Sequence Analysis and Molecular Characterization of Clonorchis sinensis Hexokinase,an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors of CsHK to interfere with glycolysis in C. sinensis.     

Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.     

β乳球蛋白基因（βlg）的表达调控及其应用

β乳球蛋白（BLG）是反刍动物乳汁中的主要乳清蛋白,BLG表达受到βlg核心启动子,LCR,MAR等顺式作用元件和激素,转录因子等反式作用因子的调控,利用βlg启动子已在乳腺成功表达外源基因,但乳腺组织特异性表达外源基因时尚存在异位表达,差异表达,表达水平低和表达受位置效应影响等问题,构建表达载体时充分考虑βlg启动子和远端调控元件,有可能使上源基因获得,高效,特异的表达。     

A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes

Clonorchiasis caused by Clonorchis sinensis is a fish-borne parasitic disease which is endemic in a number of countries. Using the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of C. sinensis as genetic markers, a pair of C. sinensis-specific primers was designed and used to establish a specific PCR assay for the diagnosis of C. sinensis infection in humans, cats and fish. This approach allowed the specific identification of C. sinensis after optimizing amplification conditions, with no amplicons being amplified from related heterogeneous DNA samples, and sequencing of amplicons confirmed the identity of the sequences amplified. The detection limit of this assay was 1.03 pg of adult C. sinensis, 1.1 metacercariae per gram of fish filet, and a single egg in human and cat feces. The PCR assay should provide a useful tool for the diagnosis and molecular epidemiological investigation of clonorchiasis in humans and animals.     

Cyst formation, increased anti-inflammatory cytokines and expression of chemokines support for Clonorchis sinensis infection in FVB mice

To verify the hypothesis that different pathology of Clonorchis sinensis infection by mouse strains is determined by different responses of cytokines and chemokines, we compared those responses of FVB with those of BALB/c mice. All of FVB mice infected with 30 metacercariae of C. sinensis showed cystic dilatation in the liver, whereas infected BALB/c mice did not. Mature worms were recovered from 19 of 20 liver sections of FVB mice while only one of 20 sections of BALB/c mice revealed a mature worm. In both strains the proportion of CD4⁺ T cells was lower in C. sinensis-infected than in the uninfected group. However, the proportion of CD8⁺ T cells was elevated in C. sinensis-infected from both strains compared to uninfected mice. The Th2-associated anti-inflammatory cytokines such as IL-4, IL-5 and IL-13, IL-10 and TGF-β, were significantly more produced by the lymphocytes of FVB than by those of BALB/c mice. Especially, the 2 anti-inflammatory cytokines, IL-10 and TGF-β, were presumably related with susceptibility and the development of worms in the liver. C. sinensis infected FVB mice also produced more chemokines such as RANTES and MIP-1α in the liver lymphocytes than BALB/c mice. In conclusion, the FVB mice provide the favorable niche for C. sinensis by cyst formation in the bile duct, increased production of Th2-associated anti-inflammatory cytokines and upregulation of chemokines.     

Probiotic Potential of a Lactobacillus Bacterium of Canine Faecal-Origin and Its Impact on Select Gut Health Indices and Immune Response of Dogs

The objective of the present study was to develop a probiotic of canine-origin for its potential application in pet nutrition. Accordingly, 32 lactic acid bacteria (LAB) strains were isolated from faeces of dogs, out of which 9 strains were short-listed for further in vitro testing based on the aggregation time and cell surface hydrophobicity. The results of acid-, bile- and phenol-tolerance tests indicated that out of the nine, isolate cPRO23 was having better resistance to these adverse conditions likely to be encountered in the gastrointestinal tract. The isolate also showed optimal enzymatic activities for amylase, lipase and protease. Further assessments also indicated its superiority in terms of co-aggregation and antagonistic activity against pathogenic strains of Salmonella typhimurium and Salmonella enteritidis. Subsequently, the isolate was identified through 16S rRNA sequencing and sequence homology, and designated as Lactobacillus johnsonii CPN23. The candidate probiotic was then evaluated in vivo using 15 adult Labrador dogs, divided into 3 groups, viz. CON (with no probiotics), dPRO (with Lactobacillus acidophilus NCDC 15 as a conventional dairy-origin probiotic) and cPRO (with L. johnsonii CPN23 as a canine-origin probiotic). Results of the 9-week study indicated that supplementation of cPRO improved (P < 0.05) the faecal concentration of acetate and butyrate with a concomitant reduction (P < 0.05) in faecal ammonia. The cell-mediated immune response, assessed as delayed-type hypersensitivity reaction to phytohaemagglutinin-P, was better (P < 0.05) in dogs fed cPRO as compared to the CON dogs. There were, however, no variations evident in the antibody response to sheep-erythrocytes among the three groups. It is concluded that the canine-origin L. johnsonii CPN23, in addition to possessing all the in vitro functional attributes of a candidate probiotic, also has the potential to be used as a probiotic in pet nutrition programs.     

飞虱虫疠霉杀蚜效果及开发应用前景

通过比较飞虱虫疠霉 (Pandoradelphacis)与其它几种常见蚜虫专化性病原真菌新蚜虫疠霉(P .neoaphidis)、安徽虫瘟霉 (Zoothphoraanhuiensis)和根虫瘟霉 (Z .radicans)对桃蚜 (Myzuspersicae)毒力 ,证实了该菌的杀蚜应用潜力。时间 -剂量 -死亡率模型分析表明 ,飞虱虫疠霉杀蚜的各项毒力指标与其它几种虫霉相似或略高 ,但杀蚜速率更快。同时对培养性状、产孢特性和生长适温等的比较研究发现 ,该菌具有易发酵生产、产孢能力强和温度生长范围宽等特点 ,是一种较为理想的有望研制开发成杀蚜真菌制剂的病原菌。此外 ,根据虫霉孢子主动弹射的生物学特性 ,提出了该菌的适用剂型 ,并对它们的流行学特性及其在设栽培中的应用前景进行了讨论。     

Effect of Different Levels of Selenium Supplementation on Growth Rate,Nutrient Utilization,Blood Metabolic Profile,and Immune Response in Lambs

The nutritional role of silver for the freshwater crustacean, Daphnia magna, was examined through four generations of deprivation. Silver inclusion in animal media was set at a nominal zero (employing chemicals of the highest available purity). Both reproduction (−60%) and life span (−40%) were negatively affected when compared to animals reared in the presence of 0.4 ng g⁻¹ Ag. These results strongly suggest a nutritional requirement of silver for daphnids at nanomolar concentrations.     

Genome-Wide Identification,Evolution, and Expression Profile of Aquaporin Genes in Coffea canephora in Response to Water Deficit

Aquaporins (APQs) belong to the major intrinsic protein superfamily and play a key role in the transport of water and other solutes across cell membranes. Coffea canephora is an evergreen shrub used for making instant coffees. Genome analysis of C. canephora identified 33 putative aquaporin genes assigned to five subfamilies including seven plasma membrane intrinsic proteins (PIP), 9 tonoplast intrinsic proteins (TIP), 11 NOD26-like intrinsic proteins (NIP), 3 small basic intrinsic proteins (SIP), and 3 X intrinsic proteins (XIP). Generally, the AQPs gene structure was conserved within each subfamily, with exon numbers ranging from one to five. The prediction of the aromatic/arginine selectivity filter (ar/R) and Froger’s positions indicated a noticeable difference in substrate specificity between subfamilies. Synteny analysis revealed high conservation of aquaporin genes in coffee. In silico expression analysis of the CcAQPs genes indicated that they were differentially expressed in various tissues. Members of CcPIPs and CcTIPs subfamilies were validated by real-time quantitative analysis in leaves of two genotypes of C. canephora with contrasting responses to water deficit (clone 14: drought-tolerant and clone 109A: drought-susceptible). Under severe water deficit, the relative expression of isoforms of both genes decreased in clone 14 compared with that under the irrigated condition, while clone 109A showed comparatively higher mRNA levels, with the exception of CcPIP1;2 in the stress condition. This study was the first to characterize and validate aquaporin genes in C. canephora in response to water deficit, and the findings may provide insights for biotechnological approaches to increase tolerance to drought.

     

Lactobacillus reuteri KT260178 Supplementation Reduced Morbidity of Piglets Through Its Targeted Colonization,Improvement of Cecal Microbiota Profile,and Immune Functions

Supplementing suckling piglets with Lactobacillus reuteri isolated from a homologous source improves L. reuteri colonization number in the gastrointestinal tract, which can have health benefits. This study investigated dietary L. reuteri supplementation on the growth and health—including immune status—of piglets, as well as its colonization. A total of 60 sows with similar parity and body weight were allocated into one of three groups after secretion (n = 20 each, with 10 neonatal piglets of each): untreated control, L. reuteri supplementation, and antibiotic treatment. The experimental duration was 28 days, from birth of piglets to their group transferred. For the first 7 days after birth, all neonatal piglets were fed by sows. Piglets in the L. reuteri supplementation group were administered with 1.0 ml L. reuteri fermentation broth containing 5.0 × 10⁷ CFU. From 7 to 28 days, piglets were given basal feed (control), basal feed supplemented with L. reuteri (1.0 × 10⁷ CFU/g), or aureomycin (150 mg/kg). L. reuteri colonization in the distal jejunum and ileum was increased in piglets in the L. reuteri-supplemented as compared to the control group after 28 days, as determined by fluorescence in situ hybridization and real-time PCR analysis. Total Lactobacillus and Bifidobacterium counts in the cecum were higher whereas total aerobic bacteria (Escherichia coli and Staphylococcus) counts were lower in the L. reuteri as compared to the control group. L. reuteri supplementation also improved body antioxidant status and immune function relative to control animals. Strain-specific L. reuteri administered to piglets colonizes the intestinal mucosa and improves cecal microbiota profile and whole-body antioxidant and immune status, leading to better growth and lower morbidity and mortality rates.

     

IBV核蛋白的表达纯化及在监测中应用

鸡传染性支气管炎病毒（Infectious bronchitis virus,IBV）是禽类一种变异性很强的冠状病毒,为获得高度纯化的IBV核蛋白,建立监测IBV抗体的方法,通过RT-PCR从IBV中扩增IBV-N基因,定向连接到表达载体pGEX-KG中,转化E. coli BL21（DE3）菌株,诱导获得表达产物,SDS-PAGE分析产物大小、Western blot分析其免疫学活性,通过亲和层析法获得高度纯化的表达蛋白,建立一种检测IBV抗体的方法,并应用于临床监测。结果显示,IBV-N基因全长1230bp,GST融合蛋白表达产物大小约为80kDa,表达量约占菌体总蛋白的25%,具有良好的免疫学活性;通过GST亲和层析柱成功获得纯化蛋白,以该蛋白包被酶标板,成功地建立了一种检测血清中IBV抗体的间接ELISA方法。研究表明重组N蛋白作为IBV诊断抗原,具有制备简便、特异性强、敏感性高、重复性好的特点,可用于该病的临床监测,为该病疫情监测、发病机制研究奠定了基础。     

Investigations on Modulating Effect of Vanadium Supplementation on Growth and Metabolism Through Improved Immune Response,Antioxidative Profile and Endocrine Variables in Hariana heifers

Biological Trace Element Research - This study was conducted to investigate the effect of vanadium (V) supplementation on growth, metabolism, antioxidant, and immunological and endocrine variables...     

Effect of a Potential Probiotic Candidate Enterococcus faecalis -1 on Growth Performance,Intestinal Microbiota,and Immune Response of Commercial Broiler Chickens

The probiotic effect of Enterococcus faecalis-1 (isolated from healthy chickens) on growth performance, immune response, and modulation of the intestinal m     

Brief Introduction of Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention(May 20,2009)

The Institute for Viral Diseases Control and Prevention,Chinese Center for Disease Control and Prevention(IVDC,China CDC) is the unique nationally comprehensive medical research institute of virology and national center for viral diseases control and prevention,which undertakes the responsibility for national research of viral diseases,technical guidance,consulting,and training.     

The Molecular Basis for Control of ETEC Enterotoxin Expression in Response to Environment and Host

Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhoea in humans and neonatal farm animals. Annually, 380,000 human deaths, and multi-million dollar losses in the farming industry, can be attributed to ETEC infections. Illness results from the action of enterotoxins, which disrupt signalling pathways that manage water and electrolyte homeostasis in the mammalian gut. The resulting fluid loss is treated by oral rehydration. Hence, aqueous solutions of glucose and salt are ingested by the patient. Given the central role of enterotoxins in disease, we have characterised the regulatory trigger that controls toxin production. We show that, at the molecular level, the trigger is comprised of two gene regulatory proteins, CRP and H-NS. Strikingly, this renders toxin expression sensitive to both conditions encountered on host cell attachment and the components of oral rehydration therapy. For example, enterotoxin expression is induced by salt in an H-NS dependent manner. Furthermore, depending on the toxin gene, expression is activated or repressed by glucose. The precise sensitivity of the regulatory trigger to glucose differs because of variations in the regulatory setup for each toxin encoding gene.     

大鼠脑红蛋白基因编码区的克隆、多态性分析及该基因组织表达谱研究

参考人和小鼠脑红蛋白（Neuroglobin,NGB)的cDNA序列设计简并引物,用RT－PCR方法从大鼠脑组织中扩增出大鼠NGB基因编码区的cDNA序列,该序列与小鼠NGB基因编码区的序列同尖性为96％,与人NGB基因编码区的序列同源性为88％,进一步分析表明,大鼠NGB基因编码区存在多个多态性位点;113t/c[138P],133a/g[N45D],388a/g[R130G],417t/c.该序列已被GenBank接受,登录号为AF333245,RT－PCR分析表明,该基因在大鼠脑,肝,肾,心肌和骨骼肌中均有较高水平表达,提示了其功能上的重要性。     

cDNA芯片研发及其在肥胖患者脂肪基因差异表达研究中的应用

为了加快基因功能的研究,利用已有的来源于不同组织的cDNA克隆,并通过交换和购买补充了低丰度和染色体覆盖不完全的部分cDNA,研制开发出具有相当代表性、覆盖较完全的高密度cDNA表达型基因芯片,每张芯片上含有384个质控DNA和12 630个cDNA探针,其中包括12 508个Unigene和122个表达序列标签(EST).利用这些芯片,对肥胖患者及正常人内脏脂肪组织基因表达谱进行了初步研究,并发现在肥胖患者内脏脂肪组织差异表达的基因,其中上调的有与凋亡相关的基因、与免疫有关的基因以及与能量代谢有关的基因等,而下调的主要是与脂肪酸及胆固醇合成有关的基因,对这些基因进一步的功能研究将为阐明肥胖发生机制奠定基础.