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Publications (9)12.79 Total impact

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    ABSTRACT: A variety of experiments suggest that space flight is associated with an increase in oxidative stress in organism. To explore the effects of oxidative stress on neuronal cells during microgravity, we used rat pheochromocytoma (PC12) cells as a neuronal cell model, cultured in a clinostat, which could simulate microgravity, to investigate the effects of reactive nitrogen species on protein nitration in PC12 cells during clinorotation. The effects of melatonin and quercetin on protein nitration in PC12 cells were also assayed to evaluate the possible protective role of melatonin or quercetin as an antioxidant. The results of immunological staining showed that after the 3 days' clinorotation the protein expressions of neuronal nitric oxide synthase and inducible nitric oxide synthesis were up-regulated. Our data also reflected that the concentrations of nitric oxide and nitrotyrosine were significantly increased after clinorotation, and they were reduced markedly in cells that were treated with 50 micromol/L melatonin or 0.5 micromol/L quercetin during simulated microgravity, when compared to those of control cells. These results suggest that clinorotation-induced weightlessness increases oxidative stress responses in PC12 cells, and melatonin or quercetin was shown to protect PC12 cells from oxidative damage during simulated weightlessness.
    Nitric Oxide 09/2006; 15(1):58-63. · 3.27 Impact Factor
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    ABSTRACT: 2, 4-Dinitrophenyl (DNP) is a widely used hapten in molecular biology and immunoassay fields. Considering that 2, 4-dintrophenylhydrazine (DNPH) could be used as DNA probe and bind with protein carbonyl to form a stable 2 4-dinitrophenyl (DNP) hydrazone product, on which the level of oxidative stress could be validated with a sensitive noncompetitive ELISA, we prepared DNP-aminocaproic acid and NHS-aminocaproic acid-dinitrobenzene and the conjugates between DNP and carrier proteins such as bovine thyroglobulin (BTG) and bovine serum albumin (BSA). High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, twenty stable murine monoclonal antibodies (MAbs) producing cell lines to DNP were generated. The donor mouse produced antiserum with a high titer of 1/1,280,000. Five MAbs were selected for further characterization as class and subclass. After four successive limiting dilutions, antibodies were produced by five clones with high affinities ranging from 10(10) to 10(11) M(-1). These clones were found to be of IgG(1) subclass with kappa and lambda light chain. Competitive ELISA and SPR-based sensing system for the detection of DNPH are both used to confirm the specificity of MAb (4D(9)A(9)C(2)C(2)).
    Journal of Immunological Methods 07/2006; 313(1-2):20-8. · 2.23 Impact Factor
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    ABSTRACT: Anti-melatonin monoclonal antibodies (MAbs) of high titer were prepared by coupling melatonin to bovine serum albumin with formaldehyde and by immunizing BALB/c mice with multifocal intradermal injections and by fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Five MAbs were selected for further characterization as classes and subclasses. After four successive limiting dilutions, antibodies were produced by these five clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin Ig G1 and IgG(2b) subclass with kappa light chain. A systematic study of cross-reactions with seven compounds (indole, aromatic and imidazole derivatives) showed that the antibody had a high specificity for melatonin, low reactivity with 6-hydroxymelatonin and N-acetyl-5-hydroxytryptamine, and no detectable reactivity with tryptamine, l-tryptophan, 5-methoxytryptamine and N-acetyl-L-tryptophan. The roles of the indole nucleus and the side chain in the determination of the antigenic properties of the molecule are discussed. One of the MAbs, 4C9D7, was used to establish a competitive enzyme-linked immunosorbent assay for the detection of melatonin in supernatant.
    Journal of Pineal Research 06/2006; 40(4):350-4. · 7.30 Impact Factor
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    ABSTRACT: To develop a detective method applied in online assaying of astronauts' humours using the portable online bio-molecules analyzer (POBA) based on surface plasmon resonance biosensor. An assay format was developed based on the detection of 2, 4-Dinitrophenyl-hydrazine. The bio-molecule slide was made by DNP-BSA. Range of detection and standard curve were obtained using inhibition assay. Reliability and specificity of the assay were also tested. 1) The linear range of the assay was 7.8 ng/ml-2 micrograms/ml with lower detection limit of 2.5 ng/ml; 2) Preparation of the bio-molecule slide and regeneration of the biosensor ensured detections for many samples. This assay method can be used to detect small molecules sensitively, rapidly and easily. It can be repeated with good reliability, and has a good application in space medicine.
    Hang tian yi xue yu yi xue gong cheng = Space medicine & medical engineering 05/2005; 18(2):126-9.
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    ABSTRACT: A new competitive inhibition immunoassay (group-selective immunoassay; GSI) has been developed to detect free morphine in urine with the Fab' fragments of monoclonal antibodies (MAbs) (1B(12)F(9)B(4), IgG(1), kappa, K(aff) = 9.66 x 10(10)M(-1)). At the first assay step, microtiter plates were coated with morphine-ovalbumin (M-6-S-OVA), in which free amino acids were protected by a glutaraldehyde cross-linking modification. The modification did not essentially influence the antibody-binding capacity of the immunosorbent. At the second assay step, anti-morphine MAbs' Fab' fragments, in which free amino groups were biotinylated by N-hydrosuccinimide-biotin ester, were bound to chemically modified immunosorbent. The biotin residues were then detected by the streptavidin-peroxide conjugate. This method has a sensitivity of 3.50 x 10(-15) mol/L using very little volume of sample, covering up to almost 1.20 x 10(-11) mol/L of standard concentration of morphine with good reproducibility. Standard curve prepared in urine indicated a good correlation between the concentration of morphine and the value of OD (y = 1/ax + b; r = 0.99939257, S = 0.01138127). Coefficients of variation for this immunoassay were 1.41 approximately 6.61% within-a-day assay and 2.31 approximately 8.99% between days assay. The recoveries were 94 approximately 101.4% from negative urine and 95.2 approximately 107.5% from positive urine samples, respectively. This method has application as a specific screen for morphine in drug abusers, to study the metabolism of the drug in the body, or to screen the monoclonal antibodies (MAbs) against morphine.
    Hybridoma and Hybridomics 03/2004; 23(1):69-72.
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    ABSTRACT: 3-Nitro-L-tyrosine (nitrotyrosine) has recently been considered to be useful as a biomarker of endogenous production of several reactive nitrogen species including peroxynitrite. In the present study, nitrotyrosine was coupled to human serum albumin (HSA) using a two-step glutaraldehyde method and immunized mouse with multifocal intradermal injections. Using a conventional immunization protocol, 12 stable monoclonal antibodies (MAbs) producing cell lines recognizing nitrotyrosine were obtained. Six MAbs were selected for further characterization. A study of cross-reactions with nitrotyrosine-like compounds showed that the antibodies had a high specificity for nitrotyrosine, but no detectable reactivity with L-tyrosine, p-nitro-L-phenylalanine, o-phospho-L-tyrosine or 3-amino-L-tyrosine. Using these high titer and affinity antibodies, a competitive inhibition ELISA was developed with a lower detection limit of approximately 20 nmol/L to detect both free and protein-bound nitrotyrosine in biological systems.
    Hybridoma and Hybridomics 01/2004; 22(6):401-6.
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    ABSTRACT: Space flight is associated with an increase of peroxidative damage after returning to 1 g. The effect is more pronounced after long-duration space flight and can even last for several weeks after landing. In humans there is increased lipid peroxidation in erythrocyte membranes, reduced blood antioxidants, and increased urinary excretion of 8-iso-prostaglandin F2alpha, and 8-oxo-7, 8 dihydro-2 deoxyguanosine. Isoprostane 8-iso-prostaglandin F2alpha and 8-oxo-7, 8 dihydro-2 deoxyguanosine are markers for oxidative damage to lipids and DNA, respectively. The changes are attributed to a combination of energy deficiency that occurs during flight and substrate competition for amino acids occurring between repleted muscle and other tissues during the recovery phase. The observations in humans have been complemented by studies in rodents, which showed increased production of lipid peroxidation products and decreased antioxidant enzyme activity afterflight. The changes in rodents were attributed to the stress associated with re-entry into Earth's gravity. Reducing the imbalance between the production of endogenous oxidant defenses and oxidant production by increasing the supply antioxidants in diet may lessen the severity of the postflight increase in oxidative stress.
    Hang tian yi xue yu yi xue gong cheng = Space medicine & medical engineering 01/2004; 16(6):455-8.
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    ABSTRACT: To study the changes of expression and secretion of some growth factors and ERK1/2 activation in cardiac fibroblasts under clinorotation conditions. Primary cultured neonatal rat cardiac fibroblasts were clinorotated to simulate weightlessness. The bFGF and TGF(beta)1 expressions in the cells and Ang II concentration in the medium were detected by western blotting and radioimmunoassay respectively. The expression and phosphorylated level of ERK1/2 were analyzed by western blotting. Compared to the control group, bFGF expression and Ang II concentration increased in clinorotation group, but there was no evident change in TGF(beta)1 expression. At the same time, the expression of ERK1/2 increased but their phosphorylated level decreased. Different growth factors responds to clinorotation by different ways. As one of the most important reactions of growth factor signal downstream pathways, ERK1/2 activation is inhibited in clinorotation.
    Hang tian yi xue yu yi xue gong cheng = Space medicine & medical engineering 02/2003; 16 Suppl:532-7.
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    ABSTRACT: A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin and ovalbumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol generated twenty-six stable murine monoclonal antibodies (MAbs) producing cell lines to morphine. The donor mouse produced antiserum with a high titer of 1/640,000. Twelve MAbs were selected for further characterization since they showed high sensitivities (53 pg/well to inhibit 50% of the tracer) in improved group-selective immunoassay (IGSI). The assay, which maintains high sensitivity, high precision, and a wide range of optical density (OD) values, was developed using the conjugate M-6-S-OVA to screen and characterize the anti-morphine MAbs. After four successive limiting dilutions, antibodies produced by 12 clones had high affinities ranging from 10(9) to 10(10) M(-1). These clones were found to be of Ig(G) class and IgM class with kappa and lambda light chain. Subclass determination showed that the clones produced IgG1, IgG2a, IgG3, and IgM types of antibody. One clone (2F8B11F2A12) was used to establish the calibration curve with a sensitivity of 400 pg/mL covering up to 25.6 ng/mL in urine.
    Hybridoma and Hybridomics 07/2002; 21(3):197-201.