Expression of Bt Protein in Transgenic Pest-resistant Rice

[Objective] The aim of this study was to study on expression of Bt protein in transgenic pest-resistant rice. [Method] Enzyme-linked immunosorbent assay （ELISA） was used to measure Bt protein expression in different tissues of transgenic pest-resistant rice at same growth stage. [Result] Absolute content of Bt protein from high to low was as follows： leaves 〉 immature seeds and glumes 〉 roots 〉 stems in different tissues of transgenic rice in grain-filling stage; Bt protein content of trans- genic rice changed a little in different growth stages （including tillering stage, booting stage, and grain-filling stage）; in general, its level declined a little in later growth stage, but the resistibility would not be influenced significantly. [Conclusion] The ex- periment is significant for pest prevention and transgenic rice breeding.

Preparation and Application of Monoclonal Antibodies Specific for Salicylic Acid

Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of ethylene, and more attractively, it seems to be an important natural signal molecule in the induction of systemic acquired resistance (SAR) in tobacco, cucumber and other plants([2,3]). Studies in recent years showed that SA was also intimately related to the resistance of plants to aboitic stress, for example, SA increased chilling resistance of maize seedlings. Hence, SA has been accepted as a kind of new plant hormones. Up to date, the quantification of SA usually has been performed by HPLC[4,5], which often needs a large quantity of sample and a verbose pretreatment. Compared to HPLC, immunoassays, including radio-immunoassays (RIA) and enzyme-immunoassays (EIA), are easy to perform and have been widely used in the quantification of other plant hormones, such as IAA([6]), ABA([7,8]), GAs([9]), cytokinins et al([10]), and jasmonic acid (JA)([11]), and other low-molecular-weight, none-immunogenic compounds in plants([12]). Till now, only an indirect enzyme-linked immunosorbent assay (ELISA) for SA based on polyclonal antibodies (PAbs) has been developed by our group([13]), although Bennett et al([14]) had prepared SA PAbs using 4-aminosalicylic acid linked to KLH as immunogen in goat. However, the sensitivity of the ELISA we established formerly was relatively low, and also relatively larger quantity of sample is needed than other ELISAs for plant hormones. In this paper, an ELISA for SA based on monoclonal antibody raised against SA-NH-CH2-NH-KLH was introduced, and the fluctuation of SA content in cucumber leaves after inoculated with Pseudomonas syringae pv. syringae was determined.

探讨 81 例临床分型 HIV 感染者血液样本 HIV1-RNA 载量、HIV1-P24、Anti-HIV 及 CD4+T 细胞计数检测结果的相关性

通过检测临床已知病例血样 HIV 抗原抗体含量、HIV1-RNA 及 CD4+T 细胞计数,了解 HIV1-P24 抗原及抗 HIV 抗体的含量与 HIV/AIDS 病程进展的关系,寻找监视和预测 HIV 感染者病程发展及疗效观察的敏感的免疫学指标。方法:收集81 例不同感染阶段病人血清或血浆分别检测 HIV1-P24、Anti-HIV 含量、HIV(s/co)及 CD4+淋巴细胞的数量和 HIV1-RNA 病毒载量,用统计学方法比较它们的相关性,评价它们在 HIV 感染者/AIDS 病程进展中的价值。结果:对 81 例检测结果进行统计分析:HIV1-RNA 病毒载量与 HIV1-P24 之间 U=4.02,P<0.01,数值相差非常显著;HIV1-RNA 阳性率为 0.6667,HIV1-P24阳性率为 0.1604,二者阴性/阳性符合率为 0.5185,阳性预断值均为 1.0;随着病程的发展 HIV1-RNA 病毒载量、HIV1-P24 检测结果呈现不同幅度上升趋势,而不同感染阶段相应 CD4+T 细胞计数结果呈现不同程度的下降趋势;Anti-HIV 和 HIV(s/co)具有良好的一致性,但 HIV(s/co)变化与病程长短无相关性,对病程的进展整体表现为不敏感;81 例 HIV1-P24 与 Anti-HIV检测结果:急性无症状感染期(A)HIV1-P24(18 例阴性)/Anti-HIV(18 例阳性) ;有症状感染期(B)HIV1-P24(29 例阴性,1 例阳性)/Anti-HIV(30 例阳性) ;艾滋病期(C)HIV1-P24(19 例阴性,14 例阳性)/Anti-HIV(33 例阳性) ;HIV1-P24与 Anti-HIV 检测结果同时阳性共有 15 例(其中 8 例 Cut-off:1.7-3.86;7 例 Cut-off:10.54-44.25) ,有 53.33%HIV1-P24表现为低反应性 (弱阳性) 。 结论: 用罗氏电化学发光法检测 HIV1-P24 及 Anti-HIV 的含量, 其具有结果快速准确和高灵敏性,更适合临床 HIV 感染的初筛和病程监测的需要。

Exogenous carbon monoxide attenuates inflammatory responses in the small intestine of septic mice

AIM： To determine whether the carbon monoxide （CO）-releasing molecules （CORM）-Iiberated CO sup- press inflammatory responses in the small intestine of septic mice. METHODS： The C57BL/6 mice （male, n = 36; weight 20±2 g） were assigned to four groups in three re- spective experiments. Sepsis in mice was induced by cecal ligation and puncture （CLP） （24 h）. Tricarbonyl- dichlororuthenium （Ⅱ） dimer （CORM-2） （8 mg/kg, i. v.） was administrated immediately after induction of CLP. The levels of inflammatory cytokines [interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）] in tis- sue homogenates were measured with enzyme-linked immunosorbent assay. The levels of malondialdehyde （MDA） in the tissues were determined. The levels of nitric oxide （NO） in tissue homogenate were measured and the expression levels of intercellular adhesion mol- ecule 1 （ICAM-1） and inducible nitric oxide synthase （iNOS） in the small intestine were also assessed. NO and IL-8 levels in the supernatants were determined after the human adenocarcinoma cell line Caco-2 was stimulated by lipopolysaccharide （LPS） （10 g/mL） for 4 h in vitro. RESULTS： At 24 h after CLP, histological analysis showed that the ileum and jejunum from CLP mice in- duced severe edema and sloughing of the villous tips, as well as infiltration of inflammatory cells into the mu- cosa. Semi-quantitative analysis of histological samples of ileum and jejunum showed that granulocyte infil- tration in the septic mice was significantly increased compared to that in the sham group. Administration of CORM-2 significantly decreased granulocyte infiltration. At 24 h after CLP, the tissue MDA levels in the mid- ileum and mid-jejunum significantly increased com- pared to the sham animals （103.68 ± 23.88 nmol/ml vs 39.66 ± 8.23 nmol/mL, 89.66±9.98 nmol/mL vs 32.32 ± 7.43 nmol/mL, P 〈 0.01）. In vitro administra- tion of CORM-2, tissue MDA levels were significantly decreased （50.65±11.46 nmol/mL, 59.32 ± 6.62 nmol/mL, P 〈 0.05）. Meanwhile, the tissue IL-1β and TNF-α levels in the mid-ileum significantly increased compared to the sham animals （6.66±1.09 pg/mL vs 1.67±0.45 pg/mL, 19.34±3.99 pg/mL vs 3.98 ± 0.87 pg/mL, P 〈 0.01）. In vitro administration of CORM-2, tissue IL-1β and TNF-α levels were significantly de- creased （3.87 ± 1.08 pg/mL, 10.45±2.48 pg/mL, P 〈 0.05）. The levels of NO in mid-ileum and mid-jejunum tissue homogenate were also decreased （14.69 ± 2.45 nmol/mL vs 24.36 ± 2.97 nmol/mL, 18.47 ± 2.47 nmol/mL vs 27.33 ± 3.87 nmol/mL, P 〈 0.05）. The ex- pression of iNOS and ICAM-1 in the mid-ileum of septic mice at 24 h after CLP induction significantly increased compared to the sham animals. In vitro administration of CORM-2, expression of iNOS and ICAM-1 were sig- nificantly decreased. In parallel, the levels of NO and IL-8 in the supernatants of Caco-2 stimulated by LPS was markedly decreased in CORM-2-treated Caco-2 cells （2.22 ± 0.12 nmol/mL vs 6.25±1.69 nmol/mL, 24.97 ± 3.01 pg/mL vs 49.45± 5.11 pg/mL, P 〈 0.05）. CONCLUSION： CORM-released CO attenuates the inflammatory cytokine production （IL-1β and TNF-α）, and suppress the oxidative stress in the small intestine during sepsis by interfering with protein expression of ICAM-1 and iNOS.

Secretory Expression of E2 Main Antigen Domain of CSFV C Strain and the Establishment of Indirect ELISA Assay

The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21–CodonPlus (DE3)–RIL using the pGEX-4T-1 expression vector and the soluble recombinant product was purified with Glutathione Sepharose TM4B by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 μg/well and the optimal dilution of serum was 1:80. The positive cut-off value of this ELISA assay was OD tested serum / OD negative serum≥2.1. The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.

Melatonin ameliorates experimental hepatic fibrosis induced by carbon tetrachloride in rats

AIM:To investigate the protective effects of melatonin on carbon tetrachloride(CCl4)-induced hepatic fibrosis in experimental rats.METHODS:All rats were randomly divided into normal control group,model control group treated with CCl4 for 12 wk,CCl4+NAC group treated with CCl4+NAC(100 mg/kg,i.p.)for 12 wk,CCl4+MEL-1 group treated with CCl4+melatonin(2.5 mg/kg)for 12 wk,CCl4+MEL-2 group treated with CCl4+ melatonin(5.0 mg/kg)for 12 wk,and CCl4+MEL-3 group treated with CCl4+melatonin(10 mg/kg).Rats in the treatment groups were injected subcutaneously with sterile CCl4(3 mL/kg,body weight)in a ratio of 2:3 with olive oil twice a week.Rats in normal control group received hypodermic injection of olive oil at the same dose and frequency as those in treatment groups.At the end of experiment,rats in each group were anesthetized and sacrificed.Hematoxylin and eosin(HE)staining and Van Gieson staining were used to examine changes in liver pathology.Serum activities of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and protein concentration weremeasured with routine laboratory methods using an autoanalyzer.Hydroxyproline(HYP)content in liver and malondialdehyde(MDA)and glutathione peroxidase(GPx)levels in liver homogenates were assayed by spectrophotometry.Serum hyaluronic acid(HA),laminin(LN),and procollagenⅢN-terminal peptide(PⅢNP)were determined by radioimmunoassay.RESULTS:Pathologic grading showed that the fibrogenesis was much less severe in CCl4+MEL3 group than in model control group(u=2.172,P<0.05),indicating that melatonin(10 mg/kg)can significantly ameliorate CCl4-induced hepatic fibrotic changes.The serum levels of ALT and AST were markedly lower in CCl4+MEL treatment groups(5,10 mg/kg)than in model control group(ALT:286.23 ±121.91 U/L vs 201.15±101.16 U/L and 178.67 ±103.14 U/L,P=0.028,P=0.007;AST:431.00 ±166.35 U/L vs 321.23±162.48 U/L and 292.42 ±126.23 U/L,P=0.043,P=0.013).Similarly,the serum laminin(LN)and hyaluronic acid(HA)levels and hydroxyproline(HYP)contents in liver were significantly lower in CCl4+MEL-3 group(10 mg/kg)than in model control group(LN:45.89±11.71μg/L vs 55.26± 12.30μg/L,P=0.012;HA:135.71±76.03μg/L vs 201.10±68.46μg/L,P=0.020;HYP:0.42±0.08 mg/g tissue vs 0.51±0.07 mg/g tissue,P=0.012).Moreover,treatment with melatonin(5,10 mg/kg)significantly reduced the MDA content and increased the GPx activity in liver homogenates compared with model control group(MDA:7.89±1.49 noml/mg prot vs 6.29±1.42 noml/mg prot and 6.25±2.27 noml/mg prot,respectively,P=0.015,P=0.015;GPx:49.13± 8.72 U/mg prot vs 57.38±7.65 U/mg prot and 61.39± 13.15 U/mg prot,respectively,P=0.035,P=0.003).CONCLUSION:Melatonin can ameliorate CCl4-induced hepatic fibrosis in rats.The protective effect of melatonin on hepatic fibrosis may be related to its antioxidant activities.

Expression of Angiotensin Ⅱ and Aldosterone in Radiation-induced Lung Injury

Objective Radiation-induced lung injury （RILl） is the most common, dose-limiting complication in thoracic malignancy radiotherapy. Considering its negative impact on patients and restrictions to efficacy, the mechanism of RILl was studied. Methods Wistar rats were locally irradiated with a single dose of 0, 16, and 20 Gy to the right half of the lung to establish a lung injury model. Two and six months after irradiation, the right half of the rat lung tissue was removed, and the concentrations of TGF-[31, angiotensin II, and aldosterone were determined via enzyme-linked immunosorbent assay. Results Statistical differences were observed in the expression levels of angiotensin II and aldosterone between the non-irradiation and irradiation groups. Moreover, the expression level of the angiotensin II-aldosterone system increased with increasing doses, and the difference was still observed as time progressed. Conclusions Angiotensin II-aldosterone system has an important pathophysiological function in the progression of RILI.

Mycotoxins in Brewing Grain Raw Material （Barley, Malt） in Russia

The levels of five mycotoxins （MT）： deoxynivalenol （DON）, T-2-toxin （T-2）, zearalenone （ZEA）, aflatoxin （Aft）, ochratoxin A （OTA） were measured in malting barley and malt samples by enzyme immunoassay （ELISA）, using test systems RIDASCREEN FAST （R-Biopharm, Germany）. 40 samples of malting barley, mainly from the Central part of European Russia and fewer from the Southern part of, and also some samples from Altai （Asian Russia） were analyzed during 2007-2011 years as well as 120 samples of malt from Russian malting companies. It was found that 17% of barley samples were contaminated with MT; in two cases （5%）, the MT concentration exceeded maximum allowable levels （MAL）. Among malt samples in more than half （in 56%） MT were detected, in 9% of samples, the MT concentration exceeded MAL （Aft-3 incidents, T-2-3 incidents, OTA-2 incidents, ZEA-1 incident）. Maximum levels ofmycotoxins in malt were found to be higher than those in barley. These facts support the idea about risky conditions during malting processing.

Fumonisin-Producing Fusarium from Maize Grains in Tretep, Indonesia

Fusarium species commonly occur in maize are fungal pathogen which produce mycotoxins, such as fumonisin, trichothecene and zearalenone. In this study, Fusarium species were isolated from maize kernel from Tretep, maize producer region and were identified based on microscopic- and macroscopic characters as well as molecular characters using PCR assays and the partial sequence of TEF 1-α gene （Translation Elongation Factor 1-α. The fumonisin-producing ability of these Fusarium was determined by growing them in corn medium and analyzed their fumonisin by ELISA （enzyme-linked immuno assay）. Among 9 isolates, three of them were identified as Fusarium verticillioides, two as Fusarium temperatum, two as Fusarium globosum, one as Fusarium proliJeratum and one as Fusarium subglutinans. Fusarium temperatum is similar morphologically to F. subglutinans, however, both of their differences can be found by molecular analysis. Fumonisin-producing abilities of Fusarium were determined in concentrations 20.51 pg/g-1,109.74 pg/g medium with the highest producer was identified as F. globosum.

Association Between IL-6 （Interleukin-6） and Iron Status in Rheumatoid Arthritis Patients

The study was performed to determine whether the srum concentrations of IL （interleukin）-6 are elevated in patients with RA （rheumatoid arthritis） and to investigate the relationship between IL-6 levels and iron status in RA patients. 95 serum samples were obtained, 70 of them from patients with RA who had visited the department of Rheumatology at Al-Sadder medical city in Najaf governorate （Iraq） and 25 age and sex-matched healthy controls. The authors assessed the clinical parameters of the disease, including ESR （erythrocyte sedimentation rate）, CRP （C-reactive protein）, and RF （rheumatoid factor）. Serum levels of iron and TIBC （total iron binding capacity） were measured spectrophotometrically, while TS% （transferrin saturation percentage） and transferrin concentration were calculated mathematically. Serum concentrations of IL-6 （interleukin-6） and ferritin were measured using an ELISA （enzyme-linked immunosorbent assay）. The results of serum concentration of IL-6 （interleukin-6） and ferritin were significantly elevated （P 〈 0.0001） in patients with RA compared to those of healthy controls. On the other hand, serum concentrations of iron, TIBC （total iron binding capacity）, TS% （transferrin saturation percentage） and transferrin concentration were significantly decreased in patients with RA compared with those of healthy controls. These findings suggest that anemia is the most frequent observations in patients with RA and mostly associative with increasing level of interleukin-6.

SIMPLE METHOD OF PUNCHING AND RE-LOCATING TISSUES FOR MANUAL CONSTRUCTION OF TISSUE MICROARRAY

A series of human tissue samples and cultured cell lines were formalin-fixed and paraffin-embedded. Specimen cylinder （1.2 -1.8ram） were punched by a modified bone marrow biopsy needle and arrayed on a recipient paraffin block. Microscopic analysis on the sections from this tissue microarray （ TMA ） block demonstrated that the spots of tissues and cells were well preserved, and the cultured cell samples were successfully embedded from 5 × 104 to 2 × 105 in number. These TMA sections were also suitable for immunohistochemistry and RNA in situ hybridization.

Helicobacter pylori antigen and its IgG, IgA -type specific immunocomplexes in sera from patients with Helicobacter pylori infection

OBJECTIVE: To explore the characteristics of Helicobacter pylori (H. pylori) antigen in serum and to evaluate its clinical diagnostic value. METHODS: Enzyme-linked immunosorbant assay (ELISA) was developed to detect the soluble H. pylori antigen (S-Hp) and circulatory specific H. pylori antigen immunocomplexes (Hp-IC) in serum. RESULTS: The positive rate of S-Hp was 90.91% from 66 patients with H. pylori infection, which was much greater than 0% found in 28 controls (P

Xinfeng capsule improves pulmonary function in ankylosing spondylitis patients via NF-κB-iNOS-NO signaling pathway

OBJECTIVE: To study changes in the nuclear factor-κB p65(NF-κB p65)-inducible nitric oxide synthase(i NOS)-nitric oxide(NO) signaling pathway and the effects of Xinfeng capsules(XFC) in patients with ankylosing spondylitis(AS)METHODS: One hundred twenty patients with AS were randomly divided into an XFC group and a Salazopyrin group. Sixty health subjects were included as a normal control group. In the two treatment groups, pulmonary functional parameters,forced vital capacity(FVC), forced expiratory volume in 1 second(FEV1), maximal voluntary ventilation(MVV), peak expiratory flow(PEF), forced expiratory flow at 25% of forced vital capacity(FEF25),forced expiratory flow at 50% of forced vital capacity(FEF50), and forced expiratory flow at 75% of forced vital capacity(FEF75) were determined. Enzyme linked immunosorbent assays were used for detection of the serum oxidative stress indexes,NF-κB p65, i NOS, NO, reactive oxygen species(ROS), reactive nitrogen species(RNS), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), total antioxidative capacity(TAOC) and interleukin-4(IL-4), IL-10, IL-1β, and tumor necrosis factor-α(TNF-α) contents. Westergren's method was used for determination of erythrocyte sedimentation rate(ESR). High-sensitivity C-reactive protein(Hs-CRP) was detected with a 7060 full-automatic biochemical analyzer(Hitachi, Japan).RESULTS: The clinical therapeutic effect in the XFC group was significantly superior to that in the Salazopyrin group(P<0.01). Compared with the normal control group, FEV1, MVV, PEF, FEF50, FEF75, SOD, CAT,TAOC, IL-4, IL-10 were significantly lower, and NF-κB p65, i NOS, NO, ROS, RNS, MDA, IL-1β, TNF-α, ESR,and Hs-CRP significantly higher in patients with AS(P<0.01 or P<0.05). Compared with before treatment, FEV1, MVV, PEF, FEF50, FEF75, SOD, CAT, TAOC,IL-4, and IL-10 were significantly increased, and NF-κB p65, i NOS, NO, ROS, RNS, MDA, IL-1β, TNF-α,ESR, CRP, visual analog scales(VAS), Bath ankylosing spondylitis disease active index, Bath ankylosing spondylitis functional index, and Bath ankylosing spondylitis global index significantly decreased in the two treatment groups after treatment(P<0.01 or P<0.05), with significant differences between the XFC and Salazopyrin groups(P<0.01 or P<0.05). Spearman correlation analysis indicated that FEV1, MVV, PEF, FEF50, and FEF75 were positively correlated with SOD, CAT, TAOC, IL-4, and IL-10, and were negatively correlated with NF-κB p65, i NOS,NO, ROS, RNS, MDA, IL-1β, TNF-α, ESR, and CRP.CONCLUSION: Patients with AS have local pathologic changes in the spinal cord and other joints.They also have decreased pulmonary function,which is negatively correlated with the NF-κB-i NOS-NO signaling pathway, oxidative indexes, and inflammatory factors. XFC improves rigidity and pain in spinal joints and other symptoms, laboratory indexes, and pulmonary function. The mechanism is possibly related to inhibition of the NF-κB-i NOS-NO signaling pathway.

Effect of acupotomy on nitric oxide synthase and beta-endorphin in third lumbar vertebrae transverse process syndrome model rats

OBJECTIVE： To explore the long-term effects and pain relief mechanism of acupotomy by observing changes in nitric oxide synthase （NOS） and beta-en- dorphin （~3-EP） in the hypothalamus, spinal cord, and peripheral blood of rats with third lumbar ver- tebrae （L3） transverse process syndrome. METHODS： Twenty-eight SD rats were randomly as- signed to normal, model, electroacupuncture （EA）, and acupotomy group. The last three groups were put through an operation to emulate L3 transverse process syndrome. Fourteen days after the simulation operation, EA and acupotomy treatments were applied to the respective groups. Fifty-six days afterthe simulation operation, biochemistry tests and enzyme-linked immunosorbent assay were used to measure NOS and 13-EP in the hypothalamus, spinal cord, and peripheral blood. RESULTS： Rats with the simulation operation showed significantly higher levels of NOS and II3-EP in the hypothalamus, spinal cord, and peripheral blood than those in the normal group. The EA and acupotomy groups had significantly lower levels of NOS and β-EP than those in the model group. There was no statistical difference between the EA and acupotomy groups. CONCLUSION： EA and acupotomy treatments significantly lowered NOS and β-EP levels in the hypothalamus, spinal cord, and peripheral blood and alleviated L3 transverse process syndrome.

Regulatory effects of stage-treatment with established Chinese herbal formulas on inflammatory mediators in pediatric asthma

OBJECTIVE： To explore the regulatory effects of es- tablished Chinese herbal formulas on inflammatory mediators released during asthma attacks, and to elucidate the molecular mechanism of Traditional Chinese Medicine in the treatment of asthma. METHODS： Seventy-five asthmatic children were randomly divided into a Chinese medication group （45 cases） and a Western medication control group （30 cases）. Patients in the Chinese medication group were treated with a series of established Chi- nese herbal formulas, whereas the Western medica- tion control group received a leukotriene receptor antagonist and a bronchial relaxant. Real-time PCR was used to determine the mRNA expression levels of interleukin （IL）-4, cysteinyl leukotriene receptor 1 （CysLTR1）, and interferon （IFN）-γ in peripheralblood mononuclear cells before and after treat- ment. Enzyme-linked immunosorbent assay was used to measure the peripheral blood levels of IL-4, leukotriene （LTE）-4, and INF-γ before and after treat- ment. RESULTS： After treatment, the mRNA expression levels of 11-4 and CysLTR1 were down-regulated （P〈 0.01） and the mRNA expression levels of IFN-γ were up-regulated （P〈0.05） in the Chinese medication and Western medication groups; no significant dif- ference was found between the two groups. In the Chinese medication group, IL-4 blood level was de- creased and it was significantly different from that in the Western medication group （P〈0.05）; there was also a significant increase in IFN-γ blood levels after treatment with Chinese medica- tion （P〈0.05）. There were no significant differenc- es in LTE-4 blood levels between the two groups before and after treatment （P〉0.05）. CONCLUSION： Chinese medication has a regulato- ry effect on leukotriene receptor gene expression and the imbalance of Th1/Th2 immune cells dur- ing asthma attacks in pediatric patients.

Effect of Liuweibuqi capsule, a Chinese patent medicine, on the JAK1/STAT3 pathway and MMP9/TIMP1 in a chronic obstructive pulmonary disease rat model

OBJECTIVE： To observe effect of Liuweibuqi Capsule, a Traditional Chinese Medicine （TCM）, on the janus kinase （JAK）/signal transducer and activator of transcription （STAT） pathway and matrix metalloproteinases （MMPs） in a chronic obstructive pulmonary disease （COPD） rat model with lung deficiency in terms of TCM＇s pattern differentiation. METHODS： Rats were randomly divided into a normal group, model group, Liuweibuqi group, Jinshuibao group, and spleen aminopeptidase group （n= 10）. Aside from the normal group, all rats were ex-posed to smoke plus lipopolysaccharide tracheal instillation to establish the COPD model with lung deficiency. Models were established after 28 days and then the normal and model groups were given normal saline （0.09 g/kg）, Liuweibuqi group was given Liuweibuqi capsule （0.35 g/kg）, Jinshuibao group was given Jinshuibao capsules （0.495 g/kg）, and the spleen group was given spleen aminopeptidase （0.33 mg/kg）, once a day for 30 days. Changes in symptoms, signs, and lung histology were observed. Lung function was measured with a spirometer. Serum cytokines were detected using enzyme-linked immunosorbent assay, and changes in the JAK/STAT pathway, MMP-9, and MMPs inhibitor 1 （TIMP1） were detected by immunohistochemistry, RT-PCR, and western blotting, respectively.RESULTS： Compared with the normal group, lung tissue was damaged, and lung function was reduced in the model control group. Additionally, the levels of interleukin （IL）-1β, y interferon （IFN-γ）, and IL-6 were higher, while IL-4 and IL-10 were lower in the model control group than those in the normal group. The expressions of JAK1, STAT3, ρ-STAT3, and MMP-9 mRNA and protein in lung tissue were higher, and TIMP1 mRNA and protein was lower in the model group compared with the normal group. After treatment, compared with the model group, the expression of inflammatory cytokines was lower in each treatment group, and expressions of JAK/ STAT pathway, MMPs were lower. Compared with the positive control groups, the Jinshuibao and spleen aminopeptidase groups, lung function was better, and JAK1, STAT3, and p-STAT3 protein were lower and TIMP1 was higher in the Liuweibuqi group.CONCLUSION： Liuweibuqi capsules can improve the symptoms of COPD possibly by regulating the expression of the JAK1/STAT3 pathway and MMP9/ TIMP1.

Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp

A novel monoclonal antibody （MAb） against oxytetracycline （OTC） was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay （ELISA）-based detection system. An OTC-bovine serum albumin （BSA） conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA （icELISA） was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration （IC50） and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an ICs0 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross- reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118% for an intra-assay and 96%-113% for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9% and 5.5%-14.9%, respectively.

High throughput monoclonal antibody generation by immunizing multiple antigens

Recognizing proteins via the production of highly specific monoclonal antibodies(mAbs)is crucial to identifying proteins for proteomic research.However,traditional mAb generation is time-consuming with low efficiency.In this study,we assessed the high throughput method of producing mAbs by immunizing mice with multiple antigens in order to obtain hybridomas against these multiple antigens in one cell fusion.We selected eight proteins that play important roles in human physiological or pathological processes.These proteins were mixed and simultaneously administered to one mouse.We observed the immunizing period for 10 d,and determined the effect of liquid medium and semi-solid medium in hybridoma generation.As a result,all eight immunogens induced antibodies in the immunized mouse in one cell fusion,we obtained hybridomas specific to all eight proteins by enzyme-linked immuno sorbent assay(ELISA)screening,hybridomas against five out of eight showed specific positive in Western-blotting assays.This indicates that we generated mAbs specific to eight proteins in one cell fusion,greatly increasing the efficiency of mAb generation.Furthermore,we observed that hybridomas selected from the liquid medium and semi-solid medium showed different reactivity to antigens.Our study established high-throughput and time-saving methods for production of mAbs.These results provide alternative approaches for increasing the efficacy of mAb generation.