Synthesis and Expression in Escherichia coli of a Human Neutrophil Activating Protein-1/Interleukin-8 Gene

The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was shown to be identical to that of the ntive protein.

Role of Activator Protein-1 in the Transcription of Interleukin-5 Gene Regulated by Protein Kinase C Signal in Asthmatic Human TLymphocytes

Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.

Clinical significance of dynamic detection for serum levels of MCP-1,TNF-α and IL-8 in patients with acute pancreatitis

Objective:To observe dynamic changes of levels of monocyte chemotactic protein-1(MCP-1),tumor necrosis factor-α(TNF-α) and interleukin-8(IL-8) in patients with acute pancreatitis and to investigate its evaluation value on the severity of acute pancreatitis.Methods:A total of 109 patients with acute pancreatitis admitted were divided into mild acute pancreatitis group(MAP group,42 cases),moderately severe acute pancreatitis(MSAP group,35 cases)and severe acute pancreatitis(SAP group,32 cases).ELISA was used to detect the serum levels of MCP-1,TNF-α and IL-8 of patients at day 1,day 4 and day 7 of admission to hospital.Results:The serum levels of MCP-1,TNF-α and IL-8 from MAP group,MSAP group and SAP group at day 1 of admission to hospital all significantly increased.There was a significant difference between MAP group and control group,MSAP group and MAP group,SAP group and MSAP group(P<0.05).The serum concentrations of IL-8 from MASP group and SAP group obviously increased at day 1,and there was significant difference between MASP group and MAP group,SAP group and MSAP group(P<0.05),while the difference between MAP group and control group was not obvious(P>0.05);The serum concentrations of MCP-1,TNF-α and IL-8 from MAP group all reached the highest level at day 4,which were significantly higher than the detection levels at day 1.In MSAP group and SAP group,the serum concentrations of MCP-1,TNF-α and IL-8 were the highest at day 1,which were significantly higher than the detection levels at day 4 and 7.At each detecting timing,the serum concentrations of MCP-1,TNF-α and IL-8 from MSAP group and SAP group were all higher than those of MAP group and MSAP group,respectively.Conclusions:The dynamic changes of serum levels of MCP-1,TNF-α and IL-8 in patients with acute pancreatitis have their rules,and the change rule of MAP group was different with that of MSAP and SAP group,which showed the reference value for the diagnosis and illness severity evaluation of acute pancreatitis.

人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因的修正及修正基因在大肠杆菌中的表达

本文采用寡核苷酸介导的定位诱变技术修正了人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因中出现的合成错误,在该基因编码区的201位插入了原来缺失的G残基,从而使之恢复正确读框。经对修正基因进行DNA全序列测定,表明定位诱变的结果符合设计要求。在此基础上,利用原核高效表达载体pBV220在P_RP_L串联启动子的控制下在大肠杆菌中对该基因进行了表达,并测定了重组人嗜中性白细胞活化蛋白-1/白细胞介素-8的嗜中性白细胞趋化活性。本工作为开展人嗜中性白细胞活化蛋白-1/白细胞介素-8基因工程及蛋白质工程的研究奠定了基础。

Benzydamine inhibits osteoclast differentiation and bone resorption via down-regulation of interleukin-1β expression

Bone diseases such as osteoporosis and periodontitis are induced by excessive osteoclastic activity,which is closely associated with inflammation.Benzydamine(BA)has been used as a cytokine-suppressive or non-steroidal anti-inflammatory drug that inhibits the production of proinflammatory cytokines or prostaglandins.However,its role in osteoclast differentiation and function remains unknown.Here,we explored the role of BA in regulating osteoclast differentiation and elucidated the underlying mechanism.BA inhibited osteoclast differentiation and strongly suppressed interleukin-1β(IL-1β)production.BA inhibited osteoclast formation and bone resorption when added to bone marrowderived macrophages and differentiated osteoclasts,and the inhibitory effect was reversed by IL-1βtreatment.The reporter assay and the inhibitor study of IL-1βtranscription suggested that BA inhibited nuclear factor-κB and activator protein-1 by regulating IκB kinase,extracellular signal regulated kinase and P38,resulting in the down-regulation of IL-1βexpression.BA also promoted osteoblast differentiation.Furthermore,BA protected lipopolysaccharide-and ovariectomy-induced bone loss in mice,suggesting therapeutic potential against inflammation-induced bone diseases and postmenopausal osteoporosis.

EXPERIMENTAL WORK and RESEARCH Effect of Reduqing (热毒清) on TNF-α,IL-1,IL-6,IL-8 and PAF Levels in Endotoxin-Induced DIC Model of Rabbits

Objective: To study the mechanism of anti-endotoxemic effect of Reduqing Injection (RDQ)and to explore the essence of traditional Chinese 'heat-clearing and detoxifying therapy'. Methods: A disseminated intravascular coagulation (DIC) model was made in rabbits by intravenous injection with E. colt endotoxin. Increased plasma levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8) and platelet activating factor (PAF), as well as the ex-vivo LPS-induced interleukin-1 (IL-1 ) production in peripheral blood monocytes were observed in model (M) group. Results: Same parameters were significantly lower in RDQ and dexamethasone (DXM) groups than those in the M group (P < 0. 01, P < 0. 05). Histopathological examination showed severe damage of the liver, lungs and kidneys in the rabbits of M group, in contrast, only mild affects were seen in the RDQ and DXM groups. Conclusions: RDQ exhibits protective effect on rabbits against endotoxin-induced DIC. The suppression of cytokines and inflammatory mediator PAF by RDQ may play a central role in the inhibition of endotoxin-induced DIC cascade.

Bioactive proteins in healthy pregnancies and preeclampsia： relevance to hypertension and proteinuria

Background Bioactive proteins, such as cytokines and chemokines, have not been systematically evaluated in healthy and preeclamptic pregnancies. We aimed to investigate the difference of these proteins between healthy and preeclamptic pregnancies in order to help clarify their potential roles in the pathogenesis of hypertension and proteinuria in preeclampsia. Methods Samples of amniotic fluid and maternal/umbilical cord blood were collected from normal pregnancies and women with preeclampsia for examination of bioactive proteins. Fifty-three pregnant women were enrolled in this study. Of them, 30 pregnant women were recruited as healthy controls, and 23 pregnant women were diagnosed with preeclampsia. An antibody array was used to screen for higher levels of cytokines and related proteins in amniotic fluid than in the blood samples, and these proteins were then selected for quantification by immunoassay. Results Interleukin-1 receptor 4, hepatocyte growth factor, and urokinase plasminogen activator receptor were significantly elevated in the blood of preec^ampsia patients. In particular, intedeukin-1 receptor 4 was 8-fold higher in preeclampsia patients than in the healthy pregnancies. Moreover, in cord blood samples hepatocyte growth factor and interleukin-8 were significantly higher in preeclampsia patients. Conclusions Because of the biologic activities, Interleukin-1 receptor 4, hepatocyte growth factor, urokinase plasminogen activator receptor and interleukin-8 in maternal and/or cord blood could play a role in the pathogenesis of hypertension and proteinuria in preeclampsia.