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Biomedical subjects

Jing Li

Publications and source records attributed to Jing Li.

At least 217 records · Page 12Linked to original sources
PubMed2005

Effect of the cyclin-dependent kinases inhibitor p27 on resistance of ovarian cancer multicellular spheroids to anticancer chemotherapy.

PURPOSE: A low proliferating fraction in solid tumors limits the effectiveness of cell-cycle-dependent chemotherapeutic agents. To understand the molecular basis of such resistance, we examined the expression of the cyclin-dependent kinases inhibitor p27, and relationship with drug resistance and P-gp expression in ovarian cancer multicellular spheroids. METHODS: We cultured ovarian cancer cells (A2780 and CAOV3) as multicellular spheroids and examined the expression of p27 and P-glycoprotein (P-gp) by western blot, flow cytometry and confocal. We also analyzed the cell-cycle distribution by flow cytometry. In addition, trypan blue exclusion testing and cell apoptosis analysis were used to detect the sensitivity to Taxol. RESULTS: When transferred from monolayer to three-dimensional culture, a consistent upregulation of p27 protein and P-gp protein was observed in ovarian cancer cell lines. Compared with monolayer cells, there was a significant increase of G0-G1 phase cells and decrease of S and G2-M phase cells in spheroid cells. Aggregates of cells showed higher cell viability than monolayer cells. Antisense oligodeoxynucleotide (ASON) -mediated downregulation of p27 reduced intercellular adhesion, increased cell proliferation, downregulated P-gp expression and sensitized cells to Taxol. CONCLUSIONS: Our results implicate that p27 serves as a regulator of drug resistance in ovarian tumors. ASON-mediated alteration of p27 reverses resistance of ovarian cancer to anticancer agents that are associated with increased sensitivity of ovarian cancer cells to chemotherapeutic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice.

AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system. METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 microL phosphate buffered saline (PBS) at an interval of 3 wk. Group 1: PBS only; group 2: gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminal fragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95 degrees heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination. RESULTS: The average titer of serum anti-HBsAg antibody in the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay. CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.

Animals↗
PubMed2005

Effect of tempol on renal cyclooxygenase expression and activity in experimental diabetes in the rat.

Renal cyclooxygenase (COX)-2 expression is increased in the streptozotocin (STZ)-diabetic rat and is associated with enhanced renal prostaglandin release in response to arachidonic acid (AA). Endoperoxide-mediated vasoconstrictor responses to AA were also enhanced in the diabetic rat kidney. Because oxidative stress is increased in diabetes and has been shown to induce COX-2, we assessed its contribution to prostaglandin release by treating diabetic rats with tempol (120 mg/kg/day) for 28 days. Release of AA-stimulated prostaglandins PGE(2) and 6-ketoPGF(1alpha) from the isolated perfused kidney was used as an index of COX activity, and Western analysis was used to determine COX-2 protein expression. In untreated diabetic rats, the release of prostaglandins in response to AA was markedly enhanced; the increase in release of both 6-ketoPGF(1alpha) and PGE(2) after AA was twice that in control rats. Renal cortical COX-2 expression in diabetic rats was 3-fold that of control rats. Tempol treatment reduced the AA-stimulated release of prostaglandins to levels seen in control rats; this was associated with reduced expression of COX-2 protein to levels not different from that in control rats. However, the enhanced vasoconstrictor response to AA in diabetic rats was unaffected by tempol treatment but abolished by inhibition of COX-1 with SC58560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole]. The addition of tempol to the perfusate of kidneys from diabetic and control rats had only a slight effect on prostaglandin release. We conclude that oxidative stress is an integral component of the mechanism involved in the induction of renal COX-2 in diabetes.

Animals↗
PubMed2005

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry.

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.

Animals↗
PubMed2005

T-cell receptor (TCR)/CD3 is involved in sulfated polymannuroguluronate (SPMG)-induced T lymphocyte activation.

Sulfated polymannuroguluronate (SPMG) has entered the Phase II clinical trial as the first anti-acquired immune deficiency syndrome (AIDS) drug candidate in China. Proliferation assays showed that SPMG was effective at enhancing the proliferative response of T lymphocytes either with or without concanavalin A (ConA) stimulation. Flow cytometry (FCM) and fluorescence microscope examination revealed the significant binding of SPMG to T lymphocytes. The significant engagement of SPMG with TCR/CD3 complex was verified by competitive inhibition assay and one of the SPMG binding proteins purified by affinity chromatography from thymocyte membrane preparations was further confirmed to be CD3 component of TCR/CD3 complex via Western blotting analysis. In addition, SPMG was demonstrated to dramatically interact with ConA in a multivalent manner by surface plasmon resonance (SPR) assay. Notably, the concomitant presence of ConA and SPMG facilitated each other's binding to T cells. Together, the simultaneous interactions of SPMG with TCR/CD3 and with ConA can be highly proposed to facilitate the cross-linking of these molecules, and thus favoring costimulatory signaling, which serves to well explain the immunopotentiation and anti-human immunodeficiency virus (HIV) activities of SPMG.

Animals↗
PubMed2005

Preparation and characterization of SARS in-house reference antiserum.

A reference antiserum for SARS is in urgent need in the development of SARS vaccine and other serological test of SARS research. Convalescent serum was collected from clinical confirmed patient. ELISA, Western-blotting and neutralization assay detected specific antibody against SARS coronavirus. This antiserum was prepared as in-house reference antiserum, freeze-dried and sealed in ampoules. The potency of this reference antiserum is defined to be 52.7 U after extensive calibration. Further, collaborative studies for the evaluation of this serum are needed in order to satisfy the requirements for international reference antiserum.

Animals↗
PubMed2005

Repertoire diversification in mice with an IgH-locus-targeted transgene for the rearranged VH domain of a physiologically selected anti-ssDNA antibody.

To test the fate of developing B cells with autoreactive receptor components, we studied mice homozygous for a knock-in transgene coding the VH domain of an IgM ssDNA-binding antibody. The transgene has unmutated C57 BL/6 V gene segments. Homozygous knock-in mice developed normal numbers of spleen and bone marrow B cells and normal serum Ig concentrations, and had the same low level of serum anti-ssDNA antibody as non-transgenic mice. Mature B cells expressed the transgene, and it underwent mutation and class switching. In young knock-in animals, nearly all IgM and some IgG cDNA clones from bone marrow and spleen contained the transgene V(H)D(H)J(H), with few or no mutations. In many IgM clones from older animals, however, and many IgG clones from both young and old mice, VH domains were revised by productive replacement with a new V(H)D(H) segment. VL segments were diverse. Immunized homozygous knock-in mice produced serum antibodies to polysaccharide, nucleic acid and protein antigens. Monoclonal IgM and IgG antibodies to nucleic acids used either transgenic or revised VH domains; but all of 20 IgG monoclonal antibodies to thyroglobulin used revised VH domain genes. Thus, B cells expressing an autoreactive (ssDNA-binding) VH domain did progress through development and were precursors for cells producing IgM and IgG, but underwent extensive VH gene revision in diversification of antibody responses.

Animals↗
PubMed2005

The WRKY family of transcription factors in rice and Arabidopsis and their origins.

WRKY transcription factors, originally isolated from plants contain one or two conserved WRKY domains, about 60 amino acid residues with the WRKYGQK sequence followed by a C2H2 or C2HC zinc finger motif. Evidence is accumulating to suggest that the WRKY proteins play significant roles in responses to biotic and abiotic stresses, and in development. In this research, we identified 102 putative WRKY genes from the rice genome and compared them with those from Arabidopsis. The WRKY genes from rice and Arabidopsis were divided into three groups with several subgroups on the basis of phylogenies and the basic structure of the WRKY domains (WDs). The phylogenetic trees generated from the WDs and the genes indicate that the WRKY gene family arose during evolution through duplication and that the dramatic amplification of rice WRKY genes in group III is due to tandem and segmental gene duplication compared with those of Arabidopsis. The result suggests that some of the rice WRKY genes in group III are evolutionarily more active than those in Arabidopsis, and may have specific roles in monocotyledonous plants. Further, it was possible to identify the presence of WRKY-like genes in protists (Giardia lamblia and Dictyostelium discoideum) and green algae Chlamydomonas reinhardtii through database research, demonstrating the ancient origin of the gene family. The results obtained by alignments of the WDs from different species and other analysis imply that domain gain and loss is a divergent force for expansion of the WRKY gene family, and that a rapid amplification of the WRKY genes predate the divergence of monocots and dicots. On the basis of these results, we believe that genes encoding a single WD may have been derived from the C-terminal WD of the genes harboring two WDs. The conserved intron splicing positions in the WDs of higher plants offer clues about WRKY gene evolution, annotation, and classification.

Amino Acid Motifs↗
PubMed2005

A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D.

The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser(82) in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.

Amino Acid Motifs↗
PubMed2005

Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation.

The E2F transcription factor family plays a crucial and well established role in cell cycle progression. Deregulation of E2F activities in vivo leads to developmental defects and cancer. Based on current evidence in the field, mammalian E2Fs can be functionally categorized into either transcriptional activators (E2F1, E2F2, and E2F3a) or repressors (E2F3b, E2F4, E2F5, E2F6, and E2F7). We have identified a novel E2F family member, E2F8, which is conserved in mice and humans and has its counterpart in Arabidopsis thaliana (E2Ls). Interestingly, E2F7 and E2F8 share unique structural features that distinguish them from other mammalian E2F repressor members, including the presence of two distinct DNA-binding domains and the absence of DP-dimerization, retinoblastoma-binding, and transcriptional activation domains. Similar to E2F7, overexpression of E2F8 significantly slows down the proliferation of primary mouse embryonic fibroblasts. These observations, together with the fact that E2F7 and E2F8 can homodimerize and are expressed in the same adult tissues, suggest that they may have overlapping and perhaps synergistic roles in the control of cellular proliferation.

Amino Acid Sequence↗
PubMed2005

Trypsin secretion and turnover in patients with acute pancreatitis.

Studies in humans have shown that pancreatic enzyme secretion is reduced during acute pancreatitis. It is not known, however, whether the reduction is due to impaired synthesis or disruption of the secretory pathway. The rate of secretion and turnover of trypsin was measured in 12 patients with acute pancreatitis of variable etiology and severity (median Ranson's score 2.5, range 0-5, 4 with severe necrotizing disease) and eight healthy volunteers by 4-h primed/continuous intravenous infusions of 1-(13)C-labeled l-leucine, and collection of pancreatic secretions by duodenal perfusion and sampling. Trypsin secretion was reduced from 476 +/- 73 to 153 +/- 60 U/h (means +/- SE, P = 0.005) in acute pancreatitis, with the greatest reductions being observed in patients with necrotizing disease (32 +/- 7 U/h, P = 0.003). The time for newly labeled trypsin to first appear in digestive juice was not, however, delayed in pancreatitis patients (87.2 +/- 11.1 vs. 94.7 +/- 4.9 min); on the contrary, there was an early appearance of newly labeled trypsin at 30 min in patients with severe necrotizing pancreatitis (P < 0.05). Calculated zymogen pool turnover was unchanged, but pool size was decreased (P = 0.01). Despite low rates of luminal secretion, trypsin continues to be synthesized in patients with acute pancreatitis. Our findings could be explained by post-Golgi leakage of enzymes from acinar cells or by loss of synthetic function in some cells with preservation in others.

Adolescent↗
PubMed2005

Thermal decomposition kinetics of the Pb0.25Ba0.75(TNR).H2O complex.

Studies of the non-isothermal decomposition of Pb(0.25)Ba(0.75)(TNR).H(2)O (TNR=2,4,6-trinitro-1,3-dihydroxy-benzene) were carried out by means of TG-DTA, DSC and IR techniques. The thermal decomposition mechanism and the associated kinetics have been investigated. The kinetic parameters were obtained from the analysis of the DSC curves by integral and differential methods. The most probable kinetic model function of the dehydration reaction of Pb(0.25)Ba(0.75)(TNR).H(2)O were suggested by comparison of the kinetic parameters.

Barium Compounds↗
PubMed2005

Monitoring of integrin-mediated adhesion of human ovarian cancer cells to model protein surfaces by quartz crystal resonators: evaluation in the impedance analysis mode.

The quartz crystal microbalance (QCM) was used to monitor specific, integrin-mediated adhesion of human ovarian cancer cells to distinct extracellular matrix (ECM) proteins immobilized on gold-coated quartz crystal resonators. The QCM was operated in the impedance analysis mode, where frequency shift as well as bandwidth are accessible on a broad range of overtones. The increase in bandwidth caused by covering the quartz resonator with cells was reproducible and largely independent of overtone order, whereas the frequency shift displayed some variability. Thus the bandwidth proved to be the more robust parameter for sensing cell adhesive events. The bandwidth increased in proportion to the number of seeded cells to the quartz crystal as long as the number was below 150,000 cells/ml. Comparing the resonance parameters on different harmonics, one finds that viscoelastic modeling with homogeneous layer systems cannot reproduce the results: lateral heterogeneity has to be taken into account. The differences in adhesive strength of human ovarian cancer cells towards selected ECM proteins monitored by QCM was in good agreement with data obtained by conventional cell adhesion assays. Strong cell adhesion was observed to the ECM proteins vitronectin (VN) and fibronectin (FN), while only weak attachment occurred on laminin. In order to prove specific, integrin alphavbeta3-mediated cell adhesion to its ligands FN and VN, the cyclic integrin alphavbeta3-directed peptide c(RGDfV) was used as competitor and significantly reversed cell adhesion. Since integrin interaction with ECM proteins is dependent on the presence of bivalent cations, cell detachment was also seen after treatment of cell monolayers with the chelator ethylene-dinitro-tetra-acetic acid (EDTA). The QCM technique is a reliable method to monitor cell adsorption to ECM-pretreated surfaces in real time. It may be an alternative tool for screening specific and selective antagonists of integrin/ECM interaction.

Biosensing Techniques↗
PubMed2005

A new layered zinc phosphate templated by protonated isonicotinate, [Zn2(C6H5NO2)2(HPO4)2].

An organic-inorganic hybrid compound, poly[bis[(pyridine-4-carboxylato)zinc(II)]-di-mu3-phosphato], [Zn2(C6H5NO2)2(HPO4)2], has been hydrothermally synthesized and structurally characterized. The crystal structure consists of two types of two-dimensional layers of zinc hydrogenphosphate templated by protonated isonicotinate (ina) (or 4-pyridinecarboxylic acid), which contain two crystallographically independent centrosymmetric [Zn2(ina)2(HPO4)2] dimers as basic building units. The layers are interconnected via hydrogen-bonding and heterocyclic ring interactions.

Crystallography, X-Ray↗
PubMed2005

Engineering glutathione transferase to a novel glutathione peroxidase mimic with high catalytic efficiency. Incorporation of selenocysteine into a glutathione-binding scaffold using an auxotrophic expression system.

Glutathione peroxidase (GPx, EC 1.11.1.9) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several attempts have been made to imitate its function for mechanical study and for its pharmacological development as an antioxidant. By replacing the active site serine 9 with a cysteine and then substituting it with selenocysteine in a cysteine auxotrophic system, catalytically essential residue selenocysteine was bioincorporated into GSH-specific binding scaffold, and thus, glutathione S-transferase (GST, EC 2.5.1.18) from Lucilia cuprina was converted into a selenium-containing enzyme, seleno-LuGST1-1, by genetic engineering. Taking advantage of the important structure similarities between seleno-LuGST1-1 and naturally occurring GPx in the specific GSH binding sites and the geometric conformation for the active selenocysteine in their common GSH binding domain-adopted thioredoxin fold, the as-generated selenoenzyme displayed a significantly high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione, being comparable with those of natural GPxs. The catalytic behaviors of this engineered selenoenzyme were found to be similar to those of naturally occurring GPx. It exhibited pH and temperature-dependent catalytic activity and a typical ping-pong kinetic mechanism. Engineering GST into an efficient GPx-like biocatalyst provided new proof for the previous assumption that both GPx and GST were evolved from a common thioredoxin-like ancestor to accommodate different functions throughout evolution.

Catalysis↗
PubMed2005

[Protection of liver sinusoidal endothelial cells from hypoxia-reoxygenation induced apoptosis by alpha-1 antitrypsin in vitro].

OBJECTIVE: To investigate the effect of alpha-1 antitrypsin on ischemia-reperfusion injury of human liver sinusoidal endothelial cells (LSECs). METHODS: LSECs were cultured and put into 4 degrees C refrigerator for 12 hours and then into 37 degrees C culture box with 95% O(2) and 5% CO(2) for 2 - 6 hours to establish an experimental hypoxia-reoxygenation injury model. The LSECs were inoculated in 24-pit culture plate and L-NAME, NO inhibitor, SNAP, a NO supplier, BB3103, a matrix metalloproteinase (MMP) inhibitor, or alpha-1 antitrypsin of different concentrations were added. The LSECs were put into 4 degrees C refrigerator for 12 hours and then into 37 degrees C culture box with 5% CO(2) for 2 - 6 hours. The supernatant was collected to detect the production of nitric oxide (NO) and activity of MMPs. LSECs were cultured under conditions of presence or absence of alpha-1 antitrypsin and normal temperature and oxygen concentration, low-temperature and hypoxia, and low temperature and hypoxia -reoxygenation respectively. Then the supernatant was collected to detect the activities of MMP-2 and MMP-9 with zymography and the production of NO with determination of nitrite concentration and expression of eNOS and iNOS by immunohistochemistry. RESULTS: Immunohistochemistry showed that LSECs were alpha-1 trypsin positive, RT-PCR showed LSECs did not express alpha-1 trypsin mRNA. TUNEL staining showed that hypothermia for 12 hours caused apoptosis of LSECs, apoptosis of LSECs was more obvious after hypothermia and hypoxia-reoxygenation; however, with the presence of alpha-1 trypsin apoptosis of LSECs was significantly decreased after hypothermia and hypoxia-reoxygenation. Hypoxia-reoxygenation induced apoptosis was significantly decreased by L-NAME and BB3103 and increased by SNAP. significantly decreased the apoptosis Zymography showed a significant increase of MMP production, in the forms of proMMP-2 and proMMP-9, in LSECs, paralleling with the number of apoptotic LSECs; and alpha-1 trypsin significantly inhibited the MMP activity during hypothermia and hypoxia-reoxygenation dose-dependently. Paralleling with the number of apoptotic LSECs, the expression of iNOS and eNOS, especially the former, was significantly increased after hypothermia and hypoxia-reoxygenation. L-NAME significantly decreased and SNAP significantly increased the production of NO during hypothermia and hypoxia-reoxygenation. The NOS expression and NO production of LSECs during hypothermia and hypoxia-reoxygenation were inhibit by alpha-1 trypsin dose-dependently. CONCLUSION: alpha-1 antitrypsin protects LSEC from apoptosis during hypothermia and hypoxia-reoxygenation injury by decreasing NO production and inhibiting MMP activity.

Apoptosis↗
PubMed2005

Short tau inversion recovery and proton density-weighted fat suppressed sequences for the evaluation of osteoarthritis of the knee with a 1.0 T dedicated extremity MRI: development of a time-efficient sequence protocol.

Aim of this study was to develop a time-efficient sequence protocol for a 1.0 T dedicated MR system to be used for whole-organ scoring of osteoarthritis (OA). Thirty-four knees were examined using a protocol that included fat suppressed fast spin echo proton density weighted sequences (PDFS) in three planes plus a coronal STIR sequence. Two radiologists scored each knee by consensus for five OA features. In separate sessions, all knees were scored using three different combinations of sequences: (1) all four sequences (reference protocol, 16 min 31 s scanning time), (2) three PDFS sequences without STIR ("No STIR", 12 min 25 s scanning time) and (3) sagittal and axial PDFS sequences plus a coronal STIR sequence ("No PDFS", 11 min 49 s scanning time). Agreement of the readings using both subsets of sequences compared to the reference protocol was evaluated using weighted kappa statistics. kappa-coefficients showed good or excellent agreement for both sequence subsets in comparison to the reference protocol for all assessed features. kappa-coefficients for No PDFS/No STIR: bone marrow abnormalities (0.74/0.67), subarticular cysts (0.84/0.63), marginal osteophytes (0.77/0.71), menisci (0.75/0.79), tibial cartilage (0.71/0.78). Optimization of sequence protocols consisting of three sequences results in time savings and cost efficiency in imaging of knee OA without loss of information over a more time consuming protocol.

Female↗