Biomedical subjects

Qin Fu

Publications and source records attributed to Qin Fu.

14 recordsLinked to original sources

PubMed2007

A rapid, economical, and reproducible method for human serum delipidation and albumin and IgG removal for proteomic analysis.

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin (HSA) and immunoglobulin G (IgG) in the serum proteome. Therefore, in order to observe lower-abundance serum proteins, removal or depletion of at least these two proteins is required. However, the depletion method needs to be inexpensive and reproducible. We describe such a protocol that combines delipidation by centrifugation, IgG removal with Protein G Sepharose, and HSA depletion with sodium chloride/ethanol precipitation. The protocol is streamlined to increase reproducibility and is compatible with many proteomic platforms, including two-dimensional gel electrophoresis, and high-performance liquid chromatography either offline or coupled online with a mass spectrometer. The reproducible depletion of lipids, IgG, and HSA permits a higher load of the remaining serum proteins, facilitating the identification of disease biomarkers.

Biomarkers↗

PubMed2006

Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid.

AIM: The present study was designed to determine the possible pathway underlying the enhancement of apoptosis induced by the combined use of arsenic trioxide (As(2)O(3)) and ascorbic acid (AA). METHODS: The level of intracellular reactive oxygen species (ROS) was detected by means of flow cytometry analysis with an oxidation-sensitive fluorescent probe (6-carboxy-2',7' dichlorodihydrofluorescein diacetate) uploading. The activity of glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were detected by biochemical methods. The mitochondrial membrane potential was measured by flow cytometry analysis with rhodamine 123 staining. Bcl-2, Bax, and p17 subunit of caspase-3 were analyzed using the Western blot method. The apoptosis rate was determined by flow cytometry with annexin-V/propidium iodide staining. RESULTS: Compared with As(2)O(3) (2.0 micromol/L) treated alone, As(2)O(3) (2.0 micromol/L) in combination with AA (100 micromol/L) decreased intracellular GSH content from 101.30+/-5.76 to 81.91+/-3.12 mg/g protein, and increased ROS level from 127.61+/-5.12 to 152.60+/-5.88, which was represented by the 2, 7-dichlorofluorescein intensity. The loss of mitochondria membrane potential was increased from 1269.97+/-36.11 to 1540.52+/-52.63, which was presented by fluorescence intensity. The p17 subunit of caspase-3 expression was increased approximately 2-fold. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax were equal to that of As2O3 treated alone (P>0.05). When the ROS scavenger, N-acetyl-L-cysteine, was added to As(2)O(3) and AA combined treatment group, the apoptosis rate decreased from 15.60 %+/-1.14% to 9.48%+/-0.67%, and the ROS level decreased from 152.60+/-5.88 to 102.77+/-10.25. CONCLUSION: AA potentiated As(2)O(3)-induced apoptosis through the oxidative pathway by increasing the ROS level. This may be the result of depleting intracellular GSH. It may influence the downstream cascade following ROS, including mitochondria depolarization and caspase-3 activation. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax influenced by As(2)O(3) was not found to be potentiated by AA.

Acetylcysteine↗

PubMed2006

Proteomics and heart disease: identifying biomarkers of clinical utility.

Cardiovascular disease is the leading cause of mortality and morbidity in the industrialized world. Total worldwide deaths due to this disease are currently estimated at 17 million per year, and this number is expected to increase over the next several decades. To address this epidemic, a major effort has begun to develop new cardiovascular disease markers through the use of proteomic analysis, the global study of proteins. This review discusses strategies, recent technological advances and other issues in plasma/serum biomarker discovery for cardiovascular diseases. Emphasis lies on the needs for standardizing specimen collection, methods for reducing plasma proteome complexity to subproteomes, selection of appropriate technology platforms and strategies to evaluate candidates by multiplexed immune assays. The overall goal of this effort is to identify serum biomarkers for diagnosis, therapeutic monitoring and risk stratification of cardiovascular diseases.

Animals↗

PubMed2005

[Experimental study of bFGF modulating rabbit articular chondrocytes cultured in vitro and seeded onto polylactic acid scaffold coated with different materials].

OBJECTIVE: Culturing rabbit articular chondrocytes in vitro and seeding on polylactic acid (PLA) coated with lecithin and poly-l-lysine modulated by bFGF to find a suitable method for cartilaginous tissue engineering. METHODS: The articular chondrocytes were isolated enzymatically from the articular cartilage of young rabbits, and cultured in vitro. Collecting the chondrocytes of the third passage and seeding on three-dimensional scaffold of polylactic acid coated with lecithin and poly-l-lysine. At the same time, basic fibroblast growth factor (bFGF) was added. Proceeding series of detections when the cell-scaffold complexes were cultured more than two weeks, such as macroscopic, invert microscope, light microscope, scanning electron microscope and immunohistochemistry of collagen II. RESULTS: The cell-scaffold complexes modulate by bFGF could not only keep their original shapes, but also maintain the stable homogeneous three-dimensional distribution of chondrocytes without cell falling during the cultivation. At the same time, the complexes were gradually decreasing the consistency, however, increasing the Pliability with elasticity and lubrication surface. After the second week, the complexes were gradually reorganized into the mature engineered cartilage with typical cartilaginous histological structure with rich collagen II. CONCLUSION: bFGF can facilitate the regeneration and maturation of tissue-engineered articular cartilage.

Animals↗

PubMed2005

Improvements in two-dimensional gel electrophoresis by utilizing a low cost "in-house" neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system.

Two-dimensional gel electrophoresis (2-DE) is widely used for initial protein separation in proteomics. Commercial products using neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis ((SDS-PAGE)/(Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl, or Bis-Tris)) have greatly improved this technique, but cost and limited sizes restrict their applications. An "in-house" system is presented, resulting in better resolution, separation, and new spot visualization and improved resolution when compared to Tris-HCl gels. Their utility is demonstrated using albumin-depleted serum samples, rabbit heart left ventricle, and human immunodeficiency virus type 1 (HIV-1).

Animals↗

PubMed2005

A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis.

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.

Blood Proteins↗

PubMed2005

Hypoglycemic activity of jatrorrhizine.

The hypoglycemic activity and its mechanism of Jatrorrhizine (Jat) were studied. The normal mice and alloxan-induced hyperglycemic mice were given with different doses of Jat. Blood glucose and liver glycogen levels were determined by spectrophotometry with glucose-oxidase and iodine reagents respectively. The levels of blood lactic acid (LC) and liver lactate dehydrogenase (LDH) activity were measured to explore the effect of Jat on anaerobic glycolysis. Succinate dehydrogenase (SDH) activity in liver was measured to evaluate the effect of Jat on aerobic glycolysis in liver. It was found that Jat (50 mg/kg, 100 mg/kg) could significantly decrease blood glucose level in a dose- and time-dependent manner in both normal and alloxan-diabetic mice, increase the activity of SDH, but had no significant effects on the LC level and LDH activity. Jat could significantly reduce the content of liver glycogen in normal mice. Moreover, Jat could inhibit the platelet aggregation in rabbits in vitro in a dose-effect relationship. It was concluded that Jat induced the pronounced decrease in blood glucose in normal and hyperglycemic mice. The hypoglycemic activity of Jat may be attributed to the enhancement of aerobic glycolysis.

Animals↗

PubMed2005

Roles of nitric oxide in protective effect of berberine in ethanol-induced gastric ulcer mice.

AIM: To investigate the protective effects of berberine on ethanol-induced gastric ulcer in mice. METHODS: Gastric ulcers were induced by oral ingestion of ethanol. Nitric oxide (NO) content was measured, and mRNA expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The ulcer index (UI) at 1 h, 2 h, 3 h and 6 h after oral administration of ethanol was 23.8+/-1.4, 23.3+/-2.2, 22.3+/-1.2 and 20.8+/-1.1, respectively. The UI in the berberine-treated groups (5 mg/kg and 50 mg/kg) was less than the control group. The content of NO in the control group was 73.3+/-7.3 microL/L, 94.0+/-9.2 microL/L, 109.6+/-6.4 microL/L and 138.2+/-10.2 microL/L in gastric juice and 5.8+/-1.1 micromol/g protein, 8.3+/-1.1 micromol/g protein, 9.8+/-1.1 micromol/g protein and 11.9+/-1.2 micromol/g protein in gastric tissue at 1 h, 2 h, 3 h and 6 h, respectively, after the oral administration of ethanol. The content of NO in the berberine-treated groups (5 mg/kg and 50 mg/kg) was higher than the control group at 1 h after the oral administration of ethanol (P<0.05), and was lower at 6 h (P<0.05). Analysis by RT-PCR showed that expression of eNOS was inhibited but iNOS expression was enhanced by ethanol. However, the expression of eNOS could be enhanced and iNOS expression could be inhibited by berberine (P<0.01). CONCLUSION: Berberine could significantly protect gastric mucosa from damage by ethanol. This effect may be related to the increased expression of eNOS mRNA and inhibited expression of iNOS mRNA.

Animals↗

PubMed2004

Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation.

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-alpha, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.

Antigens, CD34↗

PubMed2004

Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore.

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3beta (GSK-3beta). Alternatively, receptor tyrosine kinase or certain G protein-coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3beta, via protein kinase B/Akt and mTOR/p70(s6k) pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3beta on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.

Animals↗

PubMed2002

Multiplexed protein profiling on microarrays by rolling-circle amplification.

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-alpha treatment. Because microarrays can accommodate approximately 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.

Antibodies↗

PubMed2002

[Experimental study on growth factor in promotion of chondrocytes proliferation].

OBJECTIVE: To study the effect of two cytokines, basic fibrolast growth factor(bFGF) and insulin-like growth factor-I(IGF-I), on cell proliferation in chondrocytes of adult rabbits. METHODS: The primary chondrocytes of adult rabbits were harvested and cultured with bFGF and IGF-I at different concentrations, respectively, as well as with the mixture of the two cytokines; the quantity of cultured chondrocytes was detected by MTT assay at the 24th, 48th and 72nd hours; and the final fold increase of different groups was measured by cell count for the 3rd passage; and the proliferation index of the groups was recorded by flowing cytometer on the 14th day. RESULTS: 1. The cultured chondrocytes with either bFGF, IGF-I or their mixture were significantly more than that of control group at the 24th, 48th and 72th hours (P < 0.01). 2. After the 3rd passage, the final folds of proliferation were significantly higher in the groups with cytokines than in the control group (P < 0.01); and the final fold with the mixture of cytokines was significantly higher than that of both IGF-I and bFGF (P < 0.01). 3. The proliferation index was significantly higher in the groups with cytokines than in the control group (P < 0.01); the proliferation index with the mixture of cytokines was significantly higher than that of both IGF-I and bFGF (P < 0.05); besides, proliferation index was higher when cytokine was applied twice than once (P < 0.05); besides, proliferation index was higher when cytokine was applied twice than once (P < 0.05). CONCLUSION: bFGF and IGF-I could promote chondrocytes proliferation of adult rabbits obviously and they are synergistic in cell proliferation.

Animals↗

PubMed2002

[The antitumor activity of Diosgenin in vivo and in vitro].

OBJECTIVE: To investigate the antitumor activity of Diosgenin in vivo and in vitro. METHOD: S-180, HepA, U14 and EAC transplant mice were given Diosgenin ig or i.p. everyday for 10 days, from the next day when they were inoculated in axilla. Tumor growth inhibit rates were calculated. Four kinds of cells, MCF, L929, A375-S2 and HeLa, were incubated respectively with Diosgenin in vitro. Tumor growth inhibit rates were also calculated. RESULT: In vivo, both ig and i.p., Diosgenin inhibited S-180, HepA, U 14 mice transplant tumor, the inhibit rates being 30%-50%, but it did not inhibit the EAC mice transplant tumor. In vitro, Diosgenin inhibited L929, HeLa, MCF cell growth, and IC50 were 1.2, 18.2, 19.8 micrograms.mL-1 respectively, but it did not significantly affect A375-S2 cells. CONCLUSION: Diosgenin has an obvious antitumor activity on S-180, HepA, U14 transplant mice in vivo and L929, HeLa, MCF cells in vitro.

Animals↗

PubMed2001

Phosphorylation of pyrimidine deoxynucleoside analog diphosphates: selective phosphorylation of L-nucleoside analog diphosphates by 3-phosphoglycerate kinase.

D-Nucleoside analogs, which are in the natural configuration, as well as the L-nucleoside analogs, are clinically relevant antiviral and anticancer agents. Metabolism of L-nucleoside analog diphosphates to the triphosphates, however, remains unexplored. Studies with recombinant nm23-H1 and -H2 isoforms indicated that L-nucleoside analog diphosphates were not phosphorylated by their nucleoside diphosphate kinase (NDPK) activity. Therefore, roles of creatine kinase, 3-phosphoglycerate kinase, and pyruvate kinase were evaluated using preparations from commercial sources and human HepG2 cells. Phosphorylation of L-OddC, L-SddC, L-Fd4C, L-FMAU, and L-ddC were compared with D-deoxynucleoside analogs, AraC, dFdC, and D-FMAU, and D-dideoxynucleoside analogs, ddC and d4T. Results based on preparations from HepG2 cells showed that L-nucleoside analog diphosphates were selectively phosphorylated by 3-phosphoglycerate kinase, whereas, D-deoxynucleoside analog diphosphates were phosphorylated by NDPK. Interestingly, ddCDP and d4TDP were substrates for creatine kinase, but were not phosphorylated by NDPK. In conclusion, it is proposed that specificity of the phosphorylating enzymes toward the nucleoside analog diphosphates is dependent on the configuration of the analog (L or D) and the presence or absence of 3'-hydroxyl group in the sugar moiety. The enzymatic process of phosphorylation of L- and D-nucleoside analog diphosphates is different in cells.

Adenosine Diphosphate↗