroactive materials, several mu-oxo-bismetalloporphyrins with different substituents on the benzene rings were compared for potentiometric response characteristics. The interaction between BF4- and bismetalloporphyrin was investigated using UV/VIS spectrophotometry. The electrode was applied to the determination of fluoroborate in electroplating solutions.

Borates↗
PubMed1999

DNA binding studies of a solvatochromic fluorescence probe 3-methoxybenzanthrone.

A fluorescence probe of 3-methoxybenzanthrone (MBA) exhibits significant solvatochromic characteristics correlated with the polarity of solvents. The interaction of the solvatochromic fluorescence probe with calf thymus DNA (ct-DNA) has been investigated. In the presence of ct-DNA the fluorescence of MBA is strongly quenched with a blue-shift of emission peak and a hypochromism in absorption spectra. The absorption spectra, fluorescence quenching and fluorescence polarization experiments show that the MBA molecule as an intercalator is inserted into the base-stacking domain of the ct-DNA double helix, and the interaction of the nucleobases with the MBA molecule causes quenching of fluorescence and hypochromism in the absorption spectra. The intrinsic binding constant and the binding site number were determined to be 1.70 x 10(5) mol l-1 in base pairs and six, respectively. The I0/I versus [ct-DNA] plot shows linear relationship in the range covering 4.3 x 10(-7)-1.02 x 10(-4) mol l-1 in base pairs which can be used for ct-DNA determination. The limit of detection was found to be 4.3 x 10(-7) mol l-1 in base pairs (0.5 microgram ml-1).

Absorption↗
PubMed1998

Organic solvent induced quenching of porous silicon photoluminescence.

A comparison has been made for the quenching effect of three classes of organic solvents on the porous silicon photoluminescence, including hydrocarbons, solvents containing oxygen and nitrogen. Among them organoamines possess the strongest quenching effect. The experiments show that the solvent molecules interact synergistically with porous silicon and the main pathways include the surface hydrophobic interaction, dipole-dipole interaction and hole trapping, the later being the dominant pathway in quenching of porous silicon photoluminescence.

Hydrocarbons↗
PubMed1997

Fluorescence quenching of ethidium ion by porphyrin cations and quaternary ammonium surfactants in the presence of DNA.

Fluorescence quenching of free and DNA-bound ethidium bromide (EB) by a number of quaternary ammonium and other compounds was studied. For free EB or bound EB at lower DNA concentration the fluorescence quenching follows the Stern-Volmer equation and at higher DNA concentration follows an exponential model. At least at low quencher concentrations the quenching efficiency varies with DNA or NaCl concentrations and is about 100 times greater for bound than free EB. The quenching pathways may involve energy transfer and conformational loosening or distortion of the DNA helix in addition to possible electron transfer.

DNA↗
PubMed1997

Highly selective iodide poly(vinyl chloride) membrane electrode based on a nickel(II) tetraazaannulene macrocyclic complex.

A PVC membrane electrode based on a nickel(II) tetraazaannulene macrocyclic complex as the carrier is described. The electrode exhibits an anti-Hofmeister selectivity sequence with a preference for iodide at pH 3.0-4.0. It has a linear response to iodide from 8.0 x 10(-6) to 1 x 10(-1) mol dm-3 with a slope of -54.7 +/- 0.2 mV per decade (20 degrees C), a satisfactory reproducibility and a rapid response time. The pH dependence of the potential response is discussed. The response mechanism of the electrode was investigated by ac impedance, quartz crystal microbalance and spectroscopic techniques. The electrode can be used for the determination of iodide in drug preparations.

Heterocyclic Compounds↗
PubMed1996

Polymer agglutination-based piezoelectric immunoassay for the determination of complement III.

A piezoelectric immunoassay technique, which is based on the detection of agglutination of antibody- or antigen-bearing polymer by an immunoreaction using a piezoelectric quartz crystal, has been developed for the determination of complement III (C3). Anti-C3 antibodies were physically adsorbed onto the carboxymethyl cellulose polymer by hydrogen bonds. The agglutination of the (anti-C3 antibody)-bearing polymer by immunoreaction caused a viscosity change of the solution, which could be monitored by a piezoelectric quartz crystal. The effect of experimental conditions such as the polymer concentration, the antibody dilution ratio and the reaction temperature on the frequency response were investigated. The linear ranges for C3 concentration determined by the end-point method and the initial rate method were 22.0-43.2 micrograms ml-1 and 22.0-49.1 micrograms ml-1, respectively. Other antigens presenting in serum did not interfere with the determination of C3. Analytical results of ten clinical specimens obtained using the developed technique were in satisfactory agreement with those given by the rate diffusion turbidimetry. With a simple regeneration method devised, the crystal can be used repeatedly with acceptable reproducibility.

Complement C3↗
PubMed1995

Piezoelectric immunosensor for the detection of immunoglobulin M.

A piezoelectric immunosensor has been developed for the determination of human IgM. The crystals are AT-cut and have a basic resonant frequency of 9 MHz. Immobilization of goat antihuman IgM antibodies to the crystals' surfaces was accomplished via a CNBr-activated copolymer coating of 2-hydroxyethyl methacrylate and methylmethacrylate. The IgM piezoelectric immunosensor can be used for the human IgM determination in the range 5-93 micrograms ml-1. The analytical results given by this approach were in satisfactory agreement with those given by the single radical immunodiffusion procedure. The sensitivity, specificity and reproducibility of this immunosensor were investigated. Further, the valent value of goat antihuman IgM antibody binding with human IgM antigen and the affinity constant of immunoreaction in this experimental system were studied. After washing with tetrahydrofuran, a crystal can be re-used 20 times without detectable loss of sensitivity.

Biosensing Techniques↗
PubMed1994

Bispecific anti-CD22/anti-CD3-ricin A chain immunotoxin is cytotoxic to Daudi lymphoma cells but not T cells in vitro and shows both A-chain-mediated and LAK-T-mediated killing.

We have generated a bispecific anti-CD22/anti-CD3 immunotoxin (IT) and determined whether it would exert both lymphokine-activated killer (LAK) T cell-mediated and ricin A chain (dgA)-mediated toxicity to Daudi tumor cells but not to T cells in vitro. One parental IT, Fab'-anti-CD22-dgA makes a potent immunotoxin for B cells, but not T cells, while the other, Fab'-anti-CD3-dgA, kills neither T nor B cells. Three mouse quadromas were generated and the bispecific Abs (BsAbs) were purified by double affinity chromatography. Two of the three purified BsAbs induced significant proliferation and IL-2 production in T cells. All three BsAbs induced LAK-T cell-mediated specific lysis of CD22+ Daudi cells. Two of the purified Ab were conjugated to dgA. Using a 51Cr release assay in the presence of LAK-T cells and Daudi target cells, the IC50s of the BsAbs were 3.5 x 10(-10) M and 9 x 10(-11) M, as compared to 2.1 x 10(-11) M and 3.2 x 10(-11) M, for their respective ITs. Hence, in the presence of LAK-T cells, the BsITs were 3- to 17-fold more cytotoxic than unconjugated BsAbs in 51Cr-release assays. Daudi cells were also treated in vitro with different mixtures of LAK-T cells, BsAbs, and BsITs and then adoptively transferred into SCID mice. As determined by the mean paralysis time of the recipients, in the presence of LAK-T cells the BsITs had impressive anti-tumor activity.

Antibodies, Bispecific↗
PubMed1993

Cancer vaccines: the perspective of the Cancer Immunology Branch, NCI.

The Cancer Immunology Branch, NCI, is supporting a great deal of exciting research relevant to cancer vaccine development. The few areas highlighted here are representative of ongoing research opportunities, but further progress depends largely on a continued infusion of investigator-initiated ideas to realize the potential of current research areas and open new ones.

Antigens, Neoplasm↗