Search

Leukocyte cell-derived chemotaxin 2 (LECT2) is a secretory cytokine that functions in many physiological and pathological processes. We used a Pichia pastoris expression system for the recombinant expression of rainbow trout LECT2. The recombinant LECT2 was purified by UNOsphere S Cation exchange and size-exclusion chromatography columns. The obtained target protein was highly pure (>96% homogeneity) and the yield was >120 mg/L of yeast cultures. An in vitro chamber assay revealed that recombinant LECT2 could induce chemotactic responses in rainbow trout head kidney-derived macrophages. Recombinant LECT2 not only enhanced macrophage respiratory burst activity and bactericidal activity, but also changed macrophage gene expression. In summary, we established a rapid and efficient method to prepare active recombinant rainbow trout LECT2 using a yeast expression system and column chromatography. Bioactive recombinant LECT2 is essential for studies on protein functions.

Objective: To observe the changes in glucose 6-phosphate dehydrogenase (G6PD) activity, cAMP and respiratory burst function in THP-1 cells exposed to high glucose and identify the possible signaling pathways to mediate these changes., Methods: THP-1 cells were treated with high glucose, high glucose plus the PKA inhibitor (PKI), or normal glucose plus Forskolin. The changes in the G6PD activity and cAMP in the exposed cells were assayed using the spectrophotometric method, and the reactive oxygen species (ROS) content in the cell culture was determined using the fluorescent probe DCFH-DA. Western blotting was employed to examine the expression of phosphorylated p47(phox) in the cells., Results: Compared with the normal control cells, the cells exposed to high glucose and to normal glucose and Forskolin showed a significantly lowered G6PD activity, ROS content and expression of phosphorylated p47(phox), but with a increased cAMP content (P<0.01). High glucose exposure in the presence of PKI caused no significant changes in G6PD activity, ROS level, phosphorylated p47(phox) or cAMP compared to those in the normal control cells (P>0.01)., Conclusion: High glucose causes inhibition of G6PD activity in THP-1 cells via activation of PKA and thus leads to respiratory burst dysfunction, which is the probable mechanism underlying the lowered leucocyte function and susceptibility to infections in diabetic patients.

Objective: To study the relationship between HPLC fingerprint chromatogram and inhibitory effect on respiratory burst of rat PMN of leaves of crataegus L., Method: HPLC fingerprint peaks of different species of hawthorn leaves were isolated and used for the effective experiment on the respiratory burst of rat PMN. The mathematic models of the relationship between the area and the effect of fingerprint peaks were established. According to the mathematic models, the HPLC fingerprint were change into bioactive fingerprint (include effective fingerprint and potency fingerprint) with the helps of mathematics, chemometrics, computer program simulation and etc., Result: The chromatogram-effect relationship of leaves of crataegus. on respiratory burst of rat PMN was established. According to this relationship, the activities of fourteen samples of leaves of crataegus. were forecasted. It was positive correlation between the expected value and the practical value. And the correlation coefficients was 0.968 (P < 0.01)., Conclusion: An all-around evaluative system, which includes not only chemical identification but also effective evaluation for traditional Chinese medicine was established. It will provide a new idea for study on fingerprint chromatogram of traditional Chinese medicine.

This study was to explore the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on neutrophil morphology, function and phenotype in patients with acute leukemia undergoing chemotherapy. Neutrophil morphology was observed under microscope with oil immersion; phagocytotic function was examined by measuring the amount of hydrogen peroxide produced by neutrophil; chemotaxis was analyzed by agarose method; oxidative burst was analyzed by flow cytometry using immunofluorescence technique; neutrophil phenotype was analyzed by flow cytometry and immunofluorescence techniques. The results showed that after rhG-CSF administration, the increased "toxic" granulation, vacuoles and Döhle bodies were observed in neutrophils of patients with acute leukemia. Compared with normal control, the functions of phagocytosis, chemotaxis, oxidative burst of neutrophil were impaired after chemotherapy, while these functions were enhanced and returned to normal level or even to be exceeded after administration of rhG-CSF. In patients with acute leukemia the neutrophil presented significantly higher expression of CD64 and CD62L than that in normal control, and a mild increase of CD64 expression and significant increase of CD62L expression were found in patients after rhG-CSF treatment. No modifications of CD16, CD32, CD14 and CD11b expression were detected in these patients before or after G-CSF administration. It is concluded that rhG-CSF administration can modify the morphology, function and phenotype of neutrophils in the patients with acute leukemia undergoing chemotherapy, and these modifications of neutrophil behavior may be supposed to be a reason for the enhancement of organism anti-infection ability.

Objective: To study the chemical constituents in roots of P. fallax and their anti-oxidation activities in vitro., Method: Column chromatographic techniques were employed for isolation and purification of chemical constituents of the plant. The structures were elucidated on the basis of the spectral evidence and the physical and chemical character. The isolated compounds were screened with four anti-oxidation models in vitro., Result: Seven xanthones, 1,7-dihydroxy-2,3-methylenedioxyxanthone (1), 1-methoxy-2,3-methylenedioxyxanthone (2), 3-hydroxy-1,2-dimethoxyxanthone (3), 1,6,7-trihydroxy-2,3-dimethoxyxanthone (4), 7-hydroxy-1-methoxy-2,3-methylenedioxyxanthone (5), 1,3-dihydroxy-2-methoxyxanthone (6) and 1,3,7-trihydroxy-2-methoxyxanthone (7), were isolated from the roots of P. fallax. And compounds 1 - 7 showed different anti-oxidation activities in the different pharmacological models., Conclusion: Compounds 2, 3, 5 and 7 were isolated from this plant for the first time. Xanthones from this plant showed anti-oxidation activities. The pharmacological activities of the pure compounds from this plant were also reported for the first time.

Objective: The effect of platelet factor four (PF4) on human neutrophil function was studied., Methods: Crystal violet dye staining, immunofluorescence labeling, PKC kit assay, NBT and HVA fluoro-spectrophotometry were applied to study the effect of PF4 on total adherence, integrin CD11b level, PKC level and respiratory burst level of resting human neutrophils and FMLP/or PMA-stimulated human neutrophils., Results: It was found that PF4 slightly increased the total adherence of resting human neutrophils. There was no change on integrin CD11b level and respiratory burst level of resting human neutrophils after interaction with PF4. However, PF4 significantly down-regulated the total adherence, integrin CD11b level and respiratory burst level of human neutrophils stimulated by FMLP/or PMA. In addition, PF4 did not influence PKC level on resting and activated human neutrophil., Conclusion: These results indicate that PF4 plays a down regulation of the function of human neutrophils. Differing from other members of classical CXC-chemokine family, the signaling of PF4 is not through PLC-PKC signal pathway.

The relationship between apoptosis of neutrophils and the change of their intracellular free Ca2+ concentration [Ca2+]i was studied. FMLP and A23187 were used to elevate the [Ca2+]i while BAPTA was used to deplete it. Fluorescence microscope, flow cytometry and gel electrophoresis were used to study the percentage of cell apoptosis and the change of f-actin during apoptosis. The results showed that the apoptosis was obviously inhibited by fMLP and A23187, while accelerated by BAPTA. The detection of f-actin showed that the f-actin depolymerized obviously during apoptosis. The elevation of [Ca2+]i inhibit the actin depolymerization while depletion of [Ca2+]i accelerated it. This result indicated that the apoptosis of neutrophil was obviously inhibited by [Ca2+]i elevation but accelerated by [Ca2+]i depletion.

In this experiment the protective effects of beta-carotene (beta-C) liposome against rat neutrophile membrane damage caused by extracellular or intracellular reactive oxygen were species (ROS) investigated by means of fluorescence labels 1, 6-diphenyl-1,3,5 hexatriane (DPH) and N-(3-pyrene) maleimide (N-(3p)M). The extracellular ROS include the singlet oxygen (1O2) generated by photoreaction of sensitizer rosebengal and the superoxide anion (O2-.) produced by xanthine/xanthine oxidase reactive system. The intracellular ROS was sensitizer rosebengal and superoxide anion (O2-.) produced by xanthine/xanthine oxidase reactive system while the intracellular oxygen was generated by stimulating neutrophile respiratory burst with opsonized zymosan. No change in the peak or shape of fluorescent excitation and emission spectra was found for either of the two labels. The results of fluorescent polarization value in the membrane showed that beta-C liposome could significantly inhibit the damage of rat neutrophile membrane protein reacted either by intracellular generated reactive oxygen species or by extracellular generated O2-. or 1O2. It could also inhibit the injury of the rat neutrophile membrane lipid reacted by extracellular reactive oxygen species. However, the protective effects of beta-C liposome against membrane lipid damage reacted by intracellular reactive oxygen species by at the same concentration as those on membrane protein were not significant, which could be partly explained by relatively higher damage level of rat neutrophile membrane lipid domain reacted by intracellular reactive oxygen species than that of the membrane protein domain.

Chemiluminescence method was used to measure (1) Active oxygen production induced by respiratory burst of polymorphonuclears (PMN) from human blood stimulated with phorbol myristate acetate (PMA); (2) Superoxide (O2-.) induced by xanthine-xanthine oxidase system; (3) Hydroxyl radicals (.OH) produced by Vit C-Cu(2+)-zymosan; and (4) The release of hydrogen peroxide (H2O2). Effects of tetramethylpyrazine on these active oxygen species were observed. The results showed respiratory burst of PMN was inhibited by tetramethylpyrazine, superoxide and hydrogen peroxide were scavenged by tetramethylpyrazine and their median inhibition concentration (IC50, mumol.L-1) were 5.6 and 7.1 respectively.

The stimulatory effect of FMLP 5 nmol.L-1, OAG 25 nmol.L-1 and calcimycin 10 nmol.L-1 on luminol-dependent chemiluminescence (CL) was observed in human neutrophils (Neu). The rest level of Neu intracellular free calcium ([Ca]i) measured by Ca(2+)-sensitive probe Quin 2/AM was calculated to be 200 +/- s 19 nmol.L-1. By the addition of FMLP 0.1 nmol.L-1 or calcimycin 0.5 nmol.L-1, the peak [Ca]i increased to 769 +/- 104 nmol.L-1 and 953 +/- 53 nmol.L-1, respectively. Ketotifen (50-300 mumol.L-1) inhibited Neu CL in a dose-dependent manner with activator FMLP. Inhibition was also seen when Neu CL was activated by calcimycin and OAG, respectively. However, ketotifen did not inhibit Neu [Ca]i increment activated by FMLP and calcimycin.

Studies were made to determine the neutrophil's phagocytosis and bactericidal function in three groups of rats (control, iron deficiency, and iron supplement). Results showed that there were significant differences in values of chemiluminescence (CL) among three groups. The values of peak CL and five minutes integrated CL were markedly decreased in neutrophils of iron-deficient rats, accounting for only 41% and 32% of the control's values respectively. These suggested that the activity of NADPH oxidase was decreased, and the function of respiratory burst of neutrophils was impaired. The activity of myeloperoxidase in the iron-deficient neutrophils was also significantly lower than that in the control cells. It constituted only 30% of the control's value, indicating that the bactericidal function of neutrophils was injured. One week after iron administration, the low values of the peak CL, the five minutes integrated CL and the activity of myeloperoxidase all went up apparently, but not reached the normal levels yet. The time the function of neutrophils in iron-deficient rats returned to normal may be related to the process of neutrophil maturation in bone marrow.