Sample records for chitinase insertion domain

  1. Biochemistry of plant class IV chitinases and fungal chitinase-modifying proteins (United States)

    Plant class IV chitinases have 2 domains, a small (3 kDa) amino-terminal domain with homology to carbohydrate binding peptides, and a larger (25 kDa) catalytic domain. The biological function of these chitinases is not known. But it is known that some pathogenic fungi secrete chitinase modifying pro...

  2. Lesion insertion in the projection domain: Methods and initial results

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    Chen, Baiyu; Leng, Shuai; Yu, Lifeng; Yu, Zhicong; Ma, Chi; McCollough, Cynthia, E-mail: [Department of Radiology, Mayo Clinic, Rochester, Minnesota 55905 (United States)


    Purpose: To perform task-based image quality assessment in CT, it is desirable to have a large number of realistic patient images with known diagnostic truth. One effective way of achieving this objective is to create hybrid images that combine patient images with inserted lesions. Because conventional hybrid images generated in the image domain fails to reflect the impact of scan and reconstruction parameters on lesion appearance, this study explored a projection-domain approach. Methods: Lesions were segmented from patient images and forward projected to acquire lesion projections. The forward-projection geometry was designed according to a commercial CT scanner and accommodated both axial and helical modes with various focal spot movement patterns. The energy employed by the commercial CT scanner for beam hardening correction was measured and used for the forward projection. The lesion projections were inserted into patient projections decoded from commercial CT projection data. The combined projections were formatted to match those of commercial CT raw data, loaded onto a commercial CT scanner, and reconstructed to create the hybrid images. Two validations were performed. First, to validate the accuracy of the forward-projection geometry, images were reconstructed from the forward projections of a virtual ACR phantom and compared to physically acquired ACR phantom images in terms of CT number accuracy and high-contrast resolution. Second, to validate the realism of the lesion in hybrid images, liver lesions were segmented from patient images and inserted back into the same patients, each at a new location specified by a radiologist. The inserted lesions were compared to the original lesions and visually assessed for realism by two experienced radiologists in a blinded fashion. Results: For the validation of the forward-projection geometry, the images reconstructed from the forward projections of the virtual ACR phantom were consistent with the images physically

  3. Insertion of tetracysteine motifs into dopamine transporter extracellular domains.

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    Deanna M Navaroli

    Full Text Available The neuronal dopamine transporter (DAT is a major determinant of extracellular dopamine (DA levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

  4. Structural and biophysical analysis of sequence insertions in the Venezuelan Equine Encephalitis Virus macro domain. (United States)

    Guillén, Jaime; Lichière, Julie; Rabah, Nadia; Beitzel, Brett F; Canard, Bruno; Coutard, Bruno


    Random transposon insertions in viral genomes can be used to reveal genomic regions important for virus replication. We used these genomic data to evaluate at the protein level the effect of such insertions on the Venezuelan Equine Encephalitis Virus nsP3 macro domain. The structural analysis showed that transposon insertions occur mainly in loops connecting the secondary structure elements. Some of the insertions leading to a temperature sensitive viral phenotype (ts) are close to the cleavage site between nsP2 and nsP3 or the ADP-ribose binding site, two important functions of the macro domain. Using four mutants mimicking the transposon insertions, we confirmed that these insertions can affect the macro domain properties without disrupting the overall structure of the protein.

  5. Crystal structures and inhibitor binding properties of plant class V chitinases: the cycad enzyme exhibits unique structural and functional features. (United States)

    Umemoto, Naoyuki; Kanda, Yuka; Ohnuma, Takayuki; Osawa, Takuo; Numata, Tomoyuki; Sakuda, Shohei; Taira, Toki; Fukamizo, Tamo


    A class V (glycoside hydrolase family 18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/β)8 barrel fold with an (α + β) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8 kcal mol(-1) . However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.

  6. Chitinases: An update

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    Rifat Hamid


    Full Text Available Chitin, the second most abundant polysaccharide in nature after cellulose, is found in the exoskeleton of insects, fungi, yeast, and algae, and in the internal structures of other vertebrates. Chitinases are enzymes that degrade chitin. Chitinases contribute to the generation of carbon and nitrogen in the ecosystem. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications, especially the chitinases exploited in agriculture fields to control pathogens. Chitinases have a use in human health care, especially in human diseases like asthma. Chitinases have wide-ranging applications including the preparation of pharmaceutically important chitooligosaccharides and N-acetyl D glucosamine, preparation of single-cell protein, isolation of protoplasts from fungi and yeast, control of pathogenic fungi, treatment of chitinous waste, mosquito control and morphogenesis, etc. In this review, the various types of chitinases and the chitinases found in different organisms such as bacteria, plants, fungi, and mammals are discussed.

  7. Evaluation of a projection-domain lung nodule insertion technique in thoracic CT (United States)

    Ma, Chi; Chen, Baiyu; Koo, Chi Wan; Takahashi, Edwin A.; Fletcher, Joel G.; McCollough, Cynthia H.; Levin, David L.; Kuzo, Ronald S.; Viers, Lyndsay D.; Vincent Sheldon, Stephanie A.; Leng, Shuai; Yu, Lifeng


    Task-based assessment of computed tomography (CT) image quality requires a large number of cases with ground truth. Inserting lesions into existing cases to simulate positive cases is a promising alternative approach. The aim of this study was to evaluate a recently-developed raw-data based lesion insertion technique in thoracic CT. Lung lesions were segmented from patient CT images, forward projected, and reinserted into the same patient CT projection data. In total, 32 nodules of various attenuations were segmented from 21 CT cases. Two experienced radiologists and 2 residents blinded to the process independently evaluated these inserted nodules in two sub-studies. First, the 32 inserted and the 32 original nodules were presented in a randomized order and each received a rating score from 1 to 10 (1=absolutely artificial to 10=absolutely realistic). Second, the inserted and the corresponding original lesions were presented side-by-side to each reader, who identified the inserted lesion and provided a confidence score (1=no confidence to 5=completely certain). For the randomized evaluation, discrimination of real versus artificial nodules was poor with areas under the receiver operative characteristic curves being 0.69 (95% CI: 0.58-0.78), 0.57 (95% CI: 0.46-0.68), and 0.62 (95% CI: 0.54-0.69) for the 2 radiologists, 2 residents, and all 4 readers, respectively. For the side-by-side evaluation, although all 4 readers correctly identified inserted lesions in 103/128 pairs, the confidence score was moderate (2.6). Our projection-domain based lung nodule insertion technique provides a robust method to artificially generate clinical cases that prove to be difficult to differentiate from real cases.

  8. Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker. (United States)

    Kawashima, Satoshi; Ikehata, Hiroki; Tada, Chihiro; Ogino, Tomohiro; Kakizaki, Hiromi; Ikeda, Mana; Fukushima, Hideto; Matsumiya, Masahiro


    Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)₈-fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

  9. Cloning, Expression and 3D Structure Prediction of Chitinase from Chitinolyticbacter meiyuanensis SYBC-H1

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    Zhikui Hao


    Full Text Available Two CHI genes from Chitinolyticbacter meiyuanensis SYBC-H1 encoding chitinases were identified and their protein 3D structures were predicted. According to the amino acid sequence alignment, CHI1 gene encoding 166 aa had a structural domain similar to the GH18 type II chitinase, and CHI2 gene encoding 383 aa had the same catalytic domain as the glycoside hydrolase family 19 chitinase. In this study, CHI2 chitinase were expressed in Escherichia coli BL21 cells, and this protein was purified by ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G-100 chromatography. Optimal activity of CHI2 chitinase occurred at a temperature of 40 °C and a pH of 6.5. The presence of metal ions Fe3+, Fe2+, and Zn2+ inhibited CHI2 chitinase activity, while Na+ and K+ promoted its activity. Furthermore, the presence of EGTA, EDTA, and β-mercaptoethanol significantly increased the stability of CHI2 chitinase. The CHI2 chitinase was active with p-NP-GlcNAc, with the Km and Vm values of 23.0 µmol/L and 9.1 mM/min at a temperature of 37 °C, respectively. Additionally, the CHI2 chitinase was characterized as an N-acetyl glucosaminidase based on the hydrolysate from chitin. Overall, our results demonstrated CHI2 chitinase with remarkable biochemical properties is suitable for bioconversion of chitin waste.

  10. Comparative genomic analysis of chitinase and chitinase-like genes in the African malaria mosquito (Anopheles gambiae.

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    Jianzhen Zhang

    Full Text Available Chitinase is an important enzyme responsible for chitin metabolism in a wide range of organisms including bacteria, yeasts and other fungi, nematodes and arthropods. However, current knowledge on chitinolytic enzymes, especially their structures, functions and regulation is very limited. In this study we have identified 20 chitinase and chitinase-like genes in the African malaria mosquito, Anopheles gambiae, through genome-wide searching and transcript profiling. We assigned these genes into eight different chitinase groupings (groups I-VIII. Domain analysis of their predicted proteins showed that all contained at least one catalytic domain. However, only seven (AgCht4, AgCht5-1, AgCht6, AgCht7, AgCht8, AgCht10 and AgCht23 displayed one or more chitin-binding domains. Analyses of stage- and tissue-specific gene expression revealed that most of these genes were expressed in larval stages. However, AgCht8 was mainly expressed in the pupal and adult stages. AgCht2 and AgCht12 were specifically expressed in the foregut, whereas AgCht13 was only expressed in the midgut. The high diversity and complexity of An. gambiae chitinase and chitinase-like genes suggest their diverse functions during different developmental stages and in different tissues of the insect. A comparative genomic analysis of these genes along with those present in Drosophila melanogaster, Tribolium castaneum and several other insect species led to a uniform classification and nomenclature of these genes. Our investigation also provided important information for conducting future studies on the functions of chitinase and chitinase-like genes in this important malaria vector and other species of arthropods.

  11. Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase



    Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20–50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq...

  12. pH-Triggered Conformational Switching along the Membrane Insertion Pathway of the Diphtheria Toxin T-Domain

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    Alexey S. Ladokhin


    Full Text Available The translocation (T-domain plays a key role in the action of diphtheria toxin and is responsible for transferring the catalytic domain across the endosomal membrane into the cytosol in response to acidification. Deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the T-domain, which is considered to be a paradigm for cell entry of other bacterial toxins, reveals general physicochemical principles underlying membrane protein assembly and signaling on membrane interfaces. Structure-function studies along the T-domain insertion pathway have been affected by the presence of multiple conformations at the same time, which hinders the application of high-resolution structural techniques. Here, we review recent progress in structural, functional and thermodynamic studies of the T-domain archived using a combination of site-selective fluorescence labeling with an array of spectroscopic techniques and computer simulations. We also discuss the principles of conformational switching along the insertion pathway revealed by studies of a series of T-domain mutants with substitutions of histidine residues.

  13. Kinetic characterization of Aspergillus niger chitinase CfcI using a HPAEC-PAD method for native chitin oligosaccharides. (United States)

    van Munster, Jolanda M; Sanders, Peter; ten Kate, Geralt A; Dijkhuizen, Lubbert; van der Maarel, Marc J E C


    The abundant polymer chitin can be degraded by chitinases (EC and β-N-acetyl-hexosaminidases (EC to oligosaccharides and N-acetyl-glucosamine (GlcNAc) monomers. Kinetic characterization of these enzymes requires product quantification by an assay method with a low detection limit, preferably compatible with the use of native, non-labeled substrates. Here we report a quantitative HPAEC-PAD method that allows fast separation of chitin oligosaccharides (COS) ranging from (GlcNac)1-6 at detection limits of 1-3 pmol and a linear range of 5-250 pmol. Quantification under intra- and interday precision conditions was performed with 2.1-5.4% relative standard deviation (RSD) and 1.2-10.3% RSD, respectively. This method was successfully used for the determination of the kinetic parameters of the Aspergillus niger chitinase CfcI with native COS. CfcI was recently shown to release GlcNAc from the reducing end of COS, a new activity for fungal chitinases. A Carbohydrate Binding Module of family 18 (CBM18) is inserted in the CfcI catalytic domain. Site directed mutagenesis was used to assess the functionality of this CfcI-CBM18: four of its key amino acids were replaced by glycine residues, yielding CfcISYNF. Comparison of the kinetic parameters of CfcI and CfcISYNF confirmed that this CBM18 is functionally involved in catalysis.

  14. Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells

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    Zong-Hai Huang; Wen-Yu Yang; Qi Cheng; Jing-Long Yu; Zhou Li; Zong-Yan Tong; Hui-Juan Song; Xiao-Yan Che


    AIM: To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. METHODS: By PCR technology, human KDR promoter gene, Escherichia coli(E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDRCDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured.RESULTS: Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone. CONCLUSION: Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.

  15. X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold. (United States)

    Ericsson, Ulrika B; Nordlund, Pär; Hallberg, B Martin


    Pseudouridine synthases catalyse the isomerisation of uridine to pseudouridine in structural RNA. The pseudouridine synthase TruD, that modifies U13 in tRNA, belongs to a recently identified and large family of pseudouridine synthases present in all kingdoms of life. We report here the crystal structure of Escherichia coli TruD at 2.0 A resolution. The structure reveals an overall V-shaped molecule with an RNA-binding cleft formed between two domains: a catalytic domain and an insertion domain. The catalytic domain has a fold similar to that of the catalytic domains of previously characterised pseudouridine synthases, whereas the insertion domain displays a novel fold.

  16. Higher-Resolution Structure of the Human Insulin Receptor Ectodomain: Multi-Modal Inclusion of the Insert Domain. (United States)

    Croll, Tristan I; Smith, Brian J; Margetts, Mai B; Whittaker, Jonathan; Weiss, Michael A; Ward, Colin W; Lawrence, Michael C


    Insulin receptor (IR) signaling is critical to controlling nutrient uptake and metabolism. However, only a low-resolution (3.8 Å) structure currently exists for the IR ectodomain, with some segments ill-defined or unmodeled due to disorder. Here, we revise this structure using new diffraction data to 3.3 Å resolution that allow improved modeling of the N-linked glycans, the first and third fibronectin type III domains, and the insert domain. A novel haptic interactive molecular dynamics strategy was used to aid fitting to low-resolution electron density maps. The resulting model provides a foundation for investigation of structural transitions in IR upon ligand binding.

  17. Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi gene and characterization of its protein

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    Wan-Fang Zhong


    Full Text Available Chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. We used touchdown PCR to clone the chitinase (Schi gene from Bacillus thuringiensis serovar sotto (Bt sotto chromosomal DNA. Our DNA sequencing analysis revealed that the Bt sotto Schi gene consists of an open reading frame (ORF of 2067 nucleotides with codes for the chitinase precursor. We also found that the putative promoter consensus sequences (the -35 and -10 regions of the Bt soto Schi gene are identical to those of the chiA71 gene from Bt Pakistani, the chiA74 gene from Bt kenyae and the ichi gene from Bt israelensis. The Schi chitinase precursor is 688 amino acids long with an estimated molecular mass of 75.75 kDa and a theoretical isoelectric point of 5.74, and contains four domains, which are, in sequence, a signal peptide, an N-terminal catalytic domain, a fibronectin type III like domain and a C-terminal chitin-binding domain. Sequence comparison and the evolutionary relationship of the Bt sotto Schi chitinase to other chitinase and chitinase-like proteins are also discussed.

  18. Chitinase Production by Streptomyces sp. ANU 6277

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    Kolla J.P. Narayana


    Full Text Available Chitinase production by a terrestrial Streptomyces sp. ANU 6277 was studied under sub-merged fermentation. Chitinase production started after 24 h of incubation and reached maximum levels after 60 h of cultivation. A high level of chitinase activity was observed in the culture medium with pH 6 at 35ºC. Culture medium amended with 1% chitin was found to be suitable for maximum production of chitinase. An optimum concentration of colloidal chitin for chitinase production was determined. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that starch and yeast extract served as good carbon and nitrogen sources to enhance chitinase yield.Chitinase was purified from crude enzyme extract by single step gel filtration by Sephadex G-100. Purified chitinase of the strain exhibited a distinct protein band near 45 kDa by means of SDS-PAGE.

  19. The zinc finger SET domain gene Prdm14 is overexpressed in lymphoblastic lymphomas with retroviral insertions at Evi32.

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    E J Dettman

    Full Text Available BACKGROUND: AKXD recombinant inbred strains of mice have proven to be very useful in the identification of potential oncogenes and tumor suppressors involved in the development of lymphoid and myeloid malignancies. In these tumors, the hematopoietic insertion of an active AKV murine leukemia virus (MuLV is associated with the onset of disease. Common sites of retroviral insertion (CIS identify genes causally associated with the development or initiation of lymphoma. METHODOLOGY: In the present study, we analyzed a previously uncharacterized CIS, Ecotropic Viral Integration Site 32 (Evi32, which is located on mouse chromosome 1. We analyzed candidate genes in the region to identify those involved in Evi32 mediated oncogenesis. RESULTS: Here we show that proviral insertion at Evi32 correlates with significant overexpression of a putative transcription factor, PR-domain containing 14 (Prdm14. Tumors with insertions at Evi32 are consistently lymphoid in nature. Prdm14 is normally expressed early in embryonic development with the highest expression in undifferentiated embryonic stem (ES cells. This study implicates Prdm14 as a proto-oncogene involved in lymphoblastic lymphoma formation.

  20. Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia

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    Kristin K. Deeb


    Full Text Available In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations.

  1. Amino substituted nitrogen heterocycle ureas as kinase insert domain containing receptor (KDR inhibitors: Performance of structure–activity relationship approaches

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    Hayriye Yilmaz


    Full Text Available A quantitative structure–activity relationship (QSAR study was performed on a set of amino-substituted nitrogen heterocyclic urea derivatives. Two novel approaches were applied: (1 the simplified molecular input-line entry systems (SMILES based optimal descriptors approach; and (2 the fragment-based simplex representation of molecular structure (SiRMS approach. Comparison with the classic scheme of building up the model and balance of correlation (BC for optimal descriptors approach shows that the BC scheme provides more robust predictions than the classic scheme for the considered pIC50 of the heterocyclic urea derivatives. Comparison of the SMILES-based optimal descriptors and SiRMS approaches has confirmed good performance of both techniques in prediction of kinase insert domain containing receptor (KDR inhibitory activity, expressed as a logarithm of inhibitory concentration (pIC50 of studied compounds.

  2. Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

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    Wen-Yu Yang; Zong-Hai Huang; Li-Jun Lin; Zhou Li; Jing-Long Yu; Hui-Juan Song; Yong Qian; Xiao-Yan Che


    AIM: To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector.METHODS: Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified.HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured.RESULTS: The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVCDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P<0.001).CONCLJSION: Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

  3. Different structure and mRNA expression of Entamoeba invadens chitinases in the encystation and excystation. (United States)

    Makioka, Asao; Kumagai, Masahiro; Hiranuka, Kazushi; Kobayashi, Seiki; Takeuchi, Tsutomu


    Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D

  4. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

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    Saray Santamaría-Hernando

    Full Text Available Proteins of the animal heme peroxidase (ANP superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20, where it was found to be involved in Ca(2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+ binding with a K(D of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821 is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of

  5. Pulmonary cryptococcosis induces chitinase in the rat

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    Casadevall Arturo


    Full Text Available Abstract Background We previously demonstrated that chronic pulmonary infection with Cryptococcus neoformans results in enhanced allergic inflammation and airway hyperreactivity in a rat model. Because the cell wall of C. neoformans consists of chitin, and since acidic mammalian chitinase (AMCase has recently been implicated as a novel mediator of asthma, we sought to determine whether such infection induces chitinase activity and expression of AMCase in the rat. Methods We utilized a previously-established model of chronic C. neoformans pulmonary infection in the rat to analyze the activity, expression and localization of AMCase. Results Our studies indicate that intratracheal inoculation of C. neoformans induces chitinase activity within the lung and bronchoalveolar lavage fluid of infected rats. Chitinase activity is also elicited by pulmonary infection with other fungi (e.g. C. albicans, but not by the inoculation of dead organisms. Enhanced chitinase activity reflects increased AMCase expression by airway epithelial cells and alveolar macrophages. Systemic cryptococcosis is not associated with increased pulmonary chitinase activity or AMCase expression. Conclusion Our findings indicate a possible link between respiratory fungal infections, including C. neoformans, and asthma through the induction of AMCase.

  6. Purification and Characterization of a Novel Thermostable Chitinase from Thermomyces lanuginosus SY2 and Cloning of Its Encoding Gene

    Institute of Scientific and Technical Information of China (English)


    A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodeeyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 min. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer (pH 2.5). The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable ehitinases so far isolated in fungi. Ca2+, Ba2+, Na2+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, Hg2+, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplieation. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.

  7. A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps

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    Etchells J Peter


    Full Text Available Abstract Background The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs. Results Here, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated. Conclusion The PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data.

  8. Aeromonas chitinase degrades chironomid egg masses. (United States)

    Laviad, Sivan; Golan, Amnon; Shaked, Tamar; Vaizel-Ohayon, Dalit; Halpern, Malka; Pick, Elah


    Chironomids are freshwater insects that undergo a complete metamorphosis of four life stages. Chironomid egg masses can be degraded by Vibrio cholerae and some Aeromonas species. Egg mass degradation by V. cholerae requires haemagglutinin protease activity. Our aim was to identify the egg mass degrading (EMD) factor secreted by Aeromonas dhkanesis 3K1C15. Following the hypothesis that the EMD factor of A. dhkanesis is also a protease, secreted proteases were screened, but none of them proved to have the same properties as the EMD factor. Using conventional protein purification methods, we found that the active fraction included chitinases. We further confirmed chitin as a building block of the egg masses. Interestingly, by supplementing bacterial growth media with chitin, we observed unexpected EMD factor activity in Aeromonas isolates that initially were not able to degrade egg masses. Accordingly, we concluded that although strain 3K1C15 secretes chitinases constitutively, most Aeromonas strains secrete chitinases inductively. Induction of chitinases in nature presumably occurs when bacteria are attached to the egg mass habitat, in which chitin is abundant. Considering that chitinases are highly conserved across bacteria phyla, we assume that the role of this enzyme in the bacteria-insect interplay could be wider than is currently thought.


    Directory of Open Access Journals (Sweden)

    Zuzana Gregorová


    Full Text Available The powdery mildew and leaf rust caused by Blumeria graminis and Puccinia recondita (respectively are common diseases of wheat throughout the world. These fungal diseases greatly affect crop productivity. Incorporation of effective and durable disease resistance is an important breeding objective for wheat improvement. We have evaluated resistance of four bread wheat (Triticum aestivum and four spelt wheat (Triticum spelta cultivars. Chitinases occurrence as well as their activity was determined in leaf tissues. There was no correlation between resistance rating and activity of chitinase. The pattern of chitinases reveals four isoforms with different size in eight wheat cultivars. A detailed understanding of the molecular events that take place during a plant–pathogen interaction is an essential goal for disease control in the future.

  10. Chitinase Activity in Healthy and Sclerotium rolfsii Infected Peanut

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    Full Text Available The objectives of this experiment were to analyze the endo- or exo-chitinase activities of healthy and Sclerotium rolfsii infected peanuts. The experiment analyzed 24 different peanut genotypes. Results of the experiment showed chromogenic dimer was the most suitable substrate for analysing chitinase activities. Both endo- and exo-chitinases activities were detected in leaf, stem, and crown tissues. Increased in chitinase activities were detected in S. rolfsii infected peanut tissues than in healthy plant. Regression analysis showed negative slope between disease intensity and chitinase activity in S. rolfsii infected peanut tissue (R2= 0.45.

  11. A broad pH range and processive chitinase from a metagenome library

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    S.S. Thimoteo

    Full Text Available Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4 present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

  12. Two forms of the membrane-bound state of the first C2 domain (C2A) of synaptotagminⅠand calcium-triggered membrane insertion

    Institute of Scientific and Technical Information of China (English)

    HE Yuhong; LI Xianghui; WANG Fu; XUE Yi; SUI Senfang


    The synaptic vesicle protein synaptotagminⅠ (sytⅠ) is a vesicle trans membrane protein present in synaptic vesicles, which has been proposed as the Ca 2+ sensor that regulates secretion. The C2A domain is the membrane proximal part of its cytoplasmic domain. The interaction between C2A and lipid bilayer has be en considered to be essential for triggering neurotransmitter release. In the pr esent work, the measurements of membrane surface tension and surface concentrati on showed that the C2A domain of sytⅠexhibited two membrane-bound states: the s urface adsorption state and the membrane insertion state. The surface absorption state formed in a Ca2+-independent manner with lower affinity, while the membra ne insertion state formed with high affinity was only found in the presence of C a2+. Both the Ca2+-independent and Ca2+-dependent sytⅠ- membrane interactions r equired anionic phospholipids, such as phosphatidylserine (PS). When expressed i nto rat pheochromocytoma (PC12) cells and human embryonic kidney (HEK-293) cells , as demonstrated by immunofluorescence staining and subcellular fractionation, most of the C2A was found at the plasma membrane, even when the cells were deple ted of Ca2+ by incubation with EGTA. These results suggested a new molecular mec hanism of sytⅠas a Ca2+ sensor in membrane fusion. Ca2+-independent surface ads orption might attach sytⅠto the release site during the docking or priming step . When intracellular Ca2+ increased, sytⅠtriggered the neurotransmitter release following the Ca2+-dependent penetration into the target membrane.

  13. Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis. (United States)

    Bonfim, Sheyla M R C; Cruz, Aline H S; Jesuino, Rosália S A; Ulhoa, Cirano J; Molinari-Madlum, Eugênia E W I; Soares, Célia M A; Pereira, Maristela


    A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.

  14. The Zα domain of fish PKZ facilitates the B-Z conformational transition of oligonucleotide DNAs with d(GC)n inserts

    Institute of Scientific and Technical Information of China (English)

    Puzhong Lu; Shoulong Deng; Youlin Zhu; Yongbin Yan; Yong Liu; Chengyu Hu


    PKZ (PKR-like) was discovered as a member of eIF2α kinase family in fish,which possesses a conserved catalytic domain of an eIF2α kinase in C-terminal and also two ZDNA-binding domains (Zα1 and Zα2) in N-terminal.PKZ can be activated through binding of Zα to Z-DNA.However,the regulatory function of PKZ Zα still remains unclear.To investigate a molecular mechanism of how PKZ Zα interacts with Z-DNA,we expressed Zα polypeptide Zα1α2 in Escherichia coli Rosetta strain and purified by affinity chromatography on Ni-NTA resin.Different lengths of oligonucleotide DNAs with various inserts,namely d(GC)n (n =6,8,10,13),d(TA)n (n =6,10),nond(GC),and non-d(TA),were designed and synthesized.Circular dichroism spectrum and gel mobility shift assays were used to investigate the effects of Zα1α2 on the conformational transition of different oligonucleotide DNAs.Results showed that oligonucleotide DNAs retained a conventional B-DNA conformation in the absence of Zα1α2.With the increasing amount of Zα1α2 titration,d(GC)n were recognized and converted to Z-DNA conformation to some degree.With increasing the repeat number (from n =6 to n =13),the tendency of conformational transition became more obvious.However,the conformation of oligonucleotides with d(TA)n inserts changed a little in the presence of Zα1α2,and Zα1α2 had no effect on conformational transition of oligonucleotides with non-d(GC) or non-d(TA) inserts.Gel mobility shift assays further showed that Zα1α2 could bind to oligonucleotide with d(GC)10.In other words,Zα1α2 can turn oligonucleotides with d(GC)n inserts into Z-DNA conformation and bind to it with high affinity.

  15. Phylogeny of chitinases and its implications for estimating horizontal gene transfer from chitinase-transgenic silver birch (Betula pendula). (United States)

    Lohtander, Katileena; Pasonen, Hanna-Leena; Aalto, Markku K; Palva, Tapio; Pappinen, Ari; Rikkinen, Jouko


    Chitinases are hydrolytic enzymes that have been employed in biotechnology in attempts to increase plants' resistance against fungal pathogens. Genetically modified plants have given rise to concerns of the spreading of transgenes into the environment through vertical or horizontal gene transfer (HGT). In this study, chitinase-like sequences from silver birch (Betula pendula) EST-libraries were identified and their phylogenetic relationships to other chitinases were studied. Phylogenetic analyses were used to estimate the frequency of historical gene transfer events of chitinase genes between plants and other organisms, and the usefulness of phylogenetic analyses as a source of information for the risk assessment of transgenic silver birch carrying a sugar beet chitinase IV gene was evaluated. Thirteen partial chitinase-like sequences, with an approximate length of 600 bp, were obtained from the EST-libraries. The sequences belonged to five chitinase classes. Some bacterial chitinases from Streptomyces and Burkholderia, as well as a chitinase from an oomycete, Phytophthora infestans, grouped together with the class IV chitinases of plants, supporting the hypothesis that some class IV chitinases in bacteria have evolved from eukaryotic chitinases via horizontal gene transfer. According to our analyses, HGT of a chitinase IV gene from eukaryotes to bacteria has presumably occurred only once. Based on this, the likelihood for the HGT of chitinase IV gene from transgenic birch to other organisms is extremely low. However, as risk is a function of both the likelihood and consequences of an event, the effects of rare HGT event(s) will finally determine the level of the risk.

  16. Characterization and antifungal activity of gazyumaru (Ficus microcarpa) latex chitinases: both the chitin-binding and the antifungal activities of class I chitinase are reinforced with increasing ionic strength. (United States)

    Taira, Toki; Ohdomari, Atsuko; Nakama, Naoya; Shimoji, Makiko; Ishihara, Masanobu


    Three chitinases, designated gazyumaru latex chitinase (GLx Chi)-A, -B, and -C, were purified from the latex of gazyumaru (Ficus microcarpa). GLx Chi-A,-B, and -C are an acidic class III (33 kDa, pI 4.0), a basic class I (32 kDa, pI 9.3), and a basic class II chitinase (27 kDa, pI > 10) respectively. GLx Chi-A did not exhibit any antifungal activity. At low ionic strength, GLx Chi-C exhibited strong antifungal activity, to a similar extent as GLx Chi-B. The antifungal activity of GLx Chi-C became weaker with increasing ionic strength, whereas that of GLx Chi-B became slightly stronger. GLx Chi-B and -C bound to the fungal cell-walls at low ionic strength, and then GLx Chi-C was dissociated from them by an escalation of ionic strength, but this was not the case for GLx Chi-B. The chitin-binding activity of GLx Chi-B was enhanced by increasing ionic strength. These results suggest that the chitin-binding domain of basic class I chitinase binds to the chitin in fungal cell walls by hydrophobic interaction and assists the antifungal action of the chitinase.

  17. Expression of a chitinase gene from Metarhizium anisopliae in tobacco plants confers resistance against Rhizoctonia solani. (United States)

    Kern, Marcelo Fernando; Maraschin, Simone de Faria; Vom Endt, Débora; Schrank, Augusto; Vainstein, Marilene Henning; Pasquali, Giancarlo


    The chit1 gene from the entomopathogenic fungus Metarhizium anisopliae, encoding the endochitinase CHIT42, was placed under the control of the CaMV 35S promoter, and the resulting construct was transferred to tobacco. Seventeen kanamycin-resistant transgenic lines were recovered, and the presence of the transgene was confirmed by polymerase chain reactions and Southern blot hybridization. The number of chit1 copies was determined to be varying from one to four. Copy number had observable effects neither on plant growth nor development. Substantial heterogeneity concerning production of the recombinant chitinase, and both general and specific chitinolytic activities were detected in leaf extracts from primary transformants. The highest chitinase activities were found in plants harboring two copies of chit1 inserts at different loci. Progeny derived from self-pollination of the primary transgenics revealed a stable inheritance pattern, with transgene segregation following a mendelian dihybrid ratio. Two selected plants expressing high levels of CHIT42 were consistently resistant to the soilborne pathogen Rhizoctonia solani, suggesting a direct relationship between enzyme activity and reduction of foliar area affected by fungal lesions. To date, this is the first report of resistance to fungal attack in plants mediated by a recombinant chitinase from an entomopathogenic and acaricide fungus.

  18. Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation.


    Watanabe, T.; Oyanagi, W; K. Suzuki; H. Tanaka


    Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharide-degrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (A1, A2, B1, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pI 4.7 (A1); Mr 69,000, pI 4.5 (A2); Mr 38,000, pI 6.6 (B1); Mr 38,000, pI 5.9 (B2); Mr 39,000, pI ...

  19. Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection. (United States)

    van den Burg, Harrold A; Harrison, Stuart J; Joosten, Matthieu H A J; Vervoort, Jacques; de Wit, Pierre J G M


    Resistance against the leaf mold fungus Cladosporium fulvum is mediated by the tomato Cf proteins which belong to the class of receptor-like proteins and indirectly recognize extracellular avirulence proteins (Avrs) of the fungus. Apart from triggering disease resistance, Avrs are believed to play a role in pathogenicity or virulence of C. fulvum. Here, we report on the avirulence protein Avr4, which is a chitin-binding lectin containing an invertebrate chitin-binding domain (CBM14). This domain is found in many eukaryotes, but has not yet been described in fungal or plant genomes. We found that interaction of Avr4 with chitin is specific, because it does not interact with other cell wall polysaccharides. Avr4 binds to chitin oligomers with a minimal length of three N-acetyl glucosamine residues. In vitro, Avr4 protects chitin against hydrolysis by plant chitinases. Avr4 also binds to chitin in cell walls of the fungi Trichoderma viride and Fusarium solani f. sp. phaseoli and protects these fungi against normally deleterious concentrations of plant chitinases. In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato, where it most likely protects the fungus against tomato chitinases, suggesting that Avr4 is a counter-defensive virulence factor.

  20. Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity. (United States)

    Yasir, Muhammad; Aslam, Zubair; Kim, Seon Won; Lee, Seon-Woo; Jeon, Che Ok; Chung, Young Ryun


    Bacterial communities and chitinase gene diversity of vermicompost (VC) were investigated to clarify the influence of earthworms on the inhibition of plant pathogenic fungi in VC. The spore germination of Fusarium moniliforme was reduced in VC aqueous extracts prepared from paper sludge and dairy sludge (fresh sludge, FS). The bacterial communities were examined by culture-dependent and -independent analyses. Unique clones selected from 16S rRNA libraries of FS and VC on the basis of restriction fragment length polymorphism (RFLP) fell into the major lineages of the domain bacteria Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria and Firmicutes. Among culture isolates, Actinobacteria dominated in VC, while almost equal numbers of Actinobacteria and Proteobacteria were present in FS. Analysis of chitinolytic isolates and chitinase gene diversity revealed that chitinolytic bacterial communities were enriched in VC. Populations of bacteria that inhibited plant fungal pathogens were higher in VC than in FS and particularly chitinolytic isolates were most active against the target fungi.

  1. Structure of chitinase D from Serratia proteamaculans reveals the structural basis of its dual action of hydrolysis and transglycosylation (United States)

    Madhuprakash, Jogi; Singh, Avinash; Kumar, Sanjit; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Podile, Appa R; Singh, Tej P


    Chitinases are known to hydrolyze chitin polymers into smaller chitooligosaccharides. Chitinase from bacterium Serratia proteamaculans (SpChiD) is found to exhibit both hydrolysis and transglycosylation activities. SpChiD belongs to family 18 of glycosyl hydrolases (GH-18). The recombinant SpChiD was crystallized and its three-dimensional structure was determined at 1.49 Å resolution. The structure was refined to an R-factor of 16.2%. SpChiD consists of 406 amino acid residues. The polypeptide chain of SpChiD adopts a (β/α)8 triosephosphate isomerase (TIM) barrel structure. SpChiD contains three acidic residues, Asp149, Asp151 and Glu153 as part of its catalytic scheme. While both Asp149 and Glu153 adopt single conformations, Asp151 is observed in two conformations. The substrate binding cleft is partially obstructed by a protruding loop, Asn30 - Asp42 causing a considerable reduction in the number of available subsites in the substrate binding site. The positioning of loop, Asn30 - Asp42 appears to be responsible for the transglycosylation activity. The structure determination indicated the presence of sulfone Met89 (SMet89). The sulfone methionine residue is located on the surface of the protein at a site where extra domain is attached in other chitinases. This is the first structure of a single domain chitinase with hydrolytic and transglycosylation activities. PMID:24380021

  2. The chitinase C gene PsChiC from Pseudomonas sp. and its synergistic effects on larvicidal activity

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    Wanfang Zhong


    Full Text Available Pseudomonas sp. strain TXG6-1, a chitinolytic gram-negative bacterium, was isolated from a vegetable field in Taixing city, Jiangsu Province, China. In this study, a Pseudomonas chitinase C gene (PsChiC was isolated from the chromosomal DNA of this bacterium using a pair of specific primers. The PsChiC gene consisted of an open reading frame of 1443 nucleotides and encoded 480 amino acid residues with a calculated molecular mass of 51.66 kDa. The deduced PsChiC amino acid sequence lacked a signal sequence and consisted of a glycoside hydrolase family 18 catalytic domain responsible for chitinase activity, a fibronectin type III-like domain (FLD and a C-terminal chitin-binding domain (ChBD. The amino acid sequence of PsChiCshowed high sequence homology (> 95% with chitinase C from Serratia marcescens. SDS-PAGE showed that the molecular mass of chitinase PsChiC was 52 kDa. Chitinase assays revealed that the chitobiosidase and endochitinase activities of PsChiCwere 51.6- and 84.1-fold higher than those of pET30a, respectively. Although PsChiC showed little insecticidal activity towards Spodoptera litura larvae, an insecticidal assay indicated that PsChiC increased the insecticidal toxicity of SpltNPV by 1.78-fold at 192 h and hastened death. These results suggest that PsChiC from Pseudomonas sp. could be useful in improving the pathogenicity of baculoviruses.

  3. Methylxanthine Inhibit Fungal Chitinases and Exhibit Antifungal Activity (United States)

    Tsirilakis, Kalliope; Kim, Christy; Vicencio, Alfin G.; Andrade, Christopher; Casadevall, Arturo; Goldman, David L.


    Chitinases are necessary for fungal cell wall remodeling and cell replication. Methylxanthines have been shown to competitively inhibit family 18 chitinases in vitro. We sought to determine the effects of methylxanthines on fungal chitinases. Fungi demonstrated variable chitinase activity and incubation with methylxanthines (0.5–10 mM) resulted in a dose-dependent decrease in this activity. All fungi tested, except for Candida spp., demonstrated growth inhibition in the presence of methylxanthines at a concentration of 10 mM. India ink staining demonstrated impaired budding and decreased cell size for methylxanthine-treated Cryptococcus neoformans. C. neoformans and Aspergillus fumigatus treated with pentoxifylline also exhibited abnormal cell morphology. In addition, pentoxifylline-treated C. neoformans exhibited increased susceptibility to calcofluor and a leaky melanin phenotype consistent with defective cell wall function. Our data suggest that a variety of fungi express chitinases and that methylxanthines have antifungal properties related to their inhibition of fungal chitinases. Our results highlight the potential utility of targeting chitinases in the development of novel antifungal therapies. PMID:21968902

  4. Genome-wide analysis of chitinase genes and their varied functions in larval moult, pupation and eclosion in the rice striped stem borer, Chilo suppressalis. (United States)

    Su, C; Tu, G; Huang, S; Yang, Q; Shahzad, M F; Li, F


    Some insect chitinases are required to degrade chitin and ensure successful metamorphosis. Although chitinase genes have been well characterized in several model insects, no reports exist for the rice striped stem borer, Chilo suppressalis, a highly destructive pest that causes huge yield losses in rice production. Here, we conducted a genome-level analysis of chitinase genes in C. suppressalis. After amplification of full-length transcripts with rapid amplification of cDNA ends, we identified 12 chitinase genes in C. suppressalis. All these genes had the conserved domains and motifs of glycoside hydrolase family 18 and grouped phylogenetically into five subgroups. C. suppressalis chitinase 1 (CsCht1) was highly expressed in late pupae, whereas CsCht3 was abundant in early pupae. Both CsCht2 and CsCht4 were highly expressed in larvae. CsCht2 was abundant specifically in the third-instar larvae and CsCht4 showed periodic high expression in 2- to 5-day-old larvae in each instar. Tissue specific expression analysis indicated that CsCht1 and CsCht3 were highly expressed in epidermis whereas CsCht2 and CsCht4 were specifically abundant in the midgut. Knockdown of CsCht1 resulted in adults with curled wings, indicating that CsCht1 might have an important role in wing expansion. Silencing of CsCht2 or CsCht4 arrested moulting, suggesting essential roles in larval development. When the expression of CsCht3 was interfered, defects in pupation occurred. Overall, we provide here the first catalogue of chitinase genes in the rice striped stem borer and have elucidated the functions of four chitinases in metamorphosis.

  5. Functional characterization of chitinase-3 reveals involvement of chitinases in early embryo immunity in zebrafish. (United States)

    Teng, Zinan; Sun, Chen; Liu, Shousheng; Wang, Hongmiao; Zhang, Shicui


    The function and mechanism of chitinases in early embryonic development remain largely unknown. We show here that recombinant chitinase-3 (rChi3) is able to hydrolyze the artificial chitin substrate, 4-methylumbelliferyl-β-D-N,N',N″-triacetylchitotrioside, and to bind to and inhibit the growth of the fungus Candida albicans, implicating that Chi3 plays a dual function in innate immunity and chitin-bearing food digestion in zebrafish. This is further corroborated by the expression profile of Chi3 in the liver and gut, which are both immune- and digestion-relevant organs. Compared with rChi3, rChi3-CD lacking CBD still retains partial capacity to bind to C. albicans, but its enzymatic and antifungal activities are significantly reduced. By contrast, rChi3-E140N with the putative catalytic residue E140 mutated shows little affinity to chitin, and its enzymatic and antifungal activities are nearly completely lost. These suggest that both enzymatic and antifungal activities of Chi3 are dependent on the presence of CBD and E140. We also clearly demonstrate that in zebrafish, both the embryo extract and the developing embryo display antifungal activity against C. albicans, and all the findings point to chitinase-3 (Chi3) being a newly-identified factor involved in the antifungal activity. Taken together, a dual function in both innate immunity and food digestion in embryo is proposed for zebrafish Chi3. It also provides a new angle to understand the immune role of chitinases in early embryonic development of animals.

  6. Calorimeter insertion

    CERN Multimedia


    Calorimeter insertion between toroids in the ATLAS experiment detector Calorimeters are surrounding the inner detector. Calorimeters will absorb and measure the energies of the most charged and neutral particles after the collisions. The saved energy in the calorimeter is detected and converted to signals that are taken out with data taking electronics.

  7. Insertion devices

    CERN Document Server

    Bahrdt, J


    The interaction of an insertion device with the electron beam in a storage ring is discussed. The radiation property including brightness, ux and polarization of an ideal and real planar and helical / elliptical device is described. The magnet design of planar, helical, quasiperiodic devices and of devices with a reduced on axis power density are resumed.

  8. Functional analysis of a chitinase gene during the larval-nymph transition in Panonychus citri by RNA interference. (United States)

    Xia, Wen-Kai; Shen, Xiao-Min; Ding, Tian-Bo; Niu, Jin-Zhi; Zhong, Rui; Liao, Chong-Yu; Feng, Ying-Cai; Dou, Wei; Wang, Jin-Jun


    Chitinases are hydrolytic enzymes that are required for chitin degradation and reconstruction in arthropods. In this study, we report a cDNA sequence encoding a putative chitinase (PcCht1) from the citrus red mite, Panonychus citri. The PcCht1 (564 aa) possessed a signal peptide, a conserver domain, and a chitin-binding domain. Structural and phylogenetic analyses found that PcCht1 had high sequence similarity to chitinases in Tetranychus urticae. Real-time quantitative PCR analyses showed that the transcript levels of PcCht1 peaked periodically in larval and nymph stages. Moreover, significant increase of PcCht1 transcript level in the larvae was observed upon the exposure of diflubenzuron. In contrast, exposures of the larvae to diflubenzuron resulted in the decreased chitin content. Furthermore, through a feeding-based RNA interference approach, we were able to reduce the PcCht1 transcript level by 59.7 % in the larvae, and consequently the treated larvae showed a very low molting rate compared with the control. Our results expanded the understanding of the important role of PcCht1 in the growth and development of P. citri.

  9. Complete subsite mapping of a "loopful" GH19 chitinase from rye seeds based on its crystal structure. (United States)

    Ohnuma, Takayuki; Umemoto, Naoyuki; Kondo, Kaori; Numata, Tomoyuki; Fukamizo, Tamo


    Crystallographic analysis of a mutated form of "loopful" GH19 chitinase from rye seeds a double mutant RSC-c, in which Glu67 and Trp72 are mutated to glutamine and alanine, respectively, (RSC-c-E67Q/W72A) in complex with chitin tetrasaccharide (GlcNAc)₄ revealed that the entire substrate-binding cleft was completely occupied with the sugar residues of two (GlcNAc)₄ molecules. One (GlcNAc)₄ molecule bound to subsites -4 to -1, while the other bound to subsites +1 to +4. Comparisons of the main chain conformation between liganded RSC-c-E67Q/W72A and unliganded wild type RSC-c suggested domain motion essential for catalysis. This is the first report on the complete subsite mapping of GH19 chitinase.

  10. A single base insertion in the putative transmembrane domain of the tyrosinase gene as a cause for tyrosinase-negative oculocutaneous albinism

    Energy Technology Data Exchange (ETDEWEB)

    Chintamaneni, C.D.; Kobayashi, Y.; Kwon, B.S. (Indiana Univ. School of Medicine, Indianapolis (United States)); Halaban, R. (Yale Univ. School of Medicine, New Haven, CT (United States)); Witkop, C.J. Jr. (Univ. of Minnesota, Minneapolis (United States))


    The authors have determined a molecular defect to be the likely basis for inactivity of the tyrosinase from a patient with tyrosinase-negative oculocutaneous albinism. A single base (thymine) was inserted in exon 5 of the tyrosinase gene following codon 471 in the putative transmembrane coding region. This insertion caused a shift in the reading frame of 19 amino acids at the 3{prime} end and introduced a premature termination signal that would be expected to truncate the protein by 21 amino acids at the carboxyl terminus. The albino tyrosinase was not recognized by antibodies directed to the carboxyl terminus of tyrosinase. Furthermore, as shown by gel electrophoresis of the immunoprecipitated protein, the tyrosinase was {approx} 3kDa smaller than normal. Similar immunoprecipitation data were obtained when cloned normal and mutant tyrosinases were expressed in COS-1 cells.

  11. Purification and Characterization of Streptomyces sp. IK Chitinase

    Directory of Open Access Journals (Sweden)

    Sebastian Margino


    Full Text Available Streptomyces sp. IK isolated from compost inoculants, could produce extra cellular chitinase in a medium containing 0.2% (w/v colloidal chitin, fermented for 96 hours at 30oC. The enzyme was purified by a combination of ammonium sulphate precipitation and DEAE-Cellulose anion-exchange chromatography. On SDS-polyacrylamide gel electrophoresis analysis, the purified enzyme showed a mass of 71 kDa. Chitinase was optimally active at pH of 6.7 and at 37oC. Km value and Vmax of the protein for colloidal chitin were 2.92 mg/ml and 4.26 ìg/h, respectively.Key words : chitinase, Streptomyces, purification, characterization

  12. Cloning, expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis

    Institute of Scientific and Technical Information of China (English)

    Qingyue Wu; Tian Liu; Qing Yang


    Chitinase catalyzes β-l,4-glycosidic linkages in chitin and has attracted research interest due to it being a potential pesticide target and an enzymatic tool for preparation of N-acetyl-β-D-glucosamine.An individual insect contains multiple genes encoding chitinases,which vary in domain architectures,expression patterns,physiological roles and biochemical properties.Herein,OfCht5,the glycoside hydrolase family 18 chitinase from the widespread lepidopteran pest Ostrinia furnacalis,was cloned,expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation.Complementary DNA sequence analysis indicated that OfCHT5 consisted of an open reading frame of 1 665-bp nucleotides.Phylogenic analysis suggested OfCht5 belongs to the Group Ⅰ insect chitinases.Expression of OfCht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol.Through two steps of purification,consisting of ammonium sulfate precipitation and metal chelating chromatography,about 7 mg of the recombinant OfCht5 was purified to homogeneity from 1 L culture supematant.OfCht5 effectively converted colloidal chitin into chitobiose,but had relatively low activity toward α-chitin.When chitooligosaccharides [(GlcNAc)n,n =3-6] were used as substrates,OfCht5 was observed to possess the highest catalytic efficiency parameter toward (GlcNAc)4 and predominantely hydrolyzed the second glycosidic bond from the non-reducing end.Together with β-N-acetyl-D-hexosaminidase OfHexl,OfCht5 achieved its highest efficiency in chitin degradation that yielded N-acetyl-β-D-glucosamine,a valuable pharmacological reagent and food supplement,within a molar concentration ratio of OfCht5 versus OfHexl in the range of 9∶1-15 ∶ 1.This work provides an alternative to existing preparation ofchitinase for pesticides and other applications.

  13. Peptide insertions in domain 4 of hbeta(c), the shared signalling receptor subunit for GM-CSF, IL3 and IL5, induce ligand-independent activation. (United States)

    Jones, K L; Bagley, C J; Butcher, C; Barry, S C; Vadas, M A; D'Andrea, R J


    A mutant form of the common beta-subunit of the GM-CSF, interleukin-3 (IL3) and IL5 receptors is activated by a 37 residue duplicated segment which includes the WSXWS motif and an adjacent, highly conserved, aliphatic/basic element. Haemopoietic expression of this mutant, hbeta(c)FIDelta, in mice leads to myeloproliferative disease. To examine the mechanism of activation of this mutant we targetted the two conserved motifs in each repeat for mutagenesis. Here we show that this mutant exhibits constitutive activity in BaF-B03 cells in the presence of mouse or human GM-CSF receptor alpha-subunit (GMRalpha) and this activity is disrupted by mutations of the conserved motifs in the first repeat. In the presence of these mutations the receptor reverts to an alternative conformation which retains responsiveness to human IL3 in a CTLL cell line co-expressing the human IL3 receptor alpha-subunit (hIL3Ralpha). Remarkably, the activated conformation is maintained in the presence of substitutions, deletions or replacement of the second repeat. This suggests that activation occurs due to insertion of extra sequence after the WSXWS motif and is not dependent on the length or specific sequence of the insertion. Thus hbeta(c) displays an ability to fold into functional receptor conformations given insertion of up to 37 residues in the membrane-proximal region. Constitutive activation most likely results from a specific conformational change which alters a dormant, inactive receptor complex, permitting functional association with GMRalpha and ligand-independent mitogenic signalling.

  14. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN. (United States)

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D


    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP.

  15. Cloning, expression, and characterization of an antifungal chitinase from Leucaena leucocephala de Wit. (United States)

    Kaomek, Mana; Mizuno, Kouichi; Fujimura, Tatsuhito; Sriyotha, Poonsook; Cairns, James R Ketudat


    Chitinase cDNAs from Leucaena leucocephala seedlings were cloned by PCR amplification with degenerate primers based on conserved class I chitinase sequences and cDNA library screening. Two closely related chitinase cDNAs were sequenced and inferred to encode precursor proteins of 323 (KB1) and 326 (KB2) amino acids. Expression of the KB2 chitinase from a pET32a plasmid in Origami (DE3) Escherichia coli produced high chitinase activity in the cell lysate. The recombinant thioredoxin fusion protein was purified and cleaved to yield a 32-kDa chitinase. The recombinant chitinase hydrolyzed colloidal chitin with endochitinase-type activity. It also inhibited growth of 13 of the 14 fungal strains tested.

  16. Nitrogen regulates chitinase gene expression in a marine bacterium

    DEFF Research Database (Denmark)

    Delpin, Marina; Goodman, A.E.


    Ammonium concentration and nitrogen source regulate promoter activity and use for the transcription of chiA, the major chitinase gene of Pseudoalteromonas sp. S91 and S91CX, an S91 transposon lacZ fusion mutant. The activity of chiA was quantified by beta-galactosidase assay of S91CX cultures con...

  17. Acidic Chitinase Limits Allergic Inflammation and Promotes Intestinal Nematode Expulsion (United States)

    Acidic mammalian chitinase (AMCase) is stereotypically induced during mammalian immune responses to helminths and allergens—yet, its precise role in immunity and inflammation is unclear. Here we show that in the lung, genetic ablation of AMCase failed to diminish type 2 inflammation against helmint...

  18. Chitinases Are Essential for Cell Separation in Ustilago maydis. (United States)

    Langner, Thorsten; Öztürk, Merve; Hartmann, Sarah; Cord-Landwehr, Stefan; Moerschbacher, Bruno; Walton, Jonathan D; Göhre, Vera


    Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.

  19. Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Abhishek, E-mail: [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Bhandari, Anita [Molecular Physiology, Zoological Institute, Christian-Albrechts-University at Kiel, Kiel (Germany); Sarde, Sandeep J. [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Goswami, Chandan [National Institute of Science Education and Research, Bhubaneswar, Orissa (India)


    Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.

  20. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. (United States)

    Watanabe, T; Kobori, K; Miyashita, K; Fujii, T; Sakai, H; Uchida, M; Tanaka, H


    Prokaryotic chitinases, class III plant chitinases, yeast chitinases, and endo-beta-N-acetylglucosaminidases share weak amino acid sequence similarities at the certain region of each enzyme. These regions have been assumed to be important for catalytic activities of the enzymes. To verify this assumption, three amino acid residues (Ser-160, Asp-200, Glu-204) in chitinase A1 of Bacillus circulans WL-12 were chosen, based on the amino acid sequence alignment of the regions sharing sequence similarity, and were replaced by site-directed mutagenesis. Kinetic parameters for 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and seven mutant chitinases. Chitinases with Glu-204-->Gln mutation and Glu-204-->Asp mutation were essentially inactive and kcat values of these chitinases were approximately 1/5,000 and 1/17,000 of that of wild-type chitinase, respectively. Asp-200-->Asn mutation decreased the kcat value to approximately 1/350 of that of the wild-type enzyme, while the Km value decreased only slightly. On the other hand, neither the kcat value nor the Km value was affected by Asp-200-->Glu mutation. Thus, it appeared that Glu-204 and Asp-200 are directly involved in the catalytic events of chitinase A1. The role of the carboxyl group of Asp-200 can be fully substituted by that of Glu residue. The Ser-160-->Ala mutant retained 10% activity of the wild-type chitinase indicating that the hydroxyl group of Ser-160 is not absolutely required for the catalytic activity. These results indicate a lysozyme-type catalytic mechanism of the chitinase.

  1. Insertions and the emergence of novel protein structure: a structure-based phylogenetic study of insertions

    Directory of Open Access Journals (Sweden)

    Blouin Christian


    Full Text Available Abstract Background In protein evolution, the mechanism of the emergence of novel protein domain is still an open question. The incremental growth of protein variable regions, which was produced by stochastic insertions, has the potential to generate large and complex sub-structures. In this study, a deterministic methodology is proposed to reconstruct phylogenies from protein structures, and to infer insertion events in protein evolution. The analysis was performed on a broad range of SCOP domain families. Results Phylogenies were reconstructed from protein 3D structural data. The phylogenetic trees were used to infer ancestral structures with a consensus method. From these ancestral reconstructions, 42.7% of the observed insertions are nested insertions, which locate in previous insert regions. The average size of inserts tends to increase with the insert rank or total number of insertions in the variable regions. We found that the structures of some nested inserts show complex or even domain-like fold patterns with helices, strands and loops. Furthermore, a basal level of structural innovation was found in inserts which displayed a significant structural similarity exclusively to themselves. The β-Lactamase/D-ala carboxypeptidase domain family is provided as an example to illustrate the inference of insertion events, and how the incremental growth of a variable region is capable to generate novel structural patterns. Conclusion Using 3D data, we proposed a method to reconstruct phylogenies. We applied the method to reconstruct the sequences of insertion events leading to the emergence of potentially novel structural elements within existing protein domains. The results suggest that structural innovation is possible via the stochastic process of insertions and rapid evolution within variable regions where inserts tend to be nested. We also demonstrate that the structure-based phylogeny enables the study of new questions relating to the

  2. Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish. (United States)

    Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J; Goswami, Chandan


    C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1'. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.

  3. Cerebrospinal fluid chitinase-3-like 2 and chitotriosidase are potential prognostic biomarkers in early multiple sclerosis

    DEFF Research Database (Denmark)

    Møllgaard, M; Vinter, Matilda Degn; Sellebjerg, F;


    BACKGROUND AND PURPOSE: The role of chitinases and chitinase-like proteins in multiple sclerosis (MS) is currently unknown; however, cerebrospinal fluid (CSF) levels of chitinase 3-like 1 (CHI3L1) predict prognosis in early MS. Whether this applies to other chitinases and chitinase-like proteins...... is yet to be established. Our objective was to investigate the potential of chitinase 3-like 2 (CHI3L2) and chitotriosidase as prognostic biomarkers in optic neuritis (ON) as the first demyelinating episode and to evaluate the ability of CHI3L2 to predict long-term MS risk and disability. METHODS......, immunoglobulin G index and leukocyte count were investigated. Long-term MS risk and disability (Expanded Disability Status Scale, Multiple Sclerosis Functional Composite components) were examined in a retrospective cohort of 78 patients with ON as the first demyelinating episode (mean follow-up 14 years...

  4. Purification and characterization of chitinase from Streptomyces violascens NRRL B2700. (United States)

    Gangwar, Mamta; Singh, Vineeta; Pandey, Asheesh Kumar; Tripathi, C K M; Mishra, B N


    Chitinase is one of the important enzymes as it is directly linked to Chitin that has wide applications in industrial, medical and commercial fields for its biocompatibility and biodegradability. Here, we report extracellular chitinase production by Streptomyces violascens NRRL B2700 under submerged fermentation condition. Chitinase production started after 10 h of incubation and reached to maximum level at 72 h of cultivation. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that maltose, xylose, fructose, lactose, soybean meal and ammonium nitrate served as good carbon and nitrogen sources to enhance chitinase yield by 1.6 to 6 fold. Medium supplemented with 1% colloidal chitin produced high chitinase concentration (0.1714 U/mg). The enzyme chitinase was purified from the culture broth by 75% ammonium sulphate precipitation, DEAE-cellulose ion-exchange and sephadex G-100 gel filtration. The molecular mass of the purified chitinase was 65 kDa as estimated by SDS-PAGE. The apparent Michaelis constant (K(m)) and the maximum rate (V(max)) of the enzyme for colloidal chitin were 1.556 mg/mL and 2.680 μM/min/mg, respectively suggested high affinity towards-chitin. Possibly, it is the first report on production of chitinase from S. violascens NRRL B2700. The findings were encouraging, especially for cost effective production, and further warrants media and purification optimization studies for enhanced yield.

  5. Isolation of bacteria producing chitinase and inhibiting growth of Rhizoctonia solani

    Institute of Scientific and Technical Information of China (English)


    @@ Five bacteria strains with higher chitinase activity were isolated by using a technique of enriched cell wall of R. solani. All of them showed inhibiting effect on the growth of R. solani. Being cultured 3 d, strain CH-1 showed higher chitinase activity on the chitin plate. The diameter of the transparent circle reached 8.7 mm (4 replications) . In the antagonistic test to R. solani in PDA plate, the circle was 18.1 mm. It was also observed that the antagonistic ability of some strains was not consistent with the chitinase activity (Table 1). It may be connected with the secretion of chitinase at different culture situations.


    Directory of Open Access Journals (Sweden)

    Urja Pandya


    Full Text Available An endospore forming Gram positive bacterium (MBCU4 was isolated from a vermicompost amended soil, and confirmed as Bacillus subtilis through the 16S rRNA sequence analysis. An extracellular chitinase was detected from this strain of B. subtilis under specific environmental condition. An attempt was made to purify the enzyme by ammonium sulfate precipitation followed by DEAE sepharose CL-6B column chromatography. The purified enzyme was demonstrated as a single band, having the molecular weight 31kDa on SDS PAGE analysis and its activity in the gel was determined by clear zone on zymogram. Further characterization of the isolated enzymes has showed that this enzyme is most active at pH 6.0 and at the optimized temperature of 50 0C. The purified chitinase exhibited high degree of antifungal activity particularly by degrading their cell wall components of plant pathogens Macrophomina phaseolina (69.0% and Rhizoctonia solani (52.0%. It infers that the chitinase produced by B. subtilis could play an important role for biopesticidal activity.

  7. Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses (United States)

    Madhuprakash, Jogi; Bobbili, Kishore Babu; Moerschbacher, Bruno M.; Singh, Tej Pal; Swamy, Musti J.; Podile, Appa Rao


    Serratia proteamaculans chitinase-D (SpChiD) has a unique combination of hydrolytic and transglycosylation (TG) activities. The TG activity of SpChiD can be used for large-scale production of chito-oligosaccharides (CHOS). The multiple activities (hydrolytic and/or chitobiase activities and TG) of SpChiD appear to be strongly influenced by the substrate-binding cleft. Here, we report the unique property of SpChiD substrate-binding cleft, wherein, the residues Tyr28, Val35 and Thr36 control chitobiase activity and the residues Trp160 and Trp290 are crucial for TG activity. Mutants with reduced (V35G and T36G/F) or no (SpChiDΔ30–42 and Y28A) chitobiase activity produced higher amounts of the quantifiable even-chain TG product with degree of polymerization (DP)-6, indicating that the chitobiase and TG activities are inversely related. In addition to its unprecedented catalytic properties, unlike other chitinases, the single modular SpChiD showed dual unfolding transitions. Ligand-induced thermal stability studies with the catalytically inactive mutant of SpChiD (E153A) showed that the transition temperature increased upon binding of CHOS with DP2–6. Isothermal titration calorimetry experiments revealed the exceptionally high binding affinities for E153A to CHOS with DP2–6. These observations strongly support that the architecture of SpChiD substrate-binding cleft adopted to control chitobiase and TG activities, in addition to usual chitinase-mediated hydrolysis. PMID:26493546

  8. Induction and purification of chitinase in Brassica napus L. ssp. oleifera infected with Phoma lingam

    DEFF Research Database (Denmark)

    Rasmussen, U.; Giese, H.; Dalgaard Mikkelsen, J.


    A pathogen-induced chitinase (EC was isolated from cotyledons of oilseed rape (Brassica napus cv. Bienvenu) 8 d after inoculation with Phoma lingam. The purified chitinase has a molecular weight of 30 kDa, and an isoelectric point of approx. 9.1. A partial amino-acid sequence obtained...

  9. Function of a recombinant Chitinase derived from a virulent Aeromonas hydrophila isolated from diseased channel catfish (United States)

    A chitinase was identified in extracellular products of a virulent Aeromonas hydrophila isolated from diseased channel catfish (Ictalurus punctatus). Bioactive recombinant chitinase (rChi-Ah) was produced in Escherichia coli. Purified rChi-Ah had optimal activity at temperature of 42°C and pH 6.5. T...

  10. Cloning and characterization of a pathogen-induced chitinase in Brassica napus

    DEFF Research Database (Denmark)

    Rasmussen, U.; Bojsen, K.; Collinge, D.B.


    A chitinase cDNA clone from rapeseed (Brassica napus L. ssp. oleifera) was isolated. The cDNA clone, ChB4, represents a previously purified and characterized basic chitinase isozyme. The longest open reading frame in ChB4 encodes a polypeptide of 268 amino acids. This polypeptide consists of a 24...

  11. Low chitinase activity in Acacia myrmecophytes: a potential trade-off between biotic and chemical defences? (United States)

    Heil, M.; Staehelin, Christian; McKey, D.

    We determined chitinase activity in leaves of four myrmecophytic and four non-myrmecophytic leguminous species at the plants' natural growing sites in Mexico. Myrmecophytic plants (or 'ant plants') have obligate mutualisms with ants protecting them against herbivores and pathogenic fungi. Plant chitinases can be considered a reliable measure of plant resistance to pathogenic fungi. The myrmecophytic Acacia species, which were colonised by mutualistic ants, exhibited at least six-fold lower levels of chitinase activity compared with the non-myrmecophytic Acacia farnesiana and three other non-myrmecophytes. Though belonging to different phylogenetic groups, the myrmecophytic Acacia species formed one distinct group in the data set, which was clearly separated from the non-myrmecophytic species. These findings allowed for comparison between two recent hypotheses that attempt to explain low chitinase activity in ant plants. Most probably, chitinases are reduced in myrmecophytic plant species because these are effectively defended indirectly due to their symbiosis with mutualistic ants.

  12. Vascular Endothelial Growth Factor Gene Polymorphism (rs2010963 and Its Receptor, Kinase Insert Domain-Containing Receptor Gene Polymorphism (rs2071559, and Markers of Carotid Atherosclerosis in Patients with Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Sebastjan Merlo


    Full Text Available Background. The current study was designed to reveal possible associations between the polymorphisms of the vascular endothelial growth factor (VEGF gene (rs2010963 and its receptor, kinase insert domain-containing receptor (KDR gene polymorphism (rs2071559, and markers of carotid atherosclerosis in patients with type 2 diabetes mellitus (T2DM. Patients and Methods. 595 T2DM subjects and 200 control subjects were enrolled. The carotid intima-media thickness (CIMT and plaque characteristics (presence and structure were assessed ultrasonographically. Biochemical analyses were performed using standard biochemical methods. Genotyping of VEGF/KDR polymorphisms (rs2010963, rs2071559 was performed using KASPar assays. Results. Genotype distributions and allele frequencies of the VEGF/KDR polymorphisms (rs2010963, rs2071559 were not statistically significantly different between diabetic patients and controls. In our study, we demonstrated an association between the rs2071559 of KDR and either CIMT or the sum of plaque thickness in subjects with T2DM. We did not, however, demonstrate any association between the tested polymorphism of VEGF (rs2010963 and either CIMT, the sum of plaque thickness, the number of involved segments, hsCRP, the presence of carotid plaques, or the presence of unstable carotid plaques. Conclusions. In the present study, we demonstrated minor effect of the rs2071559 of KDR on markers of carotid atherosclerosis in subjects with T2DM.

  13. Chitinases from the Plant Disease Biocontrol Agent, Stenotrophomonas maltophilia C3. (United States)

    Zhang, Z; Yuen, G Y; Sarath, G; Penheiter, A R


    ABSTRACT Stenotrophomonas maltophilia strain C3, a biocontrol agent of Bipolaris sorokiniana in turfgrass, produced chitinases in broth media containing chitin. Chitinases were partially purified from culture fluid by ammonium sulfate precipitation and chitin affinity chromatography. The chromatography fraction with the highest specific chitinase activity was inhibitory to conidial germination and germ-tube elongation of B. sorokiniana, but it was less inhibitory than the protein fraction or the raw culture filtrate. The fraction exhibited strong exochitinase and weak endo-chitinase activity. Optimum temperature and pH for chitinase activity were 45 to 50 degrees C and 4.5 to 5.0, respectively. Chitinase activity was inhibited by Hg(2+) and Fe(3+), but not by other metal ions or enzyme inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the chromatography fraction revealed the presence of five protein bands of 25, 32, 48, 65, and 75 kDa. Partial amino acid sequences of the 32-, 65-, and 75-kDa proteins indicated that they are homologous to known bacterial chitinases. There was no homology found in the partial amino acid sequences of the 25- and 48-kDa proteins to any known chitinases. Five chitinase-active proteins were detected in the protein and chromatography fractions by activity gels, but when each protein was extracted and re-electrophoresed separately under denaturing conditions, only 32- or 48-kDa proteins were revealed. It was concluded that strain C3 produces at least two chitinases that are antifungal.


    Directory of Open Access Journals (Sweden)

    Lu Longdou


    Full Text Available The hypocotyl and cotyledon of Brassica napus L. H165 and Brassica juncea DB3 were transformed with chitinase gene and herbicide-resistance gene by co-culture with Agrobacterium tumefacients LBA4404, and rape plants were obtained which could grow on the medium containing herbicide. The PCR result showed that exotic genes were integrated in the genome of the rape. Further study was performed to determine the impact of temperature on the transgenic rate and the differentiation of explants.

  15. Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate. (United States)

    Larsen, Tanja; Petersen, Bent O; Storgaard, Birgit G; Duus, Jens Ø; Palcic, Monica M; Leisner, Jørgen J


    Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)(6) tag. The purified enzyme hydrolyzes 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside, 4-nitrophenyl β-D-N,N',N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-β-D-glucosaminide, peptidoglycan or 4-nitrophenyl β-D-cellobioside. Enzyme activity was also characterized by directly monitoring product formation using (1)H-nuclear magnetic resonance which showed that chitin is a substrate with the release of N,N'-diacetylchitobiose. Hydrolysis occurs with the retention of configuration and the enzyme acts on only the β-anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Galβ1 → 4GlcNAcβ-O-(CH(2))(8)CONH(CH(2))(2)NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.

  16. Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action. (United States)

    Landim, Patrícia G Castro; Correia, Tuana O; Silva, Fredy D A; Nepomuceno, Denise R; Costa, Helen P S; Pereira, Humberto M; Lobo, Marina D P; Moreno, Frederico B M B; Brandão-Neto, José; Medeiros, Suelen C; Vasconcelos, Ilka M; Oliveira, José T A; Sousa, Bruno L; Barroso-Neto, Ito L; Freire, Valder N; Carvalho, Cristina P S; Monteiro-Moreira, Ana C O; Grangeiro, Thalles B


    A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu(2+) caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.

  17. Feeding tube insertion - gastrostomy (United States)

    ... this page: // Feeding tube insertion - gastrostomy To use the sharing features on this page, please enable JavaScript. A gastrostomy feeding tube insertion is the placement of a feeding ...

  18. ParasiticMeloidogyne and mutualisticAcremonium increase chitinase in tall fescue. (United States)

    Roberts, C A; Marek, S M; Niblack, T L; Karr, A L


    Tall fescue (Festuca arundinacea Schreb.) is a C-3 perennial grass noted for its persistence in harsh environments. Tall fescue persistence is enhanced byAcremonium coenophialum, a mutualistic fungal endophyte that increases resistance to drought, pathogens, and insects. This research was conducted to identify and elicit biochemical mechanism(s) that could account for tall fescue persistence. In initial studies, two cultivars known to differ in persistence were analyzed for chitinase, an antifungal hydrolase associated with disease resistance in other plants.Acremonium-infected Kentucky 31 (KY31), a persistent cultivar, and Johnstone, a nonpersistent cultivar, were inoculated with the parasitic nematode,Meloidogyne marylandi, grown for 50 days, and analyzed at 10-day intervals. Chitinase fluctuated throughout the 50-day period of seedling development, and activity was highest in the persistentAcremonium-infected KY31. In addition, chitinase was elicited by parasiticM. marylandi and expressed systemically. Subsequent studies were conducted to determine whether or not mutualisticAcremonium could increase chitinase activity. Genetically identical KY31, with and withoutAcremonium, were grown for 25 days and analyzed for chitinase at 5-day intervals. After 20 days,Acremonium-infected KY31 expressed more chitinase thanAcremonium-free KY31. We concluded that chitinase is related to tall fescue persistence; it was highest in the most persistent cultivar, increased under pathogen attack, and increased in the presence ofAcremonium, a symbiont known to enhance disease resistance.

  19. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    Energy Technology Data Exchange (ETDEWEB)

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.


    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  20. Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria. (United States)

    Li, Ying-Long; Song, Hui-Fang; Zhang, Xue-Yao; Li, Da-Qi; Zhang, Ting-Ting; Ma, En-Bo; Zhang, Jian-Zhen


    Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development.

  1. Production of chitinases with Trichoderma harzianun isolates using solid substrate fermentation

    Institute of Scientific and Technical Information of China (English)

    Viviana Nagy


    @@ Over forty Trichoderma harzianum isolates have been screened in solid substrate fermentation (SSF)for chitinase production. Strains were isolated from Asian soil and tree bark samples. Identification was performed in Canada and Austria by classical and molecular taxonomical methods.

  2. Antifungal chitinase against human pathogenic yeasts from Coprinellus congregatus. (United States)

    Yoo, Yeeun; Choi, Hyoung T


    The inky cap, Coprinellus congregatus, produces mushrooms which become autolyzed rapidly to generate black liquid droplets, in which no cell wall is detected by microscopy. A chitinase (Chi2) which is synthesized during the autolytic phase of C. congregatus inhibits the growths of Candida albicans and Cryptococcus neoformans up to 10% at the concentration of 10 μg/ml, about 50% at concentration of 20 μg/ml, and up to 95% at the concentration of 70 μg/ml. Upon treatment these yeast cells are observed to be severely deformed, with the formation of large holes in the cell wall. The two yeast species show no growth inhibition at the concentration of 5 μg/ml, which means the minimum inhibitory concentrations for both yeast species are 10 μg/ml under these experimental conditions.

  3. Partial biochemical characterization of crude extract extracellular chitinase enzyme from Bacillus subtilis B 298 (United States)

    Lestari, P.; Prihatiningsih, N.; Djatmiko, H. A.


    Extraction and characterization of extracellular chitinase from Bacillus subtilis B 298 have been done. Growth curve determination of B. subtilis B 298, production curve determination of crude extract chitinase from B. subtilis B 298, and partial biochemical characterization of crude extract chitinase have been achieved in this study. Optimum growth of B. subtilis B 298 was achieved at logarithmic phase within 9 hours incubation time, so it was used as inoculum for enzyme production. According to production curve of the enzyme, it was known that incubation time which gave the highest chitinase activity of 15 hours with activity of 6.937 U/mL respectively. Effect of various temperatures on chitinase activity showed that optimum activity was achieved at 40°C with an activity of 5.764 U/mL respectively. Meanwhile, the optimum pH for chitinase activity was achieved at pH of 5.0 with an activity of 6.813 U/mL respectively. This enzyme was then classified as metalloenzyme due to the decline of the activity by EDTA addition. All divalent cations tested acted as inhibitors.

  4. Effect of Cultural Conditions on Chitinase Production from Biocontrol Bacterium Against Aflatoxin

    Institute of Scientific and Technical Information of China (English)

    Kai Wang; Peisheng Yan; and Lixin Cao


    Chitinase is one of the most important mycolytic enzymes with industrial significance. Statistical methods are employed to optimize cultural conditions with the increased production of chitinase for the selected Serratia marcescens JPP1, which are obtained from peanut hulls in Jiangsu Province, China and exhibit antagonistic activity against aflatoxins. Using single⁃factor experiments the effects of cultural conditions ( broth content, inoculum size and rotation speed) on chitinase production from S. marcescens JPP1 are evaluated. Central composite design of Response Surface Methodology is used to optimize the levels of factors for the best yield of enzymes production. The optimized cultural conditions for obtaining the highest level of chitinase production are 23�2 mL broth content, 116 r/min rotation speed and 4�3% inoculum size. A quadratic regression model of chitinase production is built ( R2 = 0�970 9) and the verification experiments confirm its validity. The maximum chitinase production obtained after the optimization is 29�58 U/mL for a 1�4⁃fold increase.

  5. Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: their potential in antifungal biocontrol. (United States)

    Gomaa, Eman Zakaria


    Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na(+), Mg(2+), Cu(2+), and Ca(2+) caused enhancement of enzyme activities whereas they were markedly inhibited by Zn(2+), Hg(2+), and Ag(+). In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.

  6. Purification, characterization and antimicrobial activity of chitinase from marine-derived Aspergillus terreus

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    Aida M. Farag


    Full Text Available Chitinase (EC was produced from the culture filtrate of marine-derived Aspergillus terreus and purified by 65% ammonium sulphate precipitation, followed by gel filtration on Sephadex G-100 and DEAE-Sephadex A-50 ion exchange chromatography, with 5.16-fold of purification and specific activity of 182.08 U/mg protein. The molecular weight of the purified chitinase was 60 kDa, determined by a sodium dodecyl sulphate polyacrylamide gel electrophoresis. The optimum pH and temperature of purified chitinase were 5.6 and 50 °C, respectively. The chitinase enzyme was stable from pH 5 to 7.5 and stable up to 70 °C. The effect of activators and inhibitors was studied, Hg+, pb, EDTA, ethanol, methanol and acetone strongly inhibited the enzyme activity, while, metal ions such as Ca2+, Mn2+ and Na2+ highly increased chitinase activity. The purified chitinase produced by A. terreus inhibited the growth of Aspergillus niger, Aspergillus oryzae, Penicillum oxysporium, Rhizocotonia solani, Candida albicans and Fusarium solani, while did not inhibit the growth of Rhizopus oryzae. Moreover, the purified enzyme had antibacterial effects against some pathogenic bacteria such as; Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa, while, it had not any activity against Escherichia coli, Aeromonas hydrophila and Photobacterium damsela.

  7. Field performance of chitinase transgenic silver birches (Betula pendula): resistance to fungal diseases. (United States)

    Pasonen, H-L; Seppänen, S-K; Degefu, Y; Rytkönen, A; von Weissenberg, K; Pappinen, A


    A field trial of 15 transgenic birch lines expressing a sugar beet chitinase IV gene and the corresponding controls was established in southern Finland to study the effects of the level of sugar beet chitinase IV expression on birch resistance to fungal diseases. The symptoms caused by natural infections of two fungal pathogens, Pyrenopeziza betulicola (leaf spot disease) and Melampsoridium betulinum (birch rust), were analysed in the field during a period of 3 years. The lines that had shown a high level of sugar beet chitinase IV mRNA accumulation in the greenhouse also showed high sugar beet chitinase IV expression after 3 years in the field. The level of sugar beet chitinase IV expression did not significantly improve the resistance of transgenic birches to leaf spot disease. Instead, some transgenic lines were significantly more susceptible to leaf spot than the controls. The level of sugar beet chitinase IV expression did have an improving effect on most parameters of birch rust; the groups of lines showing high or intermediate transgene expression were more resistant to birch rust than those showing low expression. This result indicates that the tested transformation may provide a tool for increasing the resistance of silver birch to birch rust.

  8. Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

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    Huimin Meng


    Full Text Available Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.

  9. Purification and characterisation of a 31-kDa chitinase from the Myzus Persicae aphid: a target for hemiptera biocontrol. (United States)

    Francis, Frédéric; Saguez, Julien; Cherqui, Anas; Vandermoten, Sophie; Vincent, Charles; Versali, Marie-France; Dommès, Jacques; De Pauw, Edwin; Giordanengo, Philippe; Haubruge, Eric


    Hydrolytic enzymes involved in chitin degradation are important to allow moulting during insect development. Chitinases are interesting targets to disturb growth and develop alternative strategies to control insect pests. In this work, a chitinase from the aphid Myzus persicae was purified with a 36-fold purification rate in a three step procedure by ammonium sulphate fractionation, anion-exchange chromatography on a DEAE column and on an affinity Concanavalin A column. The purified chitinase purity assessed by 1D and 2D SDS-PAGE revealed a single band and three spots at 31 kDa, respectively. Chitinases were found to have high homologies with Concanavalins A and B, two chitinase-related proteins, a fungal endochitinase and an aphid acetylhydrolase by peptide identification by Maldi-Tof-Tof. The efficiency of two potent chitinase inhibitors, namely allosamidin and psammaplin A, was tested and showed significant rate of enzymatic inhibition.

  10. Tie rod insertion test

    CERN Multimedia



    The superconducting coil is inserted in the outer vaccum tank and supported by a set of tie rods. These tie rods are made of titanium alloy. This test reproduce the final insertion of the tie rods inside the outer vacuum tank.

  11. Two chitinase 5 genes from Locusta migratoria: molecular characteristics and functional differentiation. (United States)

    Li, Daqi; Zhang, Jianqin; Wang, Yan; Liu, Xiaojian; Ma, Enbo; Sun, Yi; Li, Sheng; Zhu, Kun Yan; Zhang, Jianzhen


    The duplication of chitinase 5 (Cht5) into two to five different genes has been reported only in mosquito species to date. Here, we report the duplication of Cht5 genes (LmCht5-1 and LmCht5-2) in the migratory locust (Locusta migratoria). Both LmCht5-1 (505 aa) and LmCht5-2 (492 aa) possess a signal peptide and a catalytic domain with four conserved motifs, but only LmCht5-1 contains a chitin-binding domain. Structural and phylogenetic analyses suggest that LmCht5-1 is orthologous to other insect Cht5 genes, whereas LmCht5-2 might be newly duplicated. Both LmCht5 genes were expressed in all tested tissues with LmCht5-1 highly expressed in hindgut and LmCht5-2 highly expressed in integument, foregut, hindgut and fat bodies. From the fourth-instar nymphs to the adults, LmCht5-1 and LmCht5-2 showed similar developmental expression patterns with transcript peaks prior to each nymphal molting, suggesting that their expression levels are similarly regulated. Treatment with 20-hydroxyecdysone (20E; the most active molting hormone) and reducing expression of EcR (ecdysone receptor gene) by RNAi increased and decreased expression of both LmCht5 genes, respectively, indicating that both genes are responsive to 20E. Although transcript level of LmCht5-2 is generally 10-fold higher than that of LmCht5-1, RNAi-mediated suppression of LmCht5-1 transcript led to severe molting defects and lethality, but such effects were not seen with RNAi of LmCht5-2, suggesting that the newly duplicated LmCht5-2 is not essential for development and survivorship of the locust.

  12. Evidence supporting a role for mammalian chitinases in efficacy of caspofungin against experimental aspergillosis in immunocompromised rats.

    Directory of Open Access Journals (Sweden)

    Patricia E B Verwer

    Full Text Available OBJECTIVES: Caspofungin, currently used as salvage therapy for invasive pulmonary aspergillosis (IPA, strangely only causes morphological changes in fungal growth in vitro but does not inhibit the growth. In vivo it has good efficacy. Therefore the question arises how this in vivo activity is reached. Caspofungin is known to increase the amount of chitin in the fungal cell wall. Mammals produce two chitinases, chitotriosidase and AMCase, which can hydrolyse chitin. We hypothesized that the mammalian chitinases play a role in the in vivo efficacy of caspofungin. METHODS: In order to determine the role of chitotriosidase and AMCase in IPA, both chitinases were measured in rats which did or did not receive caspofungin treatment. In order to understand the role of each chitinase in the breakdown of the caspofungin-exposed cells, we also exposed caspofungin treated fungi to recombinant enzymes in vitro. RESULTS: IPA in immunocompromised rats caused a dramatic increase in chitinase activity. This increase in chitinase activity was still noted when rats were treated with caspofungin. In vitro, it was demonstrated that the action of both chitinases were needed to lyse the fungal cell wall upon caspofungin exposure. CONCLUSION: Caspofungin seemed to alter the cell wall in such a way that the two chitinases, when combined, could lyse the fungal cell wall and assisted in clearing the fungal pathogen. We also found that both chitinases combined had a direct effect on the fungus in vitro.

  13. Time-Dependent Increase of Chitinase1 in APP/PS1 Double Transgenic Mice. (United States)

    Xiao, Qian; Shi, Rui; Yang, Wenxiu; Zou, Yan; Du, Yinshi; Zhang, Man; Yu, Weihua; Lü, Yang


    It is reported that chitinase1 increases in Alzheimer's disease (AD). However, the alteration of chitinase1 in the progress of AD is still unclear. Thus, we designed the present study to detect chitinase1 level in different stages of APP/PS1 double transgenic mice. Experimental models were APP/PS1 double transgenic mice with 4, 12 and 22 months. Cognitive function was detected by Morris water maze test in APP/PS1 mice as well as controls. ELISA and the quantitative RT-PCR were used to detect chitinase1 level in different groups. The study displayed that expression of chitinase1 gradually increased in a time-dependent manner in APP/PS1 mice, while there were no statistical differences among the wild-type mice in varies ages. Moreover, chitnase1 increased significantly in APP/PS1 mice aged 12 and 22 months compared with the age matched wild-type group, respectively. However, no difference of chitnase1 was found between 4 months-old APP/PS1 mice and wild-type mice. Comparing with the age matched wild type group, the consequences of mRNA on the increase in chitnase1 is in accordance with protein in APP/PS1 mice. Furthermore, Morris water maze showed that 4 months-old APP/PS1 mice have normal spatial learning and impaired spatial memory; both spatial learning and spatial memory in 12 and 22 months-old APP/PS1 mice were declined. Time-dependent increase of chitnase1 in APP/PS1 double transgenic mice indicates that the level of chitinase1 is associated with decline of cognition. Therefore, chitinase1 might be a biomarker of disease progression in AD.

  14. Chest tube insertion - slideshow (United States)

    ... presentations/100008.htm Chest tube insertion - series—Normal anatomy To use the sharing features ... pleural space is the space between the inner and outer lining of the lung. It is normally very thin, and lined only ...

  15. Chitinase mRNA levels by quantitative PCR using the single standard DNA: acidic mammalian chitinase is a major transcript in the mouse stomach.

    Directory of Open Access Journals (Sweden)

    Misa Ohno

    Full Text Available Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1 and acidic mammalian chitinase (AMCase. These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer's disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin, a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale.

  16. Acidic chitinase primes the protective immune response to gastrointestinal nematodes. (United States)

    Vannella, Kevin M; Ramalingam, Thirumalai R; Hart, Kevin M; de Queiroz Prado, Rafael; Sciurba, Joshua; Barron, Luke; Borthwick, Lee A; Smith, Allen D; Mentink-Kane, Margaret; White, Sandra; Thompson, Robert W; Cheever, Allen W; Bock, Kevin; Moore, Ian; Fitz, Lori J; Urban, Joseph F; Wynn, Thomas A


    Acidic mammalian chitinase (AMCase) is known to be induced by allergens and helminths, yet its role in immunity is unclear. Using AMCase-deficient mice, we show that AMCase deficiency reduced the number of group 2 innate lymphoid cells during allergen challenge but was not required for establishment of type 2 inflammation in the lung in response to allergens or helminths. In contrast, AMCase-deficient mice showed a profound defect in type 2 immunity following infection with the chitin-containing gastrointestinal nematodes Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. The impaired immunity was associated with reduced mucus production and decreased intestinal expression of the signature type 2 response genes Il13, Chil3, Retnlb, and Clca1. CD103(+) dendritic cells, which regulate T cell homing, were also reduced in mesenteric lymph nodes of infected AMCase-deficient mice. Thus, AMCase functions as a critical initiator of protective type 2 responses to intestinal nematodes but is largely dispensable for allergic responses in the lung.

  17. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses



    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomona...

  18. Enzymatic properties of chitinase-producing antagonistic bacterium Paenibacillus chitinolyticus with various substrates. (United States)

    Song, Yong-Su; Seo, Dong-Jun; Ju, Wan-Taek; Lee, Yong-Seong; Jung, Woo-Jin


    Various chitin substrates were used to investigate the properties of enzymes produced from the chitinase-producing bacterium Paenibacillus chitinolyticus MP-306 against phytopathogens. The MP-306 bacterium was incubated in nine culture media [crab shell powder chitin (CRS), chitin-protein complex powder (CPC), carboxymethyl-chitin powder (CMC), yeast extract only (YE), LB (Trypton, NaCl, and yeast extract), GT (Trypton, NaCl, and glucose), crab shell colloidal chitin (CSC), squid pen powder chitin (SPC), and cicada slough powder chitin (CSP)] at 30 °C for 3 days. Chitinase isozymes in CPC medium were expressed strongly as CN1, CN2, CN3, CN4, CN5, and CN6 bands on native-PAGE gels. Chitinase isozymes in CPC and CMC medium were expressed as 13 bands (CS1-CS13) on SDS-PAGE gels. Chitinase isozymes were expressed strongly on SDS-PAGE gels as two bands (CS6 and CS8) on YE and LB medium and 13 bands (CS1-CS13) on SPC medium. In crude enzyme, chitinase isozymes at pH 7 and pH 9 in chitin media appeared strongly on SDS-PAGE gels. Partial purified enzyme indicated high stability of enzyme activity at various temperatures and pHs in chitin medium, while these enzymes indicated low activity staining of enzyme on electrophoresis gels at various temperatures and pHs condition of chitin medium.

  19. Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases. (United States)

    Lonhienne, T; Baise, E; Feller, G; Bouriotis, V; Gerday, C


    Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

  20. Crystal structure of class III chitinase from pomegranate provides the insight into its metal storage capacity. (United States)

    Masuda, Taro; Zhao, Guanghua; Mikami, Bunzo


    Chitinase hydrolyzes the β-1,4-glycosidic bond in chitin. In higher plants, this enzyme has been regarded as a pathogenesis-related protein. Recently, we identified a class III chitinase, which functions as a calcium storage protein in pomegranate (Punica granatum) seed (PSC, pomegranate seed chitinase). Here, we solved a crystal structure of PSC at 1.6 Å resolution. Although its overall structure, including the structure of catalytic site and non-proline cis-peptides, was closely similar to those of other class III chitinases, PSC had some unique structural characteristics. First, there were some metal-binding sites with coordinated water molecules on the surface of PSC. Second, many unconserved aspartate residues were present in the PSC sequence which rendered the surface of PSC negatively charged. This acidic electrostatic property is in contrast to that of hevamine, well-characterized plant class III chitinase, which has rather a positively charged surface. Thus, the crystal structure provides a clue for metal association property of PSC.

  1. Characterization of two Listeria innocua chitinases of different sizes that were expressed in Escherichia coli. (United States)

    Honda, Shotaro; Wakita, Satoshi; Sugahara, Yasusato; Kawakita, Masao; Oyama, Fumitaka; Sakaguchi, Masayoshi


    Two putative chitinase genes, lin0153 and lin1996, from the nonpathogenic bacterium Listeria innocua were expressed in Escherichia coli, and the gene products were characterized. The genes were close homologs of chitinases from the pathogenic bacterium Listeria monocytogenes, in which chitinases and chitin-binding proteins play important roles in pathogenesis in mice-infection models. The purified recombinant enzymes that are different in size, LinChi78 (lin0153 product) and LinChi35 (lin1996 product)-with molecular masses of 82 and 38 kDa, including vector-derived additional sequences, respectively-exhibited optimum catalytic activity under neutral and acidic conditions at 50 °C, respectively, and were stable over broad pH (4-11) and temperature (4-40 °C) ranges. LinChi35 displayed higher k cat and K M values for 4-nitrophenyl N,N-diacetyl-β-D-chitobioside [4NP-(GlcNAc)2] than LinChi78. Both enzymes produced primarily dimers from colloidal chitin as a substrate. However, LinChi78 and LinChi35 could hydrolyze oligomeric substrates in a processive exo- and nonprocessive endo-manner, respectively, and showed different reactivity toward oligomeric substrates. Both enzymes could bind chitin beads but were different in their binding ability toward crystalline α-chitin and cellulose. The structure-function relationships of these chitinases are discussed in reference to other bacterial chitinases.

  2. An investigation of a defensive chitinase against Fusarium oxysporum in pepper leaf tissue

    Directory of Open Access Journals (Sweden)

    Khemika S. Lomthaisong


    Full Text Available Plant chitinase is classified as a PR-protein involved in a defense mechanism against a pathogen. This research aims to investigate a specific type of chitinase which is produced by pepper in response to an early defense against Fusarium oxysporum, which causes wilt disease. The changes of chitinase isozyme patterns in the inter- and intracellular fluids in the leaf of four cultivars of pepper (Capsicum annuum L. at day 1, 3, 5, 7 and 10 from fungal inoculation were analysed using SDS-PAGE in polyacrylamide gel supplemented with glycol chitin as a substrate. The levels of disease severity in the four varieties of pepper were also compared with the isozyme patterns. The results showed that the resistance of pepper to F. oxysporum attack corresponded to the expression of ~70 kDa chitinase band (Chi-3 in the intercellular fluid. Therefore, such chitinase could possibly be used as a protein marker to identify the tolerant line and as a springboard for further study of wilt disease control.

  3. Chitinase expression in Listeria monocytogenes is positively regulated by the Agr system

    DEFF Research Database (Denmark)

    Paspaliari, Dafni Katerina; Mollerup, Maria Storm; Kallipolitis, Birgitte H.;


    The food-borne pathogen Listeria monocytogenes encodes two chitinases, ChiA and ChiB, which allow the bacterium to hydrolyze chitin, the second most abundant polysaccharide in nature. Intriguingly, despite the absence of chitin in human and mammalian hosts, both of the chitinases have been deemed...

  4. Degradation of chitin and chitosan by a recombinant chitinase derived from a virulent Aeromonas hydrophila isolated from diseased channel catfish (United States)

    A chitinase was identified in extracellular products of a virulent Aeromonas hydrophila isolated from diseased channel catfish (Ictalurus punctatus). Bioactive recombinant chitinase (rChi-Ah) was produced in Escherichia coli. Purified rChi-Ah had optimal activity at temperature of 42°C and pH 6.5. T...


    NARCIS (Netherlands)



    A chitinase gene from the Gram-negative bacterium Serratia marcescens BJL200 was cloned in Lactococcus lactis subsp. lactis MG1363 and in the silage inoculum strain Lactobacillus plantarum E19b. The chitinase gene was expressed as an active enzyme at a low level in Lactococcus lactis, when cloned in

  6. Biochemical characterization of Aspergillus niger Cfcl, a glycoside hydrolase family 18 chitinase that releases monomers during substrate hydrolysis

    NARCIS (Netherlands)

    van Munster, Jolanda M.; van der Kaaij, Rachel M.; Dijkhuizen, Lubbert; van der Maarel, Marc J. E. C.


    The genome of the industrially important fungus Aspergillus niger encodes a large number of glycoside hydrolase family 18 members annotated as chitinases. We identified one of these putative chitinases, Cfcl, as a representative of a distinct phylogenetic clade of homologous enzymes conserved in all

  7. Comparing Leaf and Root Insertion

    Directory of Open Access Journals (Sweden)

    Jaco Geldenhuys


    Full Text Available We consider two ways of inserting a key into a binary search tree: leaf insertion which is the standard method, and root insertion which involves additional rotations. Although the respective cost of constructing leaf and root insertion binary search trees trees, in terms of comparisons, are the same in the average case, we show that in the worst case the construction of a root insertion binary search tree needs approximately 50% of the number of comparisons required by leaf insertion.

  8. Tissue distribution, synthesis stage, and ethylene induction of pineapple (Ananas comosus) chitinases. (United States)

    Taira, Toki; Toma, Noriko; Ichi, Marika; Takeuchi, Makoto; Ishihara, Masanobu


    We examined the tissue distribution, synthesis stage, and ethylene induction of three types of pineapple chitinase using chitinase activity gel and immunoblot analysis. Type A (acidic class III) exists in all tissues, while type B (weakly basic class I, which has strong antifungal activity) and type C (acidic class I) are localized mainly in the leaf and stem. In a pericarp, type A exists at all stages during fruit development, while type B and type C exist only at the early stage. Synthesis of type A is induced by ethylene, while that of types B and C is not affected by it. These results suggest that the physiological roles of these three types of chitinase in pineapple are different.

  9. Chitinase-like proteins with antifungal activity from emperor banana fruits. (United States)

    Ho, Vincent S M; Ng, Tzi Bun


    Two 30-kDa proteins with N-terminal sequence homology to chitinases have been isolated from fruits of the emperor banana by using a protocol that involved (NH(4))(2)SO(4) precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S and gel filtration by FPLC on Superdex 75. The proteins were adsorbed on Affi-gel blue gel and Mono S. They both inhibited mycelial growth in Fusarium oxysporum but not in Mycosphaerella arachidicola. The chitinase-like protein more strongly bound on Mono S was obtained with a slightly lower yield and exhibited a higher antifungal potency toward F. oxysporum when compared with the less strongly bound chitinase-like protein.

  10. Are mycoparasitism and chitinase production species or isolate dependent in Trichoderma ?

    Institute of Scientific and Technical Information of China (English)

    Szakacs G; Nagy V; Kovacs K


    @@ The relationship between taxonomic status of Trichoderma spp., chitinase production in solid substrate fermentation (SSF) on four media and mycoparasitism in dual culture (confrontation assay)against four plant pathogenic fungi was studied. Seventy five Trichoderma isolates belonging to 35species have been screened. The plant pathogenic fungi used in confrontation assay were Botrytis cinerea , Fusarium oxysporum f. sp. dianthi , Rhizoctonia solani and Sclerotinia sclerotiorum . The SSF media contained wheat bran, crude chitin (from crab shells, SIGMA) and salt solutions. The best performing isolates in mycoparasitism tests were Trichoderma flavofuscum, T. harzianum, T.inhamatum, T. koningii and T. strigosum. Some isolates exhibiting good mycoparasitism produced chitinase in SSF only at low or medium level. In contrary there were isolates with excellent extracellular chitinase production but their biocontrol potential did not belong to the leading group.Statistical methods have been used to evaluate the data.

  11. Isolation, purification, crystallization and preliminary crystallographic studies of chitinase from tamarind (Tamarindus indica) seeds. (United States)

    Patil, Dipak N; Datta, Manali; Chaudhary, Anshul; Tomar, Shailly; Sharma, Ashwani Kumar; Kumar, Pravindra


    A protein with chitinase activity has been isolated and purified from tamarind (Tamarindus indica) seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an approximately 34 kDa endochitinase which belongs to the acidic class III chitinase family. The protein was crystallized by the vapour-diffusion method using PEG 4000. The crystals belonged to the tetragonal space group P4(1), with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 A.

  12. Comparative molecular evolution of Trichoderma chitinases in response to mycoparasitic interactions

    DEFF Research Database (Denmark)

    Ihrmark, Katarina; Asmail, Nashwan; Ubhayasekera, Wimal;


    Certain species of the fungal genus Trichoderma are potent mycoparasites and are used for biological control of fungal diseases on agricultural crops. In Trichoderma, whole-genome sequencing reveal between 20 and 36 different genes encoding chitinases, hydrolytic enzymes that are involved in the ...... clades are observed. These observations show that Trichoderma chitinases chi18-13 and chi18-15 evolve in a manner consistent with rapid co-evolutionary interactions and identifies putative target regions involved in determining substrate-specificity....

  13. Acidic mammalian chitinase and the eye: implications for ocular inflammatory diseases

    Directory of Open Access Journals (Sweden)

    Claudio eBucolo


    Full Text Available Chitinases have an important role in the defence of organisms against chitin containing parasites. An acidic mammalian chitinase (AMCase has been detected in epithelial cells in lung tissue samples taken from patients with asthma as well as in conjunctival epithelium of patients with inflammatory ocular diseases. Particularly, elevated AMCase activity has been observed in ocular tissues of patients with vernal keratoconjunctivitis, seasonal allergic conjunctivitis, and in patients affected by dry eye syndrome. This enzyme is induced via a TH2-specific, IL-13-dependent pathway. AMCase may thus be a key mediator of IL-13-induced responses in TH2-driven inflammatory ocular diseases.

  14. Study on the character of chitinase produced by Trichoderna spp.with measuring reducing sugar

    Institute of Scientific and Technical Information of China (English)

    LIU Kai-qi; XIANG Mei-mei; LIU Ren; ZENG Yong-san; LI Hua; JIANG Xin-yin; ZHANG Yue-li


    @@ A trusty and intuitionistic method for screening chitinase produced by Trichoderma spp. was developed. 38 isolates of Trichoderma spp. were cultured in liquid medium with chitin or colloidal chitin as the sole carbon source for 4 days. The supernatant of the fermented broth was mixed with colloidal chitin and heated in water-bath at 37℃ for 30 min, then 3,5-dinitrosalicylic acid reagent (DNS) was added to the mixture, and let them react for 10 min in water-bath. According to the different colour of the mixture, the isolates of Trichoderma spp. which can produce chitinase could be screened.

  15. The Composite Insertion Electrode

    DEFF Research Database (Denmark)

    Atlung, Sven; Zachau-Christiansen, Birgit; West, Keld;


    . The theoretical basis for such electrodes is discussedand, using a simplified model, equations are derived to describe the distribution of potential and current duringdischarge/charge operation. Under the assumption that the insertion compound particles are small enough to ensureequilibrium, and that the local...... electrode potential depends linearly on the degree of insertion, these equations are solvedto obtain analytical expressions for the discharge curve. It is shown that the parameters which determine the dischargebehavior for a given discharge current are simply related to the effective ionic and electronic...

  16. Inserting the CMS solenoid

    CERN Multimedia

    Maximilien Brice


    The huge superconducting solenoid for CMS is inserted into the cryostat barrel. CMS uses the world's largest thin solenoid, in terms of energy stored, and is 12 m long, with a diameter of 6 m and weighing 220 tonnes. When turned on the magnet will produce a field strength of 4 T using superconducting niobium-titanium material at 4.5 K.

  17. Pixel detector insertion

    CERN Multimedia



    Insertion of the Pixel Tracker, the 66-million-channel device used to pinpoint the vertex of each colliding proton pair, located at the heart of the detector. The geometry of CMS is a cylinder lying on its side (22 meters long and 15 meters high in dia

  18. The Role of Chitinase Production by Stenotrophomonas maltophilia Strain C3 in Biological Control of Bipolaris sorokiniana. (United States)

    Zhang, Z; Yuen, G Y


    ABSTRACT The role of chitinase production by Stenotrophomonas maltophilia strain C3 in biological control of leaf spot on tall fescue (Festuca arundinacea), caused by Bipolaris sorokiniana, was investigated in vitro and in vivo. The filtrate of a broth culture of C3, with chitin as the carbon source, was separated into fractions. A high molecular-weight fraction (>8 kDa) was chitinolytic and more inhibitory than a low-molecular-weight, nonchitinolytic fraction to conidial germination and hyphal growth by B. sorokiniana and to leaf spot development. A protein fraction derived by ammonium sulfate precipitation and a chitinase fraction purified by chitin affinity chromatography also were chitinolytic and highly antifungal. The chitinolytic fractions caused swelling and vacuolation of conidia and discoloration, malformation, and degradation of germ tubes. When boiled, the chitinolytic fractions lost chitinase activity along with most of the antifungal properties. Two chitinase-deficient and two chitinase-reduced mutants of C3 were compared with the wild-type strain for inhibition of germination of B. sorokiniana conidia on tall fescue leaves and for suppression of leaf spot development in vivo. The mutants exhibited reduced antifungal activity and biocontrol efficacy, but did not lose all biocontrol activity. An aqueous extract of leaves colonized by wild-type C3 had higher chitinase activity than that of noncolonized leaves and was inhibitory to conidial germination. The addition of chitin to leaves along with the wild-type strain increased both chitinase and antifungal activity. The chitinase activity level of extracts from leaves colonized by a chitinase-deficient mutant of C3, with and without added chitin, was no higher than the background, and the extracts lacked antifungal activity. Chitinolysis appears to be one mechanism of biological control by strain C3, and it functions in concert with other mechanisms.

  19. Chitinolytic Bacteria Isolated from Chili Rhizosphere: Chitinase Characterization and Its Application as Biocontrol for Whitefly (Bemisia tabaci Genn.

    Directory of Open Access Journals (Sweden)

    Nisa R. Mubarik


    Full Text Available Problem statement: Chitin, a common constituent of insect exoskeleton, could be hydrolyzed by chitinase. The research was conducted to screen chitinolytic rhizobacteria isolated from rhizosphere of chilli pepper and to determine their chitinase activity in degrading chitin of whitefly, Bemisia tabaci Genn. (Hemiptera: Aleyrodidae. Whitefly is recognized as an important pest on many crops including chilli pepper. Approach: Screening and molecular identification based on 16S rRNA sequence of chitinolytic isolates, chitinase productions, measurement of chitinase activity, characterization of chitinase and effect of the chitinase treatment on whitefly were studied. Results: A total of 25 isolates of rhizobacteria formed a clear zone on solid chitin media. Two isolates, i.e., I.5 and I.21 isolates had the highest chitinolytic index. Based on sequence of 16S rRNA gene, the isolates of I.5 and I.21 were identified as Bacillus sp. and Bacillus cereus, respectively. The highest chitinolytic index and specific activity of I.5 isolate was 0.94 and 0.11 U mg-1 proteins, respectively. Maximum production of I.5 chitinase was occured after 36 h cultivation at 30°C and pH 7.0. The highest chitinolytic index and specific activity of I.21isolate was 0.75 and 0.114 U mg-1 proteins, respectively. Maximum production of I.21 chitinase was occured after 36 h cultivation at 55°C and pH 7.0. Cell culture and crude enzyme of the isolates were tested on chitin of B. tabaci and the effect was observed using a microscope and sterile water was used as a negative control. Hydrolytic observation showed that crude enzyme of I.21 isolate could degrade chitin of B. tabaci exoskeleton and the activity was better than that of I.5 isolate. Conclusion: Chitinase produced by Bacillus cereus I.21 strain has potential application as biocontrol agents for B. tabaci.

  20. Use of Metarhizium anisopliae Chitinase Genes for Genotyping and Virulence Characterization

    Directory of Open Access Journals (Sweden)

    Saliou Niassy


    Full Text Available Virulence is the primary factor used for selection of entomopathogenic fungi (EPF for development as biopesticides. To understand the genetic mechanisms underlying differences in virulence of fungal isolates on various arthropod pests, we compared the chitinase genes, chi2 and chi4, of 8 isolates of Metarhizium anisopliae. The clustering of the isolates showed various groups depending on their virulence. However, the analysis of their chitinase DNA sequences chi2 and chi4 did not reveal major divergences. Although their protein translates have been implicated in fungal virulence, the predicted protein structure of chi2 was identical for all isolates. Despite the critical role of chitin digestion in fungal infection, we conclude that chi2 and chi4 genes cannot serve as molecular markers to characterize observed variations in virulence among M. anisopliae isolates as previously suggested. Nevertheless, processes controlling the efficient upregulation of chitinase expression might be responsible for different virulence characteristics. Further studies using comparative “in vitro” chitin digestion techniques would be more appropriate to compare the quality and the quantity of chitinase production between fungal isolates.


    Directory of Open Access Journals (Sweden)

    Dominika Ďurechová


    Full Text Available Chitinases occur in dozens of genes in the individual plant species and play diverse roles in plant growth and development, and during the plant defense to biotic and abiotic stress. Here we focused on isolation and in silico characterization of regulatory sequences of chitinase gene that belongs to the first isolated gene sequences from D. rotundifolia overall. For the isolation of the 739 bp sequence of chitinase promoter the genome walking approach was applied. The authenticity of the obtained fragment(s was verified by sequencing and sequence alignment ClustalW program. The core of the chitinase promoter was predicted by Neural Network Promoter Prediction program. In total the PLACE online available database identified 66 various cis-regulatory elements in the analyzed sequence. Some of them might be potentially bound by specific transcription factors, and regulate gene expression in specific plant tissues during the plant development or upon the pathogen attack, dehydration, cold or high salinity stress. However, further analyses are needed to reveal which out of predicted cis-elements participate in the true expression profile of isolated promoter in origin and transgenic plant organism.

  2. [The study of mycolytic properties of aerobic spore-forming bacteria producing extracellular chitinases]. (United States)

    Aktuganov, G E; Melent'ev, A I; Galimzianova, N F; Shirokov, A V


    The mycolytic activity of 27 strains of antagonistic bacilli belonging to two taxonomic groups (18 strains of Bacillus subtilis and 9 strains of Paenibacillus ehimensis) capable of induced synthesis of chitinolytic enzymes was studied. Most of the B. subtilis strains neither displayed visible mycolytic effects on the phytopathogenic fungus Bipolaris sorokiniana in vitro, nor produced chitinases in the presence of an auto-claved mycelium. On the contrary, P. ehimensis strains grown under conditions favorable for induction of chitinases and other hydrolases exhibited a pronounced lytic effect on B. sorokiniana and actively grew by utilizing mycelium as the sole source of carbon and nitrogen. Comparison of the mycolytic activities of extracellular hydrolases in the studied strains demonstrated low correlation between chitinase production and the ability of the strains to degrade the cell walls of B. sorokiniana. Characterization of enzyme profiles in the studied strains revealed that beta-1,3-glucanase was a more significant factor than chitinase for determining the mycolytic potential of bacteria and their ability to utilize the mycelium of phytopathogenic fungi as a growth substrate.

  3. Draft Genome Sequence of Marine-Derived Aeromonas caviae CHZ306, a Potential Chitinase Producer Strain (United States)

    Zimpel, Cristina Kraemer; Guimaraes, Ana Marcia Sa; Pessoa, Adalberto; Rivera, Irma Nelly Gutierrez


    We report here a draft genome sequence of Aeromonas caviae CHZ306, a marine-derived bacterium with the ability to hydrolyze chitin and express high levels of chitinases. The assembly resulted in 65 scaffolds with approximately 4.78 Mb. Genomic analysis revealed different genes encoding chitin-degrading enzymes that can be used for chitin derivative production. PMID:27856589

  4. Extracellular chitinases of fluorescent pseudomonads antifungal to Fusarium oxysporum f. sp. dianthi causing carnation wilt. (United States)

    Ajit, Naosekpam Singh; Verma, Rajni; Shanmugam, V


    Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.

  5. Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, Evert; Barends, Thomas; Terwisscha van Scheltinga, Anke C.; Dijkstra, Bauke W.; Beintema, Jaap J.


    The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore, Products mere separated by HPLC and the amount of product was cal

  6. Differential aphicidal effects of chitinase inhibitors on the polyphagous homopteran Myzus persicae (Sulzer). (United States)

    Saguez, Julien; Dubois, Françoise; Vincent, Charles; Laberche, Jean-Claude; Sangwan-Norreel, Brigitte S; Giordanengo, Philippe


    Four chitinase inhibitors, cyclo-(Proline-Tyrosine), cyclo-(Histidine-Proline), allosamidin and psammaplin A, were selected for in vitro feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Artificial diets were used to provide chitinase inhibitors at 10, 50 and 100 microg mL(-1) to M. persicae. Except for cyclo-(Proline-Tyrosine), which did not modify aphid demographic parameters, chitinase inhibitors induced differential aphicidal effects on M. persicae. At all doses, cyclo-(Histidine-Proline) induced significant effects affecting daily fecundity, intrinsic rate of natural increase (r(m)) and doubling time of population. When compared with the control diet, allosamidin decreased nymph survival and daily fecundity, increasing the doubling time of population from 1 to 1.5 days. Psammaplin A was the most toxic inhibitor when delivered via artificial diet, as it induced the death of all aphids reared at 50 and 100 microg mL(-1). The results demonstrate the potential use of chitinase inhibitors as aphid management tools.


    NARCIS (Netherlands)



    A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55.5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of th

  8. Crystallization of Hevamine, an Enzyme with Lysozyme/Chitinase Activity from Hevea brasiliensis Latex

    NARCIS (Netherlands)



    Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To investiga

  9. Purification, characterization, and antifungal activity of chitinases from pineapple (Ananas comosus) leaf. (United States)

    Taira, Toki; Toma, Noriko; Ishihara, Masanobu


    Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.

  10. Expression of chitinase genes of Metarhizium anisopliae isolates in lepidopteran pests and on synthetic media. (United States)

    Bhanu Prakash, G V S; Padmaja, V; Jami, Sravan Kumar; Kirti, P B


    Pathogenecity of the well characterized entomopathogenic fungus Metarhizium anisopliae used for biocontrol of a wide range of insect pests secretes hydrolytic enzymes that degrade the host cuticle. The chitinolytic activity of high and low virulent isolates of M. anisopliae was assayed on minimal medium (MM) + colloidal chitin and MM supplemented with insect cuticles. Ex- pression pattern of four chitinase genes (chitinase (chi), chi 1, chi 2, chi 3) was profiled during pathogenic stages of the entomopathogen under in vitro and in vivo conditions. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that chitinase cDNAs were expressed during the germination of fungus under nutrient-deprived conditions. RT-PCR analysis performed for the four chitinase genes on the two insect hosts Spodoptera litura and Helicoverpa armigera at six developmental stages of the pathogen displayed up-regulation in S. litura at mycosed and conidiated condition while with H. armigera there was expression only after 48 h of incubation. Differential expression of chi, chi 1 and chi 2 genes in vitro (nitrogen rich and nitrogen limiting media) and in vivo (live insect hosts S. litura and H. armigera) implicate the role of substrate differences in pathogenesis.

  11. Use of Metarhizium anisopliae chitinase genes for genotyping and virulence characterization. (United States)

    Niassy, Saliou; Subramanian, Sevgan; Ekesi, Sunday; Bargul, Joel L; Villinger, Jandouwe; Maniania, Nguya K


    Virulence is the primary factor used for selection of entomopathogenic fungi (EPF) for development as biopesticides. To understand the genetic mechanisms underlying differences in virulence of fungal isolates on various arthropod pests, we compared the chitinase genes, chi2 and chi4, of 8 isolates of Metarhizium anisopliae. The clustering of the isolates showed various groups depending on their virulence. However, the analysis of their chitinase DNA sequences chi2 and chi4 did not reveal major divergences. Although their protein translates have been implicated in fungal virulence, the predicted protein structure of chi2 was identical for all isolates. Despite the critical role of chitin digestion in fungal infection, we conclude that chi2 and chi4 genes cannot serve as molecular markers to characterize observed variations in virulence among M. anisopliae isolates as previously suggested. Nevertheless, processes controlling the efficient upregulation of chitinase expression might be responsible for different virulence characteristics. Further studies using comparative "in vitro" chitin digestion techniques would be more appropriate to compare the quality and the quantity of chitinase production between fungal isolates.

  12. Stimulatory effects of chitinase on growth and immune defense of orange-spotted grouper (Epinephelus coioides). (United States)

    Zhang, Yanhong; Feng, Shaozhen; Chen, Jun; Qin, Chaobin; Lin, Haoran; Li, Wensheng


    Chitinase, belonging to either family 18 or family 19 of the glycosylhydrolases, hydrolyze chitin into oligosaccharides. In the present study, the cDNA fragment encoding orange-spotted grouper (Epinephelus coioides) chitinase1 was subcloned into pPIC3.5K vector and expressed in Pichia pastoris GS115. The results showed that a band with the size of about 53 kDa could be detected by SDS-PAGE and Western blot. The recombinant protein of grouper chitinase1 (rgChi1) was added into the fish diet containing shrimp shell chitin for feeding experiment lasting 8 weeks. The weight of orange-spotted grouper, fed with diets containing rgChi1 at 0, 5, 10 and 20 μg/g was calculated on the 2nd, 4th, 6th and 8th weeks, and difference in growth rates was first observed in the 6th week of the feeding period and it kept until the end of the feeding experiment. At the end of 8 weeks feeding trial, the percent weight gain (PWG), growth rate (GR) and specific growth rate (SGR) of fish fed with 10 and 20 μg rgChi1/g feed were significantly higher compared to the control group. The neuropeptide Y (NPY), growth-hormone-releasing hormone (GHRH), growth-hormone (GH), interleukin-1beta (IL-1β), cyclooxygenase-2 (COX-2), superoxide dismutase (SOD) (Cu/Zn) and SOD (Mn) mRNA expression of fish fed with diet containing 10 μg/g or/and 20 μg/g rgChi1 were obviously higher than the control group. The lysozyme (LZM) and total SOD activity of fish fed with diet containing rgChi1 at 10 and 20 μg/g were significantly higher than that of the control. The aspartate aminotransferase (AST)/glutamic oxalacetic transaminases (GOT) activity in 20 μg/g group decreased compared to the control group. These results indicated that the grouper chitinase1 was successfully produced using the P. pastoris expression system and the recombinant protein had obvious effects on growth and immune defense. The mRNA expression and protein secretion of grouper chitinase1 and chitinase2 were significantly stimulated in

  13. Expression analysis of chitinase upon challenge inoculation to Alternaria wounding and defense inducers in Brassica juncea

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    Sandhya Rawat


    Full Text Available Chitinases are the hydrolytic enzymes which belong to the pathogenesis-related (PR protein family and play an important role not only in plant defense but also in various abiotic stresses. However, only a limited number of chitinase genes have been characterised in B. juncea. In this study, we have characterised B. juncea class IV chitinase gene (accession no EF586206 in response to fungal infection, salicylic acid (SA, jasmonic acid (JA treatments and wounding. Gene expression studies revealed that the transcript levels of Bjchitinase (BjChp gene increases significantly both in local and distal tissues after Alternaria infection. Bjchitinase gene was also induced by jasmonic acid and wounding but moderately by salicylic acid. A 2.5 kb class IV chitinase promoter of this gene was isolated from B. juncea by Genome walking (accession no KF055403.1. In-silico analysis of this promoter revealed a number of conserved cis-regulatory elements related to defense, wounding and signalling molecules like SA, and JA. For validation, chitinase promoter was fused to the GUS gene, and the resultant construct was then introduced into Arabidopsis plants. Histochemical analysis of T2 transgenic Arabidopsis plants showed that higher GUS activity in leaves after fungal infection, wounding and JA treatment but weakly by SA. GUS activity was seen in meristematic tissues, young leaves, seeds and siliques. Finally investigation has led to the identification of a pathogen-inducible, developmentally regulated and organ-specific promoter. Present study revealed that Bjchitinase (BjChp promoter is induced during biotic and environmental stress and it can be used in developing finely tuned transgenics.

  14. Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens. (United States)

    Raharjo, S H; Hernandez, M O; Zhang, Y Y; Punja, Z K


    Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.

  15. Transformation of indica rice with plasmid pBGll21 containing a tobacco endo-chitinase gene I

    Institute of Scientific and Technical Information of China (English)


    @@ Several plasmids, which were suitable for cereals transformation, have been reported. In the study, rice was transformed by a new plasmid pBGll21 containing a tobacco endo-chitinase gene ( TchiB ).

  16. Chitinase but N-acetyl-β-D-glucosaminidase production correlates to the biomass decline in Penicillium and Aspergillus species. (United States)

    Pusztahelyi, Tünde; Pócsi, István


    Hydrolytic enzyme production is typical of the autolysis in filamentous fungi; however, less attention has been given to the physiological role of the enzymes. Here, the aim was to investigate the possible relation of the chitinolytic enzymes to the changes in the biomass in some filamentous fungi of high importance for pharmaceutical or food industry. In Penicillium and Aspergillus filamentous fungi, which showed different characteristics in submerged cultures, the growth and biomass decline rates were calculated and correlated to the chitinase and N-acetyl-β-D-glucosaminidase enzyme productions. Correlation was found between the biomass decrease rate and the chitinase level at the stationary growth phase; while chitinase production covariates negatively with N-acetyl-β-D-glucosaminidase activities. The chitinase production and the intensive autolysis hindered the production of N-acetyl-β-D-glucosaminidase and, therefore, could hinder the cell death in the cultures.

  17. Strong aphicidal activity of GlcNAc(β1→4)Glc disaccharides: synthesis, physiological effects, and chitinase inhibition. (United States)

    Dussouy, Christophe; Bultel, Laurent; Saguez, Julien; Cherqui, Anas; Khelifa, Mounia; Grand, Eric; Giordanengo, Philippe; Kovensky, José


    The synthesis of four GlcNAc(β1→4)Glc disaccharides containing 2-O-acetyl and/or 6-sulfate groups was performed in high yields with total 1,2-trans stereoselectivity. These disaccharides were evaluated as candidates for insect chitinase inhibition and aphicidal activity. All the compounds prepared displayed physiological effects on M. persicae aphids; however, the inhibition of chitinases of different sources (bacteria, fungus, and aphid) followed different patterns according to subtle structural characteristics.

  18. X-Ray Crystal Structure of the Full Length Human Chitotriosidase (CHIT1) Reveals Features of Its Chitin Binding Domain (United States)

    Fadel, Firas; Zhao, Yuguang; Cousido-Siah, Alexandra; Ruiz, Francesc X.; Mitschler, André; Podjarny, Alberto


    Chitinases are enzymes that catalyze the hydrolysis of chitin. Human chitotriosidase (CHIT1) is one of the two active human chitinases, involved in the innate immune response and highly expressed in a variety of diseases. CHIT1 is composed of a catalytic domain linked by a hinge to its chitin binding domain (ChBD). This latter domain belongs to the carbohydrate-binding module family 14 (CBM14 family) and facilitates binding to chitin. So far, the available crystal structures of the human chitinase CHIT1 and the Acidic Mammalian Chitinase (AMCase) comprise only their catalytic domain. Here, we report a crystallization strategy combining cross-seeding and micro-seeding cycles which allowed us to obtain the first crystal structure of the full length CHIT1 (CHIT1-FL) at 1.95 Å resolution. The CHIT1 chitin binding domain (ChBDCHIT1) structure shows a distorted β-sandwich 3D fold, typical of CBM14 family members. Accordingly, ChBDCHIT1 presents six conserved cysteine residues forming three disulfide bridges and several exposed aromatic residues that probably are involved in chitin binding, including the highly conserved Trp465 in a surface- exposed conformation. Furthermore, ChBDCHIT1 presents a positively charged surface which may be involved in electrostatic interactions. Our data highlight the strong structural conservation of CBM14 family members and uncover the structural similarity between the human ChBDCHIT1, tachycitin and house mite dust allergens. Overall, our new CHIT1-FL structure, determined with an adapted crystallization approach, is one of the few complete bi-modular chitinase structures available and reveals the structural features of a human CBM14 domain. PMID:27111557

  19. X-Ray Crystal Structure of the Full Length Human Chitotriosidase (CHIT1 Reveals Features of Its Chitin Binding Domain.

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    Firas Fadel

    Full Text Available Chitinases are enzymes that catalyze the hydrolysis of chitin. Human chitotriosidase (CHIT1 is one of the two active human chitinases, involved in the innate immune response and highly expressed in a variety of diseases. CHIT1 is composed of a catalytic domain linked by a hinge to its chitin binding domain (ChBD. This latter domain belongs to the carbohydrate-binding module family 14 (CBM14 family and facilitates binding to chitin. So far, the available crystal structures of the human chitinase CHIT1 and the Acidic Mammalian Chitinase (AMCase comprise only their catalytic domain. Here, we report a crystallization strategy combining cross-seeding and micro-seeding cycles which allowed us to obtain the first crystal structure of the full length CHIT1 (CHIT1-FL at 1.95 Å resolution. The CHIT1 chitin binding domain (ChBDCHIT1 structure shows a distorted β-sandwich 3D fold, typical of CBM14 family members. Accordingly, ChBDCHIT1 presents six conserved cysteine residues forming three disulfide bridges and several exposed aromatic residues that probably are involved in chitin binding, including the highly conserved Trp465 in a surface- exposed conformation. Furthermore, ChBDCHIT1 presents a positively charged surface which may be involved in electrostatic interactions. Our data highlight the strong structural conservation of CBM14 family members and uncover the structural similarity between the human ChBDCHIT1, tachycitin and house mite dust allergens. Overall, our new CHIT1-FL structure, determined with an adapted crystallization approach, is one of the few complete bi-modular chitinase structures available and reveals the structural features of a human CBM14 domain.

  20. Differential effect of purified spruce chitinases and beta-1,3-glucanases on the activity of elicitors from ectomycorrhizal fungi. (United States)

    Salzer, P; Hübner, B; Sirrenberg, A; Hager, A


    Two chitinases (EC and two beta-1,3-glucanases (EC were purified from the culture medium of spruce (Picea abines [L.] Karst.) cells to study their role in modifying elicitors, cell walls, growth, and hyphal morphology of ectomycorrhizal fungi. The 36-kD class I chitinase (isoelectric point [pl] 8.0) and the 28-kD chitinase (pl 8.7) decreased the activity of elicitor preparations from Hebeloma crustuliniforme (Bull. ex Fries.) Quél., Amanita muscaria (L.) Pers., and Suillus variegatus (Sw.: Fr.) O.K., as demonstrated by using the elicitor-induced extracellular alkalinization in spruce cells as a test system. In addition, chitinases released monomeric products from the walls of these ectomycorrhizal fungi. The beta-1,3-glucanases (35 kD, pl 3.7 and 3.9), in contrast, had little influence on the activity of the fungal elicitors and released only from walls of A. muscaria some polymeric products. Furthermore, chitinases alone and in combination with beta-1,3-glucanases had no effect on the growth and morphology of the hyphae. Thus, it is suggested that apoplastic chitinases in the root cortex destroy elicitors from the ectomycorrhizal fungi without damaging the fungus. By this mechanism the host plant could attenuate the elicitor signal and adjust its own defense reactions to a level allowing symbiotic interaction.

  1. Contribution of Chitinase A’s C-Terminal Vacuolar Sorting Determinant to the Study of Soluble Protein Compartmentation

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    Egidio Stigliano


    Full Text Available Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin’s tetrapeptide AFVY (AlaPheValTyr and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

  2. Metarhizium anisopliae chitinase CHIT30 is involved in heat-shock stress and contributes to virulence against Dysdercus peruvianus. (United States)

    Staats, Charley Christian; Kmetzsch, Livia; Lubeck, Irina; Junges, Angela; Vainstein, Marilene Henning; Schrank, Augusto


    Entomopathogenic fungi are able to produce several chitinases, which serve a variety of biological functions, such as fungal cell wall organization and the degradation of exogenous chitin for nutrition or insect infection processes. In this study, we analyzed the contribution of the CHIT30 chitinase from Metarhizium anisopliae in morphogenetic development and virulence as a model of chitinase function. The analysis of chi3 gene expression revealed transcript accumulation in response to heat-shock stress conditions as well as cultivation in medium supplemented with chitin. Null chi3 mutants were constructed to determine the biological role of CHIT30. No substantial differences in the secreted chitinase activity could be detected between the wild type and the Δchi3 mutant. However, both endochitinase and exochitinase activities were diminished in the mutant strain following heat-shock treatment, suggesting that CHIT30 is involved in heat-shock adaptation. Mutants lacking CHIT30 chitinase showed reduced virulence against the cotton stainer bug Dysdercus peruvianus, indicating that the CHIT30 chitinase plays a role in the infection process of M. anisopliae.


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    Balasubramanian Thiagarajan


    Full Text Available Grommet insertion the commonest surgical procedure next only to circumcision is usually performed using an operating microscope 1. Authors have been using 4 mm 0 degree nasalendoscopes to perform this procedure during the last 5 years. This is a report of their experience in using endoscope inlieu of microscope in performing this surgery. This study makes a comparative analysis of Endoscopic Grommet insertion viz a viz Microscopic Grommet insertion. For this comparative analysis one year (2009 data base of Government Stanley Medical College Chennai India was used. This study reveals that Endoscopic Grommet insertion compared favorably with Microscopic Grommet insertion in all aspects with certain obvious advantages.

  4. The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies. (United States)

    Sauter, M; Hager, A


    A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.

  5. Sinorhizobium meliloti-induced chitinase gene expression in Medicago truncatula ecotype R108-1: a comparison between symbiosis-specific class V and defence-related class IV chitinases. (United States)

    Salzer, Peter; Feddermann, Nadja; Wiemken, Andres; Boller, Thomas; Staehelin, Christian


    The Medicago truncatula (Gaertn.) ecotypes Jemalong A17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression. The pathogen-inducible class IV chitinase gene, Mtchit 4, was strongly induced during nodule formation of the ecotype Jemalong A17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit 4 expression. On the other hand, expression of the putative class V chitinase gene, Mtchit 5, was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit 5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit 5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit 4, expression of Mtchit 5 was stimulated by purified Nod factors. Conversely, Mtchit 4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit 5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit 5 (calculated molecular weight = 41,810 Da, isoelectric point pH 7.7) and Mtchit 4 (calculated molecular weight 30,527 Da, isoelectric point pH 4.9) revealed that the putative Mtchit 5 chitinase forms a separate clade within class V chitinases of plants, whereas the Mtchit 4 chitinase clusters with pathogen-induced class IV chitinases from other plants. These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit 5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and K antigens.


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    Dominika Ďurechová


    Full Text Available Round-leaf sundew (Drosera rotundifolia L. from Droseraceae family belongs among a few plant species with strong antifungal potential. It was previously shown that chitinases of carnivorous plant species may play role during the insect prey digestion, when hard chitin skeleton is being decomposed. As many phytopathogenic fungi contain chitin in their cell wall our attention in this work was focused on isolation and in silico characterization of genomic DNA sequence of sundew chitinase gene. Subsequently this gene was fused to strong constitutive CaMV35S promoter and cloned into the plant binary vector pBinPlus and tested in A. tumefaciens LBA 4404 for its stability. Next, when transgenic tobacco plants are obtained, increasing of their antifungal potential will be tested.

  7. Characterization of a novel chitinase, DkChi, from Dendrolimus kikuchii nucleopolyhedrovirus. (United States)

    Wang, Qinghua; Qu, Liangjian; Zhang, Zhilin; Wang, Yuzhu; Zhang, Yongan


    Dendrolimus kikuchii Matsumura nucleopolyhedrovirus (DkNPV) is a novel nucleopolyhedrovirus strain that has exhibited high potential as biological control agent against D. kikuchii. In this work, a 1755-bp DkChi gene with sequence homology to a chitinase gene was cloned from the genomic DNA of DkNPV using a DNA fragment library. The DkChi gene, encoding 558 residues protein with a predicted mass of 61.6 kDa, was expressed at high levels in Escherichia coli and purified by affinity chromatography. We confirmed that the prepared protein was the DkChi protein by mass spectrometry analysis. Enzyme activity analysis showed that DkChi had both endo- and exo-chitinase activities. Interestingly, the DkChi protein displayed a strong insecticidal activity against Spodoptera exigua, Hyphantria cunea, Helicoverpa armigera and Lymantria dispar. The results suggest that DkChi is a good candidate protein for significantly contributing to pest control.

  8. Chitinase activities, scab resistance, mycorrhization rates and biomass of own-rooted and grafted transgenic apple

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    Tina Schäfer


    Full Text Available This study investigated the impact of constitutively expressed Trichoderma atroviride genes encoding exochitinase nag70 or endochitinase ech42 in transgenic lines of the apple cultivar Pinova on the symbiosis with arbuscular mycorrhizal fungi (AMF. We compared the exo- and endochitinase activities of leaves and roots from non-transgenic Pinova and the transgenic lines T386 and T389. Local and systemic effects were examined using own-rooted trees and trees grafted onto rootstock M9. Scab susceptibility was also assessed in own-rooted and grafted trees. AMF root colonization was assessed microscopically in the roots of apple trees cultivated in pots with artificial substrate and inoculated with the AMF Glomus intraradices and Glomus mosseae. Own-rooted transgenic lines had significantly higher chitinase activities in their leaves and roots compared to non-transgenic Pinova. Both of the own-rooted transgenic lines showed significantly fewer symptoms of scab infection as well as significantly lower root colonization by AMF. Biomass production was significantly reduced in both own-rooted transgenic lines. Rootstock M9 influenced chitinase activities in the leaves of grafted scions. When grafted onto M9, the leaf chitinase activities of non-transgenic Pinova (M9/Pinova and transgenic lines (M9/T386 and M9/T389 were not as different as when grown on their own roots. M9/T386 and M9/T389 were only temporarily less infected by scab than M9/Pinova. M9/T386 and M9/T389 did not differ significantly from M9/Pinova in their root chitinase activities, AMF root colonization and biomass.

  9. Chitinase expression in Listeria monocytogenes is positively regulated by the Agr system.

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    Dafni Katerina Paspaliari

    Full Text Available The food-borne pathogen Listeria monocytogenes encodes two chitinases, ChiA and ChiB, which allow the bacterium to hydrolyze chitin, the second most abundant polysaccharide in nature. Intriguingly, despite the absence of chitin in human and mammalian hosts, both of the chitinases have been deemed important for infection, through a mechanism that, at least in the case of ChiA, involves modulation of host immune responses. In this study, we show that the expression of the two chitinases is subject to regulation by the listerial agr system, a homologue of the agr quorum-sensing system of Staphylococcus aureus, that has so far been implicated in virulence and biofilm formation. We demonstrate that in addition to these roles, the listerial agr system is required for efficient chitin hydrolysis, as deletion of agrD, encoding the putative precursor of the agr autoinducer, dramatically decreased chitinolytic activity on agar plates. Agr was specifically induced in response to chitin addition in stationary phase and agrD was found to regulate the amount of chiA, but not chiB, transcripts. Although the transcript levels of chiB did not depend on agrD, the extracellular protein levels of both chitinases were reduced in the ΔagrD mutant. The regulatory effect of agr on chiA is potentially mediated through the small RNA LhrA, which we show here to be negatively regulated by agr. LhrA is in turn known to repress chiA translation by binding to the chiA transcript and interfering with ribosome recruitment. Our results highlight a previously unrecognized role of the agr system and suggest that autoinducer-based regulation of chitinolytic systems may be more commonplace than previously thought.

  10. Effect of Chitinase-Producing Strain V-8 on 3ontrolling Cotton Fusarium Wilt

    Institute of Scientific and Technical Information of China (English)


    [Objective] This study aimed to screen endophytic bacteria which is antag- onistic to cotton Fusarium wilt. [Method] Fresh cotton plants collected from cotton- growing areas in Jingzhou City, Hubei Province were used as experimental materials to isolate endophytic bacteria. Through chitinase test and co-culturing both micro-or- ganisms side by side on the same PDA culture plate, antagonistic strains to cotton Fusarium wilt were screened. [Result] A total of 83 bacterial isolates were obtained from cotton plants grown in the fields, six of which were chitinase-productive bacte- ria. Through chitinase test and co-culturing both micro-organisms side by side on the same PDA culture plate, strain V-8 which had the strongest antagonistic effect on Fusarium oxysporum f. sp. vasinfectum was screened. Strain V-8 had a wider anti- fungal spectrum with certain inhibitory effect on all the six important pathogenic fungi including Fusarium oxysporum f. sp niveum; it colonized stably in the rhizospheric soil of cotton, with a colonization density of up to 6.2x10s cfu/g fifty days after inoc- ulation; the relative effect on controlling cotton Fusarium wilt in pot test was 73.2%. The Findings of this study suggested that strain V-8 had great potential for biological control of cotton Fusarium wilt and could be taken as a substantial material for the cloning of chitinase genes. [Conclusion] The results from this study provides bases for the control of cotton fusarium wilt, as well as the exploitation of endophytic bac- teria resources in cotton and the development of novel biological pesticides.

  11. Isolation and Purifi cation of Chitinase Bacillus sp. D2 Isolated from Potato Rhizosfer

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    Sebastian Margino


    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Potato Cyst Nematodes (Globodera rostochiensis is one of the important potato’s pests and caused economic looses up to 70% in the several centrals of potato plantations in Indonesia. Potato Cyst Nematodes (PCN shell component of egg shell containing chitin (inner layer and vitelline/protein (outer layer, so the purpose of research was to fi nd out of chitin degrading bacteria for controlling of egg’s PCN by cutting of their life cycle. The results showed that Bacillus sp. D2 isolated from potato rhizosphere could produce extra cellular chitinase in the medium containing of 0.20% colloidal chitin and fermented for 72 hours. Result of chitinase purifi cation using ammonium sulphate precipitation and DEAE-Cellulose ion-exchange chromatography showed a specifi c activity 2691,052 U/mg and analyzing using SDS-PAGE 12.5% resulted in molecular weight 30 kDa. The apparent Km and Vmax of chitinase towards colloidal chitin were 2 mg/ml and 2.2 μg/h, respectively.  

  12. Purification and characterization of an antifungal chitinase from Bacillus sp.SL-13

    Institute of Scientific and Technical Information of China (English)

    Chen; Shan


    Bacillus sp.SL-13 produced antifungal proteins.The growth of the plant-pathogenic fungi Rhizoctonia solani was considerably inhibited by the presence of the SL-13 culture supernatant.The proteins were purified by DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration,and the main antifungal protein was purified to be chitinase.The molecular weight of chitinase was estimated to be 36 kD by 12%SDS PAGE.The optimal pH and temperature for the chitinase was 7.0 and 50℃.It demonstrated that the enzyme was stable from pH 5 to 9 and form 40?C to 60℃.The enzyme still kept 70%activity when incubated at 121℃,0.11MPa up to 20 minutes and the enzyme is also not lost the activity when treated with protease K and ultraviolet radiation for 1.5hours.It is very suitable for the use in a relatively unstable environment,exhibiting effective biological control.

  13. Go Fly a Chitin: The Mystery of Chitin and Chitinases in Vertebrate Tissues. (United States)

    Stern, Robert


    A controversy arose decades ago whether the DG42 gene product expressed during frog embryogenesis synthesized hyaluronan or chitin. Both sets of investigators were correct. It is now possible to understand how prescient those findings were. Synthesis of a seven to nine chitin sugar chain fragment is required before hyaluronan synthesis begins. Thus, DG42 indeed synthesizes both hyaluronan and chitin. Hyaluronan turns over rapidly in vertebrate tissues, but chitin oligomers are difficult to degrade. They accumulate and can cause pathology. Chitin is a simple beta-linked repeating sugar homopolymer found prominently in the building block structures of fungi, molluscs, arthropods, and other forms of invertebrate life. It is a highly resistant insoluble material requiring chitin synthases for production and chitinases for degradation. Mysteriously, chitins and chitinases also occur in vertebrate tissues, while it had previously been assumed that no chitins were contained therein. That assumption is now challenged based on recent biochemical evidence. Chitin does accumulate in many tissues, but may be particularly toxic to neurons. Its accumulation in the brain may account for the cognitive decline found in patients with Alzheimer's disease. The DG42 observations together with the participation of chitins and chitinases in several human diseases, among which in addition to Alzheimer's disease include Gaucher's disease, asthma, and aspects of abnormal immune recognition justify a reexamination of these topics. The purpose of this review is to summarize data in order to place chitins and their attendant enzymes in a rational framework in an attempt to create a cohesive story.

  14. Facility target insert shielding assessment

    Energy Technology Data Exchange (ETDEWEB)

    Mocko, Michal [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)


    Main objective of this report is to assess the basic shielding requirements for the vertical target insert and retrieval port. We used the baseline design for the vertical target insert in our calculations. The insert sits in the 12”-diameter cylindrical shaft extending from the service alley in the top floor of the facility all the way down to the target location. The target retrieval mechanism is a long rod with the target assembly attached and running the entire length of the vertical shaft. The insert also houses the helium cooling supply and return lines each with 2” diameter. In the present study we focused on calculating the neutron and photon dose rate fields on top of the target insert/retrieval mechanism in the service alley. Additionally, we studied a few prototypical configurations of the shielding layers in the vertical insert as well as on the top.

  15. Impedance calculation for ferrite inserts

    Energy Technology Data Exchange (ETDEWEB)

    Breitzmann, S.C.; Lee, S.Y.; /Indiana U.; Ng, K.Y.; /Fermilab


    Passive ferrite inserts were used to compensate the space charge impedance in high intensity space charge dominated accelerators. They study the narrowband longitudinal impedance of these ferrite inserts. they find that the shunt impedance and the quality factor for ferrite inserts are inversely proportional to the imaginary part of the permeability of ferrite materials. They also provide a recipe for attaining a truly passive space charge impedance compensation and avoiding narrowband microwave instabilities.

  16. A plant class V chitinase from a cycad (Cycas revoluta): biochemical characterization, cDNA isolation, and posttranslational modification. (United States)

    Taira, Toki; Hayashi, Hiroko; Tajiri, Yoshiko; Onaga, Shoko; Uechi, Gen-ichiro; Iwasaki, Hironori; Ohnuma, Takayuki; Fukamizo, Tamo


    Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

  17. Development of insect resistant maize plants expressing a chitinase gene from the cotton leaf worm, Spodoptera littoralis. (United States)

    Osman, Gamal H; Assem, Shireen K; Alreedy, Rasha M; El-Ghareeb, Doaa K; Basry, Mahmoud A; Rastogi, Anshu; Kalaji, Hazem M


    Due to the importance of chitinolytic enzymes for insect, nematode and fungal growth, they are receiving attention concerning their development as biopesticides or chemical defense proteins in transgenic plants and as microbial biocontrol agents. Targeting chitin associated with the extracellular matrices or cell wall by insect chitinases may be an effective approach for controlling pest insects and pathogenic fungi. The ability of chitinases to attack and digest chitin in the peritrophic matrix or exoskeleton raises the possibility to use them as insect control method. In this study, an insect chitinase cDNA from cotton leaf worm (Spodoptera littoralis) has been synthesized. Transgenic maize plant system was used to improve its tolerance against insects. Insect chitinase transcripts and proteins were expressed in transgenic maize plants. The functional integrity and expression of chitinase in progenies of the transgenic plants were confirmed by insect bioassays. The bioassays using transgenic corn plants against corn borer (Sesamia cretica) revealed that ~50% of the insects reared on transgenic corn plants died, suggesting that transgenic maize plants have enhanced resistance against S. cretica.

  18. Domains and domain loss

    DEFF Research Database (Denmark)

    Haberland, Hartmut


    The domain concept, originally suggested by Schmidt-Rohr in the 1930’s (as credited in Fishman’s writings in the 1970s), was an attempt to sort out different areas of language use in multilingual societies, which are relevant for language choice. In Fishman’s version, domains were considered...... not described in terms of domains, and recent research e.g. about the multilingual communities in the Danish-German border area seems to confirm this....

  19. Purification and characterization of three chitinases and one beta-1,3-glucanase accumulating in the medium of cell suspension cultures of barley (Hordeum vulgare L.)

    DEFF Research Database (Denmark)

    Kragh, K.M.; Jacobsen, S.; Dalgaard Mikkelsen, J.;


    chromatography. Two of the chitinases were identified as the previously described endochitinases T and C from barley grain. The third and novel chitinase, designated K, was the major basic chitinase in the medium constituting 4% of the soluble protein. Chitinase K was found to be a 33-kDa endochitinase with p...... chitinases from barley aleurone and barley, bean and potato leaves. The purified beta-1,3-glucanase with a molecular weight (MW) of 32 kDa and pI greater-than-or-equal-to 9.8 constituted 1% of the soluble protein in the medium. Based on similar MW, pI and amino acid composition as well as identical N...

  20. Agrobacterium tumefaciens-mediated transformation of taro (Colocasia esculenta (L.) Schott) with a rice chitinase gene for improved tolerance to a fungal pathogen Sclerotium rolfsii. (United States)

    He, Xiaoling; Miyasaka, Susan C; Fitch, Maureen M M; Moore, Paul H; Zhu, Yun J


    Taro (Colocasia esculenta) is one of the most important crops in the Pacific Islands, however, taro yields have been declining in Hawaii over the past 30 years partly due to diseases caused by oomycete and fungal pathogens. In this study, an efficient Agrobacterium tumefaciens-mediated transformation method for taro is first reported. In total, approximately 200 pieces (8 g) of embryogenic calluses were infected with the super-virulent A. tumefaciens strain EHA105 harboring the plant transformation plasmid pBI121/ricchi11 that contains the rice chitinase gene ricchi11. The presence and expression of the transgene ricchi11 in six independent transgenic lines was confirmed using polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). Southern blot analysis of the six independent lines indicated that three out of six (50%) had integrated a single copy of the transgene, and the other three lines had two or three copies of the transgene. Compared to the particle bombardment transformation of taro method, which was used in the previous studies, the Agrobacterium-mediated transformation method obtained 43-fold higher transformation efficiency. In addition, these six transgenic lines via Agrobacterium may be more effective for transgene expression as a result of single-copy or low-copy insertion of the transgene than the single line with multiple copies of the transgene via particle bombardment. In a laboratory bioassay, all six transgenic lines exhibited increased tolerance to the fungal pathogen Sclerotium rolfsii, ranging from 42 to 63% reduction in lesion expansion.

  1. Optimization of nutrition factors on chitinase production from a newly isolated Chitiolyticbacter meiyuanensis SYBC-H1

    Directory of Open Access Journals (Sweden)

    Zhikui Hao


    Full Text Available The present study reports statistical medial optimization for chitinase production by a novel bacterial strain isolated from soil recently, which the name Chitinolyticbacter meiyuanensis SYBC-H1 is proposed. A sequential statistical methodology comprising of Plackett-Burman and response surface methodology (RSM was applied to enhance the fermentative production of chitinase, in which inulin was firstly used as an effective carbon source. As a result, maximum chitinase activity of 5.17 U/mL was obtained in the optimized medium, which was 15.5-fold higher than that in the basal medium. The triplicate verification experiments were performed under the optimized nutrients levels which indicated that it well agreed with the predicted value.

  2. Cloning of a chitinase gene from Ewingella americana, a pathogen of the cultivated mushroom, Agaricus bisporus

    Directory of Open Access Journals (Sweden)

    P.W. Inglis


    Full Text Available We have isolated a gene encoding a chitinase (EC from Ewingella americana, a recently described pathogen of the mushroom Agaricus bisporus. This gene, designated chiA (EMBL/Genbank/DDBJ accession number X90562, was cloned by expression screening of a plasmid-based E. americana HindIII genomic library in Escherichia coli using remazol brilliant violet-stained carboxymethylated chitin incorporated into selective medium. The chiA gene has a 918-bp ORF, terminated by a TAA codon, with a calculated polypeptide size of 33.2 kDa, likely corresponding to a previously purified and characterised 33-kDa endochitinase from E. americana. The deduced amino acid sequence shares 33% identity with chitinase II from Aeromonas sp. No. 10S-24 and 7.8% identity with a chitinase from Saccharopolyspora erythraeus. Homology to other chitinase sequences was otherwise low. The peptide sequence deduced from chiA lacks a typical N-terminal signal sequence and also lacks the chitin binding and type III fibronectin homology units common to many bacterial chitinases. The possibility that this chitinase is not primarily adapted for the environmental mineralisation of pre-formed chitin, but rather for the breakdown of nascent chitin, is discussed in the context of mushroom disease.O gene que codifica uma quitinase (EC foi isolado de Ewingella americana, recentemente descrita como patógeno do cogumelo Agaricus bisporus. Este gene, denominado chiA (EMBL/Genebank/DDBJ número de acesso X9061, foi clonado e selecionado a partir de livraria genômica construída por digestão do DNA de E. americana com HindIII e ligação em plasmídio de expressão em E. coli, utilizando meio seletivo contendo quitina carboximetilada, corada com "remazol brilliant violet'' para seleção de clones. O gene chiA apresenta uma ORF de 918 bp, código terminador TAA, tendo o tamanho do polipeptídeo sido calculado como 33,2 kDa, o qual corresponde ao tamanho de 33 kDa da endoquitinase

  3. Induced production of chitinase to enhance entomotoxicity of Bacillus thuringiensis employing starch industry wastewater as a substrate. (United States)

    Vu, Khanh Dang; Yan, S; Tyagi, R D; Valéro, J R; Surampalli, R Y


    Induced production of chitinase during bioconversion of starch industry wastewater (SIW) to Bacillus thuringiensis var. kurstaki HD-1 (Btk) based biopesticides was studied in shake flask as well as in computer-controlled fermentors. SIW was fortified with different concentrations (0%; 0.05%; 0.1%; 0.2%; 0.3% w/v) of colloidal chitin and its consequences were ascertained in terms of Btk growth (total cell count and viable spore count), chitinase, protease and amylase activities and entomotoxicity. At optimum concentration of 0.2% w/v colloidal chitin, the entomotoxicity of fermented broth and suspended pellet was enhanced from 12.4x10(9) (without chitin) to 14.4x10(9) SBU/L and from 18.2x10(9) (without chitin) to 25.1x10(9) SBU/L, respectively. Further, experiments were conducted for Btk growth in a computer-controlled 15 L bioreactor using SIW as a raw material with (0.2% w/v chitin, to induce chitinase) and without fortification of colloidal chitin. It was found that the total cell count, spore count, delta-endotoxin concentration (alkaline solubilised insecticidal crystal proteins), amylase and protease activities were reduced whereas the entomotoxicity and chitinase activity was increased with chitin fortification. The chitinase activity attained a maximum value at 24 h (15 mU/ml) and entomotoxicity of suspended pellet reached highest (26.7x10(9) SBU/L) at 36 h of fermentation with chitin supplementation of SIW. In control (without chitin), the highest value of entomotoxicity of suspended pellet (20.5x10(9) SBU/L) reached at 48 h of fermentation. A quantitative synergistic action of delta-endotoxin concentration, spore concentration and chitinase activity on the entomotoxicity against spruce budworm larvae was observed.

  4. Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression. (United States)

    Jayaraj, Jayaraman; Muthukrishnan, Subbaratnam; Liang, George H


    Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35-40 x 10(-6). The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.

  5. G protein signalling involved in host recognition and mycoparasitismrelated chitinase expression in Trichoderma atroviride

    Institute of Scientific and Technical Information of China (English)

    Susanne Zeilinger; Barbara Reithner; Kurt Brunner; Valeria Scala; Isabel Peiβl; Matteo Lorito; Robert L Mach


    @@ Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition,attack and subsequent penetration and killing of the host. Investigations on the underlying events revealed that Trichoderma responds to multiple signals from the host (e. g. lectins or other ligands such as low molecular weight components released from the host's cell wall) and host attack is accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics.Degradation of the cell wall of the host fungus is-besides glucanases and proteases-mainly achieved by chitinases. In vivo studies showed that the ech42 gene (encoding endochitinase 42) is expressed before physical contact of Trichoderma with its host, probably representing one of the earliest events in mycoparasitism, whereas Nag1 (N-acetylglucosaminidase) plays a key role in the general induction of the chitinolytic enzyme system of T. atroviride . Investigations on the responsible signal transduction pathways of T. atroviride led to the isolation of several genes encoding key components of the cAMP and MAP kinase signaling pathways, as alpha and β subunits of heterotrimeric G proteins, the regulatory subunit of cAMP-dependent protein kinase,adenylate cyclase, and three MAP kinases. Analysis of knockout mutants, generated by Agrobacterium-mediated transformation, revealed that at least two alpha-subunits of heterotrimeric G proteins are participating in mycoparasitism-related signal transduction. The Tga1 G alpha subunit was shown to be involved in mycoparasitism-related processes such as chitinase expression and overproduction of toxic secondary metabolites, whereas Tga3 was found to be completely avirulent showing defects in chitinase formation and host recognition.

  6. A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis

    DEFF Research Database (Denmark)

    Ohnuma, Takayuki; Numata, Tomoyuki; Osawa, Takuo;


    Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA......) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end...

  7. Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons. (United States)

    Allona, I; Collada, C; Casado, R; Paz-Ares, J; Aragoncillo, C


    Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.

  8. PCR-RFLP analysis of chitinase genes enables efficient genotyping of Metarhizium anisopliae var. anisopliae. (United States)

    Enkerli, Jürg; Ghormade, Vandana; Oulevey, Catherine; Widmer, Franco


    A new genotyping tool has been developed and evaluated for Metarhizium anisopliae var. anisopliae. The tool is based on Restriction Fragment Length Polymorphism (RFLP) analysis of three chitinase genes that are functionally linked to insect-pathogenicity of this fungus. It allowed for discrimination of 14 genotypes among 22 M. anisopliae var. anisopliae strains of a world wide collection. Analyses revealed that the approach may also be applicable to other Metarhizium varieties. The new tool will be useful for genetic characterization of M. anisopliae var. anisopliae strains, and it is applicable for laboratories with limited access to molecular diagnostic equipment.

  9. Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12

    Directory of Open Access Journals (Sweden)

    Ramli Aizi


    Full Text Available Abstract Background Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food. Results A gene encoding a cold-adapted chitinase (CHI II from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low

  10. Gas turbine vane cooling air insert

    Energy Technology Data Exchange (ETDEWEB)

    North, W.E.; Hultgren, K.G.; Dishman, C.D.; Van Heusden, G.S.


    This patent describes a gas turbine. It comprises turbine vanes, each of the vanes supplied with cooling air and having: an airfoil portion forming a first cavity having an insert disposed therein for directing the flow of the cooling air, the insert having first and second insert ends; a shroud portion from which the airfoil portion extends, the insert attached to the shroud portion at the first insert end; an insert extension extending through a portion of the insert and extending beyond the first insert end, the insert extension and the insert forming an annular gap therebetween separating the insert from the insert extension; a plate covering at least a portion of the shroud, the plate having a first hole formed therein through which the insert extension extends; and at least a first seal extending between the insert extension and the insert, and sealing the annular gap therebetween. This patent also describes a method of making a gas turbine. It comprises welding a first tubular insert adjacent its first end to a vane outer shroud; partially inserting a second tubular insert into the first tubular member and attaching the second tubular insert thereto; placing a plate having a hole formed therein on the outer shroud so that the hole surrounds the second tubular insert; and attaching the second tubular insert to the plate by placing a first seal between the first and second tubular inserts and attaching the first seal to each of the first and second tubular inserts, and placing a second seal between the second tubular insert and the plate and welding the second seal to the second tubular insert and the plate.

  11. Purification and characterisation of an acidic and antifungal chitinase produced by a Streptomyces sp. (United States)

    Karthik, Narayanan; Binod, Parameswaran; Pandey, Ashok


    An extremely acidic extracellular chitinase produced by a Streptomyces sp. was purified 12.44-fold by ammonium sulphate precipitation, ion-exchange chromatography and gel-permeation chromatography and further characterised. The molecular mass of the enzyme was estimated to be about 40 kDa by SDS-PAGE. The optimum pH and temperature of the purified enzyme were pH 2 and 6, and 50 °C respectively. The enzyme showed high stability in the acidic pH range of 2-6 and temperature stability of up to 50 °C. Additionally, the effect of some cations and other chemical compounds on the chitinase activity was studied. The activity of the enzyme was considerably retained under salinity conditions of up to 3%. The Km and Vmax values of the enzyme were determined to be 6.74 mg mL(-1) and 61.3 U mg(-1) respectively using colloidal chitin. This enzyme exhibited antifungal activity against phytopathogens revealing a potential biocontrol application in agriculture.

  12. Thermostable chitinase from Cohnella sp. A01: isolation and product optimization. (United States)

    Aliabadi, Nasrin; Aminzadeh, Saeed; Karkhane, Ali Asghar; Haghbeen, Kamahldin

    Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70°C, with Km and Vmax values of chitinase to be 5.6mg/mL and 0.87μmol/min, respectively. Ag(+), Co(2+), iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn(2+), Cu(2+), Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

  13. Chitinase 3-like 1 Regulates Cellular and Tissue Responses via IL-13 Receptor α2

    Directory of Open Access Journals (Sweden)

    Chuan Hua He


    Full Text Available Members of the 18 glycosyl hydrolase (GH 18 gene family have been conserved over species and time and are dysregulated in inflammatory, infectious, remodeling, and neoplastic disorders. This is particularly striking for the prototypic chitinase-like protein chitinase 3-like 1 (Chi3l1, which plays a critical role in antipathogen responses where it augments bacterial killing while stimulating disease tolerance by controlling cell death, inflammation, and remodeling. However, receptors that mediate the effects of GH 18 moieties have not been defined. Here, we demonstrate that Chi3l1 binds to interleukin-13 receptor α2 (IL-13Rα2 and that Chi3l1, IL-13Rα2, and IL-13 are in a multimeric complex. We also demonstrate that Chi3l1 activates macrophage mitogen-activated protein kinase, protein kinase B/AKT, and Wnt/β-catenin signaling and regulates oxidant injury, apoptosis, pyroptosis, inflammasome activation, antibacterial responses, melanoma metastasis, and TGF-β1 production via IL-13Rα2-dependent mechanisms. Thus, IL-13Rα2 is a GH 18 receptor that plays a critical role in Chi3l1 effector responses.

  14. Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linné) and in vitro. (United States)

    Saguez, Julien; Hainez, Romaric; Cherqui, Anas; Van Wuytswinkel, Olivier; Jeanpierre, Haude; Lebon, Gaël; Noiraud, Nathalie; Beaujean, Antony; Jouanin, Lise; Laberche, Jean-Claude; Vincent, Charles; Giordanengo, Philippe


    With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid population's growth. Artificial diets were used to provide active (1, 10, 100 and 500 microg ml(-1)) and inactive (500 microg ml(-1)) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day population's doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.

  15. Oat (Avena sativa) seed extract as an antifungal food preservative through the catalytic activity of a highly abundant class I chitinase. (United States)

    Sørensen, Hans Peter; Madsen, Lone Søvad; Petersen, Jørgen; Andersen, Jesper Tapdrup; Hansen, Anne Maria; Beck, Hans Christian


    Extracts from different higher plants were screened for the ability to inhibit the growth of Penicillium roqueforti, a major contaminating species in industrial food processing. Oat (Avena sativa) seed extracts exhibited a high degree of antifungal activity and could be used directly on rye bread to prevent the formation of P. roqueforti colonies. Proteins in the oat seed extracts were fractionated by column chromatography and proteins in fractions containing antifungal activity were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searches. Identified antifungal candidates included thaumatin-like proteins, 1,3-beta-glucanase, permatin precursor, pathogenesis-related protein type 1, and chitinases of class I and II. Class I chitinase could be specifically removed from the extracts and was found to be indispensable for 50% of the P. roqueforti inhibiting activity. The purified class I chitinase has a molecular weight of approximately 34 kDa, optimal chitinase activity at pH 7, and exists as at least two basic isoforms (pI values of 7.6 and 8.0). Partial sequencing of the class I chitinase isoforms by LC-MS/MS revealed a primary structure with high similarity to class I chitinases of wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale). Oat, wheat, barley, and rye seed extracts were compared with respect to the abundance of the class I chitinase and decrease in antifungal activity when class I chitinase is removed. We found that the oat seed class I chitinase is at least ten times more abundant than the wheat, barley, and rye homologs and that oat seed extracts are highly active toward P. roqueforti as opposed to extracts of other cereal seeds.

  16. Turnabout Is Fair Play: Herbivory-Induced Plant Chitinases Excreted in Fall Armyworm Frass Suppress Herbivore Defenses in Maize. (United States)

    Ray, Swayamjit; Alves, Patrick C M S; Ahmad, Imtiaz; Gaffoor, Iffa; Acevedo, Flor E; Peiffer, Michelle; Jin, Shan; Han, Yang; Shakeel, Samina; Felton, Gary W; Luthe, Dawn S


    The perception of herbivory by plants is known to be triggered by the deposition of insect-derived factors such as saliva and oral secretions, oviposition materials, and even feces. Such insect-derived materials harbor chemical cues that may elicit herbivore and/or pathogen-induced defenses in plants. Several insect-derived molecules that trigger herbivore-induced defenses in plants are known; however, insect-derived molecules suppressing them are largely unknown. In this study, we identified two plant chitinases from fall armyworm (Spodoptera frugiperda) larval frass that suppress herbivore defenses while simultaneously inducing pathogen defenses in maize (Zea mays). Fall armyworm larvae feed in enclosed whorls of maize plants, where frass accumulates over extended periods of time in close proximity to damaged leaf tissue. Our study shows that maize chitinases, Pr4 and Endochitinase A, are induced during herbivory and subsequently deposited on the host with the feces. These plant chitinases mediate the suppression of herbivore-induced defenses, thereby increasing the performance of the insect on the host. Pr4 and Endochitinase A also trigger the antagonistic pathogen defense pathway in maize and suppress fungal pathogen growth on maize leaves. Frass-induced suppression of herbivore defenses by deposition of the plant-derived chitinases Pr4 and Endochitinase A is a unique way an insect can co-opt the plant's defense proteins for its own benefit. It is also a phenomenon unlike the induction of herbivore defenses by insect oral secretions in most host-herbivore systems.

  17. Expression of a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic potato plants

    NARCIS (Netherlands)

    Moravcikova, J.; Matusikova, I.; Libantova, J.; Bauer, M.; Mlynarova, L.


    The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) breeding line 116/86 using Agrobacterium tumefaciens. For both transgenes the number of integrated copies and level of RNA expression wer

  18. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ


    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus whi


    NARCIS (Netherlands)



    Background: Hevamine is a member of one of several families of plant chitinases and lysozymes that are important for plant defence against pathogenic bacteria and fungi. The enzyme can hydrolyze the linear polysaccharide chains of chitin and peptidoglycan. A full understanding of the structure/funct

  20. Production of β-1,3-glucanase and chitinase of two biocon trol agents and their possible modes of action

    Institute of Scientific and Technical Information of China (English)


    Pichia membranefaciens Hansen and Candida guilliermondii(Cast) Langeronet Guerra are two antagonists of R. stolonifer on harvested nectarine and peach fruits. In this study, β-1,3-glucanase and chitinase activities of the antagonists were induced in vitro and in vivo. The highest β-1,3-glucanase activity was detected in Lilly-Barnett minimal salt medium supplemented with glucose in combination with CWP of R. stolonifer as a carbon source. The β-1,3-glucanase activity of P. membranefaciens reached the maximum level,being 114.0 SU (specific activity unit), and that of C. guilliermondii reached 103.2 SU. The lowest β-1,3-glucanase activity was observed in the medium containing glucose as sole carbon source. P. membranefaciens was able to produce significantly higher levels of chitinase (exochitinase and endochitinase) in vitro than C. guilliermondii grown in Czapeck minimal medium. An increase in β-1,3-glucanase and chitinase activity was also triggered by wounding, adding of carbon sources and yeast cells. The results showed that both β-1,3-glucanase and chitinase from P. membranefaciens and C. guilliermondii exhibited some effects on controlling R.stolonifer, and might have a synergistic activity against R.stolonifer.

  1. Stabilization of insertion electrodes for lithium batteries.

    Energy Technology Data Exchange (ETDEWEB)

    Thackeray, M. M.


    This paper discusses the techniques that are being employed to stabilize LiMn{sub 2}O{sub 4} spinel and composite Li{sub x}MnO{sub 2} positive electrodes. The critical role that spinel domains play in stabilizing these electrodes for operation at both 4 V and 3 V is highlighted. The concept of using an intermetallic electrode MM{prime} where M is an active alloying element and M{prime} is an inactive element (or elements) is proposed as an alternative negative electrode (to carbon) for lithium-ion cells. An analogy to metal oxide insertion electrodes, such as MnO{sub 2}, in which Mn is the electrochemically active ion and O is the inactive ion, is made. Performance data are given for the copper-tin electrode system, which includes the intermetallic phases eta-Cu{sub 6}Sn{sub 5} and Li{sub 2}CuSn.

  2. Gene Insertion Patterns and Sites (United States)

    Vain, Philippe; Thole, Vera

    During the past 25 years, the molecular analysis of transgene insertion patterns and sites in plants has greatly contributed to our understanding of the mechanisms underlying transgene integration, expression, and stability in the nuclear genome. Molecular characterization is also an essential step in the safety assessment of genetically modified crops. This chapter describes the standard experimental procedures used to analyze transgene insertion patterns and loci in cereals and grasses transformed using Agrobacterium tumefaciens or direct transfer of DNA. Methods and protocols enabling the determination of the number and configuration of transgenic loci via a combination of inheritance studies, polymerase chain reaction, and Southern analyses are presented. The complete characterization of transgenic inserts in plants is, however, a holistic process relying on a wide variety of experimental approaches. In this chapter, these additional approaches are not detailed but references to relevant bibliographic records are provided.

  3. An insertion algorithm for catabolizability

    CERN Document Server

    Blasiak, Jonah


    Motivated by our recent work relating canonical bases to combinatorics of Garsia-Procesi modules \\cite{B}, we give an insertion algorithm that computes the catabolizability of the insertion tableau of a standard word. This allows us to characterize catabolizability as the statistic on words invariant under Knuth transformations, certain (co)rotations, and a new operation called a catabolism transformation. We also prove a Greene's Theorem-like characterization of catabolizability, and a result about how cocyclage changes catabolizability, strengthening a similar result in \\cite{SW}.

  4. Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial. (United States)

    Pasonen, Hanna-Leena; Lu, Jinrong; Niskanen, Anna-Maija; Seppänen, Sanna-Kaisa; Rytkönen, Anna; Raunio, Janne; Pappinen, Ari; Kasanen, Risto; Timonen, Sari


    Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.

  5. Concepts for stereoselective acrylate insertion

    KAUST Repository

    Neuwald, Boris


    Various phosphinesulfonato ligands and the corresponding palladium complexes [{((PaO)PdMeCl)-μ-M}n] ([{( X1-Cl)-μ-M}n], (PaO) = κ2- P,O-Ar2PC6H4SO2O) with symmetric (Ar = 2-MeOC6H4, 2-CF3C6H4, 2,6-(MeO)2C6H3, 2,6-(iPrO)2C 6H3, 2-(2′,6′-(MeO)2C 6H3)C6H4) and asymmetric substituted phosphorus atoms (Ar1 = 2,6-(MeO)2C6H 3, Ar2 = 2′-(2,6-(MeO)2C 6H3)C6H4; Ar1 = 2,6-(MeO)2C6H3, Ar2 = 2-cHexOC 6H4) were synthesized. Analyses of molecular motions and dynamics by variable temperature NMR studies and line shape analysis were performed for the free ligands and the complexes. The highest barriers of ΔGa = 44-64 kJ/mol were assigned to an aryl rotation process, and the flexibility of the ligand framework was found to be a key obstacle to a more effective stereocontrol. An increase of steric bulk at the aryl substituents raises the motional barriers but diminishes insertion rates and regioselectivity. The stereoselectivity of the first and the second methyl acrylate (MA) insertion into the Pd-Me bond of in situ generated complexes X1 was investigated by NMR and DFT methods. The substitution pattern of the ligand clearly affects the first MA insertion, resulting in a stereoselectivity of up to 6:1 for complexes with an asymmetric substituted phosphorus. In the consecutive insertion, the stereoselectivity is diminished in all cases. DFT analysis of the corresponding insertion transition states revealed that a selectivity for the first insertion with asymmetric (P aO) complexes is diminished in the consecutive insertions due to uncooperatively working enantiomorphic and chain end stereocontrol. From these observations, further concepts are developed. © 2012 American Chemical Society.

  6. Conversion of α-chitin substrates with varying particle size and crystallinity reveals substrate preferences of the chitinases and lytic polysaccharide monooxygenase of Serratia marcescens. (United States)

    Nakagawa, Yuko S; Eijsink, Vincent G H; Totani, Kazuhide; Vaaje-Kolstad, Gustav


    Industrial depolymerization of chitinous biomass generally requires numerous steps and the use of deleterious substances. Enzymatic methods provide an alternative, but fundamental knowledge that could direct potential development of industrial enzyme cocktails is scarce. We have studied the contribution of monocomponent chitinases (ChiA, -B, and -C) and the lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens on depolymerization of α-chitin substrates with varying particle size and crystallinity that were generated using a converge mill. For all chitinases activity was positively correlated to a decline in particle size and crystallinity. Especially ChiC, the only nonprocessive endochitinase from the S. marcescens chitinolytic machinery, benefited from mechanical pretreatment. Combining the chitinases revealed clear synergies for all substrates tested. CBP21, the chitin-active LPMO from S. marcescens, increased solubilization of substrates with high degrees of crystallinity when combined with each of the three chitinases, but this synergy was reduced upon decline in crystallinity.

  7. 水稻和拟南芥中几丁质酶的分析%Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    许凤华; 范成明; 何月秋


    Chitinases (EC3.2.1.14), found in a wide range of organisms, catalyze the hydrolysis of chitin and play a major role in defense mechanisms against fungal pathogens. The alignment and typical domains were analyzed using basic local alignment search tool (BLAST) and simple modular architecture research tool (SMART), respectively. On the basis of the annotations of rice (Oryza sativa L.) and Arabidopsis genomic sequences and using the bio-software SignalP3.0, TMHMM2.0, TargetPl.1, and big-Pi Predictor, 25 out of 37 and 16 out of 24 open reading frames (ORFs) with chitinase activity from rice and Arabidopsis, respectively, were predicted to have signal peptides (SPs), which have an average of 24.8 amino acids at the N-terminal region. Some of the chitinases were secreted extracellularly, whereas some were located in the vacuole. The phylogenic relationship was analyzed with 61 ORFs and 25 known chitinases and they were classified into 6 clusters using Clustal X and MEGA3.1. This classification is not completely consistent when compared with the traditional system that classifies the chitinases into 7 classes. The frequency of distribution of amino acid residues was distinct in different clusters. The contents of alanine, glycine, serine, and leucine were very high in each cluster, whereas the contents of methionine, histidine, tryptophan, and cysteine were lower than 20%. Each cluster had distinct amino acid characteristics. Alanine, valine, leucine, cysteine, serine, and lysine were rich in Clusters Ⅰ to Ⅵ, respectively.%几丁质酶(EC3.2.1.14)是一种降解几丁质的糖苷酶,广泛存在于各种生物体中,并在植物中对病原真菌起重要抗性作用.首先通过BLAST在GenBank中对其同源性进行搜索,用SMART分析其结构.基于水稻和拟南芥的基因组注释,借助4个生物学软件(SignalP3.0,TMHMM2.0,TargetP1.1 and big-Pi Predictor),分析了水稻所有37条和拟南芥所有24条几丁质酶序列,发现有些几丁质酶都分

  8. Transformation Fava Beans by Agrobacterium using Chitinase, Glucanase and CryIA (b genes

    Directory of Open Access Journals (Sweden)

    A.H Gorji


    Full Text Available In this study two plasmid vectors that are appropriate for plant transformation were made by preparation of gene cassettes for β-1, 3-glucanase from barley, chitinase from bean and cryIA (b from Bacillus thuringiensis (BT. Each of these genes were cloned under the control of the CaMV35S  promoter and the Nos terminator in pBI121 binary vector. pBI-Chi  and pBI-Glu and recombinant plasmid vectors were constructed via cloning of chitinase , β-1,3- glucanase and cryIA (b genes, respectively, instead of the gus gene in T-DNA region of pBI121 vector. Construction of pBI-ChiGlu recombinant plasmid vector was performed by means of cloning both of the complete chitinase and glucanase gene cassettes in pBI121 vector, with the intention of production synergistic effects against fungal infection.pBI-ChiBt  recombinant plasmid vector containing both of the complete chitinase and Bt gene cassettes was also constructed in order to contemporaneous plants resistance to pest pathogens and fungal in a single transformation event. pBI-ChiGlu and pBI-ChiBt that have been  introduced into the A. tumefaciens strain LBA4404 that was subsequently used for  transformation. Results indicate that embryogenic calli are well appropriate as objective material for Agrobacterium tumefaciens-mediated transformation in Faba bean.Seventeen well established shoots  were transferred to new MLS medium including suitable  antibiotics.Finally six independent transgenic plants were successfully rooted on kanamycin-containing  selection media and then transferred to soil after 20 days .Four plants out of six putative transgenic plants displaied to contain the end part of the chit transgene and nos terminator.The corresponding  piece, 700 bp of the chit gene, was amplified using specific primer.These putative transgenic plants were also be measured for the presence of the bgn13.1 and cryIA (b genes by PCR using specific primers.Two  pieces with expected sizes (1221 bp and 640

  9. Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase. (United States)

    Sticher, L; Hofsteenge, J; Neuhaus, J M; Boller, T; Meins, F


    The fungicidal class I chitinases (EC are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

  10. Structural investigation of a novel N-acetyl glucosamine binding chi-lectin which reveals evolutionary relationship with class III chitinases. (United States)

    Patil, Dipak N; Datta, Manali; Dev, Aditya; Dhindwal, Sonali; Singh, Nirpendra; Dasauni, Pushpanjali; Kundu, Suman; Sharma, Ashwani K; Tomar, Shailly; Kumar, Pravindra


    The glycosyl hydrolase 18 (GH18) family consists of active chitinases as well as chitinase like lectins/proteins (CLPs). The CLPs share significant sequence and structural similarities with active chitinases, however, do not display chitinase activity. Some of these proteins are reported to have specific functions and carbohydrate binding property. In the present study, we report a novel chitinase like lectin (TCLL) from Tamarindus indica. The crystal structures of native TCLL and its complex with N-acetyl glucosamine were determined. Similar to the other CLPs of the GH18 members, TCLL lacks chitinase activity due to mutations of key active site residues. Comparison of TCLL with chitinases and other chitin binding CLPs shows that TCLL has substitution of some chitin binding site residues and more open binding cleft due to major differences in the loop region. Interestingly, the biochemical studies suggest that TCLL is an N-acetyl glucosamine specific chi-lectin, which is further confirmed by the complex structure of TCLL with N-acetyl glucosamine complex. TCLL has two distinct N-acetyl glucosamine binding sites S1 and S2 that contain similar polar residues, although interaction pattern with N-acetyl glucosamine varies extensively among them. Moreover, TCLL structure depicts that how plants utilize existing structural scaffolds ingenuously to attain new functions. To date, this is the first structural investigation of a chi-lectin from plants that explore novel carbohydrate binding sites other than chitin binding groove observed in GH18 family members. Consequently, TCLL structure confers evidence for evolutionary link of lectins with chitinases.

  11. Partial purification, characterization, and kinetic studies of a low-molecular-weight, alkali-tolerant chitinase enzyme from Bacillus subtilis JN032305, A potential biocontrol strain. (United States)

    Shivakumar, Srividya; Karmali, Anika Nayak; Ruhimbana, Charles


    A new alkalophilic low-molecular-mass chitinase of 14 kD from the potent biocontrol agent Bacillus subtilis JN032305 was partially purified and enzymology of the chitinase was studied. The enzyme showed optimal pH of 9.0 and temperature of 50°C. The enzyme was found stable during the 60-min incubation at 50 °C. The chitinase was inhibited by group specific agents like IAA, DAN, TLCK, and SDS and metal ions Mg(2+), Ca(2+), Fe(2+), Mn(2+), Ba(2+), and Hg(2+), whereas Zn(2+) did not show significant inhibitory effect against the chitinase. PMSF partially inhibited the enzyme. Substrates specificity tests indicated that the enzyme showed 75% of relative activity on glycol chitin, 58% on carboxymethylcellulose (CMC), 33% on chitin flakes, and 166% laminarin compared to that on colloidal chitin. The enzyme also hydrolyzed 4-methylumbelliferyl-N-acetyl-D-glucosaminide, indicating its chitobiase activity. The chitinase of this study has broad specificity, which could hydrolyze not only the glycosidic bond in GlcNAc-GlcNAc but also that of related carbohydrates with glycosidic linkages. The partially purified chitinase not only showed antifungal activity against Rhizoctonia solani and Colletotrichum gloeosporioides, two potent phytopathogens of chilli, but also increased the germination of chilli seeds when infected with the two potent phytopathogenic fungi.

  12. The first LHC insertion quadrupole

    CERN Multimedia


    An important milestone was reached in December 2003 at the CERN Magnet Assembly Facility. The team from the Accelerator Technology - Magnet and Electrical Systems group, AT-MEL, completed the first special superconducting quadrupole for the LHC insertions which house the experiments and major collider systems. The magnet is 8 metres long and contains two matching quadrupole magnets and an orbit corrector, a dipole magnet, used to correct errors in quadrupole alignment. All were tested in liquid helium and reached the ultimate performance criteria required for the LHC. After insertion in the cryostat, the superconducting magnet will be installed as the Q9 quadrupole in sector 7-8, the first sector of the LHC to be put in place in 2004. Members of the quadrupole team, from the AT-MEL group, gathered around the Q9 quadrupole at its inauguration on 12 December 2003 in building 181.

  13. Insertion device calculations with mathematica

    Energy Technology Data Exchange (ETDEWEB)

    Carr, R. [Stanford Synchrotron Radiation Lab., CA (United States); Lidia, S. [Univ. of California, Davis, CA (United States)


    The design of accelerator insertion devices such as wigglers and undulators has usually been aided by numerical modeling on digital computers, using code in high level languages like Fortran. In the present era, there are higher level programming environments like IDL{reg_sign}, MatLab{reg_sign}, and Mathematica{reg_sign} in which these calculations may be performed by writing much less code, and in which standard mathematical techniques are very easily used. The authors present a suite of standard insertion device modeling routines in Mathematica to illustrate the new techniques. These routines include a simple way to generate magnetic fields using blocks of CSEM materials, trajectory solutions from the Lorentz force equations for given magnetic fields, Bessel function calculations of radiation for wigglers and undulators and general radiation calculations for undulators.

  14. Inserting Agility in System Development (United States)


    Agile IT Acquisition, IT Box, Scrum Inserting Agility in System Development Matthew R. Kennedy and Lt Col Dan Ward, USAF With the fast-paced nature...1,700 individuals and 71 countries, found Scrum and eXtreme Programming to be the most widely followed method- ologies (VersionOne, 2007). Other...University 259 Defense ARJ, July 2012, Vol. 19 No. 3 : 249–264 Scrum Scrum is a framework used for project management, which is

  15. HTS Insert Magnet Design Study

    CERN Document Server

    Devaux, M; Fleiter, J; Fazilleau, P; Lécrevisse, T; Pes, C; Rey, J-M; Rifflet, J-M; Sorbi, M; Stenvall, A; Tixador, P; Volpini, G


    Future accelerator magnets will need to reach higher field in the range of 20 T. This field level is very difficult to reach using only Low Temperature Superconductor materials whereas High Temperature Superconductors (HTS) provide interesting opportunities. High current densities and stress levels are needed to design such magnets. YBCO superconductor indeed carries large current densities under high magnetic field and provides good mechanical properties especially when produced using the IBAD approach. The HFM EUCARD program studies the design and the realization of an HTS insert of 6 T inside a Nb3Sn dipole of 13T at 4.2 K. In the2HTS insert, engineering current densities higher than 250 MA/m under 19 T are required to fulfill the specifications. The stress level is also very severe. YBCO IBAD tapes theoretically meet these challenges from presented measurements. The insert protection is also a critical because HTS materials show low quench propagation velocities and the coupling with the Nb3Sn magnet make...

  16. Induction by chromium ions of chitinases and polyamines in barley (Hordeum vulgare L.) and rape (Brassica napus L. ssp. oleifera)

    DEFF Research Database (Denmark)

    Jacobsen, S.; Hauschild, M.Z.; Rasmussen, U.


    (III) at concentrations of 10-50-mu-g/ml did not significantly alter the concentrations of polyamines nor the chitinase activities in either species, however, at 100-mu-g/ml in barley Cr(III) induced an increase in putrescine concentration after 6 days of exposure. The induction of chitinases and the increases...... in the putrescine level caused by Cr(VI) but not by Cr(III) (10-50-mu-g/ml) exposure is similar in the two species suggesting an analogous defense system in both mono- and di-cotyledonous plants. The anionic form of Cr(VI) seems to be more potent than the cationic form of Cr(III). Grain of barley plants grown...

  17. Enhancing plant disease suppression by Burkholderia vietnamiensis through chromosomal integration of Bacillus subtilis chitinase gene chi113. (United States)

    Zhang, Xinjian; Huang, Yujie; Harvey, Paul R; Ren, Yan; Zhang, Guangzhi; Zhou, Hongzi; Yang, Hetong


    Burkholderia vietnamiensis P418 is a plant growth-promoting rhizobacteria. A chitinase gene from Bacillus subtilis was cloned and stably integrated into the chromosome of using the transposon delivery vector, pUTkm1. Chitinase activity was detected in recombinant P418-37 but not in wild type P418. Recombinant P418-37 retained the in vitro growth rate, N(2)-fixation and phosphate and potassium-solubilizing characteristics of the wild type. P418-37 significantly (P Bipolaris sorokiniana, Verticillium dahliae and Gaeumannomyces graminis var. tritici compared with P418. In planta disease suppression assays indicated that P418-37 significantly (P < 0.05) enhanced suppression of wheat sheath blight (R. cerealis), cotton Fusarium wilt (F. oxysporium f.sp. vasinfectum) and tomato gray mould (Botrytis cinerea), relative to the wild type.

  18. Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor


    Hirose, T.; Maita, N; Gouda, H.; Koseki, J; Yamamoto, T; Sugawara, A; H. Nakano; Hirono, S; Shiomi, K.; Watanabe, T; Taniguchi, H; Sharpless, KB; Omura, S; Sunazuka, T


    Several in situ click chemistry studies have been reported. To date, there is evidence to indicate that proteins act as mold between azide and alkyne fragments by X-ray analysis of protein–ligand complexes. However, only “postclick” structural evidence has been available. We succeeded in obtaining crystal structures of a chitinase complexed with an azide inhibitor and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating triazole formation in chitinas...

  19. Expression and polyclonal antibody preparation of chitinase gene in Tetranychus urticae Koch%二斑叶螨几丁质酶基因的原核表达及多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    张道伟; 陈静; 张正玲; 曾燕玲; 郭玉双


    The sequence of part chitinase gene of Tetranychus urticae was amplified by PCR from cDNA. The sequence length of this domain was 786 bp. Then, the gene was cloned into pET-32a (+) prokaryotic expressive vector, and the constructed recombinant plasmids pET-32a-TuChi was transformed into the host bacteria E. coli BL21 (DE3). About 45 ku fusion protein was abundantly expressed at 4 h after the recombinant vector was induced with 0.6 mmol·L-1 IPTG. The fusion protein was purified on a Ni+affinity column. The purified chitinase protein was used to immunize New Zealand rabbits for preparing polyclonal antibody with specificity as defined by Western Blot. ELISA analysis showed that the titer of the polyclonal antibody was 1��80 000, polyclonal antibody that was prepared had a high titer and specificity, and the results laid the foundation to further study the function of the chitinase in Tetranychus urticae.%以制备的二斑叶螨cDNA为模板,克隆二斑叶螨几丁质酶SeChi基因功能结构域催化区片段,大小为786 bp。将该序列片段克隆到表达载体pET-32a (+)中,获得多克隆原核表达载体pET-32a-TuChi。重组质粒经酶切测序鉴定后转化大肠杆菌BL21(DE3),经0.6 mmol·L-1 IPTG诱导4 h后高效表达约45 ku可溶性重组蛋白;经His亲和层析洗脱和浓缩获得高纯度的重组蛋白免疫新西兰大白兔制备TuChi多克隆抗体。制备的多克隆抗体经Western Blot证实能特异性地识别几丁质酶而不与非特异性蛋白结合。ELISA分析表明制备的抗体效价达1��80000,效价较高,为进一步研究二斑叶螨几丁质酶相关功能奠定基础。

  20. Field errors in hybrid insertion devices

    Energy Technology Data Exchange (ETDEWEB)

    Schlueter, R.D. [Lawrence Berkeley Lab., CA (United States)


    Hybrid magnet theory as applied to the error analyses used in the design of Advanced Light Source (ALS) insertion devices is reviewed. Sources of field errors in hybrid insertion devices are discussed.

  1. Progress of Researching Chitinase of Gypsy Moth%舞毒蛾几丁质酶的研究进展

    Institute of Scientific and Technical Information of China (English)

    王绥冬; 宋志芳; 张常; 李瑶; 范晓军


    The gypsy moth that is a worldwide forestry pests are distributed in China's provinces. Gypsy moth larvae eat the leaves of fi-uit trees, willow trees, which have serious harm to forestry safety. The article was described the hazards of the gypsy moth, Chitinase relevant knowledge and research status of the gypsy moth Chitinase, and expounded the prospect of application of Chitinase to combat gypsy moth.%舞毒蛾是一种世界性林业害虫,在我国各省均有分布。舞毒蛾幼虫主要蚕食果树、柳树等树木的树叶.严重危害林业安全。文章介绍了舞毒蛾的危害、几丁质酶的相关知识及舞毒蛾几丁质酶的研究现状.并对应用几丁质酶防治舞毒蛾的前景进行了论述。

  2. JT/LJT connector insert material evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Baca, J.R.F.


    Different insert (insulator) materials are undergoing evaluation to replace the Fiberite E-3938 BE96 material currently used. Also being evaluated is the reconfiguration of the insert and metal shell-edge geometries for the purpose of reducing the alleged interference principally responsible for insert damage.

  3. The insertion of human apolipoprotein H into phospholipid membranes: a monolayer study. (United States)

    Wang, S X; Cai, G P; Sui, S F


    Apolipoprotein H (ApoH) is a plasma glycoprotein isolated from human serum. The interactions of ApoH with lipid membrane were reported to be essential for its physiological and pathogenic roles. In this paper we studied the ability of ApoH to insert into phospholipid membranes using the monolayer approach. The results show that ApoH is surface active and can insert into the lipid monolayers. The insertion ability of ApoH is stronger when a higher content of negatively charged lipids is present in the membrane. The acidic-pH and low-ionic-strength conditions will also enhance ApoH insertion, but these factors may not have much influence on the final insertion ability of ApoH, suggesting that, in the mechanism of ApoH insertion, not only electrostatic forces, but also hydrophobic interactions, are evidently involved. Modification by heat inactivation and reduction/alkylation does not change the critical insertion pressure (pic) of ApoH, suggesting a stable domain, maybe a linear sequence motif, but not the native three-dimensional structure of ApoH, is responsible for its insertion. The extent to which insertion of ApoH into phospholipid membranes may facilitate the 'immune cleaning' of plasma liposomes is discussed.

  4. The Correlation between Chitin and Acidic Mammalian Chitinase in Animal Models of Allergic Asthma

    Directory of Open Access Journals (Sweden)

    Chia-Rui Shen


    Full Text Available Asthma is the result of chronic inflammation of the airways which subsequently results in airway hyper-responsiveness and airflow obstruction. It has been shown that an elicited expression of acidic mammalian chitinase (AMCase may be involved in the pathogenesis of asthma. Our recent study has demonstrated that the specific suppression of elevated AMCase leads to reduced eosinophilia and Th2-mediated immune responses in an ovalbumin (OVA-sensitized mouse model of allergic asthma. In the current study, we show that the elicited expression of AMCase in the lung tissues of both ovalbumin- and Der P2-induced allergic asthma mouse models. The effects of allergic mediated molecules on AMCase expression were evaluated by utilizing promoter assay in the lung cells. In fact, the exposure of chitin, a polymerized sugar and the fundamental component of the major allergen mite and several of the inflammatory mediators, showed significant enhancement on AMCase expression. Such obtained results contribute to the basis of developing a promising therapeutic strategy for asthma by silencing AMCase expression.

  5. Activity, stability and folding analysis of the chitinase from Entamoeba histolytica. (United States)

    Muñoz, Patricia L A; Minchaca, Alexis Z; Mares, Rosa E; Ramos, Marco A


    Human amebiasis, caused by the parasitic protozoan Entamoeba histolytica, remains as a significant public health issue in developing countries. The life cycle of the parasite compromises two main stages, trophozoite and cyst, linked by two major events: encystation and excystation. Interestingly, the cyst stage has a chitin wall that helps the parasite to withstand harsh environmental conditions. Since the amebic chitinase, EhCHT1, has been recognized as a key player in both encystation and excystation, it is plausible to consider that specific inhibition could arrest the life cycle of the parasite and, thus, stop the infection. However, to selectively target EhCHT1 it is important to recognize its unique biochemical features to have the ability to control its cellular function. Hence, to gain further insights into the structure-function relationship, we conducted an experimental approach to examine the effects of pH, temperature, and denaturant concentration on the enzymatic activity and protein stability. Additionally, dependence on in vivo oxidative folding was further studied using a bacterial model. Our results attest the potential of EhCHT1 as a target for the design and development of new or improved anti-amebic therapeutics. Likewise, the potential of the oxidoreductase EhPDI, involved in oxidative folding of amebic proteins, was also confirmed.

  6. Drosophila Chitinase 2 is expressed in chitin producing organs for cuticle formation. (United States)

    Pesch, Yanina-Yasmin; Riedel, Dietmar; Behr, Matthias


    The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. Recently we identified a key region for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex, which is required for chitin-matrix maturation and preservation. Here we present evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. We show that Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. We further show that in Cht2 knockdown larvae the assembly zone is rudimentary, preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, our findings suggest that Cht2 is involved in chitin formation also in other insects.

  7. Functional properties of a chitinase promoter from cabbage (Brassica oleracea var.capitata)

    Institute of Scientific and Technical Information of China (English)



    The 5'-region of the chitinase gene cabch29,derived from Brassica oleracea var.capitata,has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants.Different 5'-deletion fragments were linked to reporter gene β-glucuronidase (GUS) as translational fusions,and the expression of these chimeric genes was analyzed in vegetative organs and tissues.Sequences up to-651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues.The addition of further upstream sequences(-651 to-1284) enhanced expression level,and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding.Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-gus fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment.The location of GUS activity appears to be cell-specific,being highest in vascular cells and epidermal cells of stem,leaf and roots.Meanwhile,the temporal and spatial expression of cabch29-GUS fusion gene has been investigated.Among the different vegetative organs,a high level of GUS activity was observed in stem and a moderate one in roots;whereas,wounding stress led to a high level of GUS in stem and moderate one in leaf.

  8. Applications of Plackett–Burman and Central Composite Design for the Optimization of Novel Brevundimonas diminuta KT277492 Chitinase Production, Investigation of its Antifungal Activity

    Directory of Open Access Journals (Sweden)

    E. Ashour Warda

    Full Text Available ABSTRACT Biological control strategy which can damage chitin, a vital component of pathogenic fungi and arthropods promises a safe solution for many fungal problems. And it’s more favorable than chemicals which increase health risks and environmental problems. Thus, the chitinase producers appear potential candidates of biological control of pathogenic fungi. Brevundimonus diminuta KT277492 is a new isolate that has been isolated recently from Egyptian soil. Significant factors that affecting the chitinase enzyme production were studied and optimized using Plackett-Burman and Response Surface Methodology (RSM. As a result, maximum production of chitinase enzyme was 832.87 IUL-1, this result presented about 8.767-fold increase in the enzyme production. In the last phase of the study, partially purified chitinase enzyme obtained from B. diminuta KT277492 was tested against two pathogenic fungi and the results showed good inhibitory activity against A. alternata and F. solani with IZD of 31±0.25 and 25±0.91 mm respectively. Finally, obtained results indicated the value of optimization process and the optimized chitinase enzyme could be an excellent choice in application of food and biotechnology as a biofungicide. This reflects the necessity of studying the characteristics and kinetics of the enzyme in the forthcoming study.

  9. Aspartate embedding depth affects pHLIP's insertion pKa. (United States)

    Fendos, Justin; Barrera, Francisco N; Engelman, Donald M


    We have used the pHlow insertion peptide (pHLIP) family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When the pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when the pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To examine this protonation mechanism further, we created pHLIP sequence variants in which the two spanning aspartates (D14 and D25) were moved up or down in the sequence. We hypothesized that the aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to determine whether arginine snorkeling modulates the insertion pKa by affecting the aspartate depth. Our results indicate that both types of mutations change the insertion pKa, supporting the idea that the aspartate depth is a participating parameter in determining the pH dependence. We also show that pHLIP's resistance to aggregation can be altered with our mutations, identifying a new criterion for improving the performance of pHLIP in vivo when targeting acidic disease tissues such as cancer and inflammation.

  10. Sequential cooling insert for turbine stator vane

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Russel B


    A sequential flow cooling insert for a turbine stator vane of a small gas turbine engine, where the impingement cooling insert is formed as a single piece from a metal additive manufacturing process such as 3D metal printing, and where the insert includes a plurality of rows of radial extending impingement cooling air holes alternating with rows of radial extending return air holes on a pressure side wall, and where the insert includes a plurality of rows of chordwise extending second impingement cooling air holes on a suction side wall. The insert includes alternating rows of radial extending cooling air supply channels and return air channels that form a series of impingement cooling on the pressure side followed by the suction side of the insert.

  11. A HTS dipole insert coil constructed

    CERN Document Server

    Ballarino, A; Rey, J M; Stenvall, A; Sorbi, M; Tixador, P


    This report is the deliverable report 7.4.1 “A HTS dipole insert coil constructed“. The report has three parts: “Design report for the HTS dipole insert”, “One insert pancake prototype coil constructed with the setup for a high field test”, and “All insert components ordered”. The three report parts show that, although the insert construction will be only completed by end 2013, all elements are present for a successful completion and that, given the important investments done by the participants, there is a full commitment of all of them to finish the project

  12. Mining of unexplored habitats for novel chitinases - chiA as a helper gene proxy in metagenomics

    DEFF Research Database (Denmark)

    Cretoiu, Mariana Silvia; Kielak, Anna Maria; Abu Al-Soud, Waleed


    the existence across habitats of core bacterial communities responsible for chitin assimilation irrespective of ecosystem origin. Conversely, there were habitat-specific differences. In addition, a suite of sequences were obtained that are as yet unregistered in the chitinase database. In terms of chiA gene...... abundance and diversity, typical low-abundance/diversity versus high-abundance/diversity habitats was distinguished. From the combined data, we selected chitin-amended agricultural soil, the rhizosphere of the Arctic plant Oxyria digyna and the freshwater sponge Ephydatia fluviatilis as the most promising...

  13. Multipurpose Transposon-Insertion Libraries in Yeast. (United States)

    Kumar, Anuj


    Libraries of transposon-insertion alleles constitute powerful and versatile tools for large-scale analysis of yeast gene function. Transposon-insertion libraries are constructed most simply through mutagenesis of a plasmid-based genomic DNA library; modification of the mutagenizing transposon by incorporation of yeast selectable markers, recombination sites, and an epitope tag enables the application of insertion alleles for phenotypic screening and protein localization. In particular, yeast genomic DNA libraries have been mutagenized with modified bacterial transposons carrying the URA3 marker, lox recombination sites, and sequence encoding multiple copies of the hemagglutinin (HA) epitope. Mutagenesis with these transposons has yielded a large resource of insertion alleles affecting nearly 4000 yeast genes in total. Through well-established protocols, these insertion libraries can be introduced into the desired strain backgrounds and the resulting insertional mutants can be screened or systematically analyzed. Relative to alternative methods of UV irradiation or chemical mutagenesis, transposon-insertion alleles can be easily identified by PCR-based approaches or high-throughput sequencing. Transposon-insertion libraries also provide a cost-effective alternative to targeted deletion approaches, although, in contrast to start-codon to stop-codon deletions, insertion alleles might not represent true null-mutants. For protein-localization studies, transposon-insertion alleles can provide encoded epitope tags in-frame with internal codons; in many cases, these transposon-encoded epitope tags can provide a more accurate localization for proteins in which terminal sequences are crucial for intracellular targeting. Thus, overall, transposon-insertion libraries can be used quickly and economically and have a particular utility in screening for desired phenotypes and localization patterns in nonstandard genetic backgrounds.

  14. Marginal adaptation of ceramic inserts after cementation

    NARCIS (Netherlands)

    Ozcan, M; Pfeiffer, P; Nergiz, [No Value


    The advantage of using ceramic inserts is to prevent major drawbacks of composite resins such as polymerization shrinkage, wear and microleakage. This in vitro study evaluated the marginal adaptation of two approximal ceramic insert systems after cementation to the cavities opened with ultrasonic ti


    Institute of Scientific and Technical Information of China (English)

    王斌; 董莉


    Acupuncture needle insertion always results in pain in the local area due to stimulating the free nerve endings—algesireceptors of the skin. In spite of mildness, this pain may induce many patients' fright, and thus, hinders more extensive application of acupuncture. In the present paper, the author introduces some methods for reducing needle insertion induced pain.

  16. An Elementary Account of Needle Insertion

    Institute of Scientific and Technical Information of China (English)

    张文兵; 霍则军


    @@ Based on the authors' clinical and personal experiences, several pain-inducing factors easily to be ignored by the operators when quick needle insertion is applied, and the authors' first invented slow painless needle insertion method are introduced in the article.

  17. Long-distance effects of insertional mutagenesis.

    Directory of Open Access Journals (Sweden)

    Ruchi Singhal

    Full Text Available BACKGROUND: Most common systems of genetic engineering of mammalian cells are associated with insertional mutagenesis of the modified cells. Insertional mutagenesis is also a popular approach to generate random alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance. METHODOLOGY/PRINCIPAL FINDINGS: We observed that, in the context of a lentiviral construct, a transcript, which is initiated at an internal CMV promoter/enhancer region and incorporates a splice donor site, is able to extend past a collinear viral LTR and trap exons of host genes, while the polyadenylation signal, which is naturally present in the LTR, is spliced out. Unexpectedly, when a vector, which utilizes this phenomenon, was used to produce mutants with elevated activity of NF-κB, we found mutants, which owed their phenotype to the effect of the insert on a gene located tens or even hundreds of kilobases away from the insertion site. This effect did not result from a CMV-driven transcript, but was sensitive to functional suppression of the insert. Interestingly, despite the long-distance effect, expression of loci most closely positioned to the insert appeared unaffected. CONCLUSIONS/SIGNIFICANCE: We concluded that a polyadenylation signal in a retroviral LTR, when occurring within an intron, is an inefficient barrier against the formation of a hybrid transcript, and that a vector containing a strong enhancer may selectively affect the function of genes far away from its insertion site. These phenomena have to be considered when experimental or therapeutic transduction is performed. In particular, the long-distance effects of insertional mutagenesis bring into question the relevance of the lists of disease-associated retroviral integration

  18. Central Solenoid Insert Technical Specification

    Energy Technology Data Exchange (ETDEWEB)

    Martovetsky, Nicolai N [ORNL; Smirnov, Alexandre [ORNL


    The US ITER Project Office (USIPO) is responsible for the ITER central solenoid (CS) contribution to the ITER project. The Central Solenoid Insert (CSI) project will allow ITER validation the appropriate lengths of the conductors to be used in the full-scale CS coils under relevant conditions. The ITER Program plans to build and test a CSI to verify the performance of the CS conductor. The CSI is a one-layer solenoid with an inner diameter of 1.48 m and a height of 4.45 m between electric terminal ends. The coil weight with the terminals is approximately 820 kg without insulation. The major goal of the CSI is to measure the temperature margin of the CS under the ITER direct current (DC) operating conditions, including determining sensitivity to load cycles. Performance of the joints, ramp rate sensitivity, and stability against thermal or electromagnetic disturbances, electrical insulation, losses, and instrumentation are addressed separately and therefore are not major goals in this project. However, losses and joint performance will be tested during the CSI testing campaign. The USIPO will build the CSI that will be tested at the Central Solenoid Model Coil (CSMC) Test Facility at the Japan Atomic Energy Agency (JAEA), Naka, Japan. The industrial vendors (the Suppliers) will report to the USIPO (the Company). All approvals to proceed will be issued by the Company, which in some cases, as specified in this document, will also require the approval of the ITER Organization. Responsibilities and obligations will be covered by respective contracts between the USIPO, called Company interchangeably, and the industrial Prime Contractors, called Suppliers. Different stages of work may be performed by more than one Prime Contractor, as described in this specification. Technical requirements of the contract between the Company and the Prime Contractor will be covered by the Fabrication Specifications developed by the Prime Contractor based on this document and approved by

  19. A predictive model of intein insertion site for use in the engineering of molecular switches.

    Directory of Open Access Journals (Sweden)

    James Apgar

    Full Text Available Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54. In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch.

  20. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato. (United States)

    Mamta; Reddy, K R K; Rajam, M V


    Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera.

  1. Abundant class III acidic chitinase homologue in tamarind (Tamarindus indica) seed serves as the major storage protein. (United States)

    Rao, Devavratha H; Gowda, Lalitha R


    The phyla Leguminosae contains protease inhibitors, lectins, chitinases, and glycohydrolases as major defense proteins in their seeds. Electrophoretic analysis of the seed proteins of tamarind ( Tamarindus indica L.), an agri-waste material, indicated the unusual presence of two major proteins comparable to overexpression of recombinant proteins. These proteins were identified by amino-terminal analysis to be (1) Kunitz-type trypsin inhibitor and (2) class III endochitinase (34000 Da). These two proteins were purified to apparent homogeneity by a single-step chitin bead affinity chromatography and characterized. The Kunitz inhibitor was specific toward inhibiting trypsin with a stoichiometry of 1:1. The 33000 +/- 1000 Da protein, accounting for >50% of the total seed protein, is an acidic glycoprotein exhibiting a very low endotype hydrolytic activity toward chitin derivatives. SDS-PAGE followed by densitometry of tamarind seed germination indicates the disappearance of the chitinase with the concomitant appearance of a cysteine endopeptidase. On the basis of its abundance, accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity, this 34000 Da protein designated "tamarinin" physiologically serves as the major storage protein.

  2. An acidic class III chitinase in sugar beet: induction by Cercospora beticola, characterization, and expression in transgenic tobacco plants. (United States)

    Nielsen, K K; Mikkelsen, J D; Kragh, K M; Bojsen, K


    An acidic chitinase (SE) was found to accumulate in leaves of sugar beet (Beta vulgaris) during infection with Cercospora beticola. Two isoforms, SE1 and SE2, with MW of 29 kDa and pI of approximately 3.0 were purified to homogeneity. SE2 is an endochitinase that also exhibits exochitinase activity, i.e., it is capable of hydrolyzing chito-oligosaccharides, including chitobiose, into N-acetyl-glucosamine. Partial amino acid sequence data for SE2 were used to obtain a cDNA clone by polymerase chain reaction. The clone was used to isolate a cDNA clone encoding SE2. The deduced amino acid sequence for SE2 is 58-67% identical to the class III chitinases from cucumber, Arabidopsis, and tobacco. A transient induction of SE2 mRNA during the early stages of infection with C. beticola is much stronger in tolerant plants than in susceptible plants. Transgenic tobacco (Nicotiana benthamiana) plants constitutively accumulate SE2 protein in the intercellular space of their leaves. In a preliminary infection experiment, the transgenic plants did not show increase in resistance against C. nicotianae.

  3. Discovery and identification of candidate genes from the chitinase gene family for Verticillium dahliae resistance in cotton. (United States)

    Xu, Jun; Xu, Xiaoyang; Tian, Liangliang; Wang, Guilin; Zhang, Xueying; Wang, Xinyu; Guo, Wangzhen


    Verticillium dahliae, a destructive and soil-borne fungal pathogen, causes massive losses in cotton yields. However, the resistance mechanism to V. dahilae in cotton is still poorly understood. Accumulating evidence indicates that chitinases are crucial hydrolytic enzymes, which attack fungal pathogens by catalyzing the fungal cell wall degradation. As a large gene family, to date, the chitinase genes (Chis) have not been systematically analyzed and effectively utilized in cotton. Here, we identified 47, 49, 92, and 116 Chis from four sequenced cotton species, diploid Gossypium raimondii (D5), G. arboreum (A2), tetraploid G. hirsutum acc. TM-1 (AD1), and G. barbadense acc. 3-79 (AD2), respectively. The orthologous genes were not one-to-one correspondence in the diploid and tetraploid cotton species, implying changes in the number of Chis in different cotton species during the evolution of Gossypium. Phylogenetic classification indicated that these Chis could be classified into six groups, with distinguishable structural characteristics. The expression patterns of Chis indicated their various expressions in different organs and tissues, and in the V. dahliae response. Silencing of Chi23, Chi32, or Chi47 in cotton significantly impaired the resistance to V. dahliae, suggesting these genes might act as positive regulators in disease resistance to V. dahliae.

  4. Cerebrospinal fluid levels of chitinase 3-like 1 and neurofilament light chain predict multiple sclerosis development and disability after optic neuritis

    DEFF Research Database (Denmark)

    Modvig, S; Degn, M; Roed, H;


    -term disability after optic neuritis (ON). METHODS: Eighty-six patients with ON as a first demyelinating event were included retrospectively. Magnetic resonance imaging (MRI), CSF leukocytes, immunoglobulin G index and oligoclonal bands were registered. CSF levels of chitinase-3-like-1, osteopontin, neurofilament...

  5. A new chitinase-like xylanase inhibitor protein (XIP from coffee (Coffea arabica affects Soybean Asian rust (Phakopsora pachyrhizi spore germination

    Directory of Open Access Journals (Sweden)

    Mehta Angela


    Full Text Available Abstract Background Asian rust (Phakopsora pachyrhizi is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP from coffee (Coffea arabica (CaclXIP leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. Results A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP from coffee (Coffea arabica (CaclXIP, was isolated from leaves. The amino acid sequence predicts a (β/α8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18, and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. Conclusions Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

  6. Sequential cooling insert for turbine stator vane (United States)

    Jones, Russell B; Krueger, Judson J; Plank, William L


    A sequential impingement cooling insert for a turbine stator vane that forms a double impingement for the pressure and suction sides of the vane or a triple impingement. The insert is formed from a sheet metal formed in a zigzag shape that forms a series of alternating impingement cooling channels with return air channels, where pressure side and suction side impingement cooling plates are secured over the zigzag shaped main piece. Another embodiment includes the insert formed from one or two blocks of material in which the impingement channels and return air channels are machined into each block.

  7. A computational analysis of the binding mode of closantel as inhibitor of the Onchocerca volvulus chitinase: insights on macrofilaricidal drug design (United States)

    Segura-Cabrera, Aldo; Bocanegra-García, Virgilio; Lizarazo-Ortega, Cristian; Guo, Xianwu; Correa-Basurto, José; Rodríguez-Pérez, Mario A.


    Onchocerciasis is a leading cause of blindness with at least 37 million people infected and more than 120 million people at risk of contracting the disease; most (99%) of this population, threatened by infection, live in Africa. The drug of choice for mass treatment is the microfilaricidal Mectizan® (ivermectin); it does not kill the adult stages of the parasite at the standard dose which is a single annual dose aimed at disease control. However, multiple treatments a year with ivermectin have effects on adult worms. The discovery of new therapeutic targets and drugs directed towards the killing of the adult parasites are thus urgently needed. The chitinase of filarial nematodes is a new drug target due to its essential function in the metabolism and molting of the parasite. Closantel is a potent and specific inhibitor of chitinase of Onchocerca volvulus (OvCHT1) and other filarial chitinases. However, the binding mode and specificity of closantel towards OvCHT1 remain unknown. In the absence of a crystallographic structure of OvCHT1, we developed a homology model of OvCHT1 using the currently available X-ray structures of human chitinases as templates. Energy minimization and molecular dynamics (MD) simulation of the model led to a high quality of 3D structure of OvCHIT1. A flexible docking study using closantel as the ligand on the binding site of OvCHIT1 and human chitinases was performed and demonstrated the differences in the closantel binding mode between OvCHIT1 and human chitinase. Furthermore, molecular dynamics simulations and free-energy calculation were employed to determine and compare the detailed binding mode of closantel with OvCHT1 and the structure of human chitinase. This comparative study allowed identification of structural features and properties responsible for differences in the computationally predicted closantel binding modes. The homology model and the closantel binding mode reported herein might help guide the rational development of

  8. Density and composition of an insect population in a field trial of chitinase transgenic and wild-type silver birch (Betula pendula) clones. (United States)

    Vihervuori, Liisa; Pasonen, Hanna-Leena; Lyytikäinen-Saarenmaa, Päivi


    Fifteen silver birch (Betula pendula Roth) lines carrying a sugar beet chitinase IV gene and eight wild-type birch clones were grown in a field trial. The composition and density of the insect population and the leaf damage caused by insects were monitored and compared between transgenic and wild-type trees. The most abundant insect group in all trees was aphids, and the variation in total insect densities was mainly explained by the variation in aphid densities. Insect densities were generally higher in the transgenic than in the control trees, indicating that the expression of the sugar beet chitinase IV gene had an influence on the suitability of birch leaves to aphids. The level of leaf damage was higher among transgenic than among control trees. Chewing damage was the most common type of leaf damage in all trees. The number of different damage types was higher among the wild-type clones than among the transgenic lines or their controls. The results indicate that the chitinase transgenic trees are more susceptible to aphids and suffer higher levels of leaf damage than the control trees. In the composition of the damage types, the control trees were more similar to the transgenic than to other wild-type trees, indicating that the composition was mostly linked to the genotype of the tree and not to the expression of the transgene. This study provides important information on the ecological interactions of chitinase transgenic trees in the field trial. No clear harmful effects of transgenic chitinase on the biodiversity of insect population were detected.

  9. Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

    Directory of Open Access Journals (Sweden)

    Misa Ohno


    Full Text Available YKL-40 (YKL for the first three N-terminal residues of a 40 kDa protein belongs to a group of human chitinase-like proteins (CLPs, which are similar to chitinases but lack chitinolytic activity. YKL-40 mRNA and its protein levels have been reported elevated in multiple disorders including asthma, cystic fibrosis, rheumatoid arthritis and malignant tumors. Here, we quantified the YKL-40 mRNA levels and compared them with chitinases and housekeeping genes in normal human tissues. To establish the quantitative real-time PCR (qPCR system for evaluation of relative YKL-40 mRNA levels, we constructed a human standard DNA molecule by ligating cDNAs of YKL-40, two mammalian chitinases and two housekeeping genes in a one-to-one ratio. We generated cDNAs from various normal human tissues and analyzed the YKL-40 mRNA expression levels using a qPCR system with the standard DNA. We found that YKL-40 mRNA is present widely in human tissues while its expression patterns exhibit clear tissue specificity. Highest YKL-40 mRNA levels were detected in the liver, followed by kidney, trachea and lung. The levels of YKL-40 mRNA in the kidney and liver were more than 100-times higher than those of chitotriosidase mRNA. Our study provides for the first time a comprehensive analysis of the relative expression levels of YKL-40 mRNA versus mammalian chitinases in normal human tissues.

  10. HB+ inserted into the CMS Solenoid

    CERN Multimedia

    Tejinder S. Virdee, CERN


    The first half of the barrel hadron calorimeter (HB+) has been inserted into the superconducting solenoid of CMS, in preparation for the magnet test and cosmic challenge. The operation went smoothly, lasting a couple of days.

  11. Shrink-Fit Solderable Inserts Seal Hermetically (United States)

    Croucher, William C.


    Shrink-fit stainless-steel insert in aluminum equipment housing allows electrical connectors to be replaced by soldering, without degrading hermeticity of housing or connector. Welding could destroy electrostatic-sensitive components and harm housing and internal cables. Steel insert avoids problems because connector soldered directly to it rather than welded to housing. Seals between flange and housing, and between connector and flange resistant to leaks, even after mechanical overloading and thermal shocking.

  12. Identification and Antagonism Study of a Novel Chitinase-producing Bacterium Burkholderia Sp.C3 against Phytopathogenic Fungi

    Institute of Scientific and Technical Information of China (English)

    金虹; TAO Yong


    Through a modified agar well diffusion assay, antagonism of a novel chitinase-producing strain C3 against the phytopathogenic fungi including Phoma wasabiae Yokogi,Heterostrophus, Exserohilum Turcicum, Curwularia (Walk) Boed, Thantephorus cucumris, Fusarium graminearum was tested. The data showed that the crude cxtracts of strain C3 had stable antifungal activity in the range of pH 5.0 to pH 8.0. The active components were heat labile and sensitive to proteinase K. A series of experiments supported that the compound responsible for inhibitory activity appeared to be ehitinase. The 16s rDNA analysis indicated that C3 was subject to genus Burkholderia. Pbenotypic characterization of C3 was also consisted with the result of molecular identification.

  13. Domain crossing

    DEFF Research Database (Denmark)

    Schraefel, M. C.; Rouncefield, Mark; Kellogg, Wendy


    In CSCW, how much do we need to know about another domain/culture before we observe, intersect and intervene with designs. What optimally would that other culture need to know about us? Is this a “how long is a piece of string” question, or an inquiry where we can consider a variety of contexts a...

  14. The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chunyi; Li, Qi [Division of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, No. 74, Linjiang Road, Yuzhong District, Chongqing 400010 (China); Zhou, Xiangdong, E-mail: [Division of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, No. 74, Linjiang Road, Yuzhong District, Chongqing 400010 (China); Kolosov, Victor P.; Perelman, Juliy M. [Far Eastern Scientific Center of Physiology and Pathology of Respiration, Siberian Branch, Russian Academy of Medical Sciences, Blagoveshchensk (Russian Federation)


    Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5AC in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. - Highlights: • MUC5AC is the major secreted mucin in chronic inflammatory airway diseases. • YKL-40 is a prototype of the chitinase-like protein in mammals. • YKL-40 is an active player in chronic inflammatory airway diseases. • YKL-40 can increase MUC5AC production via PAR2-mediated pathway. • FAK is another candidate to mediate YKL-40-induced MUC5AC overexpression.

  15. Allozyme-specific modification of a maize seed chitinase by a protein secreted by the fungal pathogen Stenocarpella maydis. (United States)

    Naumann, Todd A; Wicklow, Donald T


    Stenocarpella maydis causes both dry-ear rot and stalk rot of maize. Maize inbred lines have varying levels of resistance to ear rot caused by S. maydis. The genetic basis of resistance appears to rely on multiple genetic factors, none of which are known. The commonly used stiff-stalk inbred line B73 has been shown to be strongly susceptible to ear rot caused by S. maydis. Here, we report that the ChitA protein alloform from B73, ChitA-F, encoded by a known allele of the chiA gene, is susceptible to modification by a protein (Stm-cmp) secreted by S. maydis. We also identify a new allele of chiA (from inbred line LH82) which encodes ChitA-S, an alloform of ChitA that is resistant to Stm-cmp modification. Chitinase zymogram analysis of seed from a commercial field showed the presence of both ChitA alloforms in healthy ears, and showed that ChitA-F but not ChitA-S was modified in ears rotted by S. maydis. The ChitA-F protein was purified from inbred line B73 and ChitA-S from LH82. ChitA-F was modified more efficiently than ChitA-S by S. maydis protein extracts in vitro. The chiA gene from LH82 was cloned and sequenced. It is a novel allele that encodes six polymorphisms relative to the known allele from B73. This is the first demonstration that the susceptibility to modification of a fungal targeted plant chitinase differs among inbred lines. These findings suggest that the LH82 chiA allele may be a specific genetic determinant that contributes to resistance to ear rot caused by S. maydis whereas the B73 allele may contribute to susceptibility.

  16. MD-simulations of Beta-Amyloid Protein Insertion Efficiency and Kinetics into Neuronal Membrane Mimics (United States)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan


    Early interaction events of beta-amyloid (A β) peptides with the neuronal membranes play a key role in the pathogenesis of Alzheimer's disease. We have used all-atom MD simulations to study the protein insertion efficiency and kinetics of monomeric A β40 and A β42 into phosphatidylcholine lipid bilayers (PC) with and without 40 mole% cholesterol (CHOL) that mimic the cholesterol-enriched and depleted lipid nanodomains of the neuronal plasma membranes. Independent replicates of 200-ns simulations of each protein pre-inserted in the upper lipid layer were generated. In PC bilayers, only 25% of A β40 and 50% of A β42 in the replicates showed complete insertion into the lower lipid layer, whereas the percentages increased to 50% and 100%, respectively, in PC/CHOL bilayers, providing evidence that cholesterol improves the protein insertion efficiency into the bilayers. The rate of protein insertion was proportional to the hydrophobic, transmembrane helix length of the inserted peptide and depended on the cholesterol content. We propose that the lysine snorkeling and C-terminus anchoring of A β to the PC headgroups at the upper and lower lipid/water interfaces represent the dual-transmembrane stabilization mechanisms of A β in the neuronal membrane domains.

  17. Hamstring tendons insertion - an anatomical study

    Directory of Open Access Journals (Sweden)

    Cristiano Antonio Grassi


    Full Text Available OBJECTIVE: To study the anatomy of the hamstring tendons insertion and anatomical rela-tionships. METHODS: Ten cadaver knees with medial and anterior intact structures were selected. The dissection was performed from anteromedial access to exposure of the insertion of the flexor tendons (FT, tibial plateau (TP and tibial tuberosity (TT. A needle of 40 × 12 and a caliper were used to measure the distance of the tibial plateau of the knee flexor tendons insertion at 15 mm from the medial border of the patellar tendon and tibial tuberosity to the insertion of the flexor tendons of the knee. The angle between tibial plateau and the insertion of the flexor tendons of the knee (A-TP-FT was calculated using Image Pro Plus software. RESULTS: The mean distance TP-FT was 41 ± 4.6 mm. The distance between the TT-FT was 6.88 ± 1 mm. The (A-TP-FT was 20.3 ± 4.9°. CONCLUSION: In the anterior tibial flexor tendons are about 40 mm from the plateau with an average of 20°.

  18. Regulation of chitinase genes expression in bacteria%细菌几丁质酶基因的表达调控

    Institute of Scientific and Technical Information of China (English)

    谢池楚; 贾海云; 陈月华


    Chitinases, which can hydrolyze chitin, occur in a wide range of microorganisms including viruses, bacteria, and fungi. The derivatives of chitin are potentially useful in several areas such as food processing, medicines, and biological control in agriculture. Some bacteria can uptake and utilize chitin as carbon source by secreting chitinase. The chitin is degraded into chito-oligosaccharides [(GlcNAc)n] or N-acetylglucosamine (GlcNAc) by chitinases, and then the chitin derivatives are transferred into cells by specific transport systems of bacteria. The intracellular chitin derivatives activate or suppress the transcription of a series of chi genes and affect the amount of chitinase. The expression of chitinase genes are strictly regulated by various regulatory factors and responsive cw-acting elements. The present review will focus on the transport system and the regulation of chitinase genes expression in bacteria.%几丁质酶可以降解几丁质,广泛存在于各类微生物中.几丁质的降解产物几丁寡糖在医药、食品及农业生防领域有很重要的应用价值及广泛的应用前景.细菌在利用几丁质时,需要先分泌几丁质酶,将几丁质降解成几丁寡糖或单体,再通过特异的转运系统送进细胞而被利用.胞内的几丁质降解产物作为特定的信号分子,可以激活或阻遏相应chi基因的转录,从而影响细菌几丁质酶的合成.在各种调节蛋白及应答元件的参与下,细菌几丁质酶的合成受到精密的控制,文章以链霉菌和大肠杆菌为代表综述了细菌在转运系统和基因表达两个层面上控制几丁质酶合成的最新研究进展.

  19. HaSNPV几丁质酶的分离纯化与毒理学研究%Purification and Toxicological Studies of HaSNPV Chitinase

    Institute of Scientific and Technical Information of China (English)

    惠有为; 夏天; 赵亚玲; 贾静芳


    Recombinant bacterium GS115-pPIC 9k-ChiA 4.0 fermented chitinase, which was purified by ammomum sulfate precipitation, dialysis , DEAE Sepharose Fast Flow ion exchange chromatography and Sephadex G-1OO gel filtration chromatography, obtained electrophoretically pure chitinase. Rate of recovery was 19. 64% , and purification factor was 17. 74. Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus ( HaSNPV) chitinase toxicological studies carried out by contact toxicity and stomach toxicity, chitinase liquefied Plutella xylostella larvae cell and caused death. The effect and the concentration of chitinase is proportional to time, and the effect caused by stomach toxicity was more intense than by action of contact toxicity. The result of antibacterial experiments which were performed on Helminthosporium sativum , Tomato botrytis cinerea , Tobacco brown spot , and Colletotrichum gloeosporioides , indicated that chitinase can inhibit fungal by enzymatic hydrolysis of chitin in fungal cell wall.%利用重组工程茵GSl 15-pPIC 9k-ChiA 4.0发酵几丁质酶粗液,经(NH4)2SO4盐析、透析、DEAE SepIlarose Fast F1ow离子交换层析以及Sephadex G-100凝胶过滤层析分离纯化,获得电泳纯的棉铃虫单粒包埋型核型多角体病毒(HasNPV)几丁质酶,回收率为19.64%,纯化17.74倍;经HaSNPV几丁质酶毒理学研究,通过触杀作用和胃毒作用,几丁质酶使小菜蛾幼虫细胞液化,导致死亡,其作用效果与几丁质酶浓度和作用时间成正比,且胃毒作用大于触杀作用;经小麦根腐、烟草赤星、番茄灰霉和苹果炭疽等植物病原真菌的抑菌实验,表明几丁质酶可以通过酶解真菌细胞壁中的几丁质达到抑茵作用.

  20. Trusted Domain

    DEFF Research Database (Denmark)

    Hjorth, Theis Solberg; Torbensen, Rune


    In the digital age of home automation and with the proliferation of mobile Internet access, the intelligent home and its devices should be accessible at any time from anywhere. There are many challenges such as security, privacy, ease of configuration, incompatible legacy devices, a wealth...... remote access via IP-based devices such as smartphones. The Trusted Domain platform fits existing legacy technologies by managing their interoperability and access controls, and it seeks to avoid the security issues of relying on third-party servers outside the home. It is a distributed system...... of wireless standards, limited resources of embedded systems, etc. Taking these challenges into account, we present a Trusted Domain home automation platform, which dynamically and securely connects heterogeneous networks of Short-Range Wireless devices via simple non-expert user. interactions, and allows...

  1. On the Insertion Time of Cuckoo Hashing

    CERN Document Server

    Fountoulakis, Nikolaos; Steger, Angelika


    Cuckoo hashing is an efficient technique for creating large hash tables with high space utilization and guaranteed constant access times. There, each item can be placed in a location given by any one out of k different hash functions. In this paper we investigate further the random walk heuristic for inserting in an online fashion new items into the hash table. Provided that k > 2 and that the number of items in the table is below (but arbitrarily close) to the theoretically achievable load threshold, we show a polylogarithmic bound for the maximum insertion time that holds with high probability.

  2. RERTR-12 Insertion 2 Irradiation Summary Report

    Energy Technology Data Exchange (ETDEWEB)

    D. M. Perez; G. S. Chang; D. M. Wachs; G. A. Roth; N. E. Woolstenhulme


    The Reduced Enrichment for Research and Test Reactor (RERTR) experiment RERTR-12 was designed to provide comprehensive information on the performance of uranium-molybdenum (U-Mo) based monolithic fuels for research reactor applications.1 RERTR-12 insertion 2 includes the capsules irradiated during the last three irradiation cycles. These capsules include Z, Y1, Y2 and Y3 type capsules. The following report summarizes the life of the RERTR-12 insertion 2 experiment through end of irradiation, including as-run neutronic analysis results, thermal analysis results and hydraulic testing results.

  3. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses. (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook


    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1-CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1.

  4. STUDI PENDAHULUAN ENZIM KITINASE EXTRASELULER YANG DIHASILKAN OLEH ISOLAT BAKTERI ASAL MANADO 1 [Preliminary Study of Extracellular Chitinase Produced by Bacteria Isolated from Manado

    Directory of Open Access Journals (Sweden)

    E.Y. Purwani 1


    Full Text Available Chitinolytic bacteria were isolated from several exotic area in Manado Province. The most potential isolate, namely 13.26, was isolated from Tompaso. The isolate was cultured in the thermus medium containing colloidal chitin as a carbon source for 5 days at 55°C to produce chitinase. It was observed that chitinase was most active at 65��C and the optimum pH was 8 in boric acid-borax buffer. Ammonium sulfate (50% saturation precipitation of the protein increased the specific activity of the enzyme from 0.20 unit/mg protein (in culture supernatant to 0.28 unit/mg protein. The molecular weight as estimated by zymogram analysis was180 kDa

  5. Silencing of Target Chitinase Genes via Oral Delivery of dsRNA Caused Lethal Phenotypic Effects in Mythimna separata (Lepidoptera: Noctuidae). (United States)

    Cao, Budao; Bao, Wenhua; Wuriyanghan, Hada


    Mythimna separata walker (Lepidoptera: Noctuidae) is a polyphagous, migratory corn pest. Outbreak of M. separata has led to severe damage to corn production recently in China. RNAi (RNA interference) is a gene silencing technology applied both in model and non-model organisms, and it is especially useful for the latter in which the reverse genetic research tools are not available. RNAi approach was broadly investigated in many plant pathogens and was used for the generation of anti-pest transgenic plants. We are proposing to use this technology to silence M. separata endogenous genes, thereby, providing a biocontrol method for this insect. Feeding of dsRNAs for target Chitinase genes resulted in substantial decreases of their transcript levels in M. separata. Furthermore, silencing of target Chitinase genes led to phenotypic effects such as reduced body weight and increased mortality. Our study provided both reverse genetic research tool and potential control strategy for this insect species.

  6. Oat (Avena sativa) seed extract as an antifungal food preservative through the catalytic activity of a highly abundant class I chitinase

    DEFF Research Database (Denmark)

    Sørensen, Hans Peter; Madsen, Lone Søvad; Petersen, Jørgen;


    to prevent the formation of P. roqueforti colonies. Proteins in the oat seed extracts were fractionated by column chromatography and proteins in fractions containing antifungal activity were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searches. Identified antifungal......Extracts from different higher plants were screened for the ability to inhibit the growth of Penicillium roqueforti, a major contaminating species in industrial food processing. Oat (Avena sativa) seed extracts exhibited a high degree of antifungal activity and could be used directly on rye bread...... candidates included thaumatin-like proteins, 1,3-beta-glucanase, permatin precursor, pathogenesis-related protein type 1, and chitinases of class I and II. Class I chitinase could be specifically removed from the extracts and was found to be indispensable for 50% of the P. roqueforti inhibiting activity...

  7. Digital Image Watermarking With Random Selection of Watermark Insertion Having Adaptive Strength

    Directory of Open Access Journals (Sweden)

    G.S. Kalra


    Full Text Available We have presented an algorithm of digital image watermarking for gray scale images which we implemented in frequency domain. Before inserting the watermark, we added the Hamming codes row wise as well column wise. Two encryption techniques were implemented on the ECC inserted watermark for its security. The pixel position for inserting the watermark was calculated using starting row and column number for that 8×8 block. Pixel embedding strength is calculated using criteria that low frequency is robust in general signal processing attacks, thus choosing less value to be embedded and vice-versa. Results show that the watermarking algorithm is robust against common signal processing attacks. The algorithm is tested against multiple attacks also.

  8. Thermal Performance of the XRS Helium Insert (United States)

    Breon, Susan R.; DiPirro, Michael J.; Tuttle, James G.; Shirron, Peter J.; Warner, Brent A.; Boyle, Robert F.; Canavan, Edgar R.


    The X-Ray Spectrometer (XRS) is an instrument on the Japanese Astro-E satellite, scheduled for launch early in the year 2000. The XRS Helium Insert comprises a superfluid helium cryostat, an Adiabatic Demagnetization Refrigerator (ADR), and the XRS calorimeters with their cold electronics. The calorimeters are capable of detecting X-rays over the energy range 0.1 to 10 keV with a resolution of 12 eV. The Helium Insert completed its performance and verification testing at Goddard in January 1999. It was shipped to Japan, where it has been integrated with the neon dewar built by Sumitomo Heavy Industries. The Helium Insert was given a challenging lifetime requirement of 2.0 years with a goal of 2.5 years. Based on the results of the thermal performance tests, the predicted on-orbit lifetime is 2.6 years with a margin of 30%. This is the result of both higher efficiency in the ADR cycle and the low temperature top-off, more than compensating for an increase in the parasitic heat load. This paper presents a summary of the key design features and the results of the thermal testing of the XRS Helium Insert.

  9. Chitinase-like (CTL) and cellulose synthase (CESA) gene expression in gelatinous-type cellulosic walls of flax (Linum usitatissimum L.) bast fibers. (United States)

    Mokshina, Natalia; Gorshkova, Tatyana; Deyholos, Michael K


    Plant chitinases (EC and chitinase-like (CTL) proteins have diverse functions including cell wall biosynthesis and disease resistance. We analyzed the expression of 34 chitinase and chitinase-like genes of flax (collectively referred to as LusCTLs), belonging to glycoside hydrolase family 19 (GH19). Analysis of the transcript expression patterns of LusCTLs in the stem and other tissues identified three transcripts (LusCTL19, LusCTL20, LusCTL21) that were highly enriched in developing bast fibers, which form cellulose-rich gelatinous-type cell walls. The same three genes had low relative expression in tissues with primary cell walls and in xylem, which forms a xylan type of secondary cell wall. Phylogenetic analysis of the LusCTLs identified a flax-specific sub-group that was not represented in any of other genomes queried. To provide further context for the gene expression analysis, we also conducted phylogenetic and expression analysis of the cellulose synthase (CESA) family genes of flax, and found that expression of secondary wall-type LusCESAs (LusCESA4, LusCESA7 and LusCESA8) was correlated with the expression of two LusCTLs (LusCTL1, LusCTL2) that were the most highly enriched in xylem. The expression of LusCTL19, LusCTL20, and LusCTL21 was not correlated with that of any CESA subgroup. These results defined a distinct type of CTLs that may have novel functions specific to the development of the gelatinous (G-type) cellulosic walls.

  10. Chitinase-like (CTL and cellulose synthase (CESA gene expression in gelatinous-type cellulosic walls of flax (Linum usitatissimum L. bast fibers.

    Directory of Open Access Journals (Sweden)

    Natalia Mokshina

    Full Text Available Plant chitinases (EC and chitinase-like (CTL proteins have diverse functions including cell wall biosynthesis and disease resistance. We analyzed the expression of 34 chitinase and chitinase-like genes of flax (collectively referred to as LusCTLs, belonging to glycoside hydrolase family 19 (GH19. Analysis of the transcript expression patterns of LusCTLs in the stem and other tissues identified three transcripts (LusCTL19, LusCTL20, LusCTL21 that were highly enriched in developing bast fibers, which form cellulose-rich gelatinous-type cell walls. The same three genes had low relative expression in tissues with primary cell walls and in xylem, which forms a xylan type of secondary cell wall. Phylogenetic analysis of the LusCTLs identified a flax-specific sub-group that was not represented in any of other genomes queried. To provide further context for the gene expression analysis, we also conducted phylogenetic and expression analysis of the cellulose synthase (CESA family genes of flax, and found that expression of secondary wall-type LusCESAs (LusCESA4, LusCESA7 and LusCESA8 was correlated with the expression of two LusCTLs (LusCTL1, LusCTL2 that were the most highly enriched in xylem. The expression of LusCTL19, LusCTL20, and LusCTL21 was not correlated with that of any CESA subgroup. These results defined a distinct type of CTLs that may have novel functions specific to the development of the gelatinous (G-type cellulosic walls.

  11. A critical appraisal of medication package inserts

    Directory of Open Access Journals (Sweden)

    Pranjit Narzaree


    Full Text Available Introduction: Package Inserts (PIs refers to officially specified document that accompanies a drug for relevant, updated and unbiased information for rational drug use based on regulatory guidelines as per section 6.2 and 6.3 of schedule D of Indian Drug and cosmetic Act 1945. But some studies had shown non-uniformity with suboptimal level of informations which frequently can lead to medication errors. Hence this study was conducted to evaluate the completeness of PIs.Aim: To critically evaluate package inserts of allopathic medicines.Material and Methods: 100 allopathic drug PIs were collected from pharmacies in Rohtak and were checked for the presence of each heading as per schedule D criteria, followed by scrutiny of the information included under the heading. Indian guidelines were also compared with US FDA guidelines for PIs.Scoring of package inserts: The informations were evaluated for completeness and scored as 1 if present otherwise scored as zero for no information or partial information. Scores for each heading were calculated by totaling the scores of all the package inserts. The total scores were expressed as absolute numbers and percentages.Results:  On an average PIs analyzed for the completeness of the criteria scored 10 (Mean± SD = 9.73±2.48 out of 16. Absence of common layout and headings caused inconvenience. In comparison to US FDA guidelines it lacked, disclaimer statement, boxed warning, revision date, approval date, toll-free number etc.Conclusion: PIs don’t seem to be serving effectively because of multiple deficiencies like completeness, uniformity, absence of headings.Keywords: Critical appraisal, package inserts.

  12. Optimalisation of expression conditions for production of round-leaf sundew chitinase (Drosera rotundifolia L. in three E. coli expression strains

    Directory of Open Access Journals (Sweden)

    Miroslav Rajninec


    Full Text Available Round-leaf sundew (Drosera rotundifolia L., family Droseraceae, genus Drosera, is one of a few plant species with a strong antifungal potential. Chitinases of carnivorous plants play an important role in decomposition of chitin-containing cell structures of insect prey. The cell wall of many phytopathogenic fungi also contains chitin, which can be utilized by chitinases, thus round-leaf sundew represents an interesting gene source for plant biotechnology. The purpose of this study was to compare the suitability of 3 different E. coli expression strains (E. coli BL21- CodonPlus® (DE3-RIPL, E. coli ArcticExpress (DE3RIL and E. coli SHuffle® T7 for production and isolation of heterologous round-leaf sundew chitinase (DrChit. Results showed that the recombinant protein was successfully expressed in all three strains, but occurred in insoluble protein fraction. To get the DrChit protein into soluble protein fraction some modifications concerning to induction temperatures and concentration of the IPTG inductor were tested. In addition, composition of lysis buffer has been modified with supplementation of strong non-ionic detergents, Triton® X100 and Tween® 20, respectively. As these modifications didn’t increase the amount of the DrChit protein in soluble fraction, therefore, its isolation under denaturing conditions and subsequent refolding for activity assays is recommended.

  13. Wide-range antifungal antagonism of Paenibacillus ehimensis IB-X-b and its dependence on chitinase and beta-1,3-glucanase production. (United States)

    Aktuganov, G; Melentjev, A; Galimzianova, N; Khalikova, E; Korpela, T; Susi, P


    Previously, we isolated a strain of Bacillus that had antifungal activity and produced lytic enzymes with fungicidal potential. In the present study, we identified the bacterium as Paenibacillus ehimensis and further explored its antifungal properties. In liquid co-cultivation assays, P. ehimensis IB-X-b decreased biomass production of several pathogenic fungi by 45%-75%. The inhibition was accompanied by degradation of fungal cell walls and alterations in hyphal morphology. Residual medium from cultures of P. ehimensis IB-X-b inhibited fungal growth, indicating the inhibitors were secreted into the medium. Of the 2 major lytic enzymes, chitinases were only induced by chitin-containing substrates, whereas beta-1,3-glucanase showed steady levels in all carbon sources. Both purified chitinase and beta-1,3-glucanase degraded cell walls of macerated fungal mycelia, whereas only the latter also degraded cell walls of intact mycelia. The results indicate synergism between the antifungal action mechanisms of these enzymes in which beta-1,3-glucanase is the initiator of the cell wall hydrolysis, whereas the degradation process is reinforced by chitinases. Paenibacillus ehimensis IB-X-b has pronounced antifungal activity with a wide range of fungi and has potential as a biological control agent against plant pathogenic fungi.


    Directory of Open Access Journals (Sweden)

    Shibani Devi


    Full Text Available INTRODUCTION According to National Family Health Survey-3, Indian women have 13% unmet need for contraception and according to District Level Household & Facility Survey-3, it is 21.3% in the postpartum period. Postpartum intrauterine contraceptive device insertion - both immediately post-placental delivery and somewhat later, but within 48 hours after delivery are options which merit consideration as the woman is likely to have a high motivation for accepting contraception and the healthcare centre provides a convenient setting for insertion of IUCD. AIM Comparison of efficacy and complications of IUCD insertions in post-placental with interval period: 6-month followup. METHOD This perspective study was conducted among 100 women: - 50 women had IUCD inserted within 10 minutes of placental delivery and 50 had insertion more than 6 weeks after delivery. They were followed till 6 months post insertion and were compared regarding early and late complications, continuation rates and expulsion rates. RESULT At the end of six months, we found higher occurrence of lower abdominal pain, heavy menstrual bleeding in case of interval insertion as compared to post-placental insertion which was statistically significant (p value-0.04 & 0.007 respectively. However, the expulsion rates of post-placental IUCD were somewhat elevated (14% compared to interval insertions (2%. Continuation rates at the end of 6 months in both the groups were 82% and 86% respectively which is comparable. CONCLUSION Post-placental IUCD is thus found to be an ideal method to meet the unmet need of postpartum women as it is easily accessible and convenient for both women and their health care providers, is associated with less discomfort and fewer side effects and allow women to obtain safe, long acting, highly effective contraception while still in the health care system.

  15. Structural and Histone Binding Ability Characterizations of Human PWWP Domains

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)


    The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

  16. Perpendicular Magnetization Behavior of Low- Temperature Ordered FePt Films with Insertion of Ag Nanolayers

    Directory of Open Access Journals (Sweden)

    Da-Hua Wei


    Full Text Available FePt-Ag nanocomposite films with large perpendicular magnetic anisotropy have been fabricated by alternate-atomic-layer electron beam evaporation onto MgO(100 substrates at the low temperature of 300 °C. Their magnetization behavior and microstructure have been studied. The surface topography was observed and varied from continuous to nanogranular microstructures with insertion of Ag nanolayers into Fe/Pt bilayer films. The measurement of angular-dependent coercivity showed a tendency of the domain-wall motion as a typical peak behavior shift toward more like a coherent Stoner-Wohlfarth rotation type with the insertion of Ag nanolayers into the FePt films. On the other hand, the inter-grain interaction was determined from a Kelly-Henkel plot. The FePt film without insertion of Ag nanolayers has a positive δM, indicating strong exchange coupling between neighboring grains, whereas the FePt film with insertion of Ag nanolayers has a negative δM, indicating that inter-grain exchange coupling is weaker, thus leading to the presence of dipole interaction in the FePt–Ag nanogranular films. The magnetic characteristic measurements confirmed that the perpendicular magnetization reversal behavior and related surface morphology of low-temperature-ordered FePt(001 nanogranular films can be systematically controlled by the insertion of Ag nanolayers into the FePt system for next generation magnetic storage medium applications.

  17. Comparison of conventional Injection Mould Inserts to Additively Manufactured Inserts using Life Cycle Assessment

    DEFF Research Database (Denmark)

    Hofstätter, Thomas; Bey, Niki; Mischkot, Michael


    Polymer Additive Manufacturing can be used to produce soft tooling inserts for injection moulding. Compared to conventional tooling, the energy and time consumption during production are significantly lower. As the life time of such inserts is significantly shorter than the life time of traditional...... of their potential environmental impact and yield throughout the development and pilot phase. Insert geometry is particularly advantageous for pilot production and small production sizes. In this research, Life Cycle Assessment is used to compare the environmental impact of soft tooling by Additive Manufacturing...... (using Digital Light Processing) and three traditional methods for the manufacture of inserts (milling of brass, steel, and aluminium) for injection moulds during the pre-production phase....

  18. Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon; Heo, Won Il; Kim, Kyung Won; Kim, Kyu Earn [Department of Pediatrics and Institute of Allergy, Severance Medical Research Institute, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of); Sohn, Myung Hyun, E-mail: [Department of Pediatrics and Institute of Allergy, Severance Medical Research Institute, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of)


    Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally

  19. Development of ocular inserts for cattle. (United States)

    Greer, R T; Ryoo, J P


    Ring shaped ocular inserts have been developed to administer a therapeutic level of tylosin tartrate throughout a five day period to treat pinkeye in cattle. The inserts are based on polyvinyl chloride rings which are dip coated with a copolymer containing the antibiotic (tylosin tartrate). Scanning electron microscope (SEM) characterization of surfaces has been of value to evaluate the presence and extent of surface flaws in the hydrogel coating, and to contribute to improvement in fabrication of the rings to insure the establishment of satisfactory seals at joints, uniformity of microporosity and cross sections, and the absence of significant cracking or flaking. In vitro release rates were determined using thin layer chromatography techniques, and rates were seen to be above a few micrograms of antibiotic per hour for experiments as long as nine days at simulated tear rates as high as 2 milliliters per hour.

  20. Perception and Action in Teleoperated Needle Insertion. (United States)

    Nisky, I; Pressman, A; Pugh, C M; Mussa-Ivaldi, F A; Karniel, A


    We studied the effect of delay on perception and action in contact with a force field that emulates elastic soft tissue with a rigid nonlinear boundary. Such a field is similar to forces exerted on a needle during teleoperated needle insertion. We found that delay causes motor underestimation of the stiffness of this nonlinear soft tissue, without perceptual change. These experimental results are supported by simulation of a simplified mechanical model of the arm and neural controller, and a model for perception of stiffness, which is based on regression in the force-position space. In addition, we show that changing the gain of the teleoperation channel cancels the motor effect of delay without adding perceptual distortion. We conclude that it is possible to achieve perceptual and motor transparency in virtual one-dimensional remote needle insertion task.

  1. Anionic phospholipids modulate peptide insertion into membranes. (United States)

    Liu, L P; Deber, C M


    While the insertion of a hydrophobic peptide or membrane protein segment into the bilayer can be spontaneous and driven mainly by the hydrophobic effect, anionic lipids, which comprise ca. 20% of biological membranes, provide a source of electrostatic attractions for binding of proteins/peptides into membranes. To unravel the interplay of hydrophobicity and electrostatics in the binding of peptides into membranes, we designed peptides de novo which possess the typical sequence Lys-Lys-Ala-Ala-Ala-X-Ala-Ala-Ala-Ala-Ala-X-Ala-Ala-Trp-Ala-Ala-X-Ala-Al a-Ala-Lys-Lys-Lys-Lys-amide, where X residues correspond to "guest" residues which encompass a range of hydrophobicity (Leu, Ile, Gly, and Ser). Circular dichroism spectra demonstrated that peptides were partially (40-90%) random in aqueous buffer but were promoted to form 100% alpha-helical structures by anionic lipid micelles. In neutral lipid micelles, only the relatively hydrophobic peptides (X = L and I) spontaneously adopted the alpha-helical conformation, but when 25% of negatively charged lipids were mixed in to mimic the content of anionic lipids in biomembranes, the less hydrophobic (X = S and G) peptides then formed alpha-helical conformations. Consistent with these findings, fluorescence quenching by the aqueous-phase quencher iodide indicated that in anionic (dimyristoylphosphatidylglycerol) vesicles, the peptide Trp residue was buried in the lipid vesicle hydrophobic core, while in neutral (dimyristoylphosphatidylcholine) vesicles, only hydrophobic (X = L and I) peptides were shielded from the aqueous solution. Trp emission spectra of peptides in the presence of phospholipids doxyl-labeled at the 5-, 7-, 10-, 12-, and 16-fatty acid positions implied not only a transbilayer orientation for inserted peptides but also that mixed peptide populations (transbilayer + surface-associated) may arise. Overall results suggest that for hydrophobic peptides with segmental threshold hydrophobicity below that which

  2. [Historical evolution of package inserts in Brazil]. (United States)

    Caldeira, Telma Rodrigues; Neves, Eugênio Rodrigo Zimmer; Perini, Edson


    In Brazil, package inserts provide key information on pharmaceuticals. The current study analyzes the evolution of package inserts and the impact on this process by scientific research and development, globalization of information, and various health policies. The study began with a retrospective review of Brazilian health legislation until 1920, the year when the National Public Health Department was created. The analysis of documents on the evolution of health regulation in Brazil began with the Brazilian Pharmaceutical Collection-Health Rulings. The second stage of the study involved a search of standards and norms in VISALEGIS: Health Surveillance Legislation, Portal for Legislation from the National Congressional Information System and the Health Legislation System. Package inserts became an important vehicle for information in the country and underwent important regulatory changes in the latter half of the 20th century. From 1946 to 2006, the number of mandatory items increased, with more in-depth description. However, the standardization of information for medicines with the same active ingredient failed to materialize, despite its importance and the various legal initiatives in this direction.

  3. App-assisted external ventricular drain insertion. (United States)

    Eftekhar, Behzad


    The freehand technique for insertion of an external ventricular drain (EVD) is based on fixed anatomical landmarks and does not take individual variations into consideration. A patient-tailored approach based on augmented-reality techniques using devices such as smartphones can address this shortcoming. The Sina neurosurgical assist (Sina) is an Android mobile device application (app) that was designed and developed to be used as a simple intraoperative neurosurgical planning aid. It overlaps the patient's images from previously performed CT or MRI studies on the image seen through the device camera. The device is held by an assistant who aligns the images and provides information about the relative position of the target and EVD to the surgeon who is performing EVD insertion. This app can be used to provide guidance and continuous monitoring during EVD placement. The author describes the technique of Sina-assisted EVD insertion into the frontal horn of the lateral ventricle and reports on its clinical application in 5 cases as well as the results of ex vivo studies of ease of use and precision. The technique has potential for further development and use with other augmented-reality devices.

  4. Beamline Insertions Manager at Jefferson Lab

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Michael C. [Jefferson Lab., Newport News, VA (United States)


    The beam viewer system at Jefferson Lab provides operators and beam physicists with qualitative and quantitative information on the transverse electron beam properties. There are over 140 beam viewers installed on the 12 GeV CEBAF accelerator. This paper describes an upgrade consisting of replacing the EPICS-based system tasked with managing all viewers with a mixed system utilizing EPICS and high-level software. Most devices, particularly the beam viewers, cannot be safely inserted into the beam line during high-current beam operations. Software is partly responsible for protecting the machine from untimely insertions. The multiplicity of beam-blocking and beam-vulnerable devices motivates us to try a data-driven approach. The beamline insertions application components are centrally managed and configured through an object-oriented software framework created for this purpose. A rules-based engine tracks the configuration and status of every device, along with the beam status of the machine segment containing the device. The application uses this information to decide on which device actions are allowed at any given time.

  5. .Gov Domains API (United States)

    General Services Administration — This dataset offers the list of all .gov domains, including state, local, and tribal .gov domains. It does not include .mil domains, or other federal domains outside...

  6. Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor. (United States)

    Hirose, Tomoyasu; Maita, Nobuo; Gouda, Hiroaki; Koseki, Jun; Yamamoto, Tsuyoshi; Sugawara, Akihiro; Nakano, Hirofumi; Hirono, Shuichi; Shiomi, Kazuro; Watanabe, Takeshi; Taniguchi, Hisaaki; Sharpless, K Barry; Omura, Satoshi; Sunazuka, Toshiaki


    The Huisgen cycloaddition of azides and alkynes, accelerated by target biomolecules, termed "in situ click chemistry," has been successfully exploited to discover highly potent enzyme inhibitors. We have previously reported a specific Serratia marcescens chitinase B (SmChiB)-templated syn-triazole inhibitor generated in situ from an azide-bearing inhibitor and an alkyne fragment. Several in situ click chemistry studies have been reported. Although some mechanistic evidence has been obtained, such as X-ray analysis of [protein]-["click ligand"] complexes, indicating that proteins act as both mold and template between unique pairs of azide and alkyne fragments, to date, observations have been based solely on "postclick" structural information. Here, we describe crystal structures of SmChiB complexed with an azide ligand and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating syn-triazole formation, which allows generation of its own distinct inhibitor. We have also performed density functional theory calculations based on the X-ray structure to explore the acceleration of the Huisgen cycloaddition by SmChiB. The density functional theory calculations reasonably support that SmChiB plays a role by the cage effect during the pretranslation and posttranslation states of selective syn-triazole click formation.

  7. CytR Is a Global Positive Regulator of Competence, Type VI Secretion, and Chitinases in Vibrio cholerae. (United States)

    Watve, Samit S; Thomas, Jacob; Hammer, Brian K


    The facultative pathogen Vibrio cholerae transitions between its human host and aquatic reservoirs where it colonizes chitinous surfaces. Growth on chitin induces expression of chitin utilization genes, genes involved in DNA uptake by natural transformation, and a type VI secretion system that allows contact-dependent killing of neighboring bacteria. We have previously shown that the transcription factor CytR, thought to primarily regulate the pyrimidine nucleoside scavenging response, is required for natural competence in V. cholerae. Through high-throughput RNA sequencing (RNA-seq), we show that CytR positively regulates the majority of competence genes, the three type VI secretion operons, and the four known or predicted chitinases. We used transcriptional reporters and phenotypic analysis to determine the individual contributions of quorum sensing, which is controlled by the transcription factors HapR and QstR; chitin utilization that is mediated by TfoX; and pyrimidine starvation that is orchestrated by CytR, toward each of these processes. We find that in V. cholerae, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.

  8. Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity. (United States)

    Filippi, Antonio; Petrussa, Elisa; Rajcevic, Uros; Čurin Šerbec, Vladka; Passamonti, Sabina; Renzone, Giovanni; Scaloni, Andrea; Zancani, Marco; Vianello, Angelo; Braidot, Enrico


    In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab) was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR) class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

  9. CytR Is a Global Positive Regulator of Competence, Type VI Secretion, and Chitinases in Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Samit S Watve

    Full Text Available The facultative pathogen Vibrio cholerae transitions between its human host and aquatic reservoirs where it colonizes chitinous surfaces. Growth on chitin induces expression of chitin utilization genes, genes involved in DNA uptake by natural transformation, and a type VI secretion system that allows contact-dependent killing of neighboring bacteria. We have previously shown that the transcription factor CytR, thought to primarily regulate the pyrimidine nucleoside scavenging response, is required for natural competence in V. cholerae. Through high-throughput RNA sequencing (RNA-seq, we show that CytR positively regulates the majority of competence genes, the three type VI secretion operons, and the four known or predicted chitinases. We used transcriptional reporters and phenotypic analysis to determine the individual contributions of quorum sensing, which is controlled by the transcription factors HapR and QstR; chitin utilization that is mediated by TfoX; and pyrimidine starvation that is orchestrated by CytR, toward each of these processes. We find that in V. cholerae, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.

  10. Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Antonio Filippi


    Full Text Available In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

  11. Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP). (United States)

    Gutiérrez-Román, Martha Ingrid; Dunn, Michael F; Tinoco-Valencia, Raunel; Holguín-Meléndez, Francisco; Huerta-Palacios, Graciela; Guillén-Navarro, Karina


    With the goal of understanding the chitinolytic mechanism of the potential biological control strain Serratia marcescens CFFSUR-B2, genes encoding chitinases ChiA, ChiB and ChiC, chitobiase (Chb) and chitin binding protein (CBP) were cloned, the protein products overexpressed in Escherichia coli as 6His-Sumo fusion proteins and purified by affinity chromatography. Following affinity tag removal, the chitinolytic activity of the recombinant proteins was evaluated individually and in combination using colloidal chitin as substrate. ChiB and ChiC were highly active while ChiA was inactive. Reactions containing both ChiB and ChiC showed significantly increased N-acetylglucosamine trimer and dimer formation, but decreased monomer formation, compared to reactions with either enzyme alone. This suggests that while both ChiB and ChiC have a general affinity for the same substrate, they attack different sites and together degrade chitin more efficiently than either enzyme separately. Chb and CBP in combination with ChiB and ChiC (individually or together) increased their chitinase activity. We report for the first time the potentiating effect of Chb on the activity of the chitinases and the synergistic activity of a mixture of all five proteins (the three chitinases, Chb and CBP). These results contribute to our understanding of the mechanism of action of the chitinases produced by strain CFFSUR-B2 and provide a molecular basis for its high potential as a biocontrol agent against fungal pathogens.

  12. New cardiac retractor for epicardial electrode insertion via subxiphoid approach. (United States)

    Sakamoto, T; Arai, H; Suzuki, A


    A new retractor for the insertion of epicardial screw-in electrodes is described. We have found that this instrument can be easily applied to the heart and gives excellent exposure for electrode insertion.

  13. Experimental testing of exchangeable cutting inserts cutting ability


    Čep, Robert; Janásek, Adam; Čepová Lenka; Petrů, Jana; Ivo HLAVATÝ; Car, Zlatan; Hatala, Michal


    The article deals with experimental testing of the cutting ability of exchangeable cutting inserts. Eleven types of exchangeable cutting inserts from five different manufacturers were tested. The tested cutting inserts were of the same shape and were different especially in material and coating types. The main aim was both to select a suitable test for determination of the cutting ability of exchangeable cutting inserts and to design such testing procedure that could make it possible...

  14. Commercial Generic Bioprocessing Apparatus Science Insert - 03 (United States)

    Moreno, Nancy; Stodieck, Louis; Cushing, Paula; Stowe, Mark; Hamilton, Mary Ann; Werner, Ken


    Commercial Generic Bioprocessing Apparatus Science Insert - 03 (CSI-03) is the third set of investigations in the CSI program series. The CSI program provides the K-12 community opportunities to utilize the unique microgravity environment of the International Space Station as part of the regular classroom to encourage learning and interest in science, technology, engineering and math. CSI-03 will examine the complete life cycle of the painted lady butterfly and the ability of an orb weaving spider to spin a web, eat and remain healthy in space.

  15. Compiler-assisted static checkpoint insertion (United States)

    Long, Junsheng; Fuchs, W. K.; Abraham, Jacob A.


    This paper describes a compiler-assisted approach for static checkpoint insertion. Instead of fixing the checkpoint location before program execution, a compiler enhanced polling mechanism is utilized to maintain both the desired checkpoint intervals and reproducible checkpoint 1ocations. The technique has been implemented in a GNU CC compiler for Sun 3 and Sun 4 (Sparc) processors. Experiments demonstrate that the approach provides for stable checkpoint intervals and reproducible checkpoint placements with performance overhead comparable to a previously presented compiler assisted dynamic scheme (CATCH) utilizing the system clock.

  16. Phoenix's Probe Inserted in Martian Soil (United States)


    The Phoenix Mars lander's robotic-arm camera took this image of the spacecraft's thermal and electrical-conductivity probe (TECP) inserted into Martian soil on day 149 of the mission. Phoenix landed on Mars' northern plains on May 25, 2008, landing. The robotic-arm camera acquired this image at 16:02:41 local solar time. The camera pointing was elevation -72.6986 degrees and azimuth 2.1093 degrees. The Phoenix mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  17. T-tube insertion for sclerotic subglottic stenosis. (United States)

    Goto, Taichiro; Kato, Ryoichi


    T-tube insertion is effective treatment for subglottic stenosis, but it is generally difficult due to bending of the T-tube. In a 52-year-old woman with relapsing polychondritis, a T-tube was inserted after predilatation using Hegar dilators. We describe the details of our T-tube insertion methods for sclerotic subglottic stenosis.

  18. Transposon insertion mapping with PIMMS – Pragmatic Insertional Mutation Mapping System.

    Directory of Open Access Journals (Sweden)

    Adam eBlanchard


    Full Text Available The PIMMS (Pragmatic Insertional Mutation Mapping System pipeline has been developed for simple conditionally essential genome discovery experiments in bacteria. Capable of using raw sequence data files alongside a FASTA sequence of the reference genome and GFF file, PIMMS will generate a tabulated output of each coding sequence with corresponding mapped insertions accompanied with normalised results enabling streamlined analysis. This allows for a quick assay of the genome to identify conditionally essential genes on a standard desktop computer prioritising results for further investigation.Availability: The PIMMS script, manual and accompanying test data is freely available at

  19. Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro

    Institute of Scientific and Technical Information of China (English)

    GUO Shu-yuan; LI Jie; CHEN Zhen; HE Kang-lai


    Domain I of the activated Crystal protein from Bacillus thuringiensis has a sevenα-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Cry1Ie is toxic to Asian corn borer, Ostrinia furnacalis (Guenée), and plays important roles in insect biological control. The domain I from Cry1Ie has been expressed and puriifed in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Cry1Ie-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Cry1Ie-domain I plays no role in receptor binding as it had a nonspeciifc binding with the brush border membrane vesicles of the Asian corn borer.

  20. A Diverse Range of Bacterial and Eukaryotic Chitinases Hydrolyzes the LacNAc (Galβ1–4GlcNAc) and LacdiNAc (GalNAcβ1–4GlcNAc) Motifs Found on Vertebrate and Insect Cells* (United States)

    Frederiksen, Rikki F.; Yoshimura, Yayoi; Storgaard, Birgit G.; Paspaliari, Dafni K.; Petersen, Bent O.; Chen, Kowa; Larsen, Tanja; Duus, Jens Ø.; Ingmer, Hanne; Bovin, Nicolai V.; Westerlind, Ulrika; Blixt, Ola; Palcic, Monica M.; Leisner, Jørgen J.


    There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1–4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1–4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1–4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1–6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1–6 bond in LacNAcβ1–6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis. PMID:25561735

  1. A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galβ1-4GlcNAc) and LacdiNAc (GalNAcβ1-4GlcNAc) motifs found on vertebrate and insect cells. (United States)

    Frederiksen, Rikki F; Yoshimura, Yayoi; Storgaard, Birgit G; Paspaliari, Dafni K; Petersen, Bent O; Chen, Kowa; Larsen, Tanja; Duus, Jens Ø; Ingmer, Hanne; Bovin, Nicolai V; Westerlind, Ulrika; Blixt, Ola; Palcic, Monica M; Leisner, Jørgen J


    There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1-4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1-6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1-6 bond in LacNAcβ1-6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.

  2. Polymeric Biodegradable Stent Insertion in the Esophagus

    Directory of Open Access Journals (Sweden)

    Kai Yang


    Full Text Available Esophageal stent insertion has been used as a well-accepted and effective alternative to manage and improve the quality of life for patients diagnosed with esophageal diseases and disorders. Current stents are either permanent or temporary and are fabricated from either metal or plastic. The partially covered self-expanding metal stent (SEMS has a firm anchoring effect and prevent stent migration, however, the hyperplastic tissue reaction cause stent restenosis and make it difficult to remove. A fully covered SEMS and self-expanding plastic stent (SEPS reduced reactive hyperplasia but has a high migration rate. The main advantage that polymeric biodegradable stents (BDSs have over metal or plastic stents is that removal is not require and reduce the need for repeated stent insertion. But the slightly lower radial force of BDS may be its main shortcoming and a post-implant problem. Thus, strengthening support of BDS is a content of the research in the future. BDSs are often temporarily effective in esophageal stricture to relieve dysphagia. In the future, it can be expect that biodegradable drug-eluting stents (DES will be available to treat benign esophageal stricture, perforations or leaks with additional use as palliative modalities for treating malignant esophageal stricture, as the bridge to surgery or to maintain luminal patency during neoadjuvant chemoradiation.

  3. Phoenix Conductivity Probe Inserted in Martian Soil (United States)


    This series of six images from the Robotic Arm Camera on NASA's Phoenix Mars Lander records the first time that the four spikes of the lander's thermal and electrical conductivity probe were inserted into Martian soil. The images were taken on July 8, 2008, during the Phoenix mission's 43rd Martian day, or sol, since landing. The insertion visible from the shadows cast on the ground on that sol was a validation test of the procedure. The spikes on the probe are about 1.5 centimeters or half an inch long. The science team will use the probe tool to assess how easily heat and electricity move through the soil from one spike to another. Such measurements can provide information about frozen or unfrozen water in the soil. The probe is mounted on the 'knuckle' of Phoenix's Robotic Arm. It has already been used for assessing water vapor in the atmosphere when it is held above the ground. The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is led by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  4. Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: a study involving site-directed mutagenesis, HPLC, and real-time ESI-MS. (United States)

    Taira, Toki; Fujiwara, Maho; Dennhart, Nicole; Hayashi, Hiroko; Onaga, Shoko; Ohnuma, Takayuki; Letzel, Thomas; Sakuda, Shohei; Fukamizo, Tamo


    Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5-10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of +1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC(50) value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from -3 to -1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.

  5. Insertion of coherence requests for debugging a multiprocessor (United States)

    Blumrich, Matthias A.; Salapura, Valentina


    A method and system are disclosed to insert coherence events in a multiprocessor computer system, and to present those coherence events to the processors of the multiprocessor computer system for analysis and debugging purposes. The coherence events are inserted in the computer system by adding one or more special insert registers. By writing into the insert registers, coherence events are inserted in the multiprocessor system as if they were generated by the normal coherence protocol. Once these coherence events are processed, the processing of coherence events can continue in the normal operation mode.

  6. A buffer insertion and simultaneous sizing timing optimization algorithm

    Institute of Scientific and Technical Information of China (English)

    Yin Guoli; Lin Zhenghui


    A path-based timing optimization algorithm for buffer insertion and simultaneous sizing is proposed.Firstly, candidate buffer insertion location and buffer size for each branch in a given routing path were obtained via localized timing optimization. Then, through evaluating each potential insertion against design objectives, potential optimal buffer insertion locations and sizes for the whole routing tree were determined. At last, by removing redundant buffer insertion operations which do not maximize S ( so ), given timing requirements are finally fulfilled through minimum number of buffers.

  7. 一个新型的棉花几丁质酶基因%A Novel Cotton Gene Encoding a New Class of Chitinase

    Institute of Scientific and Technical Information of China (English)

    李骥; 刘进元


    在对外源水杨酸处理的低酚品系棉花(Gossypium hirsutum cv.CRI13)(中棉13)幼苗进行RT-PCR差异分析并分离出一个cDNA片段的基础上,采用电子克隆和分子克隆相结合的方法克隆出该片段的全长序列,并对其cDNA和核DNA序列进行分析,结果显示该基因是一个新型的几丁质酶基因,命名为GhChia7.推测的GhChia7氨基酸序列与Ⅰ和Ⅱ型几丁质酶的相同性仅有30%,其结构特征也与已鉴定的Ⅰ~Ⅳ有差异,是一个新型的几丁质酶(Ⅶ型).Northern杂交结果显示,该基因在棉花幼苗的根中以及棉花纤维中信号较强,且在棉花子叶中受水杨酸诱导表达,用7.5 mmol/L水杨酸处理18 h后mRNA水平达到最大值.本研究的结果显示GhChia7可能会在棉花抗病防卫反应中发挥重要作用.%A novel chitinase gene (GhChia7) was isolated from salicylic acid (SA)-treated cottoncotyledons and characterized by DNA sequence analysis of its cDNA and genomic DNA clone. The deducedamino acid sequence, designated as class Ⅶ chitinase, shares about 30% identity to class Ⅰ or Ⅱ chitinases,and does not correspond to any of the previously characterized classes Ⅰ -Ⅵ chitinases. Northern blot-ting analysis showed that the transcripts of GhChia7were abundant both in cotton fibers and in the roots of theseedlings. The accumulation of GhChia7mRNA in SA-treated cotyledons reached maximum at 7.5 mmol/L concentration after 18 h. Results indicate that GhChia7might play an important role in cotton' s activedefense response.

  8. Domain structure of Lassa virus L protein. (United States)

    Brunotte, Linda; Lelke, Michaela; Hass, Meike; Kleinsteuber, Katja; Becker-Ziaja, Beate; Günther, Stephan


    The 200-kDa L protein of arenaviruses plays a central role in viral genome replication and transcription. This study aimed at providing evidence for the domain structure of L protein by combining bioinformatics with a stepwise mutagenesis approach using the Lassa virus minireplicon system. Potential interdomain linkers were predicted using various algorithms. The prediction was challenged by insertion of flexible sequences into the predicted linkers. Insertion of 5 or 10 amino acid residues was tolerated at seven sites (S407, G446, G467, G774, G939, S1952, and V2074 in Lassa virus AV). At two of these sites, G467 and G939, L protein could be split into an N-terminal and a C-terminal part, which were able to trans-complement each other and reconstitute a functional complex upon coexpression. Coimmunoprecipitation studies revealed physical interaction between the N- and C-terminal domains, irrespective of whether L protein was split at G467 or G939. In confocal immunofluorescence microscopy, the N-terminal domains showed a dot-like, sometimes perinuclear, cytoplasmic distribution similar to that of full-length L protein, while the C-terminal domains were homogenously distributed in cytoplasm. The latter were redistributed into the dot-like structures upon coexpression with the corresponding N-terminal domain. In conclusion, this study demonstrates two interdomain linkers in Lassa virus L protein, at G467 and G939, suggesting that L protein is composed of at least three structural domains spanning residues 1 to 467, 467 to 939, and 939 to 2220. The first domain seems to mediate accumulation of L protein into cytoplasmic dot-like structures.

  9. Interactions of a family 18 chitinase with the designed inhibitor HM508 and its degradation product, chitobiono-delta-lactone. (United States)

    Vaaje-Kolstad, Gustav; Vasella, Andrea; Peter, Martin G; Netter, Catharina; Houston, Douglas R; Westereng, Bjørge; Synstad, Bjørnar; Eijsink, Vincent G H; van Aalten, Daan M F


    We describe enzymological and structural analyses of the interaction between the family 18 chitinase ChiB from Serratia marcescens and the designed inhibitor N,N'-diacetylchitobionoxime-N-phenylcarbamate (HM508). HM508 acts as a competitive inhibitor of this enzyme with a K(i) in the 50 microM range. Active site mutants of ChiB show K(i) values ranging from 1 to 200 microM, providing insight into some of the interactions that determine inhibitor affinity. Interestingly, the wild type enzyme slowly degrades HM508, but the inhibitor is essentially stable in the presence of the moderately active D142N mutant of ChiB. The crystal structure of the D142N-HM508 complex revealed that the two sugar moieties bind to the -2 and -1 subsites, whereas the phenyl group interacts with aromatic side chains that line the +1 and +2 subsites. Enzymatic degradation of HM508, as well as a Trp --> Ala mutation in the +2 subsite of ChiB, led to reduced affinity for the inhibitor, showing that interactions between the phenyl group and the enzyme contribute to binding. Interestingly, a complex of enzymatically degraded HM508 with the wild type enzyme showed a chitobiono-delta-lactone bound in the -2 and -1 subsites, despite the fact that the equilibrium between the lactone and the hydroxy acid forms in solution lies far toward the latter. This shows that the active site preferentially binds the (4)E conformation of the -1 sugar, which resembles the proposed transition state of the reaction.

  10. The impact of acute aerobic exercise on chitinase 3-like protein 1 and intelectin-1 expression in obesity. (United States)

    Huang, Chun-Jung; Slusher, Aaron L; Whitehurst, Michael; Wells, Marie; Maharaj, Arun; Shibata, Yoshimi


    Chitinase 3-like 1 (CHI3L1) and intelectin 1 (ITLN-1) recognize microbial N-acetylglucosamine polymer and galactofuranosyl carbohydrates, respectively. Both lectins are highly abundant in plasma and seem to play pro- and anti-inflammatory roles, respectively, in obesity and inflammatory-related illnesses. The aim of this study was to examine whether plasma levels of these lectins in obese subjects are useful for monitoring inflammatory conditions immediately influenced by acute aerobic exercise. Plasma interleukin-6, a pro-inflammatory cytokine, was also examined. Twenty-two (11 obese and 11 normal-weight) healthy subjects, ages 18-30 years, were recruited to perform a 30 min bout of acute aerobic exercise at 75% VO2max. We confirmed higher baseline levels of plasma CHI3L1, but lower ITLN-1, in obese subjects than in normal-weight subjects. The baseline levels of CHI3L1 were negatively correlated with cardiorespiratory fitness (relative VO2max). However, when controlled for BMI, the relationship between baseline level of CHI3L1 and relative VO2max was no longer observed. While acute aerobic exercise elicited an elevation in these parameters, we found a lower ITLN-1 response in obese subjects compared to normal-weight subjects. Our study clearly indicates that acute aerobic exercise elicits a pro-inflammatory response (e.g. CHI3L1) with a lower anti-inflammatory effect (e.g. ITLN-1) in obese individuals. Furthermore, these lectins could be predictors of outcome of exercise interventions in obesity-associated inflammation.

  11. Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Daniel J Hoover

    Full Text Available The glycoprotein YKL-40 (CHI3L1 is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and

  12. Glucanases and chitinases as causal agents in the protection of Acacia extrafloral nectar from infestation by phytopathogens. (United States)

    González-Teuber, Marcia; Pozo, María J; Muck, Alexander; Svatos, Ales; Adame-Alvarez, Rosa M; Heil, Martin


    Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii x Nicotiana sanderae) contains "nectarins," proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.

  13. Plasma chitinase 3-like 1 is persistently elevated during first month after minimally invasive colorectal cancer resection

    Institute of Scientific and Technical Information of China (English)

    HMC Shantha Kumara; David Gaita; Hiromichi Miyagaki; Xiaohong Yan; Sonali AC Hearth; Linda Njoh; Vesna Cekic; Richard L Whelan


    AIM: To assess blood chitinase 3-like 1(CHi3L1) levels for 2 mo after minimally invasive colorectal resection(MICR) for colorectal cancer(CRC). METHODS: CRC patients in an Institutional Review Board approved data/plasma bank who underwent elective MICR for whom preoperative(PreO p), early postoperative(PostO p), and 1 or more late PostO p samples [postoperative day(POD) 7-27] available were included. Plasma CHi3L1 levels(ng/m L) were determined in duplicate by enzyme linked immunosorbent assay. RESULTS: PreOp and PostOp plasma sample were available for 80 MICR cancer patients for the study. The median PreOp CHi3L1 level was 56.8 CI: 41.9-78.6 ng/mL(n = 80). Significantly elevated(P < 0.001) median plasma levels(ng/mL) over PreOp levels were detected on POD1(667.7 CI: 495.7, 771.7; n = 79), POD 3(132.6 CI: 95.5, 173.7; n = 76), POD7-13(96.4 CI: 67.7, 136.9; n = 62), POD14-20(101.4 CI: 80.7, 287.4; n = 22), and POD 21-27(98.1 CI: 66.8, 137.4; n = 20, P = 0.001). No significant difference in plasma levels were noted on POD27-41. CONCLUSION: Plasma CHi3L1 levels were significantly elevated for one month after MICR. Persistently elevated plasma CHi3L1 may support the growth of residual tumor and metastasis.

  14. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily. (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael


    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  15. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    Directory of Open Access Journals (Sweden)

    Marc Lenoir


    Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  16. NMR assignments of the peptidyl-prolyl cis-trans isomerase domain of trigger factor from E. coli. (United States)

    Huang, Chih-Ting; Hsu, Shang-Te Danny


    Trigger factor (TF) is a highly conserved multi-domain molecular chaperone in bacteria. It binds via its ribosome binding domain (RBD) to the ribosomal tunnel exit and facilitates co-translational folding of a broad range of protein substrates primarily through interactions with the substrate binding domain (SBD) adjacent to the RBD. Within the SBD, a peptidyl-prolyl cis-trans isomerase (PPIase) domain is inserted leading to an unusual domain insertion, which may provide stabilizing effect to the highly plastic SBD. Here we report the near complete NMR assignments of TF PPIase providing the basis for subsequent structural and folding in the context of the chaperone activity of TF.

  17. Polymorphic Alu insertions among Mayan populations. (United States)

    Herrera, R J; Rojas, D P; Terreros, M C


    The Mayan homeland within Mesoamerica spans five countries: Belize, El Salvador, Guatemala, Honduras and Mexico. There are indications that the people we call the Maya migrated from the north to the highlands of Guatemala as early as 4000 B.C. Their existence was village-based and agricultural. The culture of these Preclassic Mayans owes much to the earlier Olmec civilization, which flourished in the southern portion of North America. In this study, four different Mayan groups were examined to assess their genetic variability. Ten polymorphic Alu insertion (PAI) loci were employed to ascertain the genetic affinities among these Mayan groups. North American, African, European and Asian populations were also examined as reference populations. Our results suggest that the Mayan groups examined in this study are not genetically homogeneous.

  18. Impact of Orthodontic Decompensation on Bone Insertion

    Directory of Open Access Journals (Sweden)

    Fabio Pinto Guedes


    Full Text Available There has always been concern in determining the relationship between orthodontic tooth movement and the consequent biological costs to the periodontium and tooth root. The possibility of evaluating the tooth and bone morphology by CBCT allows more accurate analysis of qualitative and quantitative aspects of these processes. This paper presents a case report of a 20-year-old male patient with Class III malocclusion and hyperdivergent facial pattern, who was surgically treated. A significant amount of labial movement of mandibular incisors was performed during orthodontic treatment before surgery. CBCT was used for evaluation of buccal and lingual bone plates before and after tooth decompensation. The changes in the bone insertion level of maxillary and mandibular incisors in the present case encourage a reflection on the treatment protocol in individuals with dentoskeletal discrepancies.

  19. Phoenix Conductivity Probe Inserted into Martian Soil (United States)


    NASA's Phoenix Mars Lander inserted the four needles of its thermal and conductivity probe into Martian soil during the 98th Martian day, or sol, of the mission and left it in place until Sol 99 (Sept. 4, 2008). The Robotic Arm Camera on Phoenix took this image on the morning of Sol 99 while the probe's needles were in the ground. The science team informally named this soil target 'Gandalf.' The thermal and conductivity probe measures how fast heat and electricity move from one needle to an adjacent one through the soil or air between the needles. Conductivity readings can be indicators about water vapor, water ice and liquid water. The probe is part of Phoenix's Microscopy, Electrochemistry and Conductivity suite of instruments. The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  20. Regulatory perspective on incomplete control rod insertions

    Energy Technology Data Exchange (ETDEWEB)

    Chatterton, M.


    The incomplete control rod insertions experienced at South Texas Unit 1 and Wolf Creek are of safety concern to the NRC staff because they represent potential precursors to loss of shutdown margin. Even before it was determined if these events were caused by the control rods or by the fuel there was an apparent correlation of the problem with high burnup fuel. It was determined that there was also a correlation between high burnup and high drag forces as well as with rod drop time histories and lack of rod recoil. The NRC staff initial actions were aimed at getting a perspective on the magnitude of the problem as far as the number of plants and the amount of fuel that could be involved, as well as the safety significance in terms of shutdown margin. As tests have been performed and data has been analyzed the focus has shifted more toward understanding the problem and the ways to eliminate it. At this time the staff`s understanding of the phenomena is that it was a combination of factors including burnup, power history and temperature. The problem appears to be very sensitive to these factors, the interaction of which is not clearly understood. The model developed by Westinghouse provides a possible explanation but there is not sufficient data to establish confidence levels and sensitivity studies involving the key parameters have not been done. While several fixes to the problem have been discussed, no definitive fixes have been proposed. Without complete understanding of the phenomena, or fixes that clearly eliminate the problem the safety concern remains. The safety significance depends on the amount of shutdown margin lost due to incomplete insertion of the control rods. Were the control rods to stick high in the core, the reactor could not be shutdown by the control rods and other means such as emergency boration would be required.

  1. Assessing the Outer Membrane Insertion and Folding of Multimeric Transmembrane β-Barrel Proteins. (United States)

    Leo, Jack C; Oberhettinger, Philipp; Linke, Dirk


    In addition to the cytoplasmic membrane, Gram-negative bacteria have a second lipid bilayer, the outer membrane, which is the de facto barrier between the cell and the extracellular milieu. Virtually all integral proteins of the outer membrane form β-barrels, which are inserted into the outer membrane by the BAM complex. Some outer membrane proteins, like the porins and trimeric autotransporter adhesins, are multimeric. In the former case, the porin trimer consists of three individual β-barrels, whereas in the latter, the single autotransporter β-barrel domain is formed by three separate polypeptides. This chapter reviews methods to investigate the folding and membrane insertion of multimeric OMPs and further explains the use of a BamA depletion strain to study the effects of the BAM complex on multimeric OMPs in E. coli.

  2. Exploring Beta-Amyloid Protein Transmembrane Insertion Behavior and Residue-Specific Lipid Interactions in Lipid Bilayers Using Multiscale MD Simulations (United States)

    Qiu, Liming; Vaughn, Mark; Cheng, Kelvin


    Beta-amyloid (Abeta) interactions with neurons are linked to Alzheimer's. Using a multiscale MD simulation strategy that combines the high efficiency of phase space sampling of coarse-grained MD (CGD) and the high spatial resolution of Atomistic MD (AMD) simulations, we studied the Abeta insertion dynamics in cholesterol-enriched and -depleted lipid bilayers that mimic the neuronal membranes domains. Forward (AMD-CGD) and reverse (CGD-AMD) mappings were used. At the atomistic level, cholesterol promoted insertion of Abeta with high (folded) or low (unfolded) helical contents of the lipid insertion domain (Lys28-Ala42), and the insertions were stabilized by the Lys28 snorkeling and Ala42-anchoring to the polar lipid groups of the bilayer up to 200ns. After the forward mapping, the folded inserted state switched to a new extended inserted state with the Lys28 descended to the middle of the bilayer while the unfolded inserted state migrated to the membrane surface up to 4000ns. The two new states remained stable for 200ns at the atomistic scale after the reverse mapping. Our results suggested that different Abeta membrane-orientation states separated by free energy barriers can be explored by the multiscale MD more effectively than by Atomistic MD simulations alone. NIH RC1-GM090897-02

  3. Posterior cruciate ligament's tibial insertions: topographic anatomy and morphometric study

    Directory of Open Access Journals (Sweden)

    Julio Cesar Gali


    Full Text Available OBJECTIVE: To provide anatomical and morphometric basis of the posterior cruciate ligament's tibial insertions in order to assist the creation of anatomical tibial tunnels, in the ligament surgical reconstruction. MATERIAL AND METHODS: The topographic anatomy and morphometry of the posterior cruciate ligament's anterolateral and posteromedial bundles' tibial insertions were analyzed in 24 anatomical knee pieces. The pieces were photographed by a digital camera and the images obtained were studied by the software ImageJ, where the bundles' insertion areas were measured in square millimeters, and the length of structures and the distances between significant points were measured in millimeters. RESULTS: In 54.2% of the knees the insertion' shape was concave; in most pieces (41.6% the form of insertion was oval. The average posterior cruciate ligament's tibial insertion total area was 88.33 ± 21.66 mm2; the average anterolateral bundle's tibial insertion area was 46.79 ± 14.10 mm2 and it was 41.54 ± 9.75 mm2 for the posteromedial bundle. CONCLUSIONS: The anterolateral bundle has a tibial insertion area larger than the posteromedial bundle; the insertion areas of those bundles in our study, were smaller than the ones found in the literature. The variations in the posterior cruciate ligament's tibial insertion area suggest that there should be an indication for anatomical reconstructions of this ligament using single or double tibial tunnels according to individual characteristics.

  4. Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad-spectrum resistance against insects, pathogens and abiotic stresses. (United States)

    Chen, Peng-Jen; Senthilkumar, Rajendran; Jane, Wann-Neng; He, Yong; Tian, Zhihong; Yeh, Kai-Wun


    Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid-expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.

  5. Molecular characterization of plantain class i chitinase gene and its expression in response to infection by Gloeosporium musarum Cke and Massee and other abiotic stimuli. (United States)

    Fan, Jianming; Wang, Hongbin; Feng, Dongru; Liu, Bin; Liu, Haiyan; Wang, Jinfa


    We have cloned a chitinase cDNA (MpChi-1) from plantain (Musa paradisiacal L) using rapid amplification of cDNA ends (RACE) according to a sequence fragment which we had cloned using the suppression subtractive hybridization (SSH) technique. The MpChi-1 encodes a protein of 326 amino acids and belongs to acidic chitinase class Ib subfamily. MpChi-1 shares high identity with rice endochitinase (XP_468714) and different each other only at three residues. Homology modelling indicated these three substitutions would not change the configuration of the activity site of the enzyme. We have expressed recombinant MpChi-1 and purified by ammonium sulphate precipitation and preparative reversed phase HPLC. The recombinant protein could hydrolyse chitin and inhibit the growth of the Gloeosporium musarum Cke and Massee in vitro. Northern blot revealed that the MpChi-1 transcripts rapidly after inoculation with G. musarum and maximum mRNA accumulation reached at 48 h. Jasmonic acid (JA) and salicylic acid (SA) could induce MpChi-1 expression, while mechanical wounding, silver nitrate and osmotic stress stimulated only a slight accumulation of MpChi-1 transcripts. Abscisic acid (ABA) could induce MpChi-1 transcript. These results suggest the MpChi-1 plays important role in the events of the hypersensitive reaction (HR).

  6. Transformation of blackgram (Vigna mungo (L.) Hepper) by barley chitinase and ribosome-inactivating protein genes towards improving resistance to Corynespora leaf spot fungal disease. (United States)

    Chopra, Rajan; Saini, Raman


    Blackgram (Vigna mungo (L.) Hepper), an important grain legume crop, is sensitive to many fungal pathogens including Corynespora cassiicola, the causal agent of corynespora leaf spot disease. In the present study, plasmid pGJ42 harboring neomycin phosphotransferase (nptII) a selectable marker gene, the barley antifungal genes chitinase (AAA56786) and ribosome-inactivating protein (RIP; AAA32951) were used for the transformation, to develop fungal resistance for the first time in blackgram. The presence and integration of transgene into the blackgram genome was confirmed by PCR and Southern analysis with an overall transformation frequency of 10.2 %. Kanamycin selection and PCR analysis of T0 progeny revealed the inheritance of transgene in Mendelian fashion (3:1). Transgenic plants (T1), evaluated for fungal resistance by in vitro antifungal assay, arrested the growth of C. cassiicola up to 25-40 % over the wild-type plants. In fungal bio-assay screening, the transgenic plants (T1) sprayed with C. cassiicola spores showed a delay in onset of disease along with their lesser extent in terms of average number of diseased leaves and reduced number and size of lesions. The percent disease protection among different transformed lines varies in the range of 27-47 % compare to control (untransformed) plants. These results demonstrate potentiality of chitinase and RIP from a heterologous source in developing fungal disease protection in blackgram and can be helpful in increasing the production of blackgram.

  7. Identification of fungus-responsive cis-acting element in the promoter of Brassica juncea chitinase gene, BjCHI1. (United States)

    Gao, Ying; Zan, Xin-Li; Wu, Xue-Feng; Yao, Lei; Chen, Yu-Ling; Jia, Shuang-Wei; Zhao, Kai-Jun


    Chitinases are a group of pathogenesis-related proteins. The Brassica juncea chitinase gene BjCHI1 is highly inducible by pathogenic fungal infection, suggesting that the promoter of BjCHI1 might contain specific cis-acting element responsive to fungal attack. To identify the fungus-responsive element in BjCHI1 promoter (BjC-P), a series of binary plant transformation vectors were constructed by fusing the BjC-P or its deletion-derivatives to β-glucuronidase (GUS) reporter gene. Expression of the GUS reporter gene was systematically assayed by a transient gene expression system in Nicotiana benthamiana leaves treated with fungal elicitor Hexa-N-Acetyl-Chitohexaose, as well as in transgenic Arabidopsis plants inoculated with fungus Botrytis cinerea. The histochemical and quantitative GUS assays showed that the W-box-like element (GTAGTGACTCAT) in the region (-668 to -657) was necessary for the fungus-response, although there were another five W-box-like elements in BjC-P. In addition, gain-of-function analysis demonstrated that the fragment (-409 to -337) coupled to the W-box-like element was needed for full magnitude of the fungal induction. These results revealed the existence of a novel regulation mechanism of W-box-like element involved in plant pathogenic resistance, and will benefit the potential application of BjC-P in engineering crops.

  8. Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyatake, Kazumasa [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Tsuji, Kunikazu, E-mail: [International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan); Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Sekiya, Ichiro [Section of Cartilage Regeneration, Tokyo Medical and Dental University, Tokyo (Japan); Muneta, Takeshi [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan)


    Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.

  9. Purification and enzymatic characterization of a chitinase from Phaseolus mungo%绿豆几丁质酶的纯化和酶学性质分析

    Institute of Scientific and Technical Information of China (English)

    汪少芸; 李邦德; 吴金鸿; 叶秀云; 饶平凡


    A chitinase was isolated from the seeds of mung bean ( Phaseolus mungo ) by the procedure containing aqueous extraction, affinity chromatography Affi - gel and ion exchange chromatography on SP- Toyopearl. The purified enzyme exhibited a single band on SDS - PAGE with a molecular weight of 30.8 kDa both in reduced and oxidized conditions, indicating it is monomeric protein. The pI was measured to be 6.3 by isoelectric focusing. This enzyme showed its optimum activity at pH 5.4, and a temperature between 40 ℃ and 50 ℃. These results demonswated the purified protein was a kind of new chitinase.%利用亲和色谱Affi-gel和离子交换色谱SP-Toyopearl从绿豆种子中分离纯化出几丁质酶.纯化的蛋白通过SDS-聚丙烯酰胺凝胶电泳鉴定达到电泳纯,分子质量为30.8 kDa.还原和非还原状态下的几丁质酶蛋白均显示单一区带,说明该几丁质酶为单倍体蛋白.通过等点聚焦法测得pI为6.3.该酶的最适pH为5.4,最适反应温度为40~50℃.

  10. The Effect of Windings on ADSL Transformer Insertion Losses

    Institute of Scientific and Technical Information of China (English)

    JIANG Xiao-na; LAN Zhong-wen; CHEN Sheng-ming; ZHANG Huai-wu; SU Hua


    Insertion loss (IL) is one of the important parameters of asymmetrical digital subscriber loop (ADSL) transformers. In different frequency bands, the factors that affect insertion loss are different. Windings mainly affect insertion loss in mid and high frequency bands.The effects of winding ways, winding wire diameter and winding turns on insertion loss were discussed. The presented experiment shows that the insertion loss of an ADSL transformer could be under 0.4 dB in mid frequency band when the winding is 30 turns, in which the ADSL transformer satisfies the requirement of total harmonic distortion (THD). Our experiments also show that the sandwich winding structure is better than the side by side winding structure and the twisted-pair winding structure, and the increase of winding diameter is one means to reduce insertion losses of an ADSL transformer in mid frequency band.

  11. Ocular inserts - Advancement in therapy of eye diseases

    Directory of Open Access Journals (Sweden)

    Anita Kumari


    Full Text Available The ocular insert represents a significant advancement in the therapy of eye disease. Ocular inserts are defined as sterile, thin, multilayered, drug-impregnated, solid or semisolid consistency devices placed into the cul-de-sac or conjuctival sac, whose size and shape are especially designed for ophthalmic application. They are composed of a polymeric support that may or may not contain a drug. The drug can later be incorporated as dispersion or a solution in the polymeric support. They offer several advantages as increased ocular residence and sustained release of medication into the eye. The insert includes a body portion sized to position within a lachrymal canaliculus of the eyelid. The inserts are classified according to their solubility as insoluble, soluble, or bioerodible inserts. The release of drug from the insert depends upon the diffusion, osmosis, and bioerosion of the drug, and this article is an attempt to present a brief about this newer drug delivery system.

  12. Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis



    The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries sugge...

  13. The LysM Receptor-Like Kinase LysM RLK1 Is Required to Activate Defense and Abiotic-Stress Responses Induced by Overexpression of Fungal Chitinases in Arabidopsis Plants

    Institute of Scientific and Technical Information of China (English)

    Yariv Brotman; Ada Viterbo; Udi Landau; Smadar Pnini; Jan Lisec; Salma Balazadeh; Bernd Mueller-Roeber; Aviah Zilberstein; Lothar Willmitzer; Ilan Chet


    Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased tolerance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinase1 (CERK1/LysM RLK1) gene,known to play a critical role in signaling defense responses induced by exogenous chitin.Arabidopsis plants overexpressing the endochitinase chit36 and hexoaminidase excy1 genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity,heavy-metal stresses,and Botrytis cinerea infection.Resistant lines,overexpressing fungal chitinases at different levels,were outcrossed to lysm rlk1 mutants.Independent homozygous hybrids lost resistance to biotic and abiotic stresses,despite enhanced chitinase activity.Expression analysis of 270 stress-related genes,including those induced by reactive oxygen species (ROS) and chitin,revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlk1 knockout mutant or the hybrids harboring the mutation.These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.

  14. 植物几丁质酶纯化、测定及应用研究进展%Review on purification, enzyme assay and application of plant chitinases

    Institute of Scientific and Technical Information of China (English)

    杨海霞; 邓建军; 张建; 赵广华


    植物几丁质酶是植物体中能够水解几丁质多聚体的一种致病性相关蛋白(Pathogenesis-related proteins).近年来对于几丁质酶的研究报道中,大量新型的植物几丁质酶被分离纯化,并建立了不同的酶活测定方法,在几丁质酶的结构及分类方面也逐步有了系统的研究.从几丁质酶的结构及分类,分离纯化以及已建立的酶活测定方法等方面取得的新进展进行了综述,并展望了几丁质酶在农业、食品生产及药用领域的应用前景.%Plant chitinases which hydrolyze the chitin is one of pathogenesis-related proteins and can be induced in resistance of plants to fungal pathogens. Thus, plant chitinases has a wide range of application as a antisepticise material. Recently, chitinases from different plants have been purified and their enzymatic activities have been assayed with varied methods. The structures and classifications, different methods on enzymatic activity assay and purification were summarized. Moreover, applications of chitinases such as in food and medicine field were prospected.

  15. Hevamine, a chitinase from the rubber tree Hevea brasiliensis, cleaves peptidoglycan between the C-1 of N-acetylglucosamine and C-4 of N-acetylmuramic acid and therefore is not a lysozyme

    NARCIS (Netherlands)

    Bokma, E; vanKoningsveld, GA; JeronimusStratingh, M; Beintema, JJ


    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. In this paper the cleavage specificity of hevamine for peptidoglycan was studied by HPLC and mass-spectrometry analysis of enzymatic digests. The results clearly showed that the enzyme c

  16. PcchiB1, encoding a class V chitinase, is affected by PcVelA and PcLaeA, and is responsible for cell wall integrity in Penicillium chrysogenum. (United States)

    Kamerewerd, Jens; Zadra, Ivo; Kürnsteiner, Hubert; Kück, Ulrich


    Penicillin production in Penicillium chrysogenum is controlled by PcVelA and PcLaeA, two components of the regulatory velvet-like complex. Comparative microarray analysis with mutants lacking PcVelA or PcLaeA revealed a set of 62 common genes affected by the loss of both components. A downregulated gene in both knockout strains is PcchiB1, potentially encoding a class V chitinase. Under nutrient-depleted conditions, transcript levels of PcchiB1 are strongly upregulated, and the gene product contributes to more than 50 % of extracellular chitinase activity. Functional characterization by generating PcchiB1-disruption strains revealed that PcChiB1 is responsible for cell wall integrity and pellet formation in P. chrysogenum. Further, fluorescence microscopy with a DsRed-labelled chitinase suggests a cell wall association of the protein. An unexpected phenotype occurred when knockout strains were grown on media containing N-acetylglucosamine as the sole C and N source, where, in contrast to the recipient, a penicillin producer strain, the mutants and an ancestral strain show distinct mycelial growth. We discuss the relevance of this class V chitinase for morphology in an industrially important fungus.

  17. Insertion torque versus mechanical resistance of mini-implants inserted in different cortical thickness (United States)

    Santos, Renata de Faria; Ruellas, Antonio Carlos de Oliveira; Fernandes, Daniel Jogaib; Elias, Carlos Nelson


    Objective This study aimed to measure insertion torque, tip mechanical resistance to fracture and transmucosal neck of mini-implants (MI) (Conexão Sistemas de PróteseT), as well as to analyze surface morphology. Methods Mechanical tests were carried out to measure the insertion torque of MIs in different cortical thicknesses, and tip mechanical resistance to fracture as well as transmucosal neck of MIs. Surface morphology was assessed by scanning electron microscopy (SEM) before and after the mechanical tests. Results Values of mechanical resistance to fracture (22.14 and 54.95 were higher and statistically different (P 0.05) to torsional fracture in the tip of MI (22.14 when 3 mm cortical thickness (16.11 and dense bone (23.95 were used. Torsional fracture of the transmucosal neck (54.95 was higher and statistically different (P implants tested presented adequate surface morphology. The resistance of mini-implants to fracture safely allows placement in 1 and 2-mm cortical thickness. However, in 3-mm cortical thickness and dense bones, pre-drilling with a bur is recommended before insertion. PMID:25162571

  18. Modeling of Porous Insertion Electrodes with Liquid Electrolyte

    DEFF Research Database (Denmark)

    West, Keld; Jacobsen, Torben; Atlung, Sven


    The dynamics of porous insertion electrodes during charge or discharge is described by a simplified mathematicalmodel, accounting for the coupled transport in electrode and electrolyte phases. A numerical method to evaluate theresponse of this model to either controlled potential or controlled cu...... is a consequence of the mobility of the ions not inserted, hence the performance of this type of electrode isoptimized by choosing electrolytes with transport number as close to unity as possible for the inserted ion....

  19. Risk factors for development of complication following peripherally inserted central


    Hakan Aydın; Gülsen Korfalı; Suna Gören; Esra Mercanoğlu Efe; Bachri Ramadan Moustafa; Tolga Yazıcı


    Objectives: Peripherally inserted central venous catheters (PICCs) are inserted into central veins through the upper extremity veins. In this retrospective study, we aimed to evaluate PICC procedures, related complications, their causes and factors influencing the success of the procedure during anaesthesia Methods: ‘Central Venous Catheterization Forms’ filled out for 850 patients in whom a PICC was inserted by residents during general anaesthesia between November 2009 and March 2013 in t...


    Directory of Open Access Journals (Sweden)

    Makbool Ali M.


    Full Text Available formation is a Package Insert. It is a printed leaflet that contains information based on regulatory guidelines for the safe and effective use of a drug. Incomplete and incorrect product information may have serious consequences including disability or death. In India, the concept of package insert is governed by the Drugs and Cosmetics Act (1940 and Rules (1945. Keeping this in mind, this study was designed to assess the presentation and completeness of drug information provided in the currently available package inserts for allopathic drugs in India. AIM To evaluate the presentation and completeness of drug information provided in the currently available package inserts for allopathic drugs in India. OBJECTIVES To evaluate drug information in package inserts according to headings mentioned in Section 6.2 and 6.3 of Schedule D, Drugs and Cosmetics Rules, 1945. MATERIAL AND METHODS Package inserts accompanying allopathic medicines were obtained from a drug store and three pharmacies around a tertiary care centre in Western India on request over a 1-month period. The package inserts were included in the study and analysed for the presentation and completeness of information according to the headings mentioned in Section 6.2 and Section 6.3 of Schedule D, The Drugs and Cosmetics Rules, 1945. RESULTS 110 package inserts were analysed in the study. None of the reviewed package insert contained all the sections as required by the Drugs and Cosmetic Rules. CONCLUSION To avoid medication errors due to deficits in drug information in package inserts, tighter monitoring of package inserts by regulatory authorities is recommended. Steps should be taken to ensure that the information in the package inserts follows a standard layout for easy and convenient comprehension.

  1. Protein domain prediction

    NARCIS (Netherlands)

    Ingolfsson, Helgi; Yona, Golan


    Domains are considered to be the building blocks of protein structures. A protein can contain a single domain or multiple domains, each one typically associated with a specific function. The combination of domains determines the function of the protein, its subcellular localization and the interacti

  2. Threaded insert for compact cryogenic-capable pressure vessels

    Energy Technology Data Exchange (ETDEWEB)

    Espinosa-Loza, Francisco; Ross, Timothy O.; Switzer, Vernon A.; Aceves, Salvador M.; Killingsworth, Nicholas J.; Ledesma-Orozco, Elias


    An insert for a cryogenic capable pressure vessel for storage of hydrogen or other cryogenic gases at high pressure. The insert provides the interface between a tank and internal and external components of the tank system. The insert can be used with tanks with any or all combinations of cryogenic, high pressure, and highly diffusive fluids. The insert can be threaded into the neck of a tank with an inner liner. The threads withstand the majority of the stress when the fluid inside the tank that is under pressure.

  3. Insertional protein engineering for analytical molecular sensing

    Directory of Open Access Journals (Sweden)

    Arís Anna


    Full Text Available Abstract The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors.

  4. Membrane binding domains


    Hurley, James H.


    Eukaryotic signaling and trafficking proteins are rich in modular domains that bind cell membranes. These binding events are tightly regulated in space and time. The structural, biochemical, and biophysical mechanisms for targeting have been worked out for many families of membrane binding domains. This review takes a comparative view of seven major classes of membrane binding domains, the C1, C2, PH, FYVE, PX, ENTH, and BAR domains. These domains use a combination of specific headgroup inter...

  5. Controlled language and information on vaccines: application to package inserts. (United States)

    Renahy, Julie; Vuitton, Dominique A; Rath, Barbara; Thomas, Izabella; de Grivel, Valerie; Cardey, Sylviane


    Any ambiguity in texts used in the communication about vaccines can not only interfere with comprehension, but also generate safety and liability issues. Within a survey on the quality of written protocols for at-risk interventional procedures and sanitary crises, we analyzed documents relating to vaccination, and among them, the "package-leaflet" of an anti-H1N1 influenza vaccine, widely disseminated to the public in 2009-2010. Among the most common mistakes, we observed that 1) language was not always adjusted to the non-specialist's level of knowledge; 2) chronology, logic, consistency, and homogeneity were often missing; 3) crucial pieces of information were disseminated all over the text, 4) use of the passive voice did not distinguish between instructions and information; 5) use of synonyms could be misleading and impair translation. We propose the use of "Controlled language" (CL) to improve the situation. By constraining lexicon, grammar and syntax, CL is a way to write documents that are clear, accurate and devoid of ambiguity. However, the set of rules necessary to write in CL is difficult to memorize. We thus developed authoring software (Rédacticiel Prolipsia) to make the creation of a CL by linguists and its use by health professionals easy and adapted to any domain. It may considerably improve the writing of vaccine package inserts/leaflets. It could be used to write information documents about vaccines and their safety, and operating procedures for professionals to prepare, store, and administer vaccines, decide upon proper indication of vaccines, and follow patients after vaccine injection.

  6. Selenocysteine biosynthesis and insertion machinery in Naegleria gruberi. (United States)

    da Silva, M T A; Caldas, V E A; Costa, F C; Silvestre, D A M M; Thiemann, O H


    Selenium (Se) is an essential trace element primarily found in selenoproteins as the 21st amino acid (selenocysteine, Sec, or U). Selenoproteins play an important role in growth and proliferation and are typically involved in cellular redox balance. Selenocysteine is encoded by an in-frame UGA codon specified by a stem-loop structure, the Sec insertion sequence element (SECIS), which, in eukaryotes, is located in the 3'-untranslated region (UTR). The availability of the Naegleria gruberi (ATCC 30224) genome sequence and the use of this organism as a model system for the pathogenic amoeba N. fowleri allowed us to investigate the Sec incorporation pathway in this primitive eukaryote. Using bioinformatics tools, we identified gene sequences encoding PSTK (O-phosphoseryl-tRNA(Sec) kinase), SepSecS (O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase), SelD/SPS2 (selenophosphate synthetase), EFSec (selenocysteine-specific elongation factor) and SBP (SECIS binding protein). These findings were confirmed by RT-PCR and by sequencing. A potential tRNA(Ser)Sec (SelC) gene and a putative selenoprotein with sequence similarity to a mitochondrial thioredoxin reductase (TR3) were also identified. Our results show that the selenocysteine incorporation machinery is indeed present in N. gruberi. Interestingly, the SelD/SPS2 gene is 2214 bp in length and contains two distinct domains. The N-terminal region shows sequence similarity to predicted methyltransferase proteins, and the C-terminal region is homologous to prokaryotic SelD/SPS2. Our results suggest the possibility of novel selenoproteins.

  7. Clinical information in drug package inserts in India

    Directory of Open Access Journals (Sweden)

    Shivkar Y


    Full Text Available Background: It is widely recognized that accurate and reliable product information is essential for the safe and effective use of medications. Pharmaceutical companies are the primary source of most drug information, including package inserts. Package inserts are printed leaflets accompanying marketed drug products and contain information approved by the regulatory agencies. Studies on package inserts in India, in 1996, had shown that crucial information was often missing and they lacked uniformity. Aim: To assess the presentation and completeness of clinically important information provided in the currently available package inserts in India. Materials and Methods: Package inserts accompanying allopathic drug products marketed by pharmaceutical companies in India were collected. These package inserts were analyzed for the content of clinically important information in various sections. Statistical Analysis: The results were expressed as absolute numbers and percentages. Results: Preliminary analyses revealed that most package inserts did contain information under headings, such as, therapeutic indications, contraindications, undesirable effects, etc., listed in the Drugs and Cosmetics Rules 1945. The findings indicated considerable improvement in package inserts since 1996. However, on critical evaluation it was revealed that clinically important information was not well presented and was often incomplete. Information with regard to pediatric and geriatric use was present in only 44% and 13% of the package inserts, respectively. Only five of the inserts had information on the most frequent adverse drug reactions associated with the drug. Also, information on interactions and overdosage was often missing. Conclusion: Although the package inserts appear to have improved over the past decade there is still a definite need to further refine the clinical information contained, to minimize the risks to patients. This could be brought about by self

  8. Structural and histone binding ability characterizations of human PWWP domains.

    Directory of Open Access Journals (Sweden)

    Hong Wu

    Full Text Available BACKGROUND: The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. METHODOLOGY/PRINCIPAL FINDINGS: The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. CONCLUSIONS: PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle. Both the canonical β-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web

  9. Macroscopic and microscopic observations of needle insertion into gels

    NARCIS (Netherlands)

    Veen, van Youri R.J.; Jahya, Alex; Misra, Sarthak


    Needle insertion into soft tissue is one of the most common medical interventions. This study provides macroscopic and microscopic observations of needle–gel interactions. A gelatin mixture is used as a soft-tissue simulant. For the macroscopic studies, system parameters, such as insertion velocity,

  10. 17 CFR 240.12b-14 - Riders; inserts. (United States)


    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Riders; inserts. 240.12b-14 Section 240.12b-14 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED... Exchange Act of 1934 Formal Requirements § 240.12b-14 Riders; inserts. Riders shall not be used. If...

  11. 17 CFR 270.8b-14 - Riders; inserts. (United States)


    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Riders; inserts. 270.8b-14 Section 270.8b-14 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.8b-14 Riders; inserts. Riders shall not be...

  12. 17 CFR 260.7a-20 - Riders; inserts. (United States)


    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Riders; inserts. 260.7a-20...) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Formal Requirements § 260.7a-20 Riders; inserts. Riders shall not be used. If the application, statement or report is typed on a printed form, and...

  13. Load beam unit replaceable inserts for dry coal extrusion pumps (United States)

    Saunders, Timothy; Brady, John D.


    A track assembly for a particulate material extrusion pump according to an exemplary aspect of the present disclosure includes a link assembly with a roller bearing. An insert mounted to a load beam located such that the roller bearing contacts the insert.

  14. Enhanced citrate production through gene insertion in Aspergillus niger

    DEFF Research Database (Denmark)

    Jongh, Wian de; Nielsen, Jens


    The effect of inserting genes involved in the reductive branch of the tricarboxylic acid (TCA) cycle on citrate production by Aspergillus niger was evaluated. Several different genes were inserted individually and in combination, i.e. malate dehydrogenase (mdh2) from Saccharomyces cerevisiae, two...

  15. A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction

    Directory of Open Access Journals (Sweden)

    Jakobsdottir Klara B


    Full Text Available Abstract Background The jellyfish green fluorescent protein (GFP can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. Results We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, αs. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. Conclusion This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

  16. The Evolutionary Relationship of the Domain Architectures in the RhoGEF-containing Proteins

    Institute of Scientific and Technical Information of China (English)

    Qing-Lan Sun; Hong-Jun Zhou; Kui Lin


    Domain insertions and deletions lead to variations in the domain architectures of the proteins from their common ancestor. In this work, we investigated four groups of the RhoGEF-containing proteins from different organisms with domain architectures RhoGEF-PH-SH3, SH3-RhoGEF-PH, RhoGEF-PH, and SH3-RhoGEF defined in the Pfam database. The phylogenetic trees were constructed using each individual domain and/or the combinations of all the domains. The phylogenetic analysis suggests that RhoGEF-PH-SH3 and SH3-RhoGEF-PH might have evolved from RhoGEF-PH through the insertion of SH3 independently, while SH3-RhoGEF of proteins in fruit fly might have evolved from SH3-RhoGEF-PH by the degeneration of PH domain.

  17. Domains via Graphs

    Institute of Scientific and Technical Information of China (English)

    ZHANG Guoqiang; CHEN Yixiang


    This paper provides a concrete and simple introduction to two pillars of domain theory: (1) solving recursive domain equations, and (2) universal and saturated domains. Our exposition combines Larsen and Winskel's idea on solving domain equations using information systems with Girard's idea of stable domain theory in the form of coherence spaces, or graphs.Detailed constructions are given for universal and even homogeneous objects in two categories of graphs: one representing binary complete, prime algebraic domains with complete primes covering the bottom; the other representing ω-algebraic, prime algebraic lattices. The backand-forth argument in model theory helps to enlighten the constructions.

  18. Analysis of the Sherlock II tip location system for inserting peripherally inserted central venous catheters. (United States)

    Lelkes, Valdis; Kumar, Abhishek; Shukla, Pratik A; Contractor, Sohail; Rutan, Thomas


    Peripherally inserted central catheters (PICCs) are frequently placed at the bedside. The purpose of our study was to evaluate the efficacy of the Sherlock II tip location system (Bard Access Systems, Salt Lake City, UT), which offers electromagnetic detection of the PICC tip to assist the operator in guiding the tip to a desired location. We performed a retrospective review of patients who had a bedside PICC using the Sherlock II tip location system. Three hundred seventy-five of 384 patients (97.7%) had the catheter tip positioned appropriately. Our results suggest that the Sherlock II tip location system is an efficacious system for bedside PICC placement.

  19. Welding of titanium and stainless steel using the composite insert (United States)

    Cherepanov, A. N.; Mali, V. I.; Orishich, A. M.; Malikov, A. G.; Drozdov, V. O.; Malyutina, Y. N.


    The paper concerns the possibility of obtaining a lasting permanent joint of dissimilar metals: technically pure titanium and stainless steel using laser welding and an intermediate composite insert. The insert was a four-layer composition of plates of steel, copper, niobium, and titanium welded by explosion. The material layers used in the insert prevented the molten steel and titanium from mixing, which excluded the formation of brittle intermetallic compounds, such as FeTi and Fe2Ti. The optimization of explosion welding parameters provided a high quality of the four-layer composition and the absence of defects in the area of the joint of insert plates. The results of strength tests showed that values of the ultimate strength and yield of the permanent joint with the composite insert welded by explosion are comparable to the strength characteristics of titanium.

  20. Smart structures for shock wave attenuation using ER inserts (United States)

    Kim, Jaehwan; Kim, Jung-Yup; Choi, Seung-Bok; Kim, Kyung-Su


    This Paper demonstrates the possibility of shock wave attenuation propagating through a smart structure that incorporates ER insert. The wave transmission of ER inserted beam is theoretically derived using Mead & Markus model and the theoretical results are compared with the finite element analysis results. To experimentally verify the shock wave attenuation, ER insert in an aluminum plate is made and two piezoceramic disks are used as transmitter and receiver of the wave. The transmitter sends a sine pulse signal such that a component of shock wave travels through the plate structure and the receiver gets the transmitted wave signal. Wave propagation of the ER insert can be adjusted by changing the applied electric field on the ER insert. Details of the experiment are addressed and the possibility of shock wave attenuation is experimentally verified. This kind of smart structure can be used for warship and submarine hull structures to protect fragile and important equipment.

  1. Domains of laminin

    DEFF Research Database (Denmark)

    Engvall, E; Wewer, U M


    Extracellular matrix molecules are often very large and made up of several independent domains, frequently with autonomous activities. Laminin is no exception. A number of globular and rod-like domains can be identified in laminin and its isoforms by sequence analysis as well as by electron...... microscopy. Here we present the structure-function relations in laminins by examination of their individual domains. This approach to viewing laminin is based on recent results from several laboratories. First, some mutations in laminin genes that cause disease have affected single laminin domains, and some...... laminin isoforms lack particular domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. Second, laminin-like domains have now been...

  2. Role of Chitinase 3-Like-1 in Interleukin-18-Induced Pulmonary Type 1, Type 2, and Type 17 Inflammation; Alveolar Destruction; and Airway Fibrosis in the Murine Lung. (United States)

    Kang, Min-Jong; Yoon, Chang Min; Nam, Milang; Kim, Do-Hyun; Choi, Je-Min; Lee, Chun Geun; Elias, Jack A


    Chitinase 3-like 1 (Chi3l1), which is also called YKL-40 in humans and BRP-39 in mice, is the prototypic chitinase-like protein. Recent studies have highlighted its impressive ability to regulate the nature of tissue inflammation and the magnitude of tissue injury and fibroproliferative repair. This can be appreciated in studies that highlight its induction after cigarette smoke exposure, during which it inhibits alveolar destruction and the genesis of pulmonary emphysema. IL-18 is also known to be induced and activated by cigarette smoke, and, in murine models, the IL-18 pathway has been shown to be necessary and sufficient to generate chronic obstructive pulmonary disease-like inflammation, fibrosis, and tissue destruction. However, the relationship between Chi3l1 and IL-18 has not been defined. To address this issue we characterized the expression of Chi3l1/BRP-39 in control and lung-targeted IL-18 transgenic mice. We also characterized the effects of transgenic IL-18 in mice with wild-type and null Chi3l1 loci. The former studies demonstrated that IL-18 is a potent stimulator of Chi3l1/BRP-39 and that this stimulation is mediated via IFN-γ-, IL-13-, and IL-17A-dependent mechanisms. The latter studies demonstrated that, in the absence of Chi3l1/BRP-39, IL-18 induced type 2 and type 17 inflammation and fibrotic airway remodeling were significantly ameliorated, whereas type 1 inflammation, emphysematous alveolar destruction, and the expression of cytotoxic T lymphocyte perforin, granzyme, and retinoic acid early transcript 1 expression were enhanced. These studies demonstrate that IL-18 is a potent stimulator of Chi3l1 and that Chi3l1 is an important mediator of IL-18-induced inflammatory, fibrotic, alveolar remodeling, and cytotoxic responses.

  3. Controlling Posture and Vergence Eye Movements in Quiet Stance: Effects of Thin Plantar Inserts.

    Directory of Open Access Journals (Sweden)

    A Foisy

    Full Text Available The purpose of this study was to assess properties of vergence and saccade eye movements as well as posture in quiet stance, and the effects of thin plantar inserts upon postural and oculomotor control. The performances of 36 young healthy subjects were recorded by a force platform and an eye tracker in three testing conditions: without plantar stimulation, with a 3 millimetre-thick plantar insert, either a Medial or a Lateral Arch Support (MAS/LAS. The results showed a decrease of the Surface and Variance of Speed and a more posterior position of the CoP with either stimulation compared with the control condition. The fractal analysis showed a decrease with MAS. Wavelet analysis in the time-frequency domain revealed an increase in the Cancelling Time of the low frequency band with MAS. These results suggest a better stability for a lower energy cost. Concerning eye movements, the inserts influenced only vergence (not saccades: MAS caused an increase of the phasic amplitude of divergence, and conversely a decrease of the tonic amplitude. In contrast, LAS caused an increase of the tonic amplitude of convergence. Thus, MAS renders divergence less visually driven, while LAS renders convergence more visually driven. We conclude that the CNS uses the podal signal for both postural and vergence control via specific mechanisms. Plantar inserts have an influence upon posture and vergence movements in a different way according to the part of the foot sole being stimulated. These results can be useful to clinicians interested in foot or eye.

  4. Toward automated cochlear implant insertion using tubular manipulators (United States)

    Granna, Josephine; Rau, Thomas S.; Nguyen, Thien-Dang; Lenarz, Thomas; Majdani, Omid; Burgner-Kahrs, Jessica


    During manual cochlear implant electrode insertion the surgeon is at risk to damage the intracochlear fine-structure, as the electrode array is inserted through a small opening in the cochlea blindly with little force-feedback. This paper addresses a novel concept for cochlear electrode insertion using tubular manipulators to reduce risks of causing trauma during insertion and to automate the insertion process. We propose a tubular manipulator incorporated into the electrode array composed of an inner wire within a tube, both elastic and helically shaped. It is our vision to use this manipulator to actuate the initially straight electrode array during insertion into the cochlea by actuation of the wire and tube, i.e. translation and slight axial rotation. In this paper, we evaluate the geometry of the human cochlea in 22 patient datasets in order to derive design requirements for the manipulator. We propose an optimization algorithm to automatically determine the tube set parameters (curvature, torsion, diameter, length) for an ideal final position within the cochlea. To prove our concept, we demonstrate that insertion can be realized in a follow-the-leader fashion for 19 out of 22 cochleas. This is possible with only 4 different tube/wire sets.

  5. Study on Visually Design of Indexable Insert Tip

    Institute of Scientific and Technical Information of China (English)

    YAO Ji-quan; ZHAO Shu-qiang; ZHANG Shen-hou; CHEN Xin; GUO Xian-yang


    In order to improve the development efficiency of the indexable insert tip, three-dimensional parametric and visualization applications program of indexable insert tip was developed in AutoCAD VBA (Visual Basic for Applications). The software could significantly improve the modeling efficiency of indexable insert tip and the model could also be used to static analysis, dynamic analysis and thermodynamic analysis. All aspects of performance could be fully understood by the finite element analysis. The ref-erence for the blade parameters preferred is also provided.

  6. Insertion of short transmembrane helices by the Sec61 translocon. (United States)

    Jaud, Simon; Fernández-Vidal, Mónica; Nilsson, Ingmarie; Meindl-Beinker, Nadja M; Hübner, Nadja C; Tobias, Douglas J; von Heijne, Gunnar; White, Stephen H


    The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (L(n), n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-L(n)-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is snorkeling) and by partial unfolding.

  7. Industrial stator vane with sequential impingement cooling inserts (United States)

    Jones, Russell B; Fedock, John A; Goebel, Gloria E; Krueger, Judson J; Rawlings, Christopher K; Memmen, Robert L


    A turbine stator vane for an industrial engine, the vane having two impingement cooling inserts that produce a series of impingement cooling from the pressure side to the suction side of the vane walls. Each insert includes a spar with a row of alternating impingement cooling channels and return air channels extending in a radial direction. Impingement cooling plates cover the two sides of the insert and having rows of impingement cooling holes aligned with the impingement cooling channels and return air openings aligned with the return air channel.

  8. 黏质沙雷氏菌C8-8几丁质酶基因的克隆与表达%Cloning and expression of a chitinase gene from Serratia marcescens strain C8-8

    Institute of Scientific and Technical Information of China (English)

    刘邮洲; 罗楚平; 刘永锋; 陈志谊


    利用PCR方法从黏质沙雷氏菌(Serratia marcescens)C8-8中克隆到编码几丁质酶的chiA基因,大小为1692bp,推测其编码一条长563个氨基酸的多肽链,分子量约为60900.同源分析研究结果表明从C8-8中克隆的chiA基因序列与黏质沙雷氏菌株141(DQ990373.1)和14041菌株(DQ493896.1)的chiA基因序列相似性最高,达到99%.结构域分析结果表明从C8-8中克隆的chiA基因N末端(23AA)存在典型的信号肽序列,C端存在另外两个结构域,即PKD区(73AA)和几丁质酶催化区(387AA).采用大肠杆菌表达系统重组表达chiA基因,结果表明重组菌株在几丁质诱导培养基上能产生透明的水解圈.采用SDS-PAGE电泳分析,结果表明chiA重组表达产物的相对分子质量约为60000,与预测分子量大小基本一致.初步提纯后,生物活性试验结果表明该重组表达产物能水解几丁质,在几丁质培养基上产生透明的水解圈.%An open reading frame encoding chiA gene was cloned from the Serratia marcescens strain C8-8 genomic DNA by PCR, with the length of 1 692 bp. Its sequence was 99% identical with chiA sequences of Serratia marcescens 141 and 14041. Domain analysis showed that the cloned chiA gene involved a typical signal peptide sequence at N-terraination (23 AA), and PKD domain (73 AA) and chitinase catalytic domain (387 AA) at C-termination. The PCR fragment was digested and cloned into plasmid pET28a to construct plasmid pET28a-ChiA, which was then transformed into expression host Escherichia. coli DH3. The recombined strain DH3 ChiA could yield transparent hydrolyzed zone on the colloidal chi-tin plate induced by isopropyl-1 -thiogalactopyranoside (IPTG). A protein with molecular weight about 60 000 was expressed by DH3 ChiA, and could also yield a hydrolyzed rone on the colloidal chitin plate. It indicated that chiA gene from C8-8 could be utilized as a potential biological factor for control of fungi.

  9. Effect of insertion speed on tissue response and insertion mechanics of a chronically implanted silicon-based neural probe. (United States)

    Welkenhuysen, M; Andrei, A; Ameye, L; Eberle, W; Nuttin, B


    In this study, the effect of insertion speed on long-term tissue response and insertion mechanics was investigated. A dummy silicon parylene-coated probe was used in this context and implanted in the rat brain at 10 μm/s (n = 6) or 100 μm/s (n = 6) to a depth of 9 mm. The insertion mechanics were assessed by the dimpling distance, and the force at the point of penetration, at the end of the insertion phase, and after a 3-min rest period in the brain. After 6 weeks, the tissue response was evaluated by estimating the amount of gliosis, inflammation, and neuronal cell loss with immunohistochemistry. No difference in dimpling, penetration force, or the force after a 3-min rest period in the brain was observed. However, the force at the end of the insertion phase was significantly higher when inserting the probes at 100 μm/s compared to 10 μm/s. Furthermore, an expected tissue response was seen with an increase of glial and microglial reactivity around the probe. This reaction was similar along the entire length of the probe. However, evidence for a neuronal kill zone was observed only in the most superficial part of the implant. In this region, the lesion size was also greatest. Comparison of the tissue response between insertion speeds showed no differences.

  10. Bougie insertion: A common practice with underestimated dangers (United States)

    Theodorou, D.; Doulami, G.; Larentzakis, A.; Almpanopoulos, K.; Stamou, K.; Zografos, G.; Menenakos, E.


    Introduction Esophageal perforation after bariatric operations is rare. We report two cases of esophageal perforation after bariatric operations indicating the dangers of a common practice – like insertion of esophageal tubes – and we describe our management of that complication. Presentation of case A 56 year old woman who underwent laparoscopic sleeve gastrectomy and a 41 year old woman who underwent laparoscopic adjustable gastric banding respectively. In both operations a bougie has been used and led to esophageal perforation. Discussion The insertion of bougie and especially of inflated bougie is a common practice. It is an invasive procedure that in most cases is performed by the anesthesiologist team. Conclusion Bougie insertion is an invasive procedure with risks and should always be attempted under direct supervision of surgical team or should be inserted by a surgeon. PMID:22288051

  11. Trajectory generation for robotic needle insertion in soft tissue. (United States)

    Abolhassani, Niki; Patel, Rajni; Moallem, Mehrdad


    Accurate needle insertion in soft, inhomogeneous tissue has been a major concern in several recent studies involving robot-assisted percutaneous therapies. In procedures that involve multiple needle insertions such as transrectal ultrasound guided prostate brachytherapy, it is important to reduce tissue deformation before puncture and during insertion. In order to reduce this deformation, we have studied the effect of different trajectories for a 2-DOF robot performing needle insertion in soft tissue. We have compared tissue deformation and infinitesimal force per tissue displacement for different trajectories. According to the results of our experiments, infinitesimal force per displacement is a useful parameter for online trajectory update. Our proposed position/force controller is shown to provide considerable improvement in performance with regard to tissue deformation before puncture.

  12. Spontaneous Radiation Emission from Short, High Field Strength Insertion Devices

    Energy Technology Data Exchange (ETDEWEB)

    Geoffrey Krafft


    Since the earliest papers on undulaters were published, it has been known how to calculate the spontaneous emission spectrum from ''short'' undulaters when the magnetic field strength parameter is small compared to unity, or in ''single'' frequency sinusoidal undulaters where the magnetic field strength parameter is comparable to or larger than unity, but where the magnetic field amplitude is constant throughout the undulater. Fewer general results have been obtained in the case where the insertion device is both short, i.e., the magnetic field strength parameter changes appreciably throughout the insertion device, and the magnetic field strength is high enough that ponderomotive effects, radiation retardation, and harmonic generation are important physical phenomena. In this paper a general method is presented for calculating the radiation spectrum for short, high-field insertion devices. It is used to calculate the emission from some insertion device designs of recent interest.

  13. The use of dimorphic Alu insertions in human DNA fingerprinting

    Energy Technology Data Exchange (ETDEWEB)

    Novick, G.E.; Gonzalez, T.; Garrison, J.; Novick, C.C.; Herrera, R.J. [Florida International Univ., Miami, FL (United States). Dept. of Biological Sciences; Batzer, M.A. [Lawrence Livermore National Lab., CA (United States); Deininger, P.L. [Louisiana State Univ., New Orleans, LA (United States). Medical Center


    We have characterized certain Human Specific Alu Insertions as either dimorphic (TPA25, PV92, APO), sightly dimorphic (C2N4 and C4N4) or monomorphic (C3N1, C4N6, C4N2, C4N5, C4N8), based on studies of Caucasian, Asian, American Black and African Black populations. Our approach is based upon: (1) PCR amplification using primers directed to the sequences that flank the site of insertion of the different Alu elements studied; (2) gel electrophoresis and scoring according to the presence or absence of an Alu insertion in one or both homologous chromosomes; (3) allelic frequencies calculated and compared according to Hardy-Weinberg equilibrium. Our DNA fingerprinting procedure using PCR amplification of dimorphic Human Specific Alu insertions, is stable enough to be used not only as a tool for genetic mapping but also to characterize populations, study migrational patterns and track the inheritance of human genetic disorders.

  14. Modified Seldinger technique for the insertion of standard chest tubes. (United States)

    Altman, E; Ben-Nun, A; Curtis, W; Best, L A


    Closed tube thoracostomy is a standard procedure for the evacuation of air, blood, or other materials from the pleural space. This paper describes a modification of the Seldinger technique that facilitates chest tube insertion. Either a Nelaton or Thieman catheter is threaded into the side drainage hole and out the tip of a standard Argyle-type chest tube. After using the clamp to insert the catheter into the pleural space through a previously dissected tract, the catheter serves as a guide over which the chest tube is inserted. The technique is simple to use, effective, and safe. It employs standard, inexpensive materials to insert chest tubes in such a way as to minimize the potential traumatic complications inherent in other techniques.

  15. A new insertion sequence for incremental Delaunay triangulation

    Institute of Scientific and Technical Information of China (English)

    Jian-Fei Liu; Jin-Hui Yan; S.H.Lo


    Incremental algorithm is one of the most popular procedures for constructing Delaunay triangulations (DTs).However,the point insertion sequence has a great impact on the amount of work needed for the construction of DTs.It affects the time for both point location and structure update,and hence the overall computational time of the triangulation algorithm.In this paper,a simple deterministic insertion sequence is proposed based on the breadth-first-search on a Kd-tree with some minor modifications for better performance.Using parent nodes as search-hints,the proposed insertion sequence proves to be faster and more stable than the Hilbert curve order and biased randomized insertion order (BRIO),especially for non-uniform point distributions over a wide range of benchmark examples.

  16. Lithium Insertion In Silicon Nanowires: An ab Initio Study

    KAUST Repository

    Zhang, Qianfan


    The ultrahigh specific lithium ion storage capacity of Si nanowires (SiNWs) has been demonstrated recently and has opened up exciting opportunities for energy storage. However, a systematic theoretical study on lithium insertion in SiNWs remains a challenge, and as a result, understanding of the fundamental interaction and microscopic dynamics during lithium insertion is still lacking. This paper focuses on the study of single Li atom insertion into SiNWs with different sizes and axis orientations by using full ab initio calculations. We show that the binding energy of interstitial Li increases as the SiNW diameter grows. The binding energies at different insertion sites, which can be classified as surface, intermediate, and core sites, are quite different. We find that surface sites are energetically the most favorable insertion positions and that intermediate sites are the most unfavorable insertion positions. Compared with the other growth directions, the [110] SiNWs with different diameters always present the highest binding energies on various insertion locations, which indicates that [110] SiNWs are more favorable by Li doping. Furthermore, we study Li diffusion inside SiNWs. The results show that the Li surface diffusion has a much higher chance to occur than the surface to core diffusion, which is consistent with the experimental observation that the Li insertion in SiNWs is layer by layer from surface to inner region. After overcoming a large barrier crossing surface-to-intermediate region, the diffusion toward center has a higher possibility to occur than the inverse process. © 2010 American Chemical Society.

  17. Hollow polymer microneedles array resistance and insertion tests



    Microneedles are developed in order to become the transdermal administration method of the future. They however still face numerous challenges. This paper addresses the challenge to effectively insert the microneedle arrays into membranes. A recently proposed model membrane and test method for microneedles insertion, published in International Journal of Pharmaceutics, is used in this aim. A moulded 4 by 4 hollow polymer microneedle array developed at the Université Libre de Bruxelles is test...

  18. Equilibrium insertion of nanoscale objects into phospholipid bilayers

    CERN Document Server

    Pogodin, Sergey


    Certain membrane proteins, peptides, nanoparticles and nanotubes have rigid structure and fixed shape. They are often viewed as spheres and cylinders with certain surface properties. Single Chain Mean Field theory is used to model the equilibrium insertion of nanoscale spheres and rods into the phospholipid bilayer. The equilibrium structures and the resulting free energies of the nano-objects in the bilayer allow to distinguish different orientations in the bilayer and estimate the energy barrier of insertion.

  19. Blind bedside insertion of small bowel feeding tubes.

    LENUS (Irish Health Repository)

    Duggan, SN


    The use of Naso-Jejunal (NJ) feeding is limited by difficulty in feeding tube placement. Patients have traditionally required transfer to Endoscopy or Radiology for insertion of small bowel feeding tubes, with clear resource implications. We hypothesised that the adoption of a simple bedside procedure would be effective and reduce cost. Clinical nutrition and nurse specialist personnel were trained in the 10\\/10\\/10 method of blind bedside NJ insertion.

  20. Mechanism for electrochemical hydrogen insertion in carbonaceous materials (United States)

    Qu, Deyang

    The mechanism for safe and reversible storage of hydrogen in porous carbonaceous materials by electrochemical decomposition of water in alkaline electrolyte is proposed. Atomic H was found to be inserted into the microdomains of defective graphene layers. Hydrogen storage capacity increases with increasing interlayer distance between carbon sheets. Hydrogen insertion in carbonaceous materials occurs at ambient conditions. Static potential acts as an electrochemical valve which can retain the hydrogen in the carbon structure, thus preventing leakage during storage.

  1. Templated sequence insertion polymorphisms in the human genome (United States)

    Onozawa, Masahiro; Aplan, Peter


    Templated Sequence Insertion Polymorphism (TSIP) is a recently described form of polymorphism recognized in the human genome, in which a sequence that is templated from a distant genomic region is inserted into the genome, seemingly at random. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; Class 1 TSIPs show features of insertions that are mediated via the LINE-1 ORF2 protein, including 1) target-site duplication (TSD), 2) polyadenylation 10-30 nucleotides downstream of a “cryptic” polyadenylation signal, and 3) preference for insertion at a 5’-TTTT/A-3’ sequence. In contrast, class 2 TSIPs show features consistent with repair of a DNA double-strand break via insertion of a DNA “patch” that is derived from a distant genomic region. Survey of a large number of normal human volunteers demonstrates that most individuals have 25-30 TSIPs, and that these TSIPs track with specific geographic regions. Similar to other forms of human polymorphism, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases.

  2. Polyubiquitin insertions and the phylogeny of Cercozoa and Rhizaria. (United States)

    Bass, David; Moreira, David; López-García, Purificación; Polet, Stephane; Chao, Ema E; von der Heyden, Sophie; Pawlowski, Jan; Cavalier-Smith, Thomas


    A single or double amino acid insertion at the monomer-monomer junction of the universal eukaryotic protein polyubiquitin is unique to Cercozoa and Foraminifera, closely related 'core' phyla in the protozoan infrakingdom Rhizaria. We screened 11 other candidate rhizarians for this insertion: Radiozoa (polycystine and acantharean radiolaria), a 'microheliozoan', and Apusozoa; all lack it, supporting suggestions that Foraminifera are more closely related to Cercozoa than either is to other eukaryotes. The insertion's size was ascertained for 12 additional Cercozoa to help resolve their basal branching order. The earliest branching Cercozoa generally have a single amino acid insertion, like all Foraminifera, but a large derived clade consisting of all Monadofilosa except Metopion, Helk-esimastix, and Cercobodo agilis has two amino acids, suggesting one doubling event and no reversions to a single amino acid. Metromonas and Sainouron, cercozoans of uncertain position, have a double insertion, suggesting that they belong in Monadofilosa. An alternative interpretation, suggested by the higher positions for Metopion and Cercobodo on Bayesian trees compared with most distance trees, cannot be ruled out, i.e. that the second insertion took place earlier, in the ancestral filosan, and was followed by three independent reversions to a single amino acid in Chlorarachnea, Metopion and Cercobodo.

  3. Drug transport in HEMA conjunctival inserts containing precipitated drug particles. (United States)

    Gupta, Chhavi; Chauhan, Anuj


    This paper focuses on exploring the mechanism of cyclosporine A transport in hydroxyethyl methacrylate (HEMA) rods to develop conjunctival inserts for extended ocular delivery. Cylindrical conjunctival HEMA inserts were prepared by thermal polymerization in presence of drug at high loadings to create rods containing particles of drug dispersed in the matrix. The drug release rates were measured to explore the effect of length, drug loading, crosslinking, and mixing in the release medium. Also microstructure of the inserts was characterized by SEM imaging. The inserts release the drug for a period of about a month at therapeutic rates. The rates of drug release are zero order and independent of drug loading and crosslinking for certain period of time. These effects were shown to arise due to a mass-transfer boundary layer in the fluid and a mathematical model was developed by coupling mass transfer in the insert with that in the boundary layer in the surrounding fluid. The model with diffusivity in the insert and boundary layer thickness as parameters fits the experimental data and explains all trends in release kinetics. The fitted diffusivity is about twice that obtained by direct measurements, which agreed well with the value obtained by using the Brinkman's equation but only after accounting for drug binding to the polymer.

  4. A new specifically designed forceps for chest drain insertion.

    LENUS (Irish Health Repository)

    Andrews, Emmet


    Insertion of a chest drain can be associated with serious complications. It is recommended that the drain is inserted with blunt dissection through the chest wall but there is no specific instrument to aid this task. We describe a new reusable forceps that has been designed specifically to facilitate the insertion of chest drains.A feasibility study of its use in patients who required a chest drain as part of elective cardiothoracic operations was undertaken. The primary end-point was successful and accurate placement of the drain. The operators also completed a questionnaire rating defined aspects of the procedure. The new instrument was used to insert the chest drain in 30 patients (19 male, 11 female; median age 61.5 years (range 16-81 years)). The drain was inserted successfully without the trocar in all cases and there were no complications. Use of the instrument rated as significantly easier relative to experience of previous techniques in all specified aspects. The new device can be used to insert intercostal chest drains safely and efficiently without using the trocar or any other instrument.

  5. Modeling Amyloid Beta Peptide Insertion into Lipid Bilayers

    CERN Document Server

    Mobley, D L; Singh, R R P; Maddox, M W; Longo, M J; Mobley, David L.; Cox, Daniel L.; Singh, Rajiv R. P.; Maddox, Michael W.; Longo, Marjorie L.


    Inspired by recent suggestions that the Alzheimer's amyloid beta peptide (A-beta), can insert into cell membranes and form harmful ion channels, we model insertion of the peptide into cell membranes using a Monte Carlo code which is specific at the amino acid level. We examine insertion of the regular A-beta peptide as well as mutants causing familial Alzheimer's disease. We present our results and develop the hypothesis that partial insertion into the membrane, leaving the peptide in one leaflet, increases the probability of harmful channel formation. This hypothesis can partly explain why these mutations are neurotoxic simply due to peptide insertion behavior, and also explains why, normally, A-beta 42 is more toxic to some cultured cells than A-beta 40, but the E22Q mutation reverses this effect. We further apply this model to various artificial A-beta mutants which have been examined experimentally, and offer testable experimental predictions contrasting the roles of aggregation and insertion with regard ...

  6. Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii Isolamento e caracterização de um gene de quitinase do fungo entomopatogênico Verticillium lecanii


    Yanping Zhu; Jieru Pan; Junzhi Qiu; Xiong Guan


    Entomopathogenic fungus Verticillium lecanii is a promising whitefly and aphid control agent. Chitinases secreted by this insect pathogen have considerable importance in the biological control of some insect pests. An endochitinase gene Vlchit1 from the fungus was cloned and overexpressed in Escherichia coli. The Vlchit1 gene not only contains an open reading frame (ORF) which encodes a protein of 423 amino acids (aa), but also is interrupted by three short introns. A homology modelling of Vl...

  7. Frustratingly Easy Domain Adaptation

    CERN Document Server

    Daumé, Hal


    We describe an approach to domain adaptation that is appropriate exactly in the case when one has enough ``target'' data to do slightly better than just using only ``source'' data. Our approach is incredibly simple, easy to implement as a preprocessing step (10 lines of Perl!) and outperforms state-of-the-art approaches on a range of datasets. Moreover, it is trivially extended to a multi-domain adaptation problem, where one has data from a variety of different domains.

  8. Staggered domain wall fermions

    CERN Document Server

    Hoelbling, Christian


    We construct domain wall fermions with a staggered kernel and investigate their spectral and chiral properties numerically in the Schwinger model. In some relevant cases we see an improvement of chirality by more than an order of magnitude as compared to usual domain wall fermions. Moreover, we present first results for four-dimensional quantum chromodynamics, where we also observe significant reductions of chiral symmetry violations for staggered domain wall fermions.

  9. Translation domains in multiferroics


    Meier, D; Leo, N; Jungk, T.; Soergel, E.; Becker, P.; Bohaty, L.; Fiebig, M.


    Translation domains differing in the phase but not in the orientation of the corresponding order parameter are resolved in two types of multiferroics. Hexagonal (h-) YMnO$_3$ is a split-order-parameter multiferroic in which commensurate ferroelectric translation domains are resolved by piezoresponse force microscopy whereas MnWO$_4$ is a joint-order-parameter multiferroic in which incommensurate magnetic translation domains are observed by optical second harmonic generation. The pronounced ma...

  10. Pragmatic circuits frequency domain

    CERN Document Server

    Eccles, William


    Pragmatic Circuits: Frequency Domain goes through the Laplace transform to get from the time domain to topics that include the s-plane, Bode diagrams, and the sinusoidal steady state. This second of three volumes ends with a-c power, which, although it is just a special case of the sinusoidal steady state, is an important topic with unique techniques and terminology. Pragmatic Circuits: Frequency Domain is focused on the frequency domain. In other words, time will no longer be the independent variable in our analysis. The two other volumes in the Pragmatic Circuits series include titles on DC

  11. 枯草芽孢杆菌SL-13的生防性能及其抗菌几丁质酶特性研究%Biocontrol Efficiency of Bacillus subtilis SL-13 and Characterization of an Antifungal Chitinase

    Institute of Scientific and Technical Information of China (English)

    刘燕; 陶晶; 阎豫君; 李彬; 李晖; 李春


    The seed germination and tomato seedling tests showed that Bacillus subtilis SL-13 could promote the sprouting and seedling growth of tomato. The fresh and dry weight of tomato seedlings increased 42.86% and 18.75%, respectively. The control efficacies of the SL-13 to tomato Rhizoctonia rot were 20.65% and 35.23% in the greenhouse and field, respectively. The growth of the plant-pathogenic fungus Rhizoctonia solani was considerably inhibited in the presence of the strain SL-13 culture supernatant. The main antifungal protein was detected to be chitinase through vitro assay. The chitinase was purified with DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration for further characterization. The optimal pH and temperature for the chitinase activity were 7.0 and 50 ℃, respectively. It was demonstrated that the enzyme was stable at pH 5-9 and 40-60 ℃. 70% of the enzyme activity was retained when incubated at 121 ℃ and 0.11 MPa for 20 min, and the enzyme was not sensitive to protease K and ultraviolet radiation. Thus it is suitable for effective biological control in relatively unstable environment.

  12. Visualizing domain wall and reverse domain superconductivity. (United States)

    Iavarone, M; Moore, S A; Fedor, J; Ciocys, S T; Karapetrov, G; Pearson, J; Novosad, V; Bader, S D


    In magnetically coupled, planar ferromagnet-superconductor (F/S) hybrid structures, magnetic domain walls can be used to spatially confine the superconductivity. In contrast to a superconductor in a uniform applied magnetic field, the nucleation of the superconducting order parameter in F/S structures is governed by the inhomogeneous magnetic field distribution. The interplay between the superconductivity localized at the domain walls and far from the walls leads to effects such as re-entrant superconductivity and reverse domain superconductivity with the critical temperature depending upon the location. Here we use scanning tunnelling spectroscopy to directly image the nucleation of superconductivity at the domain wall in F/S structures realized with Co-Pd multilayers and Pb thin films. Our results demonstrate that such F/S structures are attractive model systems that offer the possibility to control the strength and the location of the superconducting nucleus by applying an external magnetic field, potentially useful to guide vortices for computing application.

  13. Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B. (United States)

    Genisyuerek, Selda; Papatheodorou, Panagiotis; Guttenberg, Gregor; Schubert, Rolf; Benz, Roland; Aktories, Klaus


    Clostridium difficile toxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N-terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell-binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501-1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1-1550 and the receptor-binding domain of diphtheria toxin, caused cytotoxic effects. A large N-terminal part of toxin B (amino acids 1-829) was not essential for pore formation (measured by (86) Rb(+) release in mammalian cells). Studies using C-terminal truncation fragments of toxin B showed that amino acid residues 1-990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore-forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate-970 and glutamate-976 in pore formation of toxin B.

  14. Preprocedural ultrasound examination versus manual palpation for thoracic epidural catheter insertion

    Directory of Open Access Journals (Sweden)

    Ahmed M Hasanin


    Conclusion: Preprocedural ultrasound imaging increased the incidence of first pass success in thoracic epidural catheter insertion and reduced the catheter insertion time compared to manual palpation method.

  15. Domain Walls on Singularities

    CERN Document Server

    Halyo, Edi


    We describe domain walls that live on $A_2$ and $A_3$ singularities. The walls are BPS if the singularity is resolved and non--BPS if it is deformed and fibered. We show that these domain walls may interpolate between vacua that support monopoles and/or vortices.

  16. A Domain Analysis Bibliography (United States)


    Bauhaus , a prototype CASE workstation for D-SAPS development. [ARAN88A] Guillermo F. Arango. Domain Engineering for Software Reuse. PhD thesis...34 VITA90B: Domain Analysis within the ISEC Rapid Center 48 CMU/SEI-90-SR-3 Appendix III Alphabetical by Organization/Project BAUHAUS * ALLE87A

  17. Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356 : evidence for protein : protein interaction of two subdomains

    NARCIS (Netherlands)

    Smit, E.; Jager, D.; Martinez, B.; Tielen, F.J.; Pouwels, P.H.


    The structure of the crystallisation domain, SAN, of the S A-protein of Lactobacillus acidophilus ATCC 4356 was analysed by insertion and deletion mutagenesis, and by proteolytic treatment. Mutant S A-protein synthesised in Escherichia coli with 7-13 amino acid insertions near the N terminus or with

  18. Measurement of trocar insertion force using a piezoelectric transducer. (United States)

    Ng, Pui Shan; Sahota, Daljit Singh; Yuen, Pong Mo


    We attempted to establish a model to measure the force required for trocar insertion at laparoscopy. A 3-cm, circular transducer was constructed from piezoresistive material that changes its impedance as force is exerted on its surface. The transducer is connected by an interface box to a personal computer to record surface contact pressure digitally (pressure = force/area) profile continuously during trocar insertion. Each subject had three trocars inserted: a 10-mm trocar at the umbilicus after creation of pneumoperitoneum, and 5-mm trocars at corresponding sites on the left and right sides of the lower abdomen. All insertions were performed by the same operator using reusable trocar with a conical tip. Each subject acted as her own control. Recordings were successfully obtained from eight women. There was no instance of transducer failure. The mean (SE) peak contact surface pressure for the 10-mm and 5-mm left and right trocars were 5.3 (0.32), 6.4 (0.51), and 6.81 (0.27) pounds/square inch, respectively. Placement of the 10-mm trocar required less insertion force than placement of the 5-mm trocars. There was a strong negative correlation (r = -0.97, p trocar.

  19. Seamless lesion insertion in digital mammography: methodology and reader study (United States)

    Pezeshk, Aria; Petrick, Nicholas; Sahiner, Berkman


    Collection of large repositories of clinical images containing verified cancer locations is costly and time consuming due to difficulties associated with both the accumulation of data and establishment of the ground truth. This problem poses a significant challenge to the development of machine learning algorithms that require large amounts of data to properly train and avoid overfitting. In this paper we expand the methods in our previous publications by making several modifications that significantly increase the speed of our insertion algorithms, thereby allowing them to be used for inserting lesions that are much larger in size. These algorithms have been incorporated into an image composition tool that we have made publicly available. This tool allows users to modify or supplement existing datasets by seamlessly inserting a real breast mass or micro-calcification cluster extracted from a source digital mammogram into a different location on another mammogram. We demonstrate examples of the performance of this tool on clinical cases taken from the University of South Florida Digital Database for Screening Mammography (DDSM). Finally, we report the results of a reader study evaluating the realism of inserted lesions compared to clinical lesions. Analysis of the radiologist scores in the study using receiver operating characteristic (ROC) methodology indicates that inserted lesions cannot be reliably distinguished from clinical lesions.

  20. Heat Transfer Enhancement by Using Different Types of Inserts

    Directory of Open Access Journals (Sweden)

    S. Tabatabaeikia


    Full Text Available Heat transfer enhancement has been always a significantly interesting topic in order to develop high efficient, low cost, light weight, and small heat exchangers. The energy cost and environmental issue are also encouraging researchers to achieve better performance than the existing designs. Two of the most effective ways to achieve higher heat transfer rate in heat exchangers are using different kinds of inserts and modifying the heat exchanger tubes. There are different kinds of inserts employed in the heat exchanger tubes such as helical/twisted tapes, coiled wires, ribs/fins/baffles, and winglets. This paper presents an overview about the early studies on the improvement of the performance of thermal systems by using different kinds of inserts. Louvered strip insert had better function in backward flow compared to forward one. Modifying the shape of twisted tapes led to a higher efficiency in most of the cases excpet for perforated twisted tape and notched twisted tape. Combination of various inserts and tube with artificial roughness provided promising results. In case of using various propeller types, heat transfer enhancement was dependent on higher number of blades and blade angle and lower pitch ratio.

  1. Customizable engineered blood vessels using 3D printed inserts. (United States)

    Pinnock, Cameron B; Meier, Elizabeth M; Joshi, Neeraj N; Wu, Bin; Lam, Mai T


    Current techniques for tissue engineering blood vessels are not customizable for vascular size variation and vessel wall thickness. These critical parameters vary widely between the different arteries in the human body, and the ability to engineer vessels of varying sizes could increase capabilities for disease modeling and treatment options. We present an innovative method for producing customizable, tissue engineered, self-organizing vascular constructs by replicating a major structural component of blood vessels - the smooth muscle layer, or tunica media. We utilize a unique system combining 3D printed plate inserts to control construct size and shape, and cell sheets supported by a temporary fibrin hydrogel to encourage cellular self-organization into a tubular form resembling a natural artery. To form the vascular construct, 3D printed inserts are adhered to tissue culture plates, fibrin hydrogel is deposited around the inserts, and human aortic smooth muscle cells are then seeded atop the fibrin hydrogel. The gel, aided by the innate contractile properties of the smooth muscle cells, aggregates towards the center post insert, creating a tissue ring of smooth muscle cells. These rings are then stacked into the final tubular construct. Our methodology is robust, easily repeatable and allows for customization of cellular composition, vessel wall thickness, and length of the vessel construct merely by varying the size of the 3D printed inserts. This platform has potential for facilitating more accurate modeling of vascular pathology, serving as a drug discovery tool, or for vessel repair in disease treatment.

  2. LMA Supreme insertion by novices in manikins and patients. (United States)

    Howes, B W; Wharton, N M; Gibbison, B; Cook, T M


    The LMA Supreme has been suggested for use in emergency situations by medical personnel with no experience in endotracheal intubation. We evaluated the LMA Supreme when inserted by non-anaesthetists, firstly in a manikin and then in patients. Fifty airway novices inserted a LMA Supreme in a manikin without any complications so we proceeded to the patient phase. Fifty airway novices inserted the LMA Supreme in anaesthetised patients undergoing elective surgery. First time insertion success rate was 86% and overall insertion success rate was 100%. Mechanical ventilation was successful in all cases. Median (IQR [range]) time to establish an airway was 34 s (26-40 [18-145] s). Median (IQR [range]) pharyngeal seal pressure was 23 cmH(2)O (19-28 [13-40] cmH(2)O). There were no important complications. Results are consistent with previous studies of use of the LMA Supreme by airway experts. We conclude that the LMA supreme is suitable for use by airway novices. Further research is needed before it may be recommended for cardiopulmonary resuscitation and emergency airway use.

  3. Recombination of an intrachromosomal paracentric insertion of chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Best, R.G.; Burnett, W.J.; Brock, J.K. [Univ. of South Carolina School of Medicine, Columbia, SC (United States)] [and others


    Cytogenetic studies were initiated on a newborn female due to multiple congenital anomalies including microcephaly, clinodactyly, abnormal positioning of hands, left facial palsy, heart defect, sacral dimple, and facial dysmorphic features. Facial features were described as low set rotated ears, nystagmus, and a small, flattened nose. A structural rearrangement of the long arm of chromosome 3 was observed with a complex banding pattern. Study of parental chromosomes revealed a normal male pattern for the father, and an intrachromosomal insertion on the long arm of chromosome 3 for the mother described as 46,XX,dir ins(3)(q21q23q26.2). Further characterization of the proband`s structurally abnormal chromosome 3 revealed a karyotype best described as: 46,XX,rec(3),dupq23{r_arrow}q26.2::q21{r_arrow}q23,dir ins(3)(q21q23q26.2), which is a partial duplication of both the inserted segment as well as the intervening segment between the inserted segment and the insertion site. This would appear to be the result of a three-strand double cross-over within the insertion loop. Molecular cytogenetic studies are presently underway to further elucidate chromosome structure of the proband and her mother.

  4. Some physical and chemical indices of clique-inserted lattices (United States)

    Zhang, Zuhe


    The operation of replacing every vertex of an r-regular lattice H by a complete graph of order r is called clique-insertion, and the resulting lattice is called the clique-inserted lattice of H. For any given r-regular lattice, applying this operation iteratively, an infinite family of r-regular lattices is generated. Some interesting lattices including the 3-12-12 lattice can be constructed this way. In this paper, we recall the relationship between the spectra of an r-regular lattice and that of its clique-inserted lattice, and investigate the graph energy and resistance distance statistics. As an application, the asymptotic energy per vertex and average resistance distance of the 3-12-12 and 3-6-24 lattices are computed. We also give formulae expressing the numbers of spanning trees and dimer coverings of the kth iterated clique-inserted lattices in terms of those of the original one. Moreover, we show that new families of expander graphs can be constructed from the known ones by clique-insertion.

  5. Silver clusters insert into polymer solar cell for enhancing light absorption

    Institute of Scientific and Technical Information of China (English)

    Guolong Li; Hongyu Zhen; Zhuoyin Huang; Kan Li; Weidong Shen; Xu Liu


    As an employment of surface plasmonic effect,the consequence of insertion of a layer of Ag clusters into polymer solar cell on the enhancement of light absorption and power conversion efficiency is investigated.Optical analysis based on the finite-difference time-domain (FDTD) is performed with experiments to evaluate the effect of the interaction between the Ag clusters and incident light on light absorption in polymer solar cell Ag clusters modify the light wave vector and the electromagnetic field inside the device is redistributed and enhanced.As a result,polymer solar cells achieve an overall increase in absorption,short-circuit current density,and power conversion efficiency.

  6. Transposable element insertions in long intergenic non-coding RNA genes

    Directory of Open Access Journals (Sweden)

    Sivakumar eKannan


    Full Text Available Transposable elements (TE are abundant in mammalian genomes and appear to have contributed to the evolution of their hosts by providing novel regulatory or coding sequences. We analyzed different regions of long intergenic non-coding RNA (lincRNA genes in human and mouse genomes to systematically assess the potential contribution of TEs to the evolution of the structure and regulation of expression of lincRNA genes. Introns of lincRNA genes contain the highest percentage of TE-derived sequences, followed by exons and then promoter regions although the density of TEs is not significantly different between exons and promoters. Higher frequencies of ancient TEs in promoters and exons compared to introns implies that many lincRNA genes emerged before the split of primates and rodents. The content of TE-derived sequences in lincRNA genes is substantially higher than that in protein-coding genes, especially in exons and promoter regions. A significant positive correlation was detected between the content of TEs and evolutionary rate of lincRNAs indicating that inserted TEs are preferentially fixed in fast-evolving lincRNA genes. These results are consistent with the RIDL (Repeat Insertion Domains of LncRNAs hypothesis under which TEs have substantially contributed to the origin, evolution, and in particular functional diversification, of lincRNA genes.

  7. How to insert a nasogastric tube and check gastric position at the bedside. (United States)

    Best, Carolyn


    Rationale and key points A nasogastric tube (NG tube) can be used to aspirate stomach contents or to administer feed, medication or fluid into the stomach. ▶ A blind technique is used to insert the NG tube through the nostril, along the nasopharynx, through the oesophagus and into the stomach. ▶ It is important for nurses to be able to recognise problems that may arise when inserting a NG tube blindly, and to know what actions to take if it is suspected that the distal tip of the NG tube is not sitting in the stomach, or they are unable to identify its location. ▶ Misplacement and subsequent use of a NG tube to administer feed, medication or fluid is a 'never event' ( NHS England Patient Safety Domain 2015 ). Reflective activity Clinical skills articles can help update your practice and ensure it remains evidence based. Apply this article to your practice. Reflect on and write a short account of: 1. How you think this article will change your practice. 2. How you could use this resource to educate your colleagues. Subscribers can update their reflective accounts at

  8. HACRE1, a recently inserted copia-like retrotransposon of sunflower (Helianthus annuus L.). (United States)

    Buti, M; Giordani, T; Vukich, M; Gentzbittel, L; Pistelli, L; Cattonaro, F; Morgante, M; Cavallini, A; Natali, L


    In this paper we report on the isolation and characterization, for the first time, of a complete 6511 bp retrotransposon of sunflower. Considering its protein domain order and sequence similarity to other copia elements of dicotyledons, this retrotransposon was assigned to the copia retrotransposon superfamily and named HACRE1 (Helianthus annuus copia-like retroelement 1). HACRE1 carries 5' and 3' long terminal repeats (LTRs) flanking an internal region of 4661 bp. The LTRs are identical in their sequence except for two deletions of 7 and 5 nucleotides in the 5' LTR. Based on the sequence identity of the LTRs, HACRE1 was estimated to have inserted within the last approximately 84 000 years. The isolated sequence contains a complete open reading frame with only one complete reading frame. The absence of nonsense mutations agrees with the very high sequence identity between LTRs, confirming that HACRE1 insertion is recent. The haploid genome of sunflower (inbred line HCM) contains about 160 copies of HACRE1. This retrotransposon is expressed in leaflets from 7-day-old plantlets under different light conditions, probably in relation to the occurrence of many putative light-related regulatory cis-elements in the LTRs. However, sequenced cDNAs show less variability than HACRE1 genomic sequences, indicating that only a subset of this family is expressed under these conditions.

  9. Reliable communication over non-binary insertion/deletion channels

    CERN Document Server

    Yazdani, Raman


    We consider the problem of reliable communication over non-binary insertion/deletion channels where symbols are randomly deleted from or inserted in the transmitted sequence and all symbols are corrupted by additive white Gaussian noise. To this end, we utilize the inherent redundancy achievable in non-binary symbol sets by first expanding the symbol set and then allocating part of the bits associated with each symbol to watermark symbols. The watermark sequence, known at the receiver, is then used by a forward-backward algorithm to provide soft information for an outer code which decodes the transmitted sequence. Through numerical results and discussions, we evaluate the performance of the proposed solution and show that it leads to significant system ability to detect and correct insertions/deletions. We also provide estimates of the maximum achievable information rates of the system, compare them with the available bounds, and construct practical codes capable of approaching these limits.

  10. Polyglutamine expansion in huntingtin increases its insertion into lipid bilayers. (United States)

    Kegel, Kimberly B; Schewkunow, Vitali; Sapp, Ellen; Masso, Nicholas; Wanker, Erich E; DiFiglia, Marian; Goldmann, Wolfgang H


    An expanded polyglutamine (Q) tract (>37Q) in huntingtin (htt) causes Huntington disease. Htt associates with membranes and polyglutamine expansion in htt may alter membrane function in Huntington disease through a mechanism that is not known. Here we used differential scanning calorimetry to examine the effects of polyQ expansion in htt on its insertion into lipid bilayers. We prepared synthetic lipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine and tested interactions of htt amino acids 1-89 with 20Q, 32Q or 53Q with the vesicles. GST-htt1-89 with 53Q inserted into synthetic lipid vesicles significantly more than GST-htt1-89 with 20Q or 32Q. We speculate that by inserting more into cell membranes, mutant huntingtin could increase disorder within the lipid bilayer and thereby disturb cellular membrane function.

  11. Isolation of sequences flanking Ac insertion sites by Ac casting. (United States)

    Wang, Dafang; Peterson, Thomas


    Localizing Ac insertions is a fundamental task in studying Ac-induced mutation and chromosomal rearrangements involving Ac elements. Researchers may sometimes be faced with the situation in which the sequence flanking one side of an Ac/Ds element is known, but the other flank is unknown. Or, a researcher may have a small sequence surrounding the Ac/Ds insertion site and needs to obtain additional flanking genomic sequences. One way to rapidly clone unknown Ac/Ds flanking sequences is via a PCR-based method termed Ac casting. This approach utilizes the somatic transposition activity of Ac during plant development, and provides an efficient means for short-range genome walking. Here we describe the principle of Ac casting, and show how it can be applied to isolate Ac macrotransposon insertion sites.

  12. The strategic use of inserts in the Brazilian presidential elections

    Directory of Open Access Journals (Sweden)

    Felipe Borba


    Full Text Available The aim of this article is to analyze the communication strategies of presidential candidates during the elections held in 2006 and 2010. The focus is on the strategic component of electoral inserts and the methodology consists of investigating how candidates choose to distribute these inserts in the programming of television networks. The results indicate that the candidates pursue different strategies influenced basically by three variables: electoral legislation, their standing in polls and the difference of resources available. In parallel, the article debates the role of the regulation of electoral advertising and how this set of rules influences the level of information of campaigns, the occurrence of attacks, and party strategies. Overall, 2,993 electoral inserts were examined.

  13. Genetic aspects of targeted insertion mutagenesis in yeasts. (United States)

    Klinner, U; Schäfer, B


    Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.


    Directory of Open Access Journals (Sweden)

    Daimi S.R


    Full Text Available In view of the variable presentations of biceps brachii, a proper understanding of the anatomy of the muscle, and possible anatomical variants is a pre-requisite in managing distal biceps injury. We present the case of a 65 years old male cadaver showing variationin the insertion pattern of biceps brachii muscle unilaterally on the left arm. Although the origin of the muscle was normal, its insertion was by two separate tendons; a common and an accessory; both inserting on diverse regions of the radial tuberosity. The common tendon was formed by the tendons from short head and long head. The accessory tendon was the extension of the fleshy belly on the lateral side of the main muscle (Accessory Muscle Belly towards its lower third. Knowledge of the accessory tendon of the biceps iscrucial while performing tendon reconstruction and repair and it may also lead to unusual displacement of the bone fragment, subsequent to fracture.

  15. Optimized production and concentration of lentiviral vectors containing large inserts. (United States)

    al Yacoub, Nadya; Romanowska, Malgorzata; Haritonova, Natalie; Foerster, John


    Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10(7)/ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.


    Institute of Scientific and Technical Information of China (English)

    LIN Wen-jian


    In the present paper, the author sums up and analyzes descriptions about needle-insertion depth in Chinese classical medical book Huang Di Nei Jing (The Yellow Emperor's Internal Classic). In many chapters of Nei Jing, the needle-insertion depth is stressed to be various according to 1) the deficiency or excess of syndromes, 2) the patients' constitution, 3) the severity of disease, 4) the duration of disease, 5) the location of disease, 6) the patient's age, 7) the location of the needled acupoint, 8) the season, 9) the patient's temperament, 10) the pulse condition, 11) the state of "Deqi", and 12) the location of the running course of meridians. In addition, different kinds of diseases and different stages of diseases also need different depths of needle insertion, different manipulating skills and different stimulating quantity.

  17. Identification of Fungus Lecanicillium attenuatum and Cloning of Its Chitinase Gene LACHI1%渐狭蜡蚧菌的鉴定及其几丁质酶基因 LACHI1的克隆

    Institute of Scientific and Technical Information of China (English)

    赵洋; 陈德鑫; 王凤龙; 黄化刚; 武侠


    To clone chitinolytic genes from Lecanicillium attenuatum and provide theoretical basis for the inhibi-tion of the chitinases on egg-hatching of root knot and cyst nematode .In this study ,an nematode egg-parasitic fungus CGMCC5328 was isolated from Heterodera glycines infecting soybean in Heilongjiang Province .Based on morphological characters and molecular analysis of ITS-rDNA,the strain was identified as L.attenuatum.The chitinase gene LACHI1 from L.attenuatum was cloned using the degenerate PCR primers and RACE techniques .The analysis of the gene LA-CHI1 and its amino acid sequence was by biology software .We have cloned a chitinase gene LACHI1 from L.attenua-tum for the first time.The gene is 1 743 bp in length and contains three putative introns .The ORF of LACHI1 is 1 272 bp in size with encoding protein of 423 aa,molecular mass of 45.9 kDa and pI of 5.90.The chitinase deduced by LA-CHI1 belongs to glycosyl hydrolase family 18 chitinase .Comparison of the chitinase amino acid sequence with other chitinases from entomopathogenic fungi and nematophagous fungi revealed that the enzymes were highly similar .%克隆渐狭蜡蚧菌几丁质酶基因,为进一步明确该菌产生的几丁质酶对定居性根结类和孢囊类线虫卵孵化抑制作用提供理论依据。采用分离自黑龙江大豆孢囊线虫孢囊寄生真菌CGMCC5328,通过形态学特征及ITS序列比较分析,鉴定该菌株为渐狭蜡蚧菌。通过简并引物设计和RACE技术,克隆渐狭蜡蚧菌中几丁质酶基因,利用生物学软件分析该基因序列及其编码的氨基酸序列。首次从渐狭蜡蚧菌中克隆得到一个几丁质酶基因LACHI1,该基因DNA序列长1743 bp,含3个内含子,包含1272 bp开放阅读框,编码423个氨基酸,理论分子量45.9 kDa,等电点5.90。 LACHI1基因编码的几丁质酶,属于糖基水解酶18家族几丁质酶。同源性比对表明该菌产生的几丁质酶与昆虫寄生真菌和食线虫

  18. Effect of Light Conducting Cylindrical Inserts on Gingival Microleakage

    Directory of Open Access Journals (Sweden)

    SM. Moazzami


    Full Text Available Objective: Microleakage in the gingival floor of class II composite restorations can compromise the marginal adaptation of the filling material to the cavity edges. The aim of this study was to evaluate the effect of light conducting cylindrical inserts in decreasing the microleakage of the gingival floor in cavities 1mm below the CEJ.Materials and Methods: Eighty maxillary first molars were randomly divided into eight groups according to use of glass inserts, type of resin (Coltene unfilled resin versus Scotchbond multi purpose and filling technique (one-unit versus incremental. Proximal class II cavities were prepared in all samples with the gingival floor one millimeter below the CEJ. Etched and silan-treated glass inserts were made from 2mm cylindrical bioglass material and cavities were restored according to research protocol. The samples were subjected to 2500 thermal cycles (5-55oC, immersed in 0.5% basic fuchsin solution, embedded in epoxy resin and cut centrally and laterally (buccally or lingually in a mesiodistal direction. Microleakage was scored and collected data were statistically analyzed using Kruskal-Wallis and Mann-Whitney tests.Results: Minimal dye penetration was observed in the group that employed the incre-mental technique along with Scotchbond, with or without glass inserts. A significant difference was observed between the eight groups. In addition the use of the incremental technique and glass inserts had a significant effect on the microleakage of lateral and central sections, respectively. Application of dentin bonding agent signifi-cantly affected both sections.Conclusion: Glass inserts were effective in decreasing cervical microleakage of class II cavities restored with composite resin.

  19. Bedside prediction of right subclavian venous catheter insertion length

    Directory of Open Access Journals (Sweden)

    Yoon Ji Choi


    Full Text Available Background and objective: The present study aimed to evaluate whether right subclavian vein (SCV catheter insertion depth can be predicted reliably by the distances from the SCV insertion site to the ipsilateral clavicular notch directly (denoted as I-IC, via the top of the SCV arch, or via the clavicle (denoted as I-T-IC and I-C-IC, respectively. Method: In total, 70 SCV catheterizations were studied. The I-IC, I-T-IC, and I-C-IC distances in each case were measured after ultrasound-guided SCV catheter insertion. The actual length of the catheter between the insertion site and the ipsilateral clavicular notch, denoted as L, was calculated by using chest X-ray. Results: L differed from the I-T-IC, I-C-IC, and I-IC distances by 0.14±0.53, 2.19±1.17, and -0.45 ±0.68 cm, respectively. The mean I-T-IC distance was the most similar to the mean L (intraclass correlation coefficient = 0.89. The mean I-IC was significantly shorter than L, while the mean I-C-IC was significantly longer. Linear regression analysis provided the following formula: Predicted SCV catheter insertion length (cm = -0.037 + 0.036 × Height (cm + 0.903 × I-T-IC (cm (adjusted r2 =0.64. Conclusion: The I-T-IC distance may be a reliable bedside predictor of the optimal insertion length for a right SCV cannulation.

  20. Tension Pneumothorax and Subcutaneous Emphysema Complicating Insertion of Nasogastric Tube

    Directory of Open Access Journals (Sweden)

    Narjis AL Saif


    Full Text Available Nasogastric tube has a key role in the management of substantial number of hospitalized patients particularly the critically ill. In spite of the apparent simple insertion technique, nasogastric tube placement has its serious perhaps fatal complications which need to be carefully assessed. Pulmonary misplacement and associated complications are commonplace during nasogastric tube procedure. We present a case of tension pneumothorax and massive surgical emphysema in critically ill ventilated patient due to inadvertent nasogastric tube insertion and also discussed the risk factors, complication list, and arrays of techniques for safer tube placement.

  1. Can femoral dialysis catheter insertion cause a life threatening complication?

    Directory of Open Access Journals (Sweden)

    Nurkay Katrancıoğlu


    Full Text Available Venous catheter (VC insertion may be necessary for the patients with renal failure facing vascular access problem. Femoral VCs are commonly used for their lower complication rates especially in emergency clinics. The incidence of bleeding associated with VC is reported 0.5-1.6%, however, life threatening hemorrhage and complications requiring surgical intervention are very rare. In this manuscript, we aimed to present a case with hemolytic uremic syndrome complicated with retroperitoneal hematoma after femoral VC insertion. J Clin Exp Invest 2014; 5 (3: 472-474

  2. Insertion of lithium into electrochromic devices after completion

    Energy Technology Data Exchange (ETDEWEB)

    Berland, Brian Spencer; Lanning, Bruce Roy; Frey, Jonathan Mack; Barrett, Kathryn Suzanne; DuPont, Paul Damon; Schaller, Ronald William


    The present disclosure describes methods of inserting lithium into an electrochromic device after completion. In the disclosed methods, an ideal amount of lithium can be added post-fabrication to maximize or tailor the free lithium ion density of a layer or the coloration range of a device. Embodiments are directed towards a method to insert lithium into the main device layers of an electrochromic device as a post-processing step after the device has been manufactured. In an embodiment, the methods described are designed to maximize the coloration range while compensating for blind charge loss.

  3. Ultrasonic airborne insertion loss measurements at normal incidence (L). (United States)

    Farley, Jayrin; Anderson, Brian E


    Transmission loss and insertion loss measurements of building materials at audible frequencies are commonly made using plane wave tubes or as a panel between reverberant rooms. These measurements provide information for noise isolation control in architectural acoustics and in product development. Airborne ultrasonic sound transmission through common building materials has not been fully explored. Technologies and products that utilize ultrasonic frequencies are becoming increasingly more common, hence the need to conduct such measurements. This letter presents preliminary measurements of the ultrasonic insertion loss levels for common building materials over a frequency range of 28-90 kHz using continuous-wave excitation.

  4. Imaging of the complications of peripherally inserted central venous catheters

    Energy Technology Data Exchange (ETDEWEB)

    Amerasekera, S.S.H. [Department of Radiology, Good Hope Hospital, Sutton Coldfield, Birmingham (United Kingdom)], E-mail:; Jones, C.M.; Patel, R.; Cleasby, M.J. [Department of Radiology, Good Hope Hospital, Sutton Coldfield, Birmingham (United Kingdom)


    Peripherally inserted central catheters (PICC) are widely used to provide central venous access, often in chronically ill patients with long-term intravenous access requirements. There are a number of significant complications related to both insertion and maintenance of PICC lines, including catheter malposition, migration, venous thrombosis, and line fracture. The incidence of these complications is likely to rise as the number of patients undergoing intravenous outpatient therapy increases, with a corresponding rise in radiologist input. This paper provides an overview of the relevant peripheral and central venous anatomy, including anatomical variations, and outlines the complications of PICC lines. Imaging examples demonstrate the range of radiological findings seen in these complications.

  5. Imaging of the complications of peripherally inserted central venous catheters. (United States)

    Amerasekera, S S H; Jones, C M; Patel, R; Cleasby, M J


    Peripherally inserted central catheters (PICC) are widely used to provide central venous access, often in chronically ill patients with long-term intravenous access requirements. There are a number of significant complications related to both insertion and maintenance of PICC lines, including catheter malposition, migration, venous thrombosis, and line fracture. The incidence of these complications is likely to rise as the number of patients undergoing intravenous outpatient therapy increases, with a corresponding rise in radiologist input. This paper provides an overview of the relevant peripheral and central venous anatomy, including anatomical variations, and outlines the complications of PICC lines. Imaging examples demonstrate the range of radiological findings seen in these complications.

  6. Direct Synthesis of Telechelic Polyethylene by Selective Insertion Polymerization

    KAUST Repository

    Jian, Zhongbao


    A single-step route to telechelic polyethylene (PE) is enabled by selective insertion polymerization. PdII-catalyzed copolymerization of ethylene and 2-vinylfuran (VF) generates α,ω-di-furan telechelic polyethylene. Orthogonally reactive exclusively in-chain anhydride groups are formed by terpolymerization with carbic anhydride. Combined experimental and theoretical DFT studies reveal the key for this direct approach to telechelics to be a match of the comonomers’ different electronics and bulk. Identified essential features of the comonomer are that it is an electron-rich olefin that forms an insertion product stabilized by an additional interaction, namely a π–η3 interaction for the case of VF.

  7. Tension Pneumothorax and Subcutaneous Emphysema Complicating Insertion of Nasogastric Tube. (United States)

    Al Saif, Narjis; Hammodi, Adel; Al-Azem, M Ali; Al-Hubail, Rasheed


    Nasogastric tube has a key role in the management of substantial number of hospitalized patients particularly the critically ill. In spite of the apparent simple insertion technique, nasogastric tube placement has its serious perhaps fatal complications which need to be carefully assessed. Pulmonary misplacement and associated complications are commonplace during nasogastric tube procedure. We present a case of tension pneumothorax and massive surgical emphysema in critically ill ventilated patient due to inadvertent nasogastric tube insertion and also discussed the risk factors, complication list, and arrays of techniques for safer tube placement.

  8. Technology Insertion and Management: Options for the Canadian Forces (United States)


    L’insertion technologique et la gestion de la technologie : Options pour les Forces canadiennes », est une analyse documentaire qualitative de niveau...l’insertion technologique et son utilisation parmi les alliés du Canada et fournira certaines méthodes qui sont nécessaires pour faire une analyse ...technologique. Enfin, elle a fourni une gamme de méthodes nécessaires à une analyse coûts-avantages fondée sur l’optimisation des options en matière

  9. Shield Insertion to Minimize Noise Amplitude in Global Interconnects

    Directory of Open Access Journals (Sweden)



    Full Text Available Shield insertion is an effective technique for minimise crosstalk noise and signal delay uncertainty .To reduce the effects of coupling uniform or simultaneous shielding may be used on either or both sides of a signal line. Shields are ground or power lines placed between two signal wires to prevent direct coupling between them as the shield width increases, the noise amplitude decreases, in this paper inserting a shield line between two coupled interconnects is shown to be more effective in reducing crosstalk noise for different technology nodes .

  10. Genetic Analysis of MEFV Gene Pyrin Domain in Patients With Behçet's Disease

    Directory of Open Access Journals (Sweden)


    Full Text Available Objectives. Behçet's disease (BD is a systemic vasculitis with recurrent oral and genital ulcers and uveitis. MEFV gene, which is the main factor in familial Mediterranean fever (FMF, is also reported to be a susceptibility gene for BD. The pyrin domain of MEFV gene is a member of death-domain superfamily and has been proposed to regulate inflammatory signaling in myeloid cells. This study was designed to determine if mutations in pyrin domain of MEFV gene are involved in BD. Methods. We analyzed the pyrin domain of MEFV gene in 54 Turkish patients with BD by PCR-analysis and direct sequencing. Results. Neither deletion or insertion mutations nor point mutations in pyrin domain were found in any patient. Conclusion. Although pyrin gene mutations have been reported in patients with BD, pyrin domain is not mutated. However, alterations in other regions of MEFV gene and interaction between pyrin domains are needed to be further investigated.

  11. Definition of Metrics to Evaluate Cochlear Array Insertion Forces Performed with Forceps, Insertion Tool, or Motorized Tool in Temporal Bone Specimens

    Directory of Open Access Journals (Sweden)

    Yann Nguyen


    Full Text Available Introduction. In order to achieve a minimal trauma to the inner ear structures during array insertion, it would be suitable to control insertion forces. The aim of this work was to compare the insertion forces of an array insertion into anatomical specimens with three different insertion techniques: with forceps, with a commercial tool, and with a motorized tool. Materials and Methods. Temporal bones have been mounted on a 6-axis force sensor to record insertion forces. Each temporal bone has been inserted, with a lateral wall electrode array, in random order, with each of the 3 techniques. Results. Forceps manual and commercial tool insertions generated multiple jerks during whole length insertion related to fits and starts. On the contrary, insertion force with the motorized tool only rose at the end of the insertion. Overall force momentum was 1.16 ± 0.505 N (mean ± SD, n=10, 1.337 ± 0.408 N (n=8, and 1.573 ± 0.764 N (n=8 for manual insertion with forceps and commercial and motorized tools, respectively. Conclusion. Considering force momentum, no difference between the three techniques was observed. Nevertheless, a more predictable force profile could be observed with the motorized tool with a smoother rise of insertion forces.

  12. C-reactive protein and chitinase 3-like protein 1 as biomarkers of spatial redistribution of retinal blood vessels on digital retinal photography in patients with diabetic retinopathy. (United States)

    Cekić, Sonja; Cvetković, Tatjana; Jovanović, Ivan; Jovanović, Predrag; Pesić, Milica; Stanković Babić, Gordana; Milenković, Svetislav; Risimić, Dijana


    The aim of the study was to investigate the correlation between the levels of C-reactive protein (CRP) and chitinase 3-like protein 1 (YKL-40) in blood samples with morpohometric parameters of retinal blood vessels in patients with diabetic retinopathy. Blood laboratory examination of 90 patients included the measurement of glycemia, HbA1C, total cholesterol, LDL-C, HDL-C, triglycerides and CRP. Levels of YKL-40 were detected and measured in serum by ELISA (Micro VueYKL-40 EIA Kit, Quidel Corporation, San Diego, USA). YKL-40 correlated positively with diameter and negatively with number of retinal blood vessels. The average number of the blood vessels per retinal zone was significantly higher in the group of patients with mild non-proliferative diabetic retinopathy than in the group with severe form in the optic disc and all five retinal zones. The average outer diameter of the evaluated retinal zones and optic disc vessels was significantly higher in the group with severe compared to the group with mild diabetic retinopathy. Morphological analysis of the retinal vessels on digital fundus photography and correlation with YKL-40 may be valuable for the follow-up of diabetic retinopathy.

  13. The supermodule insertion tool of the CMS electromagnetic calorimeter and the first trial insertion of a supermodule.

    CERN Multimedia

    Maximilien Brice


    The first trial insertion of a complete Electromagnetic Calorimeter (ECAL) "supermodule" (1700 lead-tungstate crystals, with support structures, light detectors (avalanche photodiodes), readout electronics and cooling system) was performed on 1st March. This delicate operation - sliding a 2-tonne 3m-long object onto support rails (in real life these are attached to the barrel hadron calorimeter (HCAL)) - made use of a custom designed "squirrel cage". The rotatable squirrel cage allows the insertion of any supermodule into any of the 18 positions, including very fine (sub-mm) adjustments. The first supermodule will be inserted into the real HCAL later this month in preparation for the "magnet test and cosmic-ray challenge" (MTCC). In the first image the supermodule is in the centre and the alignment disks are highlighted by the flash.

  14. Conserved Domain Database (CDD) (United States)

    U.S. Department of Health & Human Services — CDD is a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins.

  15. Sequences determining the cytoplasmic localization of a chemoreceptor domain. (United States)

    Seligman, L; Bailey, J; Manoil, C


    The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure. PMID:7730259

  16. How does the Shift-insertion sort behave when the sorting elements follow a Normal distribution?

    CERN Document Server

    Pal, Mita; Mahanti, N C


    The present paper examines the behavior of Shift-insertion sort (insertion sort with shifting) for normal distribution inputs and is in continuation of our earlier work on this new algorithm for discrete distribution inputs, namely, negative binomial. Shift insertion sort is found more sensitive for main effects but not for all interaction effects compared to conventional insertion sort.

  17. Conservation of intron and intein insertion sites: implications for life histories of parasitic genetic elements

    Directory of Open Access Journals (Sweden)

    Senejani Alireza G


    Full Text Available Abstract Background Inteins and introns are genetic elements that are removed from proteins and RNA after translation or transcription, respectively. Previous studies have suggested that these genetic elements are found in conserved parts of the host protein. To our knowledge this type of analysis has not been done for group II introns residing within a gene. Here we provide quantitative statistical support from an analyses of proteins that host inteins, group I introns, group II introns and spliceosomal introns across all three domains of life. Results To determine whether or not inteins, group I, group II, and spliceosomal introns are found preferentially in conserved regions of their respective host protein, conservation profiles were generated and intein and intron positions were mapped to the profiles. Fisher's combined probability test was used to determine the significance of the distribution of insertion sites across the conservation profile for each protein. For a subset of studied proteins, the conservation profile and insertion positions were mapped to protein structures to determine if the insertion sites correlate to regions of functional activity. All inteins and most group I introns were found to be preferentially located within conserved regions; in contrast, a bacterial intein-like protein, group II and spliceosomal introns did not show a preference for conserved sites. Conclusions These findings demonstrate that inteins and group I introns are found preferentially in conserved regions of their respective host proteins. Homing endonucleases are often located within inteins and group I introns and these may facilitate mobility to conserved regions. Insertion at these conserved positions decreases the chance of elimination, and slows deletion of the elements, since removal of the elements has to be precise as not to disrupt the function of the protein. Furthermore, functional constrains on the targeted site make it more difficult

  18. Otological Findings Ten Years after Myringotomy with Tympanostomy Tube Insertion

    Directory of Open Access Journals (Sweden)

    Ali Goljanian Tabrizi


    Full Text Available Introduction: To study the long-term complications of tympanostomy tube insertion in young children 10 years after surgery.   Materials and Methods: In September 2011, the medical records of all patients who had undergone myringotomy with tympanostomy tube insertion between February 2000 and March 2001 at the two general hospitals of Isfahan University of Medical Sciences were studied. Of the 98 patients who fulfilled the inclusion criteria, 82 patients agreed to participate and were enrolled in the study. The complications of the operation were evaluated in these patients.   Results: Of the 164 ears that were operated on, myringosclerosis was found in 17.1%, atrophy of the tympanic membrane in 1.2%, permanent perforation of the tympanic membrane in 0.6% and tympanic membrane atelectasis in 0.6%. None of the patients developed cholesteatoma as a complication of tympanostomy tube insertion.   Conclusion:  Considering the low risk of serious complications after 10 years, tympanostomy tube insertion is a safe and effective treatment option in the treatment of otitis media with effusion.

  19. Insertion DNA Accelerates Meiotic Interchromosomal Recombination in Arabidopsis thaliana. (United States)

    Sun, Xiao-Qin; Li, Ding-Hong; Xue, Jia-Yu; Yang, Si-Hai; Zhang, Yan-Mei; Li, Mi-Mi; Hang, Yue-Yu


    Nucleotide insertions/deletions are ubiquitous in eukaryotic genomes, and the resulting hemizygous (unpaired) DNA has significant, heritable effects on adjacent DNA. However, little is known about the genetic behavior of insertion DNA. Here, we describe a binary transgenic system to study the behavior of insertion DNA during meiosis. Transgenic Arabidopsis lines were generated to carry two different defective reporter genes on nonhomologous chromosomes, designated as "recipient" and "donor" lines. Double hemizygous plants (harboring unpaired DNA) were produced by crossing between the recipient and the donor, and double homozygous lines (harboring paired DNA) via self-pollination. The transfer of the donor's unmutated sequence to the recipient generated a functional β-glucuronidase gene, which could be visualized by histochemical staining and corroborated by polymerase chain reaction amplification and sequencing. More than 673 million seedlings were screened, and the results showed that meiotic ectopic recombination in the hemizygous lines occurred at a frequency  >6.49-fold higher than that in the homozygous lines. Gene conversion might have been exclusively or predominantly responsible for the gene correction events. The direct measurement of ectopic recombination events provided evidence that an insertion, in the absence of an allelic counterpart, could scan the entire genome for homologous counterparts with which to pair. Furthermore, the unpaired (hemizygous) architectures could accelerate ectopic recombination between itself and interchromosomal counterparts. We suggest that the ectopic recombination accelerated by hemizygous architectures may be a general mechanism for interchromosomal recombination through ubiquitously dispersed repeat sequences in plants, ultimately contributing to genetic renovation and eukaryotic evolution.

  20. Insertion and deletion mutagenesis of the human cytomegalovirus genome

    Energy Technology Data Exchange (ETDEWEB)

    Spaete, R.R.; Mocarski, E.S.


    Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. The authors have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major ..beta.. gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this ..beta.. gene within the L-component repeats of CMV DNA. They observed high-level expression of ..beta..-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl ..beta..-D-galactoside, they generated random endpoint deletion mutants. Analysis of these mutant revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, they have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.

  1. Prototype ISR Superconducting Quadrupole for the low beta insertion.

    CERN Multimedia


    The four coils are provisionally kept together by aluminium clamps while epoxy-glass bands are wrapped around them to form a number of spacer rings.Stainless steel spacers were then inserted between these rings and the yoke quadrants. The persons are Michel Bouvier(left) and Pierre Pugin. See also7702690X.

  2. Root cause of incomplete control rod insertions at Westinghouse reactors

    Energy Technology Data Exchange (ETDEWEB)

    Ray, S. [Westinghouse, Monroeville, PA (United States)


    Within the past year, incomplete RCCA insertions have been observed on high burnup fuel assemblies at two Westinghouse PWRs. Initial tests at the Wolf Creek site indicated that the direct cause of the incomplete insertions observed at Wolf Creek was excessive fuel assembly thimble tube distortion. Westinghouse committed to the NRC to perform a root cause analysis by the end of August, 1996. The root cause analysis process used by Westinghouse included testing at ten sites to obtain drag, growth and other characteristics of high burnup fuel assemblies. It also included testing at the Westinghouse hot cell of two of the Wolf Creek incomplete insertion assemblies. A mechanical model was developed to calculate the response of fuel assemblies when subjected to compressive loads. Detailed manufacturing reviews were conducted to determine if this was a manufacturing related issue. In addition, a review of available worldwide experience was performed. Based on the above, it was concluded that the thimble tube distortion observed on the Wolf Creek incomplete insertion assemblies was caused by unusual fuel assembly growth over and above what would typically be expected as a result of irradiation exposure. It was determined that the unusual growth component is a combination of growth due to oxide accumulation and accelerated growth, and would only be expected in high temperature plants on fuel assemblies that see long residence times and high power duties.

  3. Contrast-free endoscopic stent insertion in malignant biliary obstruction

    Institute of Scientific and Technical Information of China (English)


    AIM: To present a case series of MRCP-guided endoscopic biliary stent placement, performed entirely without contrast injection.METHODS: Contrast-free endoscopic biliary drainage was attempted in 20 patients with malignant obstruction,unsuitable for resection on the basis of tumor extent or medical illness. MRCP images were used to confirm the diagnosis of tumor, to exclude other biliary diseases and to demonstrate the stenoses as well as dilation of proximal liver segments. The procedure was carried out under conscious sedation. Patients were placed in the left lateral decubitus position. The endoscope was inserted, the papilla identified and cannulated by a papillotome. A guide wire was inserted and guided deeply into the biliary tree, above the stenosis, by fluoroscopy. A papillotomy approximately 1 cm. long was performed and the papillotome was exchanged with a guiding-catheter. A 10 Fr, Amsterdam-type plastic stent,7 to 15 cm long, was finally inserted over the guide wire/guiding catheter by a pusher tube system.RESULTS: Successful stent insertion was achieved in all patients. There were no major complications. Successful drainage, with substantial reduction in bilirubin levels,was achieved in all patients.CONCLUSION: This new method of contrast-free endoscopic stenting in malignant biliary obstruction is a safe and effective method of palliation. However, a larger, randomized study comparing this new approach with the standard procedure is needed to confirm the findings of the present study.

  4. ArcGIS Tool: Inserts file name into attribute table (United States)

    US Fish and Wildlife Service, Department of the Interior — This ArcGIS model inserts a file name into a feature class attribute table. The tool allows an user to identify features by a field that reference the name of the...

  5. Surgical insertion of transmitters and telemetry methods in fisheries research

    DEFF Research Database (Denmark)

    Rub, A. Michelle Wargo; Jepsen, Niels; Liedtke, Theresa L.;


    ) will be described. Effects of surgical insertion of transmitters (ie, tagging) and aspects of the surgical implantation process where collaboration and professional exchanges among nonveterinarian researchers and veterinarians may be most fruitful will be discussed. Although this report focuses on surgical...

  6. Astronaut Scott Carpenter inserted into Aurora 7 spacecraft (United States)


    Astronaut M. Scott Carpenter, pilot of the Mercury-Atlas 7 space flight, is inserted into Aurora 7 spacecraft during the prelaunch countdown. Carpenter is assisted into the spacecraft by Astronaut John Glenn and Gunter Vendt, McDonnell Douglas pad capsule test conducter.

  7. Meningococcal septic shock after IUD insertion, a case presentation. (United States)

    Romosan, Gina; Blidisel, A; Grigoras, D; Houtsios, A; Ionac, M


    Neisseria meningitidis is a normal commensal of human mucous membranes that is no longer considered to be restricted to the nasopharynx. Due to the practice of oral sex, the mucous membranes of the cervix, urethra or anus have become a potential infection site for this bacterium. Inserting an intrauterine device (IUD), can alter the protective barrier of the endocervical mucosa, allowing for bacterial infection and systemic spread. We present a case report of a 40-year-old woman who presented with abdominal pain, spotting and fever after inserting an IUD and developed a fulminant septic shock. Blood cultures and cultures from ascites showed the presence of Neisseria meningitidis group Y. From our knowledge, there are a few cases presented in the literature of toxic shock syndrome after IUD insertion, caused by Staphylococcus aureus or Streptococcus group A, but this is the first case of meningococcal sepsis after IUD insertion described. So, even though IUDs rarely cause significant infection, physicians should consider this device as a possible source in reproductive-age women with the clinical features of sepsis.




    The paper analyses production of reusable ceramic insulating inserts applied in permanent mold casting of aluminum alloys. It presents results of manufacturing of ceramic products from synthesized materials based on wollastonite, secondary grog, aluminum slag, etc. The paper demonstrates prospects of their applying.


    Directory of Open Access Journals (Sweden)

    A. T. Volochko


    Full Text Available The paper analyses production of reusable ceramic insulating inserts applied in permanent mold casting of aluminum alloys. It presents results of manufacturing of ceramic products from synthesized materials based on wollastonite, secondary grog, aluminum slag, etc. The paper demonstrates prospects of their applying.

  10. Folding and insertion thermodynamics of the transmembrane WALP peptide

    Energy Technology Data Exchange (ETDEWEB)

    Bereau, Tristan, E-mail: [Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz (Germany); Bennett, W. F. Drew [Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada); Pfaendtner, Jim [Department of Chemical Engineering, University of Washington, Seattle, Washington 98195 (United States); Deserno, Markus [Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Karttunen, Mikko [Department of Mathematics and Computer Science & Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, MetaForum, 5600 MB Eindhoven (Netherlands)


    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA){sub n} (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide’s insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum—in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence.

  11. Proper Angle of Sono-guided Central Venous Line Insertion (United States)

    Barzegari, Hassan; Forouzan, Arash; Fahimi, Mohammad Ali; Zohrevandi, Behzad; Ghanavati, Mandana


    Introduction: Determining the proper angle for inserting central venous catheter (CV line) is of great importance for decreasing the complications and increasing success rate. The present study was designed to determine the proper angle of needle insertion for internal jugular vein catheterization. Methods: In the present case series study, candidate patients for catheterization of the right internal jugular vein under guidance of ultrasonography were studied. At the time of proper placing of the catheter, photograph was taken and Auto Cad 2014 software was used to measure the angles of the needle in the sagittal and axial planes, as well as patient’s head rotation. Result: 114 patients with the mean age of 56.96 ± 14.71 years were evaluated (68.4% male). The most common indications of catheterization were hemodialysis (55.3%) and shock state (24.6%). The mean angles of needle insertion were 102.15 ± 6.80 for axial plane, 36.21 ± 3.12 for sagittal plane and the mean head rotation angle was 40.49 ± 5.09. Conclusion: Based on the results of the present study it seems that CV line insertion under the angles 102.15 ± 6.80 degrees in the axial plane, 36.21 ± 3.12 in the sagittal plane and 40.49 ± 5.09 head rotation yield satisfactory results. PMID:27299146


    Directory of Open Access Journals (Sweden)

    L. R. Dudetskaja


    Full Text Available The paper discusses distinctive design features of casting molds and technological aspects of producing cast inserts from 5ХНМЛ pressed steel. The designs of long-life metal shell molds are described. They ensure saving of molding material, increase of accepted material and improvement of quality of castings.

  13. Eustachian tube function in children after insertion of ventilation tubes.

    NARCIS (Netherlands)

    Heerbeek, N. van; Ingels, K.J.A.O.; Snik, A.F.M.; Zielhuis, G.A.


    This study was performed to assess the effect of the insertion of ventilation tubes and the subsequent aeration of the middle ear on eustachian tube (ET) function in children. Manometric ET function tests were performed repeatedly for 3 months after the placement of ventilation tubes in 83 children

  14. Observations on rotating needle insertions using a brachytherapy robot

    Energy Technology Data Exchange (ETDEWEB)

    Meltsner, M A [Department of Medical Physics, University of Wisconsin, Madison, WI 53706 (United States); Ferrier, N J [Department of Mechanical Engineering, University of Wisconsin, Madison, WI 53706 (United States); Thomadsen, B R [Department of Medical Physics, University of Wisconsin, Madison, WI 53706 (United States)


    A robot designed for prostate brachytherapy implantations has the potential to greatly improve treatment success. Much of the research in robotic surgery focuses on measuring accuracy. However, there exist many factors that must be optimized before an analysis of needle placement accuracy can be determined. Some of these parameters include choice of the needle type, insertion velocity, usefulness of the rotating needle and rotation speed. These parameters may affect the force at which the needle interacts with the tissue. A reduction in force has been shown to decrease the compression of the prostate and potentially increase the accuracy of seed position. Rotating the needle as it is inserted may reduce frictional forces while increasing accuracy. However, needle rotations are considered to increase tissue damage due to the drilling nature of the insertion. We explore many of the factors involved in optimizing a brachytherapy robot, and the potential effects each parameter may have on the procedure. We also investigate the interaction of rotating needles in gel and suggest the rotate-cannula-only method of conical needle insertion to minimize any tissue damage while still maintaining the benefits of reduced force and increased accuracy.

  15. Folding and insertion thermodynamics of the transmembrane WALP peptide. (United States)

    Bereau, Tristan; Bennett, W F Drew; Pfaendtner, Jim; Deserno, Markus; Karttunen, Mikko


    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)n (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide's insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum-in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence.

  16. Engine valve and seat insert wear study with a simulator

    Institute of Scientific and Technical Information of China (English)

    Y.S.Wang; S.Narasimhan


    The demands on higher performance and the increasing use of alternative fuels chal-lenge engine valves now with greater wear problems than before. A seat wear simulator was builtto evaluate the compatibility and wear of valve and seat insert. The rig test results have been suc-cessfully correlated with engine test results. In this study, intake valves made from Sil 1 materialwere treated with salt bath nitride processes and tested against six different insert materials. Wearresistance of these combinations was ranked and compared to the Sil 1 valve without nitriding.The test results demonstrate that nitriding improved valve seat wear resistance. In the total valveseat recession ranking, the combination of nitrided Sil 1 valve against T 400 insert exhibited theleast total recession among the nineteen combinations of valve and insert tested. The results indi-cate that the valve seat wear mechanisms are a complex combination of adhesion and shearstrain. The nitrides in the compound layer of nitrided valves gave strong atomic bonding, higherhardness, compressive residual stresses, and possible low friction, thus resulted in the superiorwear performance.

  17. Selective insertion of sulfur dioxide reduction intermediates on graphene oxide. (United States)

    Humeres, Eduardo; Debacher, Nito A; Smaniotto, Alessandra; de Castro, Karen M; Benetoli, Luís O B; de Souza, Eduardo P; Moreira, Regina de F P M; Lopes, Cristiane N; Schreiner, Wido H; Canle, Moisés; Santaballa, J Arturo


    Graphite microparticles (d50 6.20 μm) were oxidized by strong acids, and the resultant graphite oxide was thermally exfoliated to graphene oxide sheets (MPGO, C/O 1.53). Graphene oxide was treated with nonthermal plasma under a SO2 atmosphere at room temperature. The XPS spectrum showed that SO2 was inserted only as the oxidized intermediate at 168.7 eV in the S 2p region. Short thermal shocks at 600 and 400 °C, under an Ar atmosphere, produced reduced sulfur and carbon dioxide as shown by the XPS spectrum and TGA analysis coupled to FTIR. MPGO was also submitted to thermal reaction with SO2 at 630 °C, and the XPS spectrum in the S 2p region at 164.0 eV showed that this time only the nonoxidized episulfide intermediate was inserted. Plasma and thermal treatment produced a partial reduction of MPGO. The sequence of thermal reaction followed by plasma treatment inserted both sulfur intermediates. Because oxidized and nonoxidized intermediates have different reactivities, this selective insertion would allow the addition of selective types of organic fragments to the surface of graphene oxide.

  18. Functional fluorescent protein insertions in herpes simplex virus gB report on gB conformation before and after execution of membrane fusion.

    Directory of Open Access Journals (Sweden)

    John R Gallagher


    Full Text Available Entry of herpes simplex virus (HSV into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy.

  19. Strongly Semicontinuous Domains and Semi-FS Domains

    Directory of Open Access Journals (Sweden)

    Qingyu He


    Full Text Available We are mainly concerned with some special kinds of semicontinuous domains and relationships between them. New concepts of strongly semicontinuous domains, meet semicontinuous domains and semi-FS domains are introduced. It is shown that a dcpo L is strongly semicontinuous if and only if L is semicontinuous and meet semicontinuous. It is proved that semi-FS domains are strongly semicontinuous. Some interpolation properties of semiway-below relations in (strongly semicontinuous bc-domains are given. In terms of these properties, it is proved that strongly semicontinuous bc-domains, in particular strongly semicontinuous lattices, are all semi-FS domains.

  20. Cochlear implant insertion forces in microdissected human cochlea to evaluate a prototype array. (United States)

    Nguyen, Yann; Miroir, Mathieu; Kazmitcheff, Guillaume; Sutter, Jasmine; Bensidhoum, Morad; Ferrary, Evelyne; Sterkers, Olivier; Bozorg Grayeli, Alexis


    Cochlear implant array insertion forces are potentially related to cochlear trauma. We compared these forces between a standard (Digisonic SP; Neurelec, Vallauris, France) and an array prototype (Neurelec) with a smaller diameter. The arrays were inserted by a mechatronic tool in 23 dissected human cochlea specimens exposing the basilar membrane. The array progression under the basilar membrane was filmed together with dynamic force measurements. Insertion force profiles and depth of insertion were compared. The recordings showed lower insertion forces beyond 270° of insertion and deeper insertions with the thin prototype array. This will potentially allow larger cochlear coverage with less trauma.

  1. Open Surgical Insertion of Tenkchoff Straight Catheter Without Guide Wire

    Institute of Scientific and Technical Information of China (English)

    Shi-feng Yang; Wu-jun Xue; Ai-ping Yin; Li-yi Xie; Wan-hong Lu


    Objective To compare the clinical outcomes of open surgical peritoneal dialysiscatheter(PDC) insertion with guide wireand the outcomesof PDC insertion without guide wire.Methods Data of the patients receiving open surgical Tenkchoff straight catheter insertion in our department from January 2005 to January 2011 were retrospectively analyzed.The 117 patients in whom PDC insertion was conducted with the guidance of guide wire were enrolled into group A, and the 121 cases receiving PDC insertion without guide wire wereenrolled into group B.The incidences of post-operative complications (catheter obstruction,catheter displacement, bloody dialysate, and dialysate leakage), catheter survival, and patientsurvival rates were compared between the 2 groups.Results The baseline characteristics (gender, age, body mass index, prothrombin time,activated partialthromboplastin time,platelet count,serum creatinine,follow-up time,primarydiseases, and outcomes) of the 2 groups were comparable (allP>0.05). In post-operativecomplications, only the incidence of early bloody dialysate showed significant difference, being16.2% in groupA and 7.4% in group B (P=0.04). Catheter and patient survival rates werenot significantly different between the two groups. Overweight patientsshowed a higherincidence of catheter obstruction compared with normal weight patients [16.0% (4/25) vs. 3.3% (7/213),P=0.02], but no differencesin post-operative complications werefound among overweight patientsbetween the 2 groups.Conclusions Open surgical Tenkchoff straightcatheterinsertion without guide wire does not lead to higher risk of post-operative complications and catheter removal. It may be an alternativeoption when guide wire is not available.

  2. Domains in Ferroelectric Nanostructures (United States)

    Gregg, Marty


    Ferroelectric materials have great potential in influencing the future of small scale electronics. At a basic level, this is because ferroelectric surfaces are charged, and so interact strongly with charge-carrying metals and semiconductors - the building blocks for all electronic systems. Since the electrical polarity of the ferroelectric can be reversed, surfaces can both attract and repel charges in nearby materials, and can thereby exert complete control over both charge distribution and movement. It should be no surprise, therefore, that microelectronics industries have already looked very seriously at harnessing ferroelectric materials in a variety of applications, from solid state memory chips (FeRAMs) to field effect transistors (FeFETs). In all such applications, switching the direction of the polarity of the ferroelectric is a key aspect of functional behavior. The mechanism for switching involves the field-induced nucleation and growth of domains. Domain coarsening, through domain wall propagation, eventually causes the entire ferroelectric to switch its polar direction. It is thus the existence and behavior of domains that determine the switching response, and ultimately the performance of the ferroelectric device. A major issue, associated with the integration of ferroelectrics into microelectronic devices, has been that the fundamental properties associated with ferroelectrics, when in bulk form, appear to change quite dramatically and unpredictably when at the nanoscale: new modes of behaviour, and different functional characteristics from those seen in bulk appear. For domains, in particular, the proximity of surfaces and boundaries have a dramatic effect: surface tension and depolarizing fields both serve to increase the equilibrium density of domains, such that minor changes in scale or morphology can have major ramifications for domain redistribution. Given the importance of domains in dictating the overall switching characteristics of a device

  3. Analysis of Primary Stability of Dental Implants Inserted in Different Substrates Using the Pullout Test and Insertion Torque

    Directory of Open Access Journals (Sweden)

    Nathalia Ferraz Oliscovicz


    Full Text Available The aim of the study was to evaluate mechanical behavior of implants inserted in three substrates, by measuring the pullout strength and the relative stiffness. 32 implants (Master Porous-Conexao, cylindrical, external hexagon, and surface treatment were divided into 4 groups (n=8: pig rib bone, polyurethane Synbone, polyurethane Nacional 40 PCF, and pinus wood. Implants were installed with the exact distance of 5 mm of another implant. The insertion torque (N·cm was quantified using the digital Kratos torque meter and the pullout test (N was performed by an axial traction force toward the long axis of the implant (2 min/mm through mount implant devices attached to a piece adapted to a load cell of 200 Kg of a universal testing machine (Emic DL10000. Data of insertion torque and maximum pullout force were submitted to one-way ANOVA and Bonferroni tests (α=0.05. Polyurethane Nacional 40 PCF and pinus wood showed the highest values of insertion torque and pullout force, with significant statistical difference (P<0.05 with other groups. The analysis showed stiffness materials with the highest values for primary stability.

  4. Just how versatile are domains?

    Directory of Open Access Journals (Sweden)

    Bornberg-Bauer Erich


    Full Text Available Abstract Background Creating new protein domain arrangements is a frequent mechanism of evolutionary innovation. While some domains always form the same combinations, others form many different arrangements. This ability, which is often referred to as versatility or promiscuity of domains, its a random evolutionary model in which a domain's promiscuity is based on its relative frequency of domains. Results We show that there is a clear relationship across genomes between the promiscuity of a given domain and its frequency. However, the strength of this relationship differs for different domains. We thus redefine domain promiscuity by defining a new index, DV I ("domain versatility index", which eliminates the effect of domain frequency. We explore links between a domain's versatility, when unlinked from abundance, and its biological properties. Conclusion Our results indicate that domains occurring as single domain proteins and domains appearing frequently at protein termini have a higher DV I. This is consistent with previous observations that the evolution of domain re-arrangements is primarily driven by fusion of pre-existing arrangements and single domains as well as loss of domains at protein termini. Furthermore, we studied the link between domain age, defined as the first appearance of a domain in the species tree, and the DV I. Contrary to previous studies based on domain promiscuity, it seems as if the DV I is age independent. Finally, we find that contrary to previously reported findings, versatility is lower in Eukaryotes. In summary, our measure of domain versatility indicates that a random attachment process is sufficient to explain the observed distribution of domain arrangements and that several views on domain promiscuity need to be revised.

  5. A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

    Directory of Open Access Journals (Sweden)

    V.L. Quadros


    Full Text Available Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV and an antigenically identical but cytopathic virus (cpBVDV can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98% to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

  6. Axion domain wall baryogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Daido, Ryuji; Kitajima, Naoya [Department of Physics, Tohoku University,Sendai 980-8578 (Japan); Takahashi, Fuminobu [Department of Physics, Tohoku University,Sendai 980-8578 (Japan); Kavli IPMU, TODIAS, University of Tokyo,Kashiwa 277-8583 (Japan)


    We propose a new scenario of baryogenesis, in which annihilation of axion domain walls generates a sizable baryon asymmetry. Successful baryogenesis is possible for a wide range of the axion mass and decay constant, m≃10{sup 8}–10{sup 13} GeV and f≃10{sup 13}–10{sup 16} GeV. Baryonic isocurvature perturbations are significantly suppressed in our model, in contrast to various spontaneous baryogenesis scenarios in the slow-roll regime. In particular, the axion domain wall baryogenesis is consistent with high-scale inflation which generates a large tensor-to-scalar ratio within the reach of future CMB B-mode experiments. We also discuss the gravitational waves produced by the domain wall annihilation and its implications for the future gravitational wave experiments.

  7. Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA. (United States)

    Tullman, Jennifer; Guntas, Gurkan; Dumont, Matthew; Ostermeier, Marc


    We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in a plasmid molecule. These double-stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nuclease's single-stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double-stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 β-lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates β-lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen.

  8. Chromatin states shape insertion profiles of the piggyBac, Tol2 and Sleeping Beauty transposons and murine leukemia virus. (United States)

    Yoshida, Junko; Akagi, Keiko; Misawa, Ryo; Kokubu, Chikara; Takeda, Junji; Horie, Kyoji


    DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant. Therefore, we analysed various chromatin states. PB and MLV highly correlated with Cohesin, Mediator and ESC-specific transcription factors. Notably, CTCF sites were correlated with PB but not with MLV, suggesting MLV prefers smaller promoter-enhancer loops, whereas PB insertion encompasses larger chromatin loops termed topologically associating domains. Tol2 also correlated with Cohesin and CTCF. However, correlations with ESC-specific transcription factors were weaker, suggesting that Tol2 prefers transcriptionally weak chromatin loops. Consistently, Tol2 insertions were associated with bivalent histone modifications characteristic of silent and inducible loci. SB showed minimum preference to all chromatin states, suggesting the least adverse effect on adjacent genes. These results will be useful for vector selection for various applications.

  9. Chromatin states shape insertion profiles of the piggyBac, Tol2 and Sleeping Beauty transposons and murine leukemia virus (United States)

    Yoshida, Junko; Akagi, Keiko; Misawa, Ryo; Kokubu, Chikara; Takeda, Junji; Horie, Kyoji


    DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant. Therefore, we analysed various chromatin states. PB and MLV highly correlated with Cohesin, Mediator and ESC-specific transcription factors. Notably, CTCF sites were correlated with PB but not with MLV, suggesting MLV prefers smaller promoter–enhancer loops, whereas PB insertion encompasses larger chromatin loops termed topologically associating domains. Tol2 also correlated with Cohesin and CTCF. However, correlations with ESC-specific transcription factors were weaker, suggesting that Tol2 prefers transcriptionally weak chromatin loops. Consistently, Tol2 insertions were associated with bivalent histone modifications characteristic of silent and inducible loci. SB showed minimum preference to all chromatin states, suggesting the least adverse effect on adjacent genes. These results will be useful for vector selection for various applications. PMID:28252665

  10. [Neonatology nurses' knowledge about Peripherally Inserted Central Venous Catheter]. (United States)

    Belo, Marcela Patricia Macêdo; Silva, Roberta Albuquerque Mello de Castro; Nogueira, Isis Larissa Maia; Mizoguti, Daniele Pereira; Ventura, Claudiane Maria Urbano


    The Peripherally Inserted Central Catheter (PICC) has been used as a safe venous access for infants at risk. The aim of this study was to describe the knowledge and practice of nurses from the five public Neonatal Intensive Care Units, of Recife-PE, Brazil, about the use of the PICC. The sample was comprised by 52 nurses; data were collected from January to February/2010. It was found that 64,8% of nurses did not have license for insertion of the PICC. Only two units routinely used the PICC. About the indication of the access, the accuracy was above 70%. In unit B only 8,3% of nurses reported adequate initial location of the catheter tip. It was concluded that is necessary greater incentives to train nurses to use the PICC.

  11. Convective heat transfer enhancement inside tubes using inserted helical coils (United States)

    Ali, R. K.; Sharafeldeen, M. A.; Berbish, N. S.; Moawed, M. A.


    Convective heat transfer was experimentally investigated in tubes with helical coils inserts in turbulent flow regime within Reynolds number range of 14400 ≤ Re ≤ 42900. The present work aims to extend the experimental data available on wire coil inserts to cover wire diameter ratio from 0.044 to 0.133 and coil pitch ratio from 1 to 5. Uniform heat flux was applied to the external surface of the tube and air was selected as fluid. The effects of Reynolds number and wire diameter and coil pitch ratios on the Nusselt number and friction factor were studied. The enhancement efficiency and performance criteria ranges are of (46.9-82.6%) and (100.1-128%) within the investigated range of the different parameters, respectively. Correlations are obtained for the average Nusselt number and friction factor utilizing the present measurements within the investigated range of geometrical parameters and Re.

  12. An overview of the insertion device development at SRRC

    CERN Document Server

    Hwang, C S; Fan, T C; Wang, C; Chen, J R; Chen, C T


    Five high performance insertion devices, namely W20, U10, U5, U9 and EPU5.6, have been constructed and installed in the storage ring of the Synchrotron Radiation Research Center (SRRC). Among them, the 2-m-long conventional undulator U10 and the 4-m-long elliptically polarized undulator EPU5.6 were designed and built in-house. These two devices have achieved high magnetic field quality and high spectral performance. To facilitate hard-X-ray experiments, the project of building a superconducting wavelength shifter (SWLS) and a superconducting multi-pole wiggler (SMPW) is ongoing. These two superconducting insertion devices were designed to be cryogen-free.

  13. First two barrel ECAL supermodules inserted in CMS HCAL

    CERN Multimedia



    The first two barrel "supermodules" for the CMS Electromagnetic Calorimeter (ECAL) have been inserted into the barrel hadron calorimeter (HCAL) in the experimental hall (called SX5) in Cessy in preparation for the forthcoming magnet test and cosmic challenge (MTCC). Each of the two supermodules contains 1700 lead tungstate crystals in glass-fibre alveolar support structures, with associated avalanche photodiodes (APDs, for scintillation light detection), electronics and cooling system. The barrel ECAL will consist of 36 supermodules, many of which have already been produced (see CERN Bulletin 17-18, 2006). Team from CMS ECAL, CMS Integration and CEA-DAPNIA were involved in the insertion, with the production/integration of the supermodules themselves involving many technicians, engineers and physicists from many institutes. From left to right: Olivier Teller, Maf Alidra and Lucien Veillet.

  14. Phase transitions in insertion electrodes for lithium batteries

    Energy Technology Data Exchange (ETDEWEB)

    Thackeray, M. M.


    Phase transitions that occur during lithium insertion into layered and framework structures are discussed in the context of their application as positive and negative electrodes in lithium-ion batteries. The discussion is focused on the two-dimensional structures of graphite, LiNi{sub 1{minus}x}M{sub x}O{sub 2} (M = Co, Ti and Mg), and Li{sub 1.2}V{sub 3}O{sub 8}; examples of framework structures with a three-dimensional interstitial space for Li{sup +}-ion transport include the spinel oxides and intermetallic compounds with zinc-blende-type structures. The phase transitions are discussed in terms of their tolerance to lithium insertion and extraction and to the chemical stability of the electrodes in the cell environment.

  15. Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics (United States)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan


    Atomistic details of beta-amyloid (Aβ ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long Aβ in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.

  16. Insertional hypermutation in mineral oil-induced plasmacytomas. (United States)

    Knittel, Gero; Metzner, Mirjam; Beck-Engeser, Gabriele; Kan, Ada; Ahrends, Tomasz; Eilat, Dan; Huppi, Konrad; Wabl, Matthias


    Unless stimulated by a chronic inflammatory agent, such as mineral oil, plasma cell tumors are rare in young BALB/c mice. This raises the questions: What do inflammatory tissues provide to promote mutagenesis? And what is the nature of mutagenesis? We determined that mineral oil-induced plasmacytomas produce large amounts of endogenous retroelements--ecotropic and polytropic murine leukemia virus and intracisternal A particles. Therefore, plasmacytoma formation might occur, in part, by de novo insertion of these retroelements, induced or helped by the inflammation. We recovered up to ten de novo insertions in a single plasmacytoma, mostly in genes with common retroviral integration sites. Additional integrations accompany tumor evolution from a solid tumor through several generations in cell culture. The high frequency of de novo integrations into cancer genes suggests that endogenous retroelements are coresponsible for plasmacytoma formation and progression in BALB/c mice.

  17. Invariant Quantum Algorithms for Insertion into an Ordered List

    CERN Document Server

    Farhi, E; Gutmann, S; Sipser, M; Farhi, Edward; Goldstone, Jeffrey; Gutmann, Sam; Sipser, Michael


    We consider the problem of inserting one item into a list of N-1 ordered items. We previously showed that no quantum algorithm could solve this problem in fewer than log N/(2 log log N) queries, for N large. We transform the problem into a "translationally invariant" problem and restrict attention to invariant algorithms. We construct the "greedy" invariant algorithm and show numerically that it outperforms the best classical algorithm for various N. We also find invariant algorithms that succeed exactly in fewer queries than is classically possible, and iterating one of them shows that the insertion problem can be solved in fewer than 0.53 log N quantum queries for large N (where log N is the classical lower bound). We don't know whether a o(log N) algorithm exists.

  18. Challenges for Insertion of Structural Nanomaterials in Aerospace Applications (United States)

    Sochi, Emilie J.


    In the two decades since Iijima's report on carbon nanotubes (CNT), there has been great interest in realizing the benefits of mechanical properties observed at the nanoscale in large-scale structures. The weight savings possible due to dramatic improvements in mechanical properties relative to state-of-the-art material systems can be game changing for applications like aerospace vehicles. While there has been significant progress in commercial production of CNTs, major aerospace applications that take advantage of properties offered by this material have yet to be realized. This paper provides a perspective on the technical challenges and barriers for insertion of CNTs as an emerging material technology in aerospace applications and proposes approaches that may reduce the typical timeframe for technology maturation and insertion into aerospace structures.

  19. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer (United States)

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Izaguirre, Maitane; Hernández-Lizoain, José Luis; Baixauli, Jorge; Martí, Pablo; Valentí, Víctor; Moncada, Rafael; Silva, Camilo; Salvador, Javier; Frühbeck, Gema


    Background Excess adipose tissue represents a major risk factor for the development of colon cancer with inflammation and extracellular matrix (ECM) remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC), promoting a microenvironment favorable for tumor growth. Methods Serum samples obtained from 79 subjects [26 lean (LN) and 53 obese (OB)] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (44 without CC and 35 with CC). Anthropometric measurements as well as circulating metabolites and hormones were determined. Circulating concentrations of the ECM proteins osteopontin (OPN), chitinase-3-like protein 1 (YKL-40), tenascin C (TNC) and lipocalin-2 (LCN-2) were determined by ELISA. Results Significant differences in circulating OPN, YKL-40 and TNC concentrations between the experimental groups were observed, being significantly increased due to obesity (P<0.01) and colon cancer (P<0.05). LCN-2 levels were affected by obesity (P<0.05), but no differences were detected regarding the presence or not of CC. A positive association (P<0.05) with different inflammatory markers was also detected. Conclusions To our knowledge, we herein show for the first time that obese patients with CC exhibit increased circulating levels of OPN, YKL-40 and TNC providing further evidence for the influence of obesity on CC development via ECM proteins, representing promising diagnostic biomarkers or target molecules for therapeutics. PMID:27612200

  20. Characterization of the starvation-induced chitinase CfcA and α-1,3-glucanase AgnB of Aspergillus niger. (United States)

    van Munster, Jolanda M; Dobruchowska, Justyna M; Veloo, Ruud; Dijkhuizen, Lubbert; van der Maarel, Marc J E C


    The common saprophyte Aspergillus niger may experience carbon starvation in nature as well as during industrial fermentations. Starvation survival strategies, such as conidiation or the formation of exploratory hyphae, require energy and building blocks, which may be supplied by autolysis. Glycoside hydrolases are key effectors of autolytic degradation of fungal cell walls, but knowledge on their identity and functionality is still limited. We recently identified agnB and cfcA as two genes encoding carbohydrate-active enzymes that had notably increased transcription during carbon starvation in A. niger. Here, we report the biochemical and functional characterization of these enzymes. AgnB is an α-1,3-glucanase that releases glucose from α-1,3-glucan substrates with a minimum degree of polymerization of 4. CfcA is a chitinase that releases dimers from the nonreducing end of chitin. These enzymes thus attack polymers that are found in the fungal cell wall and may have a role in autolytic fungal cell wall degradation in A. niger. Indeed, cell wall degradation during carbon starvation was reduced in the double deletion mutant ΔcfcA ΔagnB compared to the wild-type strain. Furthermore, the cell walls of the carbon-starved mycelium of the mutant contained a higher fraction of chitin or chitosan. The function of at least one of these enzymes, CfcA, therefore appears to be in the recycling of cell wall carbohydrates under carbon limiting conditions. CfcA thus may be a candidate effector for on demand cell lysis, which could be employed in industrial processes for recovery of intracellular products.

  1. Alternative splicing of basic chitinase gene PR3b in the low-nicotine mutants of Nicotiana tabacum L. cv. Burley 21 (United States)

    Ma, Haoran; Wang, Feng; Wang, Wenjing; Yin, Guoying; Zhang, Dingyu; Ding, Yongqiang; Timko, Michael P.; Zhang, Hongbo


    Two unlinked semi-dominant loci, A (NIC1) and B (NIC2), control nicotine and related alkaloid biosynthesis in Burley tobaccos. Mutations in either or both loci (nic1 and nic2) lead to low nicotine phenotypes with altered environmental stress responses. Here we show that the transcripts derived from the pathogenesis-related (PR) protein gene PR3b are alternatively spliced to a greater extent in the nic1 and nic2 mutants of Burley 21 tobacco and the nic1nic2 double mutant. The alternative splicing results in a deletion of 65 nucleotides and introduces a premature stop codon into the coding region of PR3b that leads to a significant reduction of PR3b specific chitinase activity. Assays of PR3b splicing in F2 individuals derived from crosses between nic1 and nic2 mutants and wild-type plants showed that the splicing phenotype is controlled by the NIC1 and NIC2 loci, even though NIC1 and NIC2 are unlinked loci. Moreover, the transcriptional analyses showed that the splicing patterns of PR3b in the low-nicotine mutants were differentially regulated by jasmonate (JA) and ethylene (ET). These data suggest that the NIC1 and NIC2 loci display differential roles in regulating the alternative splicing of PR3b in Burley 21. The findings in this study have provided valuable information for extending our understanding of the broader effects of the low-nicotine mutants of Burley 21 and the mechanism by which JA and ET signalling pathways post-transcriptionally regulate the activity of PR3b protein. PMID:27664270

  2. Sequence/structural analysis of xylem proteome emphasizes pathogenesis-related proteins, chitinases and β-1, 3-glucanases as key players in grapevine defense against Xylella fastidiosa

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty


    Full Text Available Background. Xylella fastidiosa, the causative agent of various plant diseases including Pierce’s disease in the US, and Citrus Variegated Chlorosis in Brazil, remains a continual source of concern and economic losses, especially since almost all commercial varieties are sensitive to this Gammaproteobacteria. Differential expression of proteins in infected tissue is an established methodology to identify key elements involved in plant defense pathways. Methods. In the current work, we developed a methodology named CHURNER that emphasizes relevant protein functions from proteomic data, based on identification of proteins with similar structures that do not necessarily have sequence homology. Such clustering emphasizes protein functions which have multiple copies that are up/down-regulated, and highlights similar proteins which are differentially regulated. As a working example we present proteomic data enumerating differentially expressed proteins in xylem sap from grapevines that were infected with X. fastidiosa. Results. Analysis of this data by CHURNER highlighted pathogenesis related PR-1 proteins, reinforcing this as the foremost protein function in xylem sap involved in the grapevine defense response to X. fastidiosa. β-1, 3-glucanase, which has both anti-microbial and anti-fungal activities, is also up-regulated. Simultaneously, chitinases are found to be both up and down-regulated by CHURNER, and thus the net gain of this protein function loses its significance in the defense response. Discussion. We demonstrate how structural data can be incorporated in the pipeline of proteomic data analysis prior to making inferences on the importance of individual proteins to plant defense mechanisms. We expect CHURNER to be applicable to any proteomic data set.

  3. The Evolution of Insertion Sequences within Enteric Bacteria


    Lawrence, J G; Ochman, H.; Hartl, D. L.


    To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and ra...

  4. The insertion loss of screens under the influence of wind

    DEFF Research Database (Denmark)

    Rasmussen, Karsten Bo; Arranz, Marta Galindo


    in the vertical wind speed profile when the wind field passes the screen. Theinfluence of turbulence is also implemented. The experimental part of the investigation relieson a scale model technique based upon a 1:25 scaling ratio and a triggered spark source. Themain results relate to the size of the insertion...... loss of a screen under windy conditions and tothe acoustic importance of the redirection of the flow before and after the screen....

  5. Evolution of implantable and insertable drug delivery systems. (United States)

    Kleiner, Lothar W; Wright, Jeremy C; Wang, Yunbing


    The paper describes the development of implantable and insertable drug delivery systems (IDDS) from their early stage in the 1960s until the current stage in the 2010s. It gives a detailed summary of non-degradable and biodegradable systems and their applications in different areas such as vascular disease treatment, birth control, cancer treatment, and eye disease treatment. It also describes the development of various implantable pump systems and some other atypical IDDS, the challenges and the future of IDDS.

  6. Non-harmful insertion of data mimicking computer network attacks

    Energy Technology Data Exchange (ETDEWEB)

    Neil, Joshua Charles; Kent, Alexander; Hash, Jr, Curtis Lee


    Non-harmful data mimicking computer network attacks may be inserted in a computer network. Anomalous real network connections may be generated between a plurality of computing systems in the network. Data mimicking an attack may also be generated. The generated data may be transmitted between the plurality of computing systems using the real network connections and measured to determine whether an attack is detected.

  7. Accurate Insertion Loss Measurements of the Juno Patch Array Antennas (United States)

    Chamberlain, Neil; Chen, Jacqueline; Hodges, Richard; Demas, John


    This paper describes two independent methods for estimating the insertion loss of patch array antennas that were developed for the Juno Microwave Radiometer instrument. One method is based principally on pattern measurements while the other method is based solely on network analyzer measurements. The methods are accurate to within 0.1 dB for the measured antennas and show good agreement (to within 0.1dB) of separate radiometric measurements.

  8. Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants


    de Montaigu Amaury; Magneschi Leonardo; Catalanotti Claudia; Yang Wenqiang; Mus Florence; Pootakham Wirulda; Gonzalez-Ballester David; Higuera Jose J; Prior Matthew; Galván Aurora; Fernandez Emilio; Grossman Arthur R


    Abstract A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment...

  9. Epigenetic Suppression of T-DNA Insertion Mutants in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Yangbin Gao; Yunde Zhao


    T-DNA insertion mutants have been widely used to define gene functions in Arabidopsis and in other plants.Here,we report an unexpected phenomenon of epigenetic suppression of T-DNA insertion mutants in Arabidopsis.When the two T-DNA insertion mutants,yucl-1 and ag-TD,were crossed together,the defects in all of the ag-TD plants in the F2 population were partially suppressed regardless of the presence of yucl-1.Conversion of ag-TD to the suppressed ag-TD (named as ag-TD*) did not follow the laws of Mendelian genetics.The ag-TD* could be stably transmitted for many generations without reverting to ag-TD,and ag-TD* had the capacity to convert ag-TD to ag-TD*.We show that epigenetic suppression of T-DNA mutants is not a rare event,but certain structural features in the T-DNA mutants are needed in order for the suppression to take place.The suppressed T-DNA mutants we observed were all intronic T-DNA mutants and the T-DNA fragments in both the trigger T-DNA as well as in the suppressed T-DNA shared stretches of identical sequences.We demonstrate that the suppression of intronic T-DNA mutants is mediated by trans-interactions between two ToDNA insertions.This work shows that caution is needed when intronic T-DNA mutants are used.

  10. Insertion devices at the Swiss Light Source (phase I)

    CERN Document Server

    Schmidt, T; Imhof, A; Patterson, B D; Patthey, L; Quitmann, C; Schulze-Briese, C; Abela, R


    The insertion devices under construction for phase I of the Swiss Light Source (SLS) are described. Five undulators and one wiggler will be installed in four straight sections of the third generation 2.4 GeV SLS storage ring, under construction at the Paul Scherrer Institute. To provide undulator radiation in the energy range from 10 eV to 18 keV, both long period and short period, small gap undulators will be installed.

  11. Sacroiliac secure corridor: analysis for safe insertion of iliosacral screws

    Directory of Open Access Journals (Sweden)

    Henrique Alves Cruz


    Full Text Available OBJECTIVE: Posterior pelvic lesions, especially of the sacral-iliac joint, have high mortality and morbidity risks. Definitive fixation is necessary for the joint stabilization, and one option is the sacral percutaneous pinning with screws. Proximity to important structures to this region brings risks to the fixation procedure; therefore, it is important to know the tridimensional anatomy of the pelvis posterior region. Deviations of the surgeon's hand of four degrees may target the screws to those structures; dimorphisms of the upper sacrum and a poor lesion reduction may redound in a screw malpositioning. This study is aimed to evaluate the dimensions of a safe surgical corridor for safe sacroiliac screw insertion and relations with age and sex of the patients. METHOD: One hundred randomly selected pelvis CTs of patients with no pelvic diseases, seen at a tertiary care teaching Hospital. Measurements were made by computer and the safest area for screw insertion was calculated by two methods. The results were expressed in mm (not in degrees, in order to be a further surgical reference. RESULTS: There was a significant size difference in the analyzed sacral vertebra, differing on a wider size in men than in women. There was no significant statistical difference between vertebral size and age. By both methods, a safe area for screw insertion could be defined. CONCLUSION: Age does not influence the width of the surgical corridor. The surgeon has a safe corridor considered narrower when inserting screws in a female pelvis than when in a male one. However, as the smallest vertebra found (feminine was considered for statics, it was concluded that this corridor is 20 mm wide in any direction, taking as a reference the centrum of the vertebra.

  12. The first insertion devices at SSRL - some personal recollections

    Energy Technology Data Exchange (ETDEWEB)

    Winick, H. [Stanford Linear Accelerator Center, CA (United States)


    The author recounts his experiences with insertion devices at the Stanford Synchrotron Radiation Laboratory. His first experiences with wigglers occured at the Cambridge Electron Accelerator, and was carried over to SSRL with the proposal for a six pole electromagnetic wiggler. Most modern undulators, and many wigglers are now designed around permanent magnets, and the origin of this transition at SSRL was rather fortuitous and humorous. It reflects some of the personality characteristics of Klaus Halbach.


    Directory of Open Access Journals (Sweden)

    Buddhadeb Ghosh


    Full Text Available A tendinous origin and fleshy insertion of palmaris longus muscle was observed in the left forearm during routine dissection which was performed on adult male cadaver in the department of Anatomy, Dr. Rajendra Prasad Government Medical College. It was having long tendinous origin from the medial epicondyle of the humerus and the surrounding deep fascia. It was fusiform at the lower middle of the forearm. The fleshy muscular insertion was noted to the flexor retinaculum and few muscular fibers interdigitate with flexor carpi ulnaris muscle and palmar aponeurosis. The length of tendon was 19 inches and fleshy muscular length was 11inches. The median nerve and ulnar nerve was covered by this fleshy insertion. This palmaris longus variation is helpful for the surgeon and the radiologist, orthopaedic, plastic surgeon during any diagnosis of the forearm because this fleshy part of muscle can compress the median nerve and ulnar nerve or it can be mistaken as a tumor or ganglion during radiological or clinical examination.

  14. Risk factors for development of complication following peripherally inserted central

    Directory of Open Access Journals (Sweden)

    Hakan Aydın


    Full Text Available Objectives: Peripherally inserted central venous catheters (PICCs are inserted into central veins through the upper extremity veins. In this retrospective study, we aimed to evaluate PICC procedures, related complications, their causes and factors influencing the success of the procedure during anaesthesia Methods: ‘Central Venous Catheterization Forms’ filled out for 850 patients in whom a PICC was inserted by residents during general anaesthesia between November 2009 and March 2013 in the operating room of Uludag University Medical Faculty Hospital were retrospectively analysed. Results: A total of 1174 procedures were evaluated. The most preferred vein for the first attempt was the right basilic vein (32.7%. Difficulty (more than two attempts with the PICC procedure was correlated with the patient’s age (p30 kg/m² (p<0.05, resident with less than 4 years of training (p=0.001, number of PICC attempts ≥2 (p<0.001, more than one resident involved in the catheterization procedure (p<0.001 and previous failed PICC procedures (p<0.001. Conclusion: We conclude that catheterization should be performed under the surveillance of a staff keeping in mind the risks of complications. In the case of failure following 2 attempts, the procedure should be handed over to a more experienced staff member. J Clin Exp Invest 2014; 5 (1: 29-35

  15. Folding and insertion thermodynamics of the transmembrane WALP peptide

    CERN Document Server

    Bereau, Tristan; Pfaendtner, Jim; Deserno, Markus; Karttunen, Mikko


    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)$_n$(L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide's insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum---in contrast to both atomistic simulations and the alternative PLUM force field. Even tho...

  16. Enhancement of Selection, Bubble and Insertion Sorting Algorithm

    Directory of Open Access Journals (Sweden)

    Muhammad Farooq Umar


    Full Text Available In everyday life there is a large amount of data to arrange because sorting removes any ambiguities and make the data analysis and data processing very easy, efficient and provides with cost less effort. In this study a set of improved sorting algorithms are proposed which gives better performance and design idea. In this study five new sorting algorithms (Bi-directional Selection Sort, Bi-directional bubble sort, MIDBiDirectional Selection Sort, MIDBidirectional bubble sort and linear insertion sort are presented. Bi-directional Selection Sort and MIDBiDirectional Selection Sort are the enhancement on basic selection sort while Bidirectional bubble sort and MIDBidirectional bubble sort are the enhancement on basic bubble sort by changing the selection and swapping mechanism of data for sorting. Enhanced sorting algorithms reduced the iteration by half and quarter times respectively. Asymptotically complexities of these algorithms are reduced to O (n2/2 and O (n2/4 from O (n2. Linear insertion sort is the enhancement of insertion sort by changing the design of algorithm (convert two loops to one loop. So asymptotically this algorithm is converted to linear time complexity from quadratic complexity. These sorting algorithms are described using C. The proposed algorithms are analyzed using asymptotic analysis and also using machine-running time and compared with their basic sorting algorithms. In this study we also discuss how the performance and complexity can be improved by optimizing the code and design.

  17. Thermodynamic study of benzocaine insertion into different lipid bilayers (United States)

    Cascales, J. J. López; Costa, S. D. Oliveira; Porasso, R. D.


    Despite the general consensus concerning the role played by sodium channels in the molecular mechanism of local anesthetics, the potency of anaesthetic drugs also seems to be related with their solubility in lipid bilayers. In this respect, this work represents a thermodynamic study of benzocaine insertion into lipid bilayers of different compositions by means of molecular dynamics simulation. Thus, the free energy profiles associated with benzocaine insertion into symmetric lipid bilayers composed of different proportions of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine were studied. From the simulation results, a maximum in the free energy (ΔG) profile was measured in the region of the lipid/solution interface. This free energy barrier appears to be very much dependent on the lipid composition of the membrane. On the other hand, the minimum free energy (ΔG) within the bilayer remained almost independent of the lipid composition of the bilayer. By repeating the study at different temperatures, it was seen how the spontaneity of benzocaine insertion into the lipid bilayer is due to an increase in the entropy associated with the process.

  18. Localized control of the orbit in the RHIC insertions

    Energy Technology Data Exchange (ETDEWEB)

    Ohnuma, S.


    It is proposed here that, for RHIC92 insertions, we remove the corrector from Ql and the beam position monitor (BPM) from Q2 in order to alleviate difficulties associated with the physical layout of the quadrupole triplet (Ql-Q2-Q3). Furthermore, it is suggested that there should be both (horizontal and vertical) types of BPMs at each end of the free space between Q3 and Q4 and between Q7 and Q8 so that one can measure the direction of the closed orbit. With this model, a localized control of the beam position and angle at the interaction point (IP) with either four or six correctors has been investigated. Similarly, a control of the orbit within an insertion for minimizing the orbit displacements at seven (or eight) BPM locations with nine (or ten) correctors in each transverse direction has been studied. Examples are given for the beta at IP = 2m, 10m, 20m, and 200m. It is shown that the design value of the integrated field strength of 0.3 T-m for each corrector should be sufficient for the tasks considered here except for some cases with extreme parameter values. At the same time, it is emphasized that the overall correction of the closed orbit for the entire ring (arcs and insertions) should be re-examined for RHIC92 lattice with the proposed arrangement of correctors and BPMS.

  19. Pneumothorax as a complication of central venous catheter insertion. (United States)

    Tsotsolis, Nikolaos; Tsirgogianni, Katerina; Kioumis, Ioannis; Pitsiou, Georgia; Baka, Sofia; Papaiwannou, Antonis; Karavergou, Anastasia; Rapti, Aggeliki; Trakada, Georgia; Katsikogiannis, Nikolaos; Tsakiridis, Kosmas; Karapantzos, Ilias; Karapantzou, Chrysanthi; Barbetakis, Nikos; Zissimopoulos, Athanasios; Kuhajda, Ivan; Andjelkovic, Dejan; Zarogoulidis, Konstantinos; Zarogoulidis, Paul


    The central venous catheter (CVC) is a catheter placed into a large vein in the neck [internal jugular vein (IJV)], chest (subclavian vein or axillary vein) or groin (femoral vein). There are several situations that require the insertion of a CVC mainly to administer medications or fluids, obtain blood tests (specifically the "central venous oxygen saturation"), and measure central venous pressure. CVC usually remain in place for a longer period of time than other venous access devices. There are situations according to the drug administration or length of stay of the catheter that specific systems are indicated such as; a Hickman line, a peripherally inserted central catheter (PICC) line or a Port-a-Cath may be considered because of their smaller infection risk. Sterile technique is highly important here, as a line may serve as a port of entry for pathogenic organisms, and the line itself may become infected with organisms such as Staphylococcus aureus and coagulase-negative Staphylococci. In the current review we will present the complication of pneumothorax after CVC insertion.

  20. Comparing Intravenous Insertion Instructional Methods with Haptic Simulators (United States)

    Malecha, Ann


    Objective. The objective of this review was to compare traditional intravenous (IV) insertion instructional methods with the use of haptic IV simulators. Design. An integrative research design was used to analyze the current literature. Data Sources. A search was conducted using key words intravenous (IV) insertion or cannulation or venipuncture and simulation from 2000 to 2015 in the English language. The databases included Academic Search Complete, CINAHL Complete, Education Resource Information Center, and Medline. Review Methods. Whittemore and Knafl's (2005) strategies were used to critique the articles for themes and similarities. Results. Comparisons of outcomes between traditional IV instructional methods and the use of haptic IV simulators continue to show various results. Positive results indicate that the use of the haptic IV simulator decreases both band constriction and total procedure time. While students are satisfied with practicing on the haptic simulators, they still desire faculty involvement. Conclusion. Combining the haptic IV simulator with practical experience on the IV arm may be the best practice for learning IV insertion. Research employing active learning strategies while using a haptic IV simulator during the learning process may reduce cost and faculty time.


    Directory of Open Access Journals (Sweden)



    Full Text Available BACKGROUND Purpose: To study the acceptance level of Post-Partum Intrauterine Contraceptive Device (PPIUCD insertion among women attending tertiary level hospital for delivery between January 2013 to July 2015 in relation to age, parity and mode of delivery, safety and their complaints/complications during followup visit. METHOD This is a retrospective study done in a tertiary care centre, Jorhat Medical College and Hospital, Assam, between January 2013 to July 2015. Women who had accepted PPIUCD after delivery (Vaginally or by Lower Segment Caesarean section were included in this study. The entire PPIUCD inserted patients were followed up to 6 weeks and 6 months after delivery. With the help of data collected, relevant parameters and data are critically analysed in our study. RESULTS Acceptance of PPIUCD showed an increasing trend, acceptance was more among patients undergoing caesarean section; 43.86% of the acceptors were in the age group of 21-25 years. More than 50% of the total acceptors in the study came for followup. The main complaints at followup were missing thread and bleeding. The main causes of removal were bleeding and pressure from family. CONCLUSION The acceptance of PPIUCD was high in this study. The PPIUCD was demonstrably safe having no serious complication reported after insertion or during followup and low rates of expulsion. The method may be particularly beneficial in our setting where women do not come for postnatal contraception counselling and usage.

  2. Development of Needle Insertion Manipulator for Central Venous Catheterization (United States)

    Kobayashi, Yo; Hong, Jaesung; Hamano, Ryutaro; Hashizume, Makoto; Okada, Kaoru; Fujie, Masakatsu G.

    Central venous catheterization is a procedure, which a doctor insert a catheter into the patient’s vein for transfusion. Since there are risks of bleeding from arterial puncture or pneumothorax from pleural puncture. Physicians are strictly required to make needle reach up into the vein and to stop the needle in the middle of vein. We proposed a robot system for assisting the venous puncture, which can relieve the difficulties in conventional procedure, and the risks of complication. This paper reports the design structuring and experimental results of needle insertion manipulator. First, we investigated the relationship between insertion force and angle into the vein. The results indicated that the judgment of perforation using the reaction force is possible in case where the needling angle is from 10 to 20 degree. The experiment to evaluate accuracy of the robot also revealed that it has beyond 0.5 mm accuracy. We also evaluated the positioning accuracy in the ultrasound images. The results displays that the accuracy is beyond 1.0 mm and it has enough for venous puncture. We also carried out the venous puncture experiment to the phantom and confirm our manipulator realized to make needle reach up into the vein.

  3. Effects of Tools Inserted through Snake-like Surgical Manipulators (United States)

    Murphy, Ryan J.; Otake, Yoshito; Wolfe, Kevin C.; Taylor, Russell H.; Armand, Mehran


    Snake-like manipulators with a large, open lumen can offer improved treatment alternatives for minimally- and less-invasive surgeries. In these procedures, surgeons use the manipulator to introduce and control flexible tools in the surgical environment. This paper describes a predictive algorithm for estimating manipulator configuration given tip position for nonconstant curvature, cable-driven manipulators using energy minimization. During experimental bending of the manipulator with and without a tool inserted in its lumen, images were recorded from an overhead camera in conjunction with actuation cable tension and length. To investigate the accuracy, the estimated manipulator configuration from the model and the ground-truth configuration measured from the image were compared. Additional analysis focused on the response differences for the manipulator with and without a tool inserted through the lumen. Results indicate that the energy minimization model predicts manipulator configuration with an error of 0.24 ± 0.22mm without tools in the lumen and 0.24 ± 0.19mm with tools in the lumen (no significant difference, p = 0.81). Moreover, tools did not introduce noticeable perturbations in the manipulator trajectory; however, there was an increase in requisite force required to reach a configuration. These results support the use of the proposed estimation method for calculating the shape of the manipulator with an tool inserted in its lumen when an accuracy range of at least 1mm is required. PMID:25571571

  4. Modeling and characterization of partially inserted electrical connector faults (United States)

    Tokgöz, ćaǧatay; Dardona, Sameh; Soldner, Nicholas C.; Wheeler, Kevin R.


    Faults within electrical connectors are prominent in avionics systems due to improper installation, corrosion, aging, and strained harnesses. These faults usually start off as undetectable with existing inspection techniques and increase in magnitude during the component lifetime. Detection and modeling of these faults are significantly more challenging than hard failures such as open and short circuits. Hence, enabling the capability to locate and characterize the precursors of these faults is critical for timely preventive maintenance and mitigation well before hard failures occur. In this paper, an electrical connector model based on a two-level nonlinear least squares approach is proposed. The connector is first characterized as a transmission line, broken into key components such as the pin, socket, and connector halves. Then, the fact that the resonance frequencies of the connector shift as insertion depth changes from a fully inserted to a barely touching contact is exploited. The model precisely captures these shifts by varying only two length parameters. It is demonstrated that the model accurately characterizes a partially inserted connector.


    Directory of Open Access Journals (Sweden)

    Anacilda Oliveira Vieira


    Full Text Available Introduction: The PICC (peripherally inserted central catheter is a long flexible catheter which is inserted through a peripheral vein, progresses through a needle introducer until the final portion of the vena cava, acquiring characteristics of a central catheter. Objective: To point out the main theoretical and scientific ideas that demonstrate the reliability, competence and ability of nurses to perform the PICC. Methodology: Systematic review of articles, which were found by searching the database scientific journals and bibliographies area. Results: The success of integration depends on the patient assessment and choice of venous access where the catheter will be positioned, and its tip should be in the middle third of the superior vena cava, or the middle third of the inferior vena cava. In neonates, which are used more frequently, proper positioning of the catheter is through nursing care in making the dressing, and the first 24 hours it should be compressive. Ideally, the PICC remains in the vein for periods longer than seven days or until the end of treatment, thus decreasing invasive procedures. Conclusion: According to the Federal Board of Nursing (COFEN, it is lawful for the insertion of PICC nurses, provided it has undergone professional training.

  6. On Binding Domains

    NARCIS (Netherlands)

    Everaert, M.B.H.


    In this paper I want to explore reasons for replacing Binding Theory based on the anaphor-pronoun dichotomy by a Binding Theory allowing more domains restricting/defining anaphoric dependencies. This will, thus, have consequences for the partitioning of anaphoric elements, presupposing more types of

  7. Domain: Labour market

    NARCIS (Netherlands)

    Oude Mulders, J.; Wadensjö, E.; Hasselhorn, H.M.; Apt, W.


    This domain chapter is dedicated to summarize research on the effects of labour market contextual factors on labour market participation of older workers (aged 50+) and identify research gaps. While employment participation and the timing of (early) retirement is often modelled as an individual deci

  8. Cellulose binding domain proteins (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy


    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  9. Normed Domains of Holomorphy

    Directory of Open Access Journals (Sweden)

    Steven G. Krantz


    Full Text Available We treat the classical concept of domain of holomorphy in ℂn when the holomorphic functions considered are restricted to lie in some Banach space. Positive and negative results are presented. A new view of the case n=1 is considered.

  10. Role of the import motor in insertion of transmembrane segments by the mitochondrial TIM23 complex. (United States)

    Popov-Čeleketić, Dušan; Waegemann, Karin; Mapa, Koyeli; Neupert, Walter; Mokranjac, Dejana


    The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. We show here that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. Furthermore, our results suggest a role for the import motor in lateral insertion. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion.

  11. Ultrasound-guided central line insertion and standard peripherally inserted catheter placement in preterm infants: Comparing results from prospective study in a single-center

    Directory of Open Access Journals (Sweden)

    Dany Antanios Al Hamod


    Full Text Available Background: Among preterm infants, the peripherally inserted central catheter (PICC is the standard line for central venous access; however, its placement exposes them to hypothermia and pain. Ultrasound (US-guided central line insertion may be less morbid than standard PICC line. Aims: To determine the ease, success rate, and morbidity associated with US-guided central line insertion in the internal jugular vein (IJV by comparing it to the standard PICC line placement. Materials and Methods: This is a single-center nonrandomized prospective study evaluating preterm infants between October 2013 and June 2014. Patients were allocated into two groups: The standard group (control group who underwent blind PICC line insertion and the intervention group who underwent a percutaneous US-guided central line insertion in the IJV. The epicutaneo-cava-catheter was used in both groups. Results: Fifty neonates were enrolled on study. A statistically difference in favor of US-IJV insertion was noted concerning the rate of successful first attempt (P < 0.001, insertion (P = 0.001, and procedure duration (P < 0.001 and number of trials (P < 0.001 compared to PICC. No difference in complications (P = 1.000 was noted. Conclusion: US guided catheterization of the IJV technique is faster than PICC line insertion with higher rates of successful first attempt and insertion, less procedure duration and fewer number of trials compared to PICC line insertion. There were no differences in complications.

  12. Sampling and TDR probe insertion in the determination of the volumetric soil water content

    Directory of Open Access Journals (Sweden)

    Teixeira W. G.


    Full Text Available Volumetric soil water content (theta can be evaluated in the field by direct or indirect methods. Among the direct, the gravimetric method is regarded as highly reliable and thus often preferred. Its main disadvantages are that sampling and laboratory procedures are labor intensive, and that the method is destructive, which makes resampling of a same point impossible. Recently, the time domain reflectometry (TDR technique has become a widely used indirect, non-destructive method to evaluate theta. In this study, evaluations of the apparent dielectric number of soils (epsilon and samplings for the gravimetrical determination of the volumetric soil water content (thetaGrav were carried out at four sites of a Xanthic Ferralsol in Manaus - Brazil. With the obtained epsilon values, theta was estimated using empirical equations (thetaTDR, and compared with thetaGrav derived from disturbed and undisturbed samples. The main objective of this study was the comparison of thetaTDR estimates of horizontally as well as vertically inserted probes with the thetaGrav values determined by disturbed and undisturbed samples. Results showed that thetaTDR estimates of vertically inserted probes and the average of horizontally measured layers were only slightly and insignificantly different. However, significant differences were found between the thetaTDR estimates of different equations and between disturbed and undisturbed samples in the thetaGrav determinations. The use of the theoretical Knight et al. model, which permits an evaluation of the soil volume assessed by TDR probes, is also discussed. It was concluded that the TDR technique, when properly calibrated, permits in situ, nondestructive measurements of q in Xanthic Ferralsols of similar accuracy as the gravimetric method.

  13. Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan. (United States)

    Bernard, Michael P; Cao, Donghui; Myers, Rebecca V; Moyle, William R


    Several excellent procedures for trapping tagged proteins have been devised, but many of these are expensive, cannot be used outside a limited pH range, fail to work in the presence of chaotropic agents, or are difficult to use. The chitin binding domain (CBD) of Bacillus circulans chitinase, which binds to chitin matrices prepared from inexpensive reagents isolated from crab shells, is an alternative tag that can be used under a variety of pH and denaturing conditions. Kits based on the interaction between the CBD and the chitin beads are available commercially. Here, we show that simultaneous treatment of microtiter plates with chitosan, a deacetylated form of chitin, and acetic anhydride produces a surface-bound film of chitin that also interacts tightly with the CBD. Chitin-coated microtiter well plates captured a CBD-tagged heterodimeric human glycoprotein hormone analog directly from mammalian cell culture media, even when present in trace amounts. Binding to the surface was stable in sodium dodecylsulfate and reversed only partially at low pH or in 8M urea at 37 degrees C. This technique appears well suited to surface attachment and permits biochemical or other analyses of molecules that can be tagged with a CBD.

  14. Time Domain Induced Polarization

    DEFF Research Database (Denmark)

    Fiandaca, Gianluca; Auken, Esben; Christiansen, Anders Vest


    Time-domain-induced polarization has significantly broadened its field of reference during the last decade, from mineral exploration to environmental geophysics, e.g., for clay and peat identification and landfill characterization. Though, insufficient modeling tools have hitherto limited the use...... of time-domaininduced polarization for wider purposes. For these reasons, a new forward code and inversion algorithm have been developed using the full-time decay of the induced polarization response, together with an accurate description of the transmitter waveform and of the receiver transfer function......%. Furthermore, the presence of low-pass filters in time-domain-induced polarization instruments affects the early times of the acquired decays (typically up to 100 ms) and has to be modeled in the forward response to avoid significant loss of resolution. The developed forward code has been implemented in a 1D...

  15. Amino substituted nitrogen heterocycle ureas as kinase insert domain containing receptor (KDR) inhibitors: Performance of structure–activity relationship approaches


    Hayriye Yilmaz; Natalia Sizochenko; Bakhtiyor Rasulev; Andrey Toropov; Yahya Guzel; Viktor Kuz'min; Danuta Leszczynska; Jerzy Leszczynski


    A quantitative structure–activity relationship (QSAR) study was performed on a set of amino-substituted nitrogen heterocyclic urea derivatives. Two novel approaches were applied: (1) the simplified molecular input-line entry systems (SMILES) based optimal descriptors approach; and (2) the fragment-based simplex representation of molecular structure (SiRMS) approach. Comparison with the classic scheme of building up the model and balance of correlation (BC) for optimal descriptors approach sho...

  16. Caractéristiques syntaxiques de la parenthèse en latin : linéarisation, délimitation et insertion

    Directory of Open Access Journals (Sweden)

    Isabelle Charnavel


    Full Text Available Le but de cet article est d’analyser les règles syntaxiques du placement de l’incidente en latin. L’incidente – ou proposition parenthétique – présente en effet le paradoxe suivant : bien qu’autonome sur un plan structurel (elle forme une proposition indépendante, elle s’insère dans une autre phrase sur un plan syntagmatique (linéairement, elle se situe à l’intérieur d’une autre proposition, l’hôte. Nous proposons de considérer que, malgré son autonomie syntaxique, l’incidente obéit à des contraintes syntaxiques précises d’insertion.Tout d’abord, nous montrons que l’incidente, toujours continue, est délimitée sur ses bords gauche et droit, bien que dépourvue de marqueurs spécifiques : le changement de proposition marque le début de l’incidente, tandis que la reprise de la phrase hôte en montre la fin, au moyen de répétitions pouvant mener à des anacoluthes.En outre, nous montrons que la place de l’incidente dans la phrase hôte suit des règles spécifiques. Premièrement, les sites d’insertion sont contraints : ils se situent à la frontière des groupes fonctionnels de l’hôte. De plus, il existe des contraintes distributionnelles entre ces sites d’insertion. Nous proposons de considérer que le domaine de l’incidente correspond au(x constituant(s sur le(squel(s porte sémantiquement l’incidente et que celle-ci se place toujours à l’intérieur ou à la périphérie de son domaine : une incidente à une proposition indépendante se place en deuxième position de son domaine, tandis qu’une incidente à un constituant occupe la frontière droite de son domaine.The aim of the paper is to analyse the syntactic rules of the incidental clause in Latin: indeed, the incidental – i.e. parenthetical sentence - exhibits the following paradox: although it is independent from a structural viewpoint (it constitutes a main clause, it is inserted in another sentence from a syntagmatic

  17. Lithium Insertion in LixMn2O4, 0

    DEFF Research Database (Denmark)

    West, Keld; Zachau-Christiansen, Birgit; Skaarup, Steen


    The electrochemical lithium insertion properties of highly crystalline LixMn2O4 are investigated in the approximate lithium insertion range 0 ... of the lithium insertion/extraction reactions is better at the higher voltages (versus Li/Li+), and particularly at the 4 V plateau. The lithium insertion/extraction reaction at the 1 V plateau although essentially reversible is associated with a significant voltage hysteresis....

  18. ALOG domains: provenance of plant homeotic and developmental regulators from the DNA-binding domain of a novel class of DIRS1-type retroposons

    Directory of Open Access Journals (Sweden)

    Iyer Lakshminarayan M


    Full Text Available Abstract Members of the Arabidopsis LSH1 and Oryza G1 (ALOG family of proteins have been shown to function as key developmental regulators in land plants. However, their precise mode of action remains unclear. Using sensitive sequence and structure analysis, we show that the ALOG domains are a distinct version of the N-terminal DNA-binding domain shared by the XerC/D-like, protelomerase, topoisomerase-IA, and Flp tyrosine recombinases. ALOG domains are distinguished by the insertion of an additional zinc ribbon into this DNA-binding domain. In particular, we show that the ALOG domain is derived from the XerC/D-like recombinases of a novel class of DIRS-1-like retroposons. Copies of this element, which have been recently inactivated, are present in several marine metazoan lineages, whereas the stramenopile Ectocarpus, retains an active copy of the same. Thus, we predict that ALOG domains help establish organ identity and differentiation by binding specific DNA sequences and acting as transcription factors or recruiters of repressive chromatin. They are also found in certain plant defense proteins, where they are predicted to function as DNA sensors. The evolutionary history of the ALOG domain represents a unique instance of a domain, otherwise exclusively found in retroelements, being recruited as a specific transcription factor in the streptophyte lineage of plants. Hence, they add to the growing evidence for derivation of DNA-binding domains of eukaryotic specific TFs from mobile and selfish elements.

  19. Epidermolytic palmoplantar keratoderma due to a novel type of keratin mutation, a 3-bp insertion in the keratin 9 helix termination motif. (United States)

    Coleman, C M; Munro, C S; Smith, F J; Uitto, J; McLean, W H


    Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by diffuse keratoderma, typically with an erythematous border. Histologically, palmoplantar epidermis shows suprabasal cytolysis and ultrastructurally, tonofilament aggregation with overlying epidermolytic hyperkeratosis. Mutations in the KRT9 gene, encoding keratin 9 (K9), a cytoskeletal protein expressed exclusively in suprabasal keratinocytes of palmoplantar epidermis, have been reported to cause EPPK. To date, all KRT9 defects reported in EPPK have been missense mutations in exon 1, which encodes the start of the alpha-helical rod domain. However, based on studies of other keratin disorders, it was postulated that mutations at the other end of the rod domain might also produce the EPPK phenotype. Here, we report the first mutation in the 2B domain of KRT9, 1362ins3, leading to an insertion of histidine in the helix termination motif of the K9 polypeptide. Insertional mutations have not been previously described in keratins. The phenotype of this case is similar to EPPK caused by 1A domain mutations, demonstrating that mutations in either of the helix boundary motif sequences of K9 are detrimental to keratin function and keratinocyte structure.

  20. The design and analysis of transposon insertion sequencing experiments. (United States)

    Chao, Michael C; Abel, Sören; Davis, Brigid M; Waldor, Matthew K


    Transposon insertion sequencing (TIS) is a powerful approach that can be extensively applied to the genome-wide definition of loci that are required for bacterial growth under diverse conditions. However, experimental design choices and stochastic biological processes can heavily influence the results of TIS experiments and affect downstream statistical analysis. In this Opinion article, we discuss TIS experimental parameters and how these factors relate to the benefits and limitations of the various statistical frameworks that can be applied to the computational analysis of TIS data.