Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling

Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as ＂bait＂： Fusobacterium nucleatum （F. nucleatum） whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans （S. mutans） whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the ＂bait＂ oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The ＂prey＂ bacterial mixtures （including known species or natural saliva samples） were added, unbound ceils were washed off after the incubation period and the remaining cells were eluted using 0.2 mol.L1 glycine. Genomic DNA was extraeted, subjeeted to 16S rRNA PCR amplification and separation of the resulting PCR produets by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.. nucleatum adhered to a variety of bacterial species including uncultivable and uneharacterized onesl This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.

Structure,Binding Characteristics,and 3D Model Prediction of a Newly Identified Odorant-Binding Protein from the Cotton Bollworm,Helicoverpa armigera (Hübner)

The full-length sequence of the odorant binding protein 5 gene,HarmOBP5,was obtained from an antennae cDNA library of cotton bollworm,Helicoverpa armigera （Hübner）.The cDNA contains a 444 bp open reading frame,encoding a protein with 147 amino acids,namely HarmOBP5.HarmOBP5 was expressed in Escherichia coli and the recombinant protein was purified by affinity chromatography.SDS-PAGE and Western blot analysis demonstrated that the purified protein can be used for further investigation of its binding characteristics.Competitive binding assays with 113 odorant chemicals indicated that HarmOBP5 has strong affinity to some special plant volatiles,including （E）-β-farnesene,ethyl butyrate,ethyl heptanoate,and acetic acid 2-methylbutyl ester.Based on three-dimensional （3D） model of AaegOBP1 from Aedes aegypti,a 3D model of HarmOBP5 was predicted.The model revealed that some key binding residues in HarmOBP5 may play important roles in odorant perception of H.armigera.This study provides clues for better understanding physiological functions of OBPs in H.armigera and other insects.

In vitro effects of buyang huanwu decoction and its ingredients on inhibiting the specific binding of ~3H-platelet activating factor to its receptor in rabbits

BACKGROUND： Pharmacologic action of traditional Chinese medicine compound is the comprehensive effect of various ingredients, and the interactions of various ingredients are closely correlated with the final effect. In order to reveal the compatibility mechanism of buyang huanwu decoction （BHD）＇s prescription in treating and preventing ischemic cerebrovascular disease, we need to explore the effect and relation of ingredients in prescription except for considering the effect of each ingredient on the whole prescription. OBJECTIVE： To study the effect of BHD and its ingredients in the prescription on the specific binding of 3H-platelet activating factor （PAF） to its receptor （PAFR）in rabbits in vitro, and to analyze the action of each ingredient in the prescription. DESIGN： A decomposed recipe study based on orthogonal test. SETTING： Guangzhou University of Traditional Chinese Medicine. MATERIALS： Five healthy adult New Zealand rabbits of either gender were provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese medicine. The prescription herbal pieces were purchased from Foshan Kangpu Pharmaceuticals Company and Jianmin Pharmaceuticals Company, and were appraised by Professor Yanchen Xu from College of Traditional Chinese Medicine, Guangzhou University of Traditional Chinese Medicine. 3H-PAF was supplied by Amersham Co,Ltd.（Specific activity： 6.475 TBq/mmol;batch number：200402）; PAF standard by Biomol Co., Ltd.（batch number： P1318V）. METHODS： This experiment was carried out in the Laboratory of Nuclear Medicine, Guangzhou University of Traditional Chinese Medicine between September and December 2004. ①The seven influencing factors were selected： such as Shenghuangqi , Dangguiwei, Chishao, Dilong, Taoren, Honghua, Chuanxiong. Each factor was divided into two levels, selected or not selected. The tests were arranged according to L8 （27） orthogonal test table. ②The specific binding of 3H-PAF to its receptors in rabbits was measured by radioligand binding assay. The inhibitory rate of the specific binding was used as an assessing index. The inhibitory action of and on 3H-PAF to PAFR binding was analyzed and compared in vitro. The inhibitory action of each ingredient in the prescription BHD on 3H-PAF to PAFR binding was investigated and compared in vitro by direct analysis and analysis of variance of orthogonal test. MAIN OUTCOME MEASURES： Effect of 8 prescriptions for L8 （27） orthogonal test table on the specific binding inhibition rate of 3H-PAF and PAFR. RESULTS： According to results of variance analysis of orthogonal test, the inhibitory action of each ingredient in the prescription BHD on 3H-PAF to PAFR binding from the highest to the lowest was in turn Honghua, ShenghuangqL Taoren, Dilong, DangguiweL Chuanxiong, Chishao. Honghua, Shenghuangqi, Taoren, Dilong, Danguiwei were major influence factors to 3H-PAF to PAFR in rabbits （F = 187.829,144.446,59.521,5.018,4.265, P 〈 0.05- 0.01）, but Chuanxiong and Chishao had not obviously inhibitory effect. The specific binding inhibition rate of prescriptions （except Shenghuangqi ） was obviously higher than that of one of prescriptions （Shenghuangqi included）. CONCLUSION： The results of orthogonal test show that Honghua, ShenghuangqL Taoren, Dilong, Dangguiwei are major influencing factors to inhibit binding of sH-PAF to PAFR in rabbits, among which, Honghua is the strongest in ingredients of prescription BHD. The results also reveal that Shenghuangqi is able to weaken the inhibitory effect and to prevent the strong inhibitory effect of blood-activating drugs in BHD.

Characterization of the chemosensory protein EforCSP3 and its potential involvement in host location by Encarsia formosa

Chemosensory proteins(CSPs) perform several functions in insects.This study performed the gene expression,ligand-binding,and molecular docking assays on the EforCSP3 identified in the parasitoid wasp Encarsia formosa,to determine whether EforCSP3 functions in olfaction,especially in host location and host preference.The results showed that EforCSP3 was highly expressed in the female head,and its relative expression was much higher in adults than in other developmental stages.The fluorescence binding assays suggested that the EforCSP3 exhibited high binding affinities to a wide range of host-related volatiles,among which dibutyl phthalate,1-octene,β-elemene,and tridecane had the strongest binding affinity with EforCSP3,besides α-humulene and β-myrcene,and should be assessed as potential attractants.Protein structure modeling and molecular docking predicted the amino acid residues of EforCSP3possibly involved in volatile binding.α-Humulene and β-myrcene attracted E.formosa in a previous study and exhibited strong binding affinities with EforCSP3 in the current study.In conclusion,EforCSP3 may be involved in semiochemical reception by E.formosa.

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera (Hübner)

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera （Hvbner） was obtained from antennal eDNA libraries and expressed in Escherichia coll. The real time quantitative PCR （RT-qPCR） results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings. Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles. The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde, whereas methyl phenylacetate, 2-decanone, 1-pentanol, carvenol, isobomeol, nerolidol, 2- nonanone and ethyl heptanoate have relatively weak binding affinity. Moreover, the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38 （αl）, 40-48 （α2）, 62-72 （α3）, 80-96 （α4）, 98-108 （α5）, and 116-119 （α6）, two pairs of disulfide bridges Cys49-Cys55, Cys75-Cys78. The two amino acid residues, Ile94 and Trpl01, may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.

Molecular and in vitro biochemical assessment of chemosensory protein 10 from brown planthopper Nilaparvata lugens at acidic pH

Chemosensory proteins(CSPs)are important molecular components of the insect olfactory system,which are involved in capturing,binding,and transporting hydrophobic odour molecules across the sensillum in sensillar lymph in regulating insect behavior.This protein family(CSPs)is also involved in many other systems that are not linked to olfactory receptors in olfactory sensilla.The brown planthopper(BPH)is a monophagous pest of rice that causes damage by sucking phloem sap and transmitting a number of diseases caused by viruses.In this study,fluorescence competitive binding assay and fluorescence quenching assay at acidic p H were performed as well as homology modelling to describe the binding affinity of Nlug CSP10.Fluorescence competitive binding assay(FCBA)demonstrated that Nlug CSP10 bound strongly to nonadecane,farnesene,and 2-tridecanone at acidic p H.The results of FCBA indicated that Nlug CSP10 bound different ligands at the physiological p H(5.0)of the bulk sensillum lymph.Fluorescence quenching assay demonstrated that Nlug CSP10 generated a stable complex with 2-tridecanone,while two ligands nonadecane and farnesene collided due to molecular collisions.The interaction of selected ligands with the modelled structure of Nlug CSP10 was also analyzed,which found the key amino acids(Gln23,Gln24,Gln25,Asn27,Met33,Ser34,Ile35,Tyr36,Asn42,Met43,Val45,Asn46,Asn93,Arg96,Ala97,Lys99,and Ala100)in Nlug CSP10 that were involved in binding of volatile compounds.The present study contributes to the binding profile of Nlug CSP10 that promotes the development of behaviorally active ligands based on BPH olfactory system.

Comparison of Different MARs(Matrix Attachment Regions) Effect on Transgene Expression

Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respectively. Sequence analysis showed that all thefragments had fairly high A/T content (73, 62 and 75%, respectively),harbored differentnumber and different type of some characteristic motifs of MARs, such as A-box and T-box,etc. The results of in vitro binding assay showed that the three MARs fragments derivedfrom different organisms could bind specifically to the matrix extracted from the tobacconuclei with different strength, which also demonstrated that these MARs fragments arefunctionally conserved during evolution. By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-bar-MARs into tobacco through Agrobacterium mediated procedures, the effects of MARs sequenceson the expression of transgenes in tobacco were investigated and compared. The GUSactivity in individual transformants showed that, comparing to the controls withoutadditional MARs, the overall transgene expression level in transformants with MARs hadbeen greatly increased while the variations in transgene expression among transformantswere decreased in different degrees. In accordance with the results of sequence analysisand in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overallexpression level.

Identification and expression profiles analysis of odorant-binding proteins in soybean aphid,Aphis glycines(Hemiptera:Aphididae)

The soybean aphid,Aphis glycines,is an extreme specialist and an important invasive pest that relies on olfaction for behaviors such as feeding,mating,and foraging.Odorant-binding proteins(OBPs)play a vital role in olfaction by binding to volatile compounds and by regulating insect sensing of the environment.In this work we used rapid amplification of complementary DNA ends technology to identify and characterize 10 genes encoding A.glycines OBPs(AglyOBPs)belonging to 3 subfamilies,including 4 classic OBPs,5 Plus-C OBPs,and one Minus-C OBP.Quantitative real-time polymerase chain reaction demonstrated variable specific expression patterns for the 10 genes based on developmental stage and aphid tssue sampled.Expression levels of 7 AglyOBPs(2,3,4,5,7,9,and 10)were highest in the 4th instar,indicating that the 4th nymphal instar is an important developmental period during which soybean aphids regulate feeding and search for host plants.Tissue-specific expression results demonstrated that AglyOBP2,7,and 9 exhibited significantly higher expression levels in antennae.Meanwhile,ligand-binding analysis of5 OBPs demonstrated binding of AglyOBP2 and AglyOBP3 to a broad spectrum of volatiles released by green leaf plants,with bias toward 6-to 8-carbon chain volatiles and strong binding of AglyOBP7 to trans-B-farnesene.Taken together,our findings build a foundation of knowledge for use in the study of molecular olfaction mechanisms and prov ide insights to guide future soybean aphid research.

Molecular and functional characterization of three odorant-binding proteins from the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana,a periodic but devastating wheat pest,relies on wheat spike volatiles as a cue in sclecing hosts for oviposition.Insect odorant-binding proteins(OBPs)are thought to play essential roles in filtering,binding and transporting hydropho-bic odorant molecules to specific receptors.To date,the molecular mechanisms underlying S.mosellana olfaction are poorly understood.Here,three S.mosellana antenna-specific OBP genes,SmosOBPII,16 and 21,were cloned and bacterially expressed.Binding properties of the recombinant proteins to 28 volatiles emitted from wheat spikes were in-vestigated using fluorescence competitive binding assays.Sequence analysis suggested that these SmosOBPs belong to the Classic OBP subfamily.Ligand-binding analysis showed that all three SmosOBPs preferentially bound alcohol,ester and ketone com-pounds,and SmosOBP11 and 16 also selectively bound terpenoid compounds.In par-ticular,the three SmosOBPs had high binding affinities(Ki<20μmol/L)to 3-hexanol and cis-3-hexenylacetate that elicited strong electroantennogram(EAG)response fromfemale antennae.In addition,SmosOBP11 displayed significantly higher binding(Ki<8μmo/L)than SmosOBP16 and 21 to l-octen-3-ol,D-panthenol,a-pinene and heptyl acetate which elicited significant EAG response,suggesting that SmosOBP11 plays a ma-jor role in recognition and transportation of these volatiles.These findings have provided important insight into the molecular mechanism by which S.mosellana specifically rec-ognizes plant volatiles for host selection,and have facilitated identification of effective volatile attractants that are potentially useful for pest monitoring and trapping.

Significance of plasma von Willebrand factor level and von Willebrand factor-cleaving protease activity in patients with chronic renal diseases

Background von Willebrand factor （vWF） mediates the initial capture of platelets to vascular subendothelium and is essential for platelet aggregation under high fluid shear stress as in arterial stenosis. On release from endothelial cells, vWF is rapidly cleaved by ADAMTS13/vWF-cleaving protease （vWF-CP）. We investigated the clinical significance of changes in plasma vWF and vWF-CP activities in chronic renal disease.Methods Plasma vWF and vWF-CP activities were measured using enzyme-linked immunosorbent assay （ELISA） and residual collagen binding assay respectively in patients with lupus nephritis （n=31）, primary nephritic syndrome （n=25）, diabetic nephropathy （n=45）, chronic glomerulonephritis （n=38） and 40 normal controls. The relation of their levels with pathological and renal status was analyzed.Results In all diseased patients the levels of vWF were significantly higher and vWF-CP activity significantly lower than the controls （both P〈0.01）. vWF in the four subgroups did not correlate with the stage of disease but correlated negatively with vWF-CP activity, vWF-CP activity was not changed two weeks after renal transplantation. Renal biopsy demonstrated that the vWF level in stage IV was higher than in stages II and III while vWF-CP activity was lower in patients with lupus nephritis. After eight-week treatment, the vWF level significantly decreased and the vWF-CP activity significantly increased in systemic lupus erythema, disease activity index 〈9, but not with index 〉9. Even though the vWF-CP activity was significantly lower in membranous nephropathy than in minimal change disease, mesangial proliferative glomerulonephritis or IgA glomerulonephritis, the vWF level was not significantly different. Conclusions The alterations of plasma vWF and vWF-CP activities were associated with different renal pathologies. Injury to endothelial cells and autoantibodies against vWF-CP activity may result in higher vWF level and lower vWF-CP activity in chronic renal disease and thus a mechanism for worsening of chronic renal disease and thrombosis.

Evaluation of von Willebrand factor-cleaving protease activity in patients with thrombotic thrombocytopenic purpura

Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.Methods The plasma or serum vWF-cp activity was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) by detecting the residual collagen binding activity (R-CBA) of von Willebrand factor (vWF) before and after digestion by vWF-cp. Multimers of vWF in plasma of patients with TTP were also analyzed by SDS-agarose electrophoresis. Moreover,the serum vWF-cp activities were compared between the patients with TTP and those with tumors.Results The coefficient of variation for intra-batch and inter-batch of the assay were 3.60% and 8.35%. The plasma and serum vWF-cp activity in healthy individuals were (78.79±9.17)% (n=30) and (79.47±10.78)% (n=53),respectively,while the plasma vWF-cp activity in 5 patients with TTP was markedly decreased [(21.83±19.98)%,P <0.001]. The unusually large vWF multimers were observed in two plasma samples of the patients with TTP. Although the vWF-cp activities in patients with benign and malignant tumors were also decreased ( P <0.03 and P <0.001,respectively),they were relatively high in comparison with that of TTP patients ( P <0.001).Conclusion Measurement of the vWF-cp activity using R-CBA is a simple and rapid method for diagnosing TTP. The vWF-cp activity in patients with TTP was markedly lower than those of patients with tumors.

Molecular and functional analysis of monoclonal antibodies in support of biologics development

Monoclonal antibody （mAb）-based therapeutics are playing an increasingly important role in the treatment or pre- vention of many important diseases such as cancers, autoimmune disorders, and infectious diseases. Multi- domain mAbs are far more complex than small molecule drugs with intrinsic heterogeneities. The critical quality attributes of a given mAb, including structure, post-trans- lational modifications, and functions at biomolecular and cellular levels, need to be defined and profiled in details during the developmental phases of a biologics. These critical quality attributes, outlined in this review, serve an important database for defining the drug properties during commercial production phase as well as post licensure life cycle managemenL Specially, the molecular characteriza- tion, functional assessment, and effector function analysis of mAbs, are reviewed with respect to the critical parame- ters and the methods used for obtaining them. The three groups of analytical methods are three essential and inte. gral facets making up the whole analytical package for a mAIPbased drug. Such a package is critically important for the licensure and the post-licensurs life cycle management of a therapeutic or prophylactic biologics. In addition, the basic principles on the evaluation of biosimilar mAbs were discussed briefly based on the recommendations by the World Health Organization.

Evaluation and clinical application of a new method for detecting ADAMTS13 activity

Background A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura （TTP）. Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, so not easily adapted to routine laboratories. ADAMTS13 cleaves von Willebrand factor （VVVF） within the domain A2, located between domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structures of VWF before and after the cleavage. Using this hypothesis we try to establish a new and simple method to determine ADAMTS13 activity. Methods First, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies, SZ-129 and SZ-125, which specifically recognize the VWF A1 and A3 domains by using a two-site sandwich ELISA. Compared with a residual-collagen binding assay （R-CBA）, plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibody in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated. Results Plasma ADAMTS13 activities in normal people and TTP, acute myocardial infarction （AMI）, and idiopathic thrombocytopenic purpura （ITP） patients determined by the new assay were （89.75±7.93）%, （17.63±18.71）%, （68.55±18.08）%, （85.83±9.84）%, respectively. Results were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays. The new assay can easily discriminate a ＂TTP plasma sample from a non-TTP plasma sample （P 〈0.01）, and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activity of ADAMTS13 autoantibody ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient. Conclusion This new and simple assay for ADAMTS13 activity could be used routinely in the clinic to determine the activity of ADAMTS13.