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Sample records for determine cleavage-site recognition

  1. Analysis of eukaryotic topoisomerase II cleavage sites in the presence of the quinolone CP-115,953 reveals drug-dependent and -independent recognition elements.

    PubMed

    Spitzner, J R; Chung, I K; Gootz, T D; McGuirk, P R; Muller, M T

    1995-08-01

    The quinolone derivative CP-115,953 [6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] has been shown to induce eukaryotic topoisomerase II-mediated breaks in DNA, producing cleavage patterns that are distinct from those induced by the anticancer drugs amsacrine, etoposide, and teniposide. High levels of the quinolone have been found to inhibit topoisomerase II activity via an interaction with the enzyme and not by DNA unwinding. Topoisomerase II cleavage sites were analyzed on nine DNA fragments, and 85 quinolone-induced sites were sequenced, as well as 86 amsacrine and 134 teniposide sites. A consensus sequence was derived for the quinolone sites that is different from those reported for other drugs; however, because topoisomerase II cleavage sites are double-stranded but not palindromic, different consensus sequences are not easily compared. For this reason, a new, double-stranded, consensus sequence method, the "unique-base analysis," was developed; this was applied to the quinolone sites as well as six other large sets of topoisomerase II sites determined in the absence or presence of drugs. For each of the seven sets of sites, conserved bases were found in the 16-base region spanning positions -6 to +10, relative to the enzyme cleavage site (DNA breakage between -1 and +1). The conserved bases were virtually identical in the regions flanking the cleavage site for all seven data sets. In contrast, the base preferences identified proximal to the cleavage sites were unique to the drug tested. These observations suggest that the selection of cleavage sites by topoisomerase II involves both enzyme-dependent and drug-dependent recognition elements. The single most preferred base in the quinolone sites was a cytosine at -1; the same preference was found with teniposide, and 60 of the 85 quinolone sites co-localized with teniposide sites.

  2. The recognition of DNA cleavage sites by porcine spleen topoisomerase II.

    PubMed

    Huang, H W; Juang, J K; Liu, H J

    1992-02-11

    The cutting sites specificity of topoisomerase II from porcine spleen were determined by a modified Sanger's DNA sequencing method. The topoisomerase II prefers to cut DNA at the 3' side of A and leave 5' protruding end with two staggering bases. Through the free energy analysis for DNA duplex, we also found that the topoisomerase II seemed cut DNA preferably at energetically unstable regions. So it is concluded that the specific DNA cutting by porcine spleen topoisomerase II has two structural recognition factors: one is to localize around the energetically unstable region and another is to act at the 3' side of A base.

  3. Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: determinants and constraints.

    PubMed

    Lai, Zon W; Hanchapola, Iresha; Steer, David L; Smith, A Ian

    2011-06-14

    ADAM17, also known as tumor necrosis factor α-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) → Glu(708) mutation and the Arg(708)Arg(710) → Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.

  4. Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and γ-Secretase

    PubMed Central

    Hogl, Sebastian; Kuhn, Peer-Hendrik; Colombo, Alessio; Lichtenthaler, Stefan F.

    2011-01-01

    Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development. PMID:21695060

  5. Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array*

    PubMed Central

    Shinkai-Ouchi, Fumiko; Koyama, Suguru; Ono, Yasuko; Hata, Shoji; Ojima, Koichi; Shindo, Mayumi; duVerle, David; Ueno, Mika; Kitamura, Fujiko; Doi, Naoko; Takigawa, Ichigaku; Mamitsuka, Hiroshi; Sorimachi, Hiroyuki

    2016-01-01

    Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10′ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5–1,710 M−1s−1. Although most sites were cleaved by both calpain-1 and −2 with a similar kcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5′. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P′-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r = 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3′, and P4′ sites, and P1-P2 cooperativity. Furthermore, using our

  6. Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

    PubMed

    Chang, Chin-I; Chen, Li-Hao; Hu, Yeh-Fang; Wu, Chia-Che; Tsai, Jyh-Ming

    2017-03-01

    Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia β-defensin. The computer-predicted Nile tilapia β-defensin ((25)ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia β-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence ((27)FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) of Nile tilapia β-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia β-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates.

    PubMed

    Guidon, P T; Perrin, D; Harrison, P

    1996-02-01

    We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.

  8. Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

    PubMed Central

    Schuster, Christopher F.; Bertram, Ralph

    2014-01-01

    Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5'-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure. In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5' ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria. PMID:25406941

  9. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    SciTech Connect

    Spannaus, Ralf; Bodem, Jochen

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  10. Sensitivity of hepatitis C virus RNA to the antiviral enzyme ribonuclease L is determined by a subset of efficient cleavage sites.

    PubMed

    Han, Jian-Qiu; Wroblewski, Gerggory; Xu, Zan; Silverman, Robert H; Barton, David J

    2004-11-01

    Ribonuclease L (RNase L) cleaves RNA predominantly at single-stranded UA and UU dinucleotides. Intriguingly, hepatitis C virus (HCV) RNAs have a paucity of UA and UU dinucleotides, and relatively interferon (IFN)-resistant strains have fewer UA and UU dinucleotides than do more IFN-sensitive strains. In this study, we found that contextual features of UA and UU dinucleotides dramatically affected the efficiency of RNase L cleavage in HCV RNA. HCV genotype la RNA was cleaved by RNase L into fragments 200-1000 bases in length, consistent with 10-50 RNase L cleavage sites within the 9650-base long viral RNA. Using primer extension, we found that HCV RNA structures with multiple single-stranded UA and UU dinucleotides were cleaved most efficiently by RNase L. UA and UU dinucleotides with 3' proximal C or G residues were cleaved infrequently, whereas UA and UU dinucleotides within dsRNA structures were not cleaved. 5'-GUAC-3' and 5'-CUUC-3' were particularly unfavorable contexts for cleavage by RNase L. More than 60% of the UA and UU dinucleotides in HCV la RNA were not cleaved by RNase L because of these contextual features. The 10-30 most efficiently cleaved sites were responsible for approximately 50%-85% of all RNase L cleavage events. Our data indicate that a relatively small number of the UA and UU dinucleotides in HCV RNA mediate the overall sensitivity of HCV RNA to cleavage by RNase L.

  11. Prediction of neuropeptide cleavage sites in insects.

    PubMed

    Southey, Bruce R; Sweedler, Jonathan V; Rodriguez-Zas, Sandra L

    2008-03-15

    The production of neuropeptides from their precursor proteins is the result of a complex series of enzymatic processing steps. Often, the annotation of new neuropeptide genes from sequence information outstrips biochemical assays and so bioinformatics tools can provide rapid information on the most likely peptides produced by a gene. Predicting the final bioactive neuropeptides from precursor proteins requires accurate algorithms to determine which locations in the protein are cleaved. Predictive models were trained on Apis mellifera and Drosophila melanogaster precursors using binary logistic regression, multi-layer perceptron and k-nearest neighbor models. The final predictive models included specific amino acids at locations relative to the cleavage sites. Correct classification rates ranged from 78 to 100% indicating that the models adequately predicted cleaved and non-cleaved positions across a wide range of neuropeptide families and insect species. The model trained on D.melanogaster data had better generalization properties than the model trained on A. mellifera for the data sets considered. The reliable and consistent performance of the models in the test data sets suggests that the bioinformatics strategies proposed here can accurately predict neuropeptides in insects with sequence information based on neuropeptides with biochemical and sequence information in well-studied species.

  12. GPS-CCD: A Novel Computational Program for the Prediction of Calpain Cleavage Sites

    PubMed Central

    Gao, Xinjiao; Ma, Qian; Ren, Jian; Xue, Yu

    2011-01-01

    As one of the most essential post-translational modifications (PTMs) of proteins, proteolysis, especially calpain-mediated cleavage, plays an important role in many biological processes, including cell death/apoptosis, cytoskeletal remodeling, and the cell cycle. Experimental identification of calpain targets with bona fide cleavage sites is fundamental for dissecting the molecular mechanisms and biological roles of calpain cleavage. In contrast to time-consuming and labor-intensive experimental approaches, computational prediction of calpain cleavage sites might more cheaply and readily provide useful information for further experimental investigation. In this work, we constructed a novel software package of GPS-CCD (Calpain Cleavage Detector) for the prediction of calpain cleavage sites, with an accuracy of 89.98%, sensitivity of 60.87% and specificity of 90.07%. With this software, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. In this regard, GPS-CCD 1.0 is considered to be a useful tool for experimentalists. The online service and local packages of GPS-CCD 1.0 were implemented in JAVA and are freely available at: http://ccd.biocuckoo.org/. PMID:21533053

  13. Signal peptide prediction based on analysis of experimentally verified cleavage sites

    PubMed Central

    Zhang, Zemin; Henzel, William J.

    2004-01-01

    A number of computational tools are available for detecting signal peptides, but their abilities to locate the signal peptide cleavage sites vary significantly and are often less than satisfactory. We characterized a set of 270 secreted recombinant human proteins by automated Edman analysis and used the verified cleavage sites to evaluate the success rate of a number of computational prediction programs. An examination of the frequency of amino acid in the N-terminal region of the data set showed a preference of proline and glutamine but a bias against tyrosine. The data set was compared to the SWISS-PROT database and revealed a high percentage of discrepancies with cleavage site annotations that were computationally generated. The best program for predicting signal sequences was found to be SignalP 2.0-NN with an accuracy of 78.1% for cleavage site recognition. The new data set can be utilized for refining prediction algorithms, and we have built an improved version of profile hidden Markov model for signal peptides based on the new data. PMID:15340161

  14. Proteomic identification of protease cleavage sites characterizes prime and non-prime specificity of cysteine cathepsins B, L, and S.

    PubMed

    Biniossek, Martin L; Nägler, Dorit K; Becker-Pauly, Christoph; Schilling, Oliver

    2011-12-02

    Cysteine cathepsins mediate proteome homeostasis and have pivotal functions in diseases such as cancer. To better understand substrate recognition by cathepsins B, L, and S, we applied proteomic identification of protease cleavage sites (PICS) for simultaneous profiling of prime and non-prime specificity. PICS profiling of cathepsin B endopeptidase specificity highlights strong selectivity for glycine in P3' due to an occluding loop blocking access to the primed subsites. In P1', cathepsin B has a partial preference for phenylalanine, which is not found for cathepsins L and S. Occurrence of P1' phenylalanine often coincides with aromatic residues in P2. For cathepsin L, PICS identifies 845 cleavage sites, representing the most comprehensive PICS profile to date. Cathepsin L specificity is dominated by the canonical preference for aromatic residues in P2 with limited contribution of prime-site selectivity determinants. Profiling of cathepsins B and L with a shorter incubation time (4 h instead of 16 h) did not reveal time-dependency of individual specificity determinants. Cathepsin S specificity was profiled at pH 6.0 and 7.5. The PICS profiles at both pH values display a high degree of similarity. Cathepsin S specificity is primarily guided by aliphatic residues in P2 with limited importance of prime-site residues.

  15. Prediction of Signal Peptide Cleavage Sites with Subsite-Coupled and Template Matching Fusion Algorithm.

    PubMed

    Zhang, Shao-Wu; Zhang, Ting-He; Zhang, Jun-Nan; Huang, Yufei

    2014-03-01

    Fast and effective prediction of signal peptides (SP) and their cleavage sites is of great importance in computational biology. The approaches developed to predict signal peptide can be roughly divided into machine learning based, and sliding windows based. In order to further increase the prediction accuracy and coverage of organism for SP cleavage sites, we propose a novel method for predicting SP cleavage sites called Signal-CTF that utilizes machine learning and sliding windows, and is designed for N-termial secretory proteins in a large variety of organisms including human, animal, plant, virus, bacteria, fungi and archaea. Signal-CTF consists of three distinct elements: (1) a subsite-coupled and regularization function with a scaled window of fixed width that selects a set of candidates of possible secretion-cleavable segment for a query secretory protein; (2) a sum fusion system that integrates the outcomes from aligning the cleavage site template sequence with each of the aforementioned candidates in a scaled window of fixed width to determine the best candidate cleavage sites for the query secretory protein; (3) a voting system that identifies the ultimate signal peptide cleavage site among all possible results derived from using scaled windows of different width. When compared with Signal-3L and SignalP 4.0 predictors, the prediction accuracy of Signal-CTF is 4-12 %, 10-25 % higher than that of Signal-3L for human, animal and eukaryote, and SignalP 4.0 for eukaryota, Gram-positive bacteria and Gram-negative bacteria, respectively. Comparing with PRED-SIGNAL and SignalP 4.0 predictors on the 32 archaea secretory proteins of used in Bagos's paper, the prediction accuracy of Signal-CTF is 12.5 %, 25 % higher than that of PRED-SIGNAL and SignalP 4.0, respectively. The predicting results of several long signal peptides show that the Signal-CTF can better predict cleavage sites for long signal peptides than SignalP, Phobius, Philius, SPOCTOPUS, Signal

  16. Specific anti-integrase abzymes from HIV-infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20-mer oligopeptides corresponding to its antigenic determinants.

    PubMed

    Odintsova, Elena S; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2013-03-01

    HIV-infected patients possess anti-integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN-Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti-IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG-mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse-phase chromatography, matrix-assisted laser desorption/ionization spectrometry, and thin-layer chromatography to analyze the cleavage products of two 20-mer OPs corresponding to these AGDs. Both OPs contained 9-10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti-IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti-IN Abzs for intact integrase was ~1000-fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN.

  17. Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus

    PubMed Central

    Wei, Ting; Chisholm, Joan

    2016-01-01

    The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase) cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa)-long hydrophobic region (termed TM2). However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G). Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3–7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s) oriented parallel to the membrane inner surface. PMID:27589230

  18. Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus.

    PubMed

    Wei, Ting; Chisholm, Joan; Sanfaçon, Hélène

    2016-01-01

    The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase) cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa)-long hydrophobic region (termed TM2). However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G). Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3-7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s) oriented parallel to the membrane inner surface.

  19. 3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

    PubMed Central

    Wirblich, C; Sibilia, M; Boniotti, M B; Rossi, C; Thiel, H J; Meyers, G

    1995-01-01

    Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size. PMID:7474137

  20. DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes.

    PubMed Central

    Janscak, P; MacWilliams, M P; Sandmeier, U; Nagaraja, V; Bickle, T A

    1999-01-01

    Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process. PMID:10228175

  1. Identification of TMPRSS6 cleavage sites of hemojuvelin

    PubMed Central

    Rausa, Marco; Ghitti, Michela; Pagani, Alessia; Nai, Antonella; Campanella, Alessandro; Musco, Giovanna; Camaschella, Clara; Silvestri, Laura

    2015-01-01

    Hemojuvelin (HJV), the coreceptor of the BMP-SMAD pathway that up-regulates hepcidin transcription, is a repulsive guidance molecule (RGMc) which undergoes a complex intracellular processing. Following autoproteolysis, it is exported to the cell surface both as a full-length and a heterodimeric protein. In vitro membrane HJV (m-HJV) is cleaved by the transmembrane serine protease TMPRSS6 to attenuate signalling and to inhibit hepcidin expression. In this study, we investigated the number and position of HJV cleavage sites by mutagenizing arginine residues (R), potential TMPRSS6 targets, to alanine (A). We analysed translation and membrane expression of HJV R mutants and the pattern of fragments they release in the culture media in the presence of TMPRSS6. Abnormal fragments were observed for mutants at arginine 121, 176, 218, 288 and 326. Considering that all variants, except HJVR121A, lack autoproteolytic activity and some (HJVR176A and HJVR288A) are expressed at reduced levels on cell surface, we identified the fragments originating from either full-length or heterodimeric proteins and defined the residues 121 and 326 as the TMPRSS6 cleavage sites in both isoforms. Using the N-terminal FLAG-tagged HJV, we showed that residue 121 is critical also in the rearrangement of the N-terminal heterodimeric HJV. Exploiting the recently reported RGMb crystallographic structure, we generated a model of HJV that was used as input structure for all-atoms molecular dynamics simulation in explicit solvent. As assessed by in silico studies, we concluded that some arginines in the von Willebrand domain appear TMPRSS6 insensitive, likely because of partial protein structure destabilization. PMID:25704252

  2. GraBCas: a bioinformatics tool for score-based prediction of Caspase- and Granzyme B-cleavage sites in protein sequences

    PubMed Central

    Backes, Christina; Kuentzer, Jan; Lenhof, Hans-Peter; Comtesse, Nicole; Meese, Eckart

    2005-01-01

    Caspases and granzyme B are proteases that share the primary specificity to cleave at the carboxyl terminal of aspartate residues in their substrates. Both, caspases and granzyme B are enzymes that are involved in fundamental cellular processes and play a central role in apoptotic cell death. Although various targets are described, many substrates still await identification and many cleavage sites of known substrates are not identified or experimentally verified. A more comprehensive knowledge of caspase and granzyme B substrates is essential to understand the biological roles of these enzymes in more detail. The relatively high variability in cleavage site recognition sequence often complicates the identification of cleavage sites. As of yet there is no software available that allows identification of caspase and/or granzyme with cleavage sites differing from the consensus sequence. Here, we present a bioinformatics tool ‘GraBCas’ that provides score-based prediction of potential cleavage sites for the caspases 1–9 and granzyme B including an estimation of the fragment size. We tested GraBCas on already known substrates and showed its usefulness for protein sequence analysis. GraBCas is available at . PMID:15980455

  3. Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

    SciTech Connect

    Craft, Willie Warren; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2005-10-10

    The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F{sub 0}, and proteolytically cleaved into the mature F{sub 1} and F{sub 2} heterodimer, following an HDLVDGVK{sub 109} motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK{sub 109} motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK{sub 109} motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein.

  4. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site.

    SciTech Connect

    Stols, L.; Gu, M. Y.; Dieckman, L.; Raffen, R.; Collart, F. R.; Donnelly, M. I.

    2002-06-01

    To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-independent cloning (LIC) is well suited to robotic cloning and expression, but few LIC vectors are available commercially. We have developed a new LIC vector, pMCSG7, that incorporates the tobacco etch virus (TEV) protease cleavage site into the leader sequence. This protease is highly specific and functions under a wide range of conditions. The new vector incorporates an N-terminal his-tag followed by the TEV protease recognition site and a SspI restriction site used for LIC. The vector functioned as expected, giving high cloning efficiencies and strong expression of proteins. Purification and cleavage of a target protein showed that the his-tag and the TEV cleavage site function properly. The protein was purified and cleaved under different conditions to simulate both plate-based screening methods and large-scale purifications for crystal production. The vector also includes a pair of adjacent, unique restriction sites that will allow insertion of additional modules between the his-tag and the cleavage site of the leader sequence to generate a family of vectors suitable for high-throughput production of proteins.

  5. Predicting caspase substrate cleavage sites based on a hybrid SVM-PSSM method.

    PubMed

    Li, Dandan; Jiang, Zhenran; Yu, Weiming; Du, Lei

    2010-12-01

    Caspases play an important role in many critical non-apoptosis processes by cleaving relevant substrates at cleavage sites. Identification of caspase substrate cleavage sites is the key to understand these processes. This paper proposes a hybrid method using support vector machine (SVM) in conjunction with position specific scoring matrices (PSSM) for caspase substrate cleavage sites prediction. Three encoding schemes including orthonormal binary encoding, BLOSUM62 matrix profile and PSSM profile of neighborhood surrounding the substrate cleavage sites were regarded as the input of SVM. The 10-fold cross validation results demonstrate that the SVM-PSSM method performs well with an overall accuracy of 97.619% on a larger dataset.

  6. Mapping of macrophage elastase cleavage sites in insoluble human skin elastin.

    PubMed

    Taddese, Samuel; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2008-06-01

    Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28-31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed.

  7. Simulative and experimental investigation on the cleavage site that generates the soluble human LOX-1.

    PubMed

    Biocca, Silvia; Arcangeli, Tania; Tagliaferri, Elisa; Testa, Barbara; Vindigni, Giulia; Oteri, Francesco; Giorgi, Alessandra; Iacovelli, Federico; Novelli, Giuseppe; Desideri, Alessandro; Falconi, Mattia

    2013-12-01

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor that mediates the recognition, the binding and internalization of ox-LDL. A truncated soluble form of LOX-1 (sLOX-1) has been identified that, at elevated levels, has been associated to acute coronary syndrome. Human sLOX-1 is the extracellular part of membrane LOX-1 which is cleaved in the NECK domain with a mechanism that has not yet been identified. Purification of human sLOX-1 has been carried out to experimentally identify the cleavage site region within the NECK domain. Molecular modelling and classical molecular dynamics simulation techniques have been used to characterize the structural and dynamical properties of the LOX-1 NECK domain in the presence and absence of the CTLD recognition region, taking into account the obtained proteolysis results. The simulative data indicate that the NECK domain is stabilized by the coiled-coil heptad repeat motif along the simulations, shows a definite flexibility pattern and is characterized by specific electrostatic potentials. The detection of a mobile inter-helix space suggests an explanation for the in vivo susceptibility of the NECK domain to the proteolytic cleavage, validating the assumption that the NECK domain sequence is composed of a coiled-coil motif destabilized in specific regions of functional significance.

  8. Cleavage Site Localization Differentially Controls Interleukin-6 Receptor Proteolysis by ADAM10 and ADAM17

    PubMed Central

    Riethmueller, Steffen; Ehlers, Johanna C.; Lokau, Juliane; Düsterhöft, Stefan; Knittler, Katharina; Dombrowsky, Gregor; Grötzinger, Joachim; Rabe, Björn; Rose-John, Stefan; Garbers, Christoph

    2016-01-01

    Limited proteolysis of the Interleukin-6 Receptor (IL-6R) leads to the release of the IL-6R ectodomain. Binding of the cytokine IL-6 to the soluble IL-6R (sIL-6R) results in an agonistic IL-6/sIL-6R complex, which activates cells via gp130 irrespective of whether the cells express the IL-6R itself. This signaling pathway has been termed trans-signaling and is thought to mainly account for the pro-inflammatory properties of IL-6. A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 are the major proteases that cleave the IL-6R. We have previously shown that deletion of a ten amino acid long stretch within the stalk region including the cleavage site prevents ADAM17-mediated cleavage, whereas the receptor retained its full biological activity. In the present study, we show that deletion of a triple serine (3S) motif (Ser-359 to Ser-361) adjacent to the cleavage site is sufficient to prevent IL-6R cleavage by ADAM17, but not ADAM10. We find that the impaired shedding is caused by the reduced distance between the cleavage site and the plasma membrane. Positioning of the cleavage site in greater distance towards the plasma membrane abrogates ADAM17-mediated shedding and reveals a novel cleavage site of ADAM10. Our findings underline functional differences in IL-6R proteolysis by ADAM10 and ADAM17. PMID:27151651

  9. Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

    PubMed Central

    Nao, Naganori; Yamagishi, Junya; Miyamoto, Hiroko; Igarashi, Manabu; Manzoor, Rashid; Ohnuma, Aiko; Tsuda, Yoshimi; Furuyama, Wakako; Shigeno, Asako; Kajihara, Masahiro; Kishida, Noriko; Yoshida, Reiko

    2017-01-01

    ABSTRACT Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. PMID:28196963

  10. Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection

    PubMed Central

    Schmelcher, Mathias; Shen, Yang; Nelson, Daniel C.; Eugster, Marcel R.; Eichenseher, Fritz; Hanke, Daniela C.; Loessner, Martin J.; Dong, Shengli; Pritchard, David G.; Lee, Jean C.; Becker, Stephen C.; Foster-Frey, Juli; Donovan, David M.

    2015-01-01

    Objectives In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs. Methods PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park–Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model. Results Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment. Conclusions Our results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target sites. PMID:25630640

  11. Amino acid substitutions in the poliovirus maturation cleavage site affect assembly and result in accumulation of provirions.

    PubMed Central

    Ansardi, D C; Morrow, C D

    1995-01-01

    The assembly of infectious poliovirus virions requires a proteolytic cleavage between an asparagine-serine amino acid pair (the maturation cleavage site) in VP0 after encapsidation of the genomic RNA. In this study, we have investigated the effects that mutations in the maturation cleavage site have on P1 polyprotein processing, assembly of subviral intermediates, and encapsidation of the viral genomic RNA. We have made mutations in the maturation cleavage site which change the asparagine-serine amino acid pair to either glutamine-glycine or threonine-serine. The mutations were created by site-directed mutagenesis of P1 cDNAs which were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses. The P1 polyproteins expressed from the recombinant vaccinia viruses were analyzed for proteolytic processing and assembly defects in cells coinfected with a recombinant vaccinia virus (VV-P3) that expresses the poliovirus 3CD protease. A trans complementation system using a defective poliovirus genome was utilized to assess the capacity of the mutant P1 proteins to encapsidate genomic RNA (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The mutant P1 proteins containing the glutamine-glycine amino acid pair (VP4-QG) and the threonine-serine pair (VP4-TS) were processed by 3CD provided in trans from VV-P3. The processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor VP4-QG were unstable and failed to assemble into subviral structures in cells coinfected with VV-P3. However, the capsid proteins derived from VP4-QG did assemble into empty-capsid-like structures in the presence of the defective poliovirus genome. In contrast, the capsid proteins derived from processing of the VP4-TS mutant assembled into subviral intermediates both in the presence and in the absence of the defective genome RNA. By a sedimentation analysis, we determined that the capsid proteins derived from the VP4-TS precursor

  12. Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif

    PubMed Central

    Luczo, Jasmina M.; Stambas, John; Durr, Peter A.; Michalski, Wojtek P.

    2015-01-01

    Summary The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio‐economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host‐pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd. PMID:26467906

  13. Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin.

    PubMed

    Nao, Naganori; Yamagishi, Junya; Miyamoto, Hiroko; Igarashi, Manabu; Manzoor, Rashid; Ohnuma, Aiko; Tsuda, Yoshimi; Furuyama, Wakako; Shigeno, Asako; Kajihara, Masahiro; Kishida, Noriko; Yoshida, Reiko; Takada, Ayato

    2017-02-14

    Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for

  14. PROSPER: An Integrated Feature-Based Tool for Predicting Protease Substrate Cleavage Sites

    PubMed Central

    Perry, Andrew J.; Akutsu, Tatsuya; Webb, Geoffrey I.; Whisstock, James C.; Pike, Robert N.

    2012-01-01

    The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using

  15. Signal peptide discrimination and cleavage site identification using SVM and NN.

    PubMed

    Kazemian, H B; Yusuf, S A; White, K

    2014-02-01

    About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model. © 2013 Published by Elsevier Ltd.

  16. Evaluation of fusion protein cleavage site sequences of Newcastle disease virus in genotype matched vaccines

    PubMed Central

    Kim, Shin-Hee; Chen, Zongyan; Yoshida, Asuka; Paldurai, Anandan; Xiao, Sa; Samal, Siba K.

    2017-01-01

    Newcastle disease virus (NDV) causes a devastating poultry disease worldwide. Frequent outbreaks of NDV in chickens vaccinated with conventional live vaccines suggest a need to develop new vaccines that are genetically matched against circulating NDV strains, such as the genotype V virulent strains currently circulating in Mexico and Central America. In this study, a reverse genetics system was developed for the virulent NDV strain Mexico/01/10 strain and used to generate highly attenuated vaccine candidates by individually modifying the cleavage site sequence of fusion (F) protein. The cleavage site sequence of parental virus was individually changed to those of the avirulent NDV strain LaSota and other serotypes of avian paramyxoviruses (APMV serotype-2, -3, -4, -6, -7, -8, and -9). In general, these mutations affected cell-to-cell fusion activity in vitro and the efficiency of the F protein cleavage and made recombinant Mexico/01/10 (rMex) virus highly attenuated in chickens. When chickens were immunized with the rMex mutant viruses and challenged with the virulent parent virus, there was reduced challenge virus shedding compared to birds immunized with the heterologous vaccine strain LaSota. Among the vaccine candidates, rMex containing the cleavage site sequence of APMV-2 induced the highest neutralizing antibody titer and completely protected chickens from challenge virus shedding. These results show the role of the F protein cleavage site sequence of each APMV type in generating genotype V-matched vaccines and the efficacy of matched vaccine strains to provide better protection against NDV strains currently circulating in Mexico. PMID:28339499

  17. Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) N-terminal sequencing of proteins and proteolytic cleavage sites by quantitative mass spectrometry.

    PubMed

    Doucet, Alain; Overall, Christopher M

    2011-01-01

    Edman degradation is a long-established technique for N-terminal sequencing of proteins and cleavage fragments. However, for accurate data analysis and amino acid assignments, Edman sequencing proceeds on samples of single proteins only and so lacks high-throughput capabilities. We describe a new method for the high-throughput determination of N-terminal sequences of multiple protein fragments in solution. Proteolytic processing can change the activity of bioactive proteins and also reveal cryptic binding sites and generate proteins with new functions (neoproteins) not found in the parent molecule. For example, extracellular matrix (ECM) protein processing often produces multiple proteolytic fragments with the generation of cryptic binding sites and neoproteins by ECM protein processing being well documented. The exact proteolytic cleavage sites need to be identified to fully understand the functions of the cleavage fragments and biological roles of proteases in vivo. However, the identification of cleavage sites in complex high molecular proteins such as those composing the ECM is not trivial. N-terminal microsequencing of proteolytic fragments is the usual method employed, but it suffers from poor resolution of sodium dodecylsulfate-polyacrylamide gel electrophoresis gels and is inefficient at identifying multiple cleavages, requiring preparation of numerous gels or membrane slices for analysis. We recently developed Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS) to overcome these limitations as a complement for N-terminal sequencing. ATOMS employs isotopic labeling and quantitative tandem mass spectrometry to identify cleavage sites in a fast and accurate manner. We successfully used ATOMS to identify nearly 100 cleavage sites in the ECM proteins laminin and fibronectin. Presented herein is the detailed step-by-step protocol for ATOMS. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis.

    PubMed

    Davy, A; SŁrensen, M B; Svendsen, I; Cameron-Mills, V; Simpson, D J

    2000-01-01

    Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.

  19. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites.

    PubMed

    Fukasawa, Yoshinori; Tsuji, Junko; Fu, Szu-Chin; Tomii, Kentaro; Horton, Paul; Imai, Kenichiro

    2015-04-01

    Mitochondria provide numerous essential functions for cells and their dysfunction leads to a variety of diseases. Thus, obtaining a complete mitochondrial proteome should be a crucial step toward understanding the roles of mitochondria. Many mitochondrial proteins have been identified experimentally but a complete list is not yet available. To fill this gap, methods to computationally predict mitochondrial proteins from amino acid sequence have been developed and are widely used, but unfortunately, their accuracy is far from perfect. Here we describe MitoFates, an improved prediction method for cleavable N-terminal mitochondrial targeting signals (presequences) and their cleavage sites. MitoFates introduces novel sequence features including positively charged amphiphilicity, presequence motifs, and position weight matrices modeling the presequence cleavage sites. These features are combined with classical ones such as amino acid composition and physico-chemical properties as input to a standard support vector machine classifier. On independent test data, MitoFates attains better performance than existing predictors in both detection of presequences and in predicting their cleavage sites. We used MitoFates to look for undiscovered mitochondrial proteins from 42,217 human proteins (including isoforms such as alternative splicing or translation initiation variants). MitoFates predicts 1167 genes to have at least one isoform with a presequence. Five-hundred and eighty of these genes were not annotated as mitochondrial in either UniProt or Gene Ontology. Interestingly, these include candidate regulators of parkin translocation to damaged mitochondria, and also many genes with known disease mutations, suggesting that careful investigation of MitoFates predictions may be helpful in elucidating the role of mitochondria in health and disease. MitoFates is open source with a convenient web server publicly available.

  20. Degradation of tropoelastin by matrix metalloproteinases--cleavage site specificities and release of matrikines.

    PubMed

    Heinz, Andrea; Jung, Michael C; Duca, Laurent; Sippl, Wolfgang; Taddese, Samuel; Ihling, Christian; Rusciani, Anthony; Jahreis, Günther; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2010-04-01

    To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P(1)'. MMP-7 shows a strong preference for Leu at P(1)', which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P(1)'. All three MMPs showed a clear preference for Pro at P(3) that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing.

  1. Studies on the preferred uracil-adenine base pair at the cleavage site of 10-23 DNAzyme by functional group modifications on adenine.

    PubMed

    Zhu, Junfei; Li, Zhiwen; Yang, Zhenjun; He, Junlin

    2015-08-01

    10-23 DNAzyme is capable of catalytically cleaving RNA substrates with the preferred cleavage sites rAU and rGU, in which the common base pair U-dA0 forms between the substrate and the DNAzyme in the cleavage reaction. Here its conservation was studied with base modifications on dA and extra functional groups introduced. The nitrogen atom at 7- or 8-position of adenine was demonstrated to be equally important for the cleavage reaction, although it is not related to the thermal stability of the base pair. Deletion of 6-amino group led to decreased stability of the base pair and a slight slower reaction rate. Extra functional groups through 6-amino group were not favorably accommodated in the cleavage site. From these modifications at the level of functional groups, it demonstrated that the base pair U-dA0 not only contributes to the recognition and binding stability, but also it is involved in the active catalytic center by its functional groups and base stacking. This kind of chemical modifications with 7-substituted 8-aza-7-deaza-2'-deoxyadenosine at dA0 is favorable for the introduction of signal molecules for mechanistic studies and biological applications, without significant loss of the catalytic function and structural destruction.

  2. Design and identification of a high efficient formic acid cleavage site for separation of fusion protein.

    PubMed

    Zhang, Huaguang; Li, Mei; Shi, Shuangfeng; Yin, Chao; Jia, Shirong; Wang, Zhixing; Liu, Yuhui

    2015-02-01

    The release of target protein with high efficiency and low cost from expressed fusion protein is a key requirement for commercial production of target proteins. To establish such a cleavage system, we have designed four formic acid (FA) cleavage sites C1 (DPDPDP), C2 (DPPDPP), C3 (DDDDPI) and C4 (IVDPNP), which was placed in between the E and G fusion protein. Four expression vectors were individually constructed and expressed in Escherichia coli. Purified proteins were reacted with a series of FA concentrations or under different temperatures followed by SDS-PAGE gel electrophoresis to verify the degree of cleavage efficiency. Results showed that the C2 was the most efficient site compared with the other three. After optimization of cleavage conditions for E-C2-G, the cleavage efficiently could reach as high as 87.3% within 2.5 h in 37% FA at 45 °C. Comparing with previous reports, a significant reduction (26%) of FA concentration at a lower temperature in a short duration of reaction (18 times less) was achieved. We believe the cleavage site of DPPDPP identified in this study can be used in the large-scale production of valuable fusion proteins to save the cost, time and energy.

  3. Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer.

    PubMed

    Starega-Roslan, Julia; Galka-Marciniak, Paulina; Krzyzosiak, Wlodzimierz J

    2015-12-15

    The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3' arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5' end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3' overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3' overhangs.

  4. A python analytical pipeline to identify prohormone precursors and predict prohormone cleavage sites.

    PubMed

    Southey, Bruce R; Sweedler, Jonathan V; Rodriguez-Zas, Sandra L

    2008-01-01

    Neuropeptides and hormones are signaling molecules that support cell-cell communication in the central nervous system. Experimentally characterizing neuropeptides requires significant efforts because of the complex and variable processing of prohormone precursor proteins into neuropeptides and hormones. We demonstrate the power and flexibility of the Python language to develop components of an bioinformatic analytical pipeline to identify precursors from genomic data and to predict cleavage as these precursors are en route to the final bioactive peptides. We identified 75 precursors in the rhesus genome, predicted cleavage sites using support vector machines and compared the rhesus predictions to putative assignments based on homology to human sequences. The correct classification rate of cleavage using the support vector machines was over 97% for both human and rhesus data sets. The functionality of Python has been important to develop and maintain NeuroPred (http://neuroproteomics.scs.uiuc.edu/neuropred.html), a user-centered web application for the neuroscience community that provides cleavage site prediction from a wide range of models, precision and accuracy statistics, post-translational modifications, and the molecular mass of potential peptides. The combined results illustrate the suitability of the Python language to implement an all-inclusive bioinformatics approach to predict neuropeptides that encompasses a large number of interdependent steps, from scanning genomes for precursor genes to identification of potential bioactive neuropeptides.

  5. LIPPRED: A web server for accurate prediction of lipoprotein signal sequences and cleavage sites

    PubMed Central

    Taylor, Paul D; Toseland, Christopher P; Attwood, Teresa K; Flower, Darren R

    2006-01-01

    Bacterial lipoproteins have many important functions and represent a class of possible vaccine candidates. The prediction of lipoproteins from sequence is thus an important task for computational vaccinology. Naïve-Bayesian networks were trained to identify SpaseII cleavage sites and their preceding signal sequences using a set of 199 distinct lipoprotein sequences. A comprehensive range of sequence models was used to identify the best model for lipoprotein signal sequences. The best performing sequence model was found to be 10-residues in length, including the conserved cysteine lipid attachment site and the nine residues prior to it. The sensitivity of prediction for LipPred was 0.979, while the specificity was 0.742. Here, we describe LipPred, a web server for lipoprotein prediction; available at the URL: http://www.jenner.ac.uk/LipPred/. LipPred is the most accurate method available for the detection of SpaseIIcleaved lipoprotein signal sequences and the prediction of their cleavage sites. PMID:17597883

  6. Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer

    PubMed Central

    Starega-Roslan, Julia; Galka-Marciniak, Paulina; Krzyzosiak, Wlodzimierz J.

    2015-01-01

    The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3′ arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5′ end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3′ overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3′ overhangs. PMID:26424848

  7. Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site

    PubMed Central

    Diederich, Sandra; Berhane, Yohannes; Embury-Hyatt, Carissa; Hisanaga, Tamiko; Handel, Katherine; Cottam-Birt, Colleen; Ranadheera, Charlene; Kobasa, Darwyn

    2015-01-01

    ABSTRACT Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin

  8. A new approach to an influenza live vaccine: modification of the cleavage site of hemagglutinin.

    PubMed

    Stech, J; Garn, H; Wegmann, M; Wagner, R; Klenk, H-D

    2005-06-01

    A promising approach to reduce the impact of influenza is the use of an attenuated, live virus as a vaccine. Using reverse genetics, we generated a mutant of strain A/WSN/33 with a modified cleavage site within its hemagglutinin, which depends on proteolytic activation by elastase. Unlike the wild-type, which requires trypsin, this mutant is strictly dependent on elastase. Both viruses grow equally well in cell culture. In contrast to the lethal wild-type virus, the mutant is entirely attenuated in mice. At a dose of 10(5) plaque-forming units, it induced complete protection against lethal challenge. This approach allows the conversion of any epidemic strain into a genetically homologous attenuated virus.

  9. DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion.

    PubMed

    Schwarz, Friedrich W; van Aelst, Kara; Tóth, Júlia; Seidel, Ralf; Szczelkun, Mark D

    2011-10-01

    DNA cleavage by the Type III Restriction-Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision.

  10. Modification of the Hemagglutinin Cleavage Site Allows Indirect Activation of Avian Influenza Virus H9N2 by Bacterial Staphylokinase

    PubMed Central

    Tse, Longping V.; Whittaker, Gary R.

    2015-01-01

    Influenza H9N2 is considered to be a low pathogenicity avian influenza (LPAI) virus that commonly infects avian species and can also infect humans. In 1996, the influenza virus, A/chicken/Korea/MS96-CE6/1996/H9N2 (MS96) was isolated from an outbreak in multiple farms in South Korea that resulted in upwards of 30% mortality in infected chickens, with the virus infecting a number of extrapulmonary tissues, indicating internal spread. However, in experimental infections, complete recovery of specific pathogen free (SPF) chickens occurred. Such a discrepancy indicated an alternative pathway for MS96 virus to gain virulence in farmed chickens. A key determinant of influenza pathogenesis is the susceptibility of the viral hemagglutinin (HA) to proteolytic cleavage/activation. Here, we identified that an amino acid substitution, Ser to Tyr found at the P2 position of the MS96 HA cleavage site optimizes cleavage by the protease plasmin (Pm). Importantly, we identified that certain Staphylococcus sp. are able to cleave and activate MS96 HA by activating plasminogen (Plg) to plasmin by use of a virulence factor, staphylokinase. Overall, these studies provide an in-vitro mechanism for bacterially mediated enhancement of influenza activation, and allow insight into the microbiological mechanisms underlying the avian influenza H9N2 outbreak in Korea in1996. PMID:25841078

  11. Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase.

    PubMed

    Zhang, X P; Sjöling, S; Tanudji, M; Somogyi, L; Andreu, D; Eriksson, L E; Gräslund, A; Whelan, J; Glaser, E

    2001-09-01

    Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.

  12. Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

    PubMed

    Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

    2016-11-01

    Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs.

  13. A Single Maturation Cleavage Site in Adenovirus Impacts Cell Entry and Capsid Assembly

    PubMed Central

    Moyer, Crystal L.; Besser, Eli S.

    2015-01-01

    ABSTRACT Proteolytic maturation drives the conversion of stable, immature virus particles to a mature, metastable state primed for cell infection. In the case of human adenovirus, this proteolytic cleavage is mediated by the virally encoded protease AVP. Protein VI, an internal capsid cement protein and substrate for AVP, is cleaved at two sites, one of which is near the N terminus of the protein. In mature capsids, the 33 residues at the N terminus of protein VI (pVIn) are sequestered inside the cavity formed by peripentonal hexon trimers at the 5-fold vertex. Here, we describe a glycine-to-alanine mutation in the N-terminal cleavage site of protein VI that profoundly impacts proteolytic processing, the generation of infectious particles, and cell entry. The phenotypic effects associated with this mutant provide a mechanistic framework for understanding the multifunctional nature of protein VI. Based on our findings, we propose that the primary function of the pVIn peptide is to mediate interactions between protein VI and hexon during virus replication, driving hexon nuclear accumulation and particle assembly. Once particles are assembled, AVP-mediated cleavage facilitates the release of the membrane lytic region at the amino terminus of mature VI, allowing it to lyse the endosome during cell infection. These findings highlight the importance of a single maturation cleavage site for both infectious particle production and cell entry and emphasize the exquisite spatiotemporal regulation governing adenovirus assembly and disassembly. IMPORTANCE Postassembly virus maturation is a cornerstone principle in virology. However, a mechanistic understanding of how icosahedral viruses utilize this process to transform immature capsids into infection-competent particles is largely lacking. Adenovirus maturation involves proteolytic processing of seven precursor proteins. There is currently no information for the role of each independent cleavage event in the generation of

  14. Avian paramyxovirus serotype 1 strains of low virulence with unusual fusion protein cleavage sites isolated from poultry species

    USDA-ARS?s Scientific Manuscript database

    Avian paramyxo-serotype-1 viruses (APMV1) with fusion cleavage sites containing two basic amino acids and a phenylalanine (F) at position 117 have been isolated from poultry species in two states from 2007-2009. The intracerebral pathogenicity indices for these viruses are of low virulence at 0.00 ...

  15. The Identity of the Nucleophile Substitution may Influence Metal Interactions with the Cleavage Site of the Minimal Hammerhead Ribozyme

    PubMed Central

    Osborne, Edith M.; Ward, W. Luke; Ruehle, Max Z.; DeRose, Victoria J.

    2010-01-01

    Potential metal interactions with the cleavage site of a minimal hammerhead ribozyme (mHHRz) were probed using 31P NMR-detected Cd2+ titration studies of HHRz constructs containing a phosphorothioate (PS) modification at the cleavage site. The mHHRz nucleophile position was replaced by either a 2′-F or a 2′-NH2 in order to block cleavage activity during the study. The 2′-F/PS cleavage site mHHRz construct, in which the 2′-F should closely imitate the atom size and electronegativity of a 2′OH, demonstrates low levels of metal ion association (<1 ppm 31P chemical shift changes). This observation indicates that having an atom size and electrostatic properties that are similar to the 2′-OH are not the governing factors in allowing metal interactions with the scissile phosphate of the mHHRz. With a 2′-NH2 substitution, a large upfield change in 31P NMR chemical shift of the phosphorothioate peak (Δ~3 ppm with 6 equivalents added Cd2+) indicates observable Cd2+ interactions with the substituted site. Since a 2′-NH2, but not a 2′-F, can serve as a metal ligand, these data suggest that a metal ion interaction with the HHRz cleavage site may include both the scissile phosphate and the 2′ nucleophile. Control samples in which the 2′-NH2/PS unit is placed either next to the mHHRz cleavage site (at U16.1), in a duplex, or in a amUPSU dinucleotide, show much weaker interactions with Cd2+. Results with these control samples indicate that simply the presence of a 2′-NH2/PS unit does not create a strong metal binding site, reinforcing the possibility that the 2′-NH2-moderated Cd-PS interaction is specific to the mHHRz cleavage site. Upfield chemical shifts of both 31P and H2′ 1H resonances in amUPSU are observed with addition of Cd2+, consistent with the predicted metal coordination to both 2′-NH2 and phosphorothioate ligands. These data suggest that metal ion association with the HHRz cleavage site may include an interaction with the 2

  16. A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3.

    PubMed

    Weber, Jonasz J; Golla, Matthias; Guaitoli, Giambattista; Wanichawan, Pimthanya; Hayer, Stefanie N; Hauser, Stefan; Krahl, Ann-Christin; Nagel, Maike; Samer, Sebastian; Aronica, Eleonora; Carlson, Cathrine R; Schöls, Ludger; Riess, Olaf; Gloeckner, Christian J; Nguyen, Huu P; Hübener-Schmid, Jeannette

    2017-03-08

    Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  17. Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

    PubMed Central

    Licitra, Beth N.; Millet, Jean K.; Regan, Andrew D.; Hamilton, Brian S.; Rinaldi, Vera D.; Duhamel, Gerald E.

    2013-01-01

    Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses. PMID:23763835

  18. Mass spectrometric identification of key proteolytic cleavage sites in statherin affecting mineral homeostasis and bacterial binding domains.

    PubMed

    Helmerhorst, Eva J; Traboulsi, Georges; Salih, Erdjan; Oppenheim, Frank G

    2010-10-01

    Human salivary statherin inhibits both primary and secondary calcium phosphate precipitation and, upon binding to hydroxyapatite, associates with a variety of oral bacteria. These functions, crucial in the maintenance of tooth enamel integrity, are located in defined regions within the statherin molecule. Proteases associated with saliva, however, cleave statherin effectively, and it is of importance to determine how statherin functional domains are affected by these events. Statherin was isolated from human parotid secretion by zinc precipitation and purified by reversed-phase high performance liquid chromatography (RP-HPLC). To characterize the proteolytic process provoked by oral proteases, statherin was incubated with whole saliva and fragmentation was monitored by RP-HPLC. The early formed peptides were structurally characterized by reversed phase liquid chromatography electrospray-ionization tandem mass spectrometry. Statherin was degraded 3.6× faster in whole saliva than in whole saliva supernatant. The main and primary cleavage sites were located in the N-terminal half of statherin, specifically after Arg(9), Arg(10), and Arg(13); after Phe(14) and Tyr(18); and after Gly(12), Gly(15), Gly(17) and Gly(19) while the C-terminal half of statherin remained intact. Whole saliva protease activities separated the charged N-terminus from the hydrophobic C-terminus, negatively impacting on full length statherin functions comprising enamel lubrication and inhibition of primary calcium phosphate precipitation. Cryptic epitopes for bacterial binding residing in the C-terminal domain were likewise affected. The full characterization of the statherin peptides generated facilitates the elucidation of their novel functional roles in the oral and gastro-intestinal environment.

  19. An MLP-based feature subset selection for HIV-1 protease cleavage site analysis.

    PubMed

    Kim, Gilhan; Kim, Yeonjoo; Lim, Heuiseok; Kim, Hyeoncheol

    2010-01-01

    In recent years, several machine learning approaches have been applied to modeling the specificity of the human immunodeficiency virus type 1 (HIV-1) protease cleavage domain. However, the high dimensional domain dataset contains a small number of samples, which could misguide classification modeling and its interpretation. Appropriate feature selection can alleviate the problem by eliminating irrelevant and redundant features, and thus improve prediction performance. We introduce a new feature subset selection method, FS-MLP, that selects relevant features using multi-layered perceptron (MLP) learning. The method includes MLP learning with a training dataset and then feature subset selection using decompositional approach to analyze the trained MLP. Our method is able to select a subset of relevant features in high dimensional, multi-variate and non-linear domains. Using five artificial datasets that represent four data types, we verified the FS-MLP performance with seven other feature selection methods. Experimental results showed that the FS-MLP is superior at high dimensional, multi-variate and non-linear domains. In experiments with HIV-1 protease cleavage dataset, the FS-MLP selected a set of 14 highly relevant features among 160 original features. On a validation set of 131 test instances, classifiers that used the 14 features showed about 95% accuracy which outperformed other seven methods in terms of accuracy and the number of features. Our experimental results indicate that the FS-MLP is effective in analyzing multi-variate, non-linear and high dimensional datasets such as HIV-1 protease cleavage dataset. The 14 relevant features which were selected by the FS-MLP provide us with useful insights into the HIV-1 cleavage site domain as well. The FS-MLP is a useful method for computational sequence analysis in general. 2009 Elsevier B.V. All rights reserved.

  20. COL1 C-propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta

    PubMed Central

    Lindahl, Katarina; Barnes, Aileen M.; Fratzl-Zelman, Nadja; Whyte, Michael P.; Hefferan, Theresa E.; Makareeva, Elena; Brusel, Marina; Yaszemski, Michael J.; Rubin, Carl-Johan; Kindmark, Andreas; Roschger, Paul; Klaushofer, Klaus; McAlister, William H.; Mumm, Steven; Leikin, Sergey; Kessler, Efrat; Boskey, Adele L.; Ljunggren, Östen; Marini, Joan C.

    2011-01-01

    Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization. PMID:21344539

  1. Evolution of the R2 Retrotransposon Ribozyme and Its Self-Cleavage Site

    PubMed Central

    Eickbush, Danna G.; Burke, William D.; Eickbush, Thomas H.

    2013-01-01

    R2 is a non-long terminal repeat retrotransposon that inserts site-specifically in the tandem 28S rRNA genes of many animals. Previously, R2 RNA from various species of Drosophila was shown to self-cleave from the 28S rRNA/R2 co-transcript by a hepatitis D virus (HDV)-like ribozyme encoded at its 5' end. RNA cleavage was at the precise 5' junction of the element with the 28S gene. Here we report that RNAs encompassing the 5' ends of R2 elements from throughout its species range fold into HDV-like ribozymes. In vitro assays of RNA self-cleavage conducted in many R2 lineages confirmed activity. For many R2s, RNA self-cleavage was not at the 5' end of the element but at 28S rRNA sequences up to 36 nucleotides upstream of the junction. The location of cleavage correlated well with the types of endogenous R2 5' junctions from different species. R2 5' junctions were uniform for most R2s in which RNA cleavage was upstream in the rRNA sequences. The 28S sequences remaining on the first DNA strand synthesized during retrotransposition are postulated to anneal to the target site and uniformly prime second strand DNA synthesis. In species where RNA cleavage occurred at the R2 5' end, the 5' junctions were variable. This junction variation is postulated to result from the priming of second strand DNA synthesis by chance microhomologies between the target site and the first DNA strand. Finally, features of R2 ribozyme evolution, especially changes in cleavage site and convergence on the same active site sequences, are discussed. PMID:24066021

  2. Rice tungro spherical virus polyprotein processing: identification of a virus-encoded protease and mutational analysis of putative cleavage sites.

    PubMed

    Thole, V; Hull, R

    1998-07-20

    Rice tungro spherical virus encodes a large polyprotein containing motifs with sequence similarity to viral serine-like proteases and RNA polymerases. Polyclonal antisera raised against domains of the putative protease and polymerase in fusion with glutathione S-transferase detected a protein of about 35 kDa and, in very low amounts, a protein of about 70 kDa, respectively, in extracts from infected plants. In in vitro transcription/translation systems and in Escherichia coli we demonstrated a proteolytic activity in the C-terminal region of the polyprotein. This protease rapidly cleaved its polyprotein precursors in vitro. Mutating a potential cleavage site located N-terminal to the protease domain, Gln2526-Asp2527, diminished processing. The transversion mutation at the putative C-terminal cleavage site of the protease, at Gln2852-Ala2853, led to a delayed and partial processing.

  3. Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.

    PubMed

    Burster, Timo; Marin-Esteban, Viviana; Boehm, Bernhard O; Dunn, Shannon; Rotzschke, Olaf; Falk, Kirsten; Weber, Ekkehard; Verhelst, Steven H L; Kalbacher, Hubert; Driessen, Christoph

    2007-11-15

    Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

  4. Analysis of cleavage-site patterns in protein precursor sequences with a perceptron-type neural network.

    PubMed

    Schneider, G; Röhlk, S; Wrede, P

    1993-07-30

    A method for feature extraction from protein sequences has been developed which is based on an artificial neural filter system. Amino acid sequences are analyzed with regard to physicochemical residue properties. This alternative representation of a sequence allows an interpretation of the networks' weight values in a comprehensive and biochemically meaningful way by displaying the optimized network weights in Hinton diagrams. Signal peptidase cleavage sites of E.coli periplasmic proteins, human mitochondrial precursors and chloroplast precursors from spinach have been investigated. The network for E.coli periplasmic protein precursors classified both training and test data with 100% accuracy. The interpretation of its network weights clearly confirms the "-3,-1 rule" and the existence of a hydrophobic core region starting at position -6. Further striking features and dominant positions can be found for all three types of cleavage sites.

  5. Characterization of an H4N2 influenza virus from Quails with a multibasic motif in the hemagglutinin cleavage site.

    PubMed

    Wong, Sook-San; Yoon, Sun-Woo; Zanin, Mark; Song, Min-Suk; Oshansky, Christine; Zaraket, Hassan; Sonnberg, Stephanie; Rubrum, Adam; Seiler, Patrick; Ferguson, Angela; Krauss, Scott; Cardona, Carol; Webby, Richard J; Crossley, Beate

    2014-11-01

    The cleavage motif in the hemagglutinin (HA) protein of highly pathogenic H5 and H7 subtypes of avian influenza viruses is characterized by a peptide insertion or a multibasic cleavage site (MBCS). Here, we isolated an H4N2 virus from quails (Quail/CA12) with two additional arginines in the HA cleavage site, PEKRRTR/G, forming an MBCS-like motif. Quail/CA12 is a reassortant virus with the HA and neuraminidase (NA) gene most similar to a duck-isolated H4N2 virus, PD/CA06 with a monobasic HA cleavage site. Quail/CA12 required exogenous trypsin for efficient growth in culture and caused no clinical illness in infected chickens. Quail/CA12 had high binding preference for α2,6-linked sialic acids and showed higher replication and transmission ability in chickens and quails than PD/CA06. Although the H4N2 virus remained low pathogenic, these data suggests that the acquisition of MBCS in the field is not restricted to H5 or H7 subtypes.

  6. Fusion expression and purification of four disulfide-rich peptides reveals enterokinase secondary cleavage sites in animal toxins.

    PubMed

    Chen, Zongyun; Han, Song; Cao, Zhijian; Wu, Yingliang; Zhuo, Renxi; Li, Wenxin

    2013-01-01

    Animal toxins are powerful tools for testing the pharmacological, physiological, and structural characteristics of ion channels, proteases, and other receptors. However, most animal toxins are disulfide-rich peptides that are difficult to produce functionally. Here, a glutathione S-transferase (GST) fusion expression strategy was used to produce four recombinant animal toxin peptides, ChTX, StKTx23, BmP01, and ImKTx1, with different isoelectric points from 4.7 to 9.2. GST tags were removed by enterokinase, a widely used and effective commercial protease that cleaves after lysine at the cleavage site DDDDK. Using this strategy, two disulfide-rich animal toxins ChTX and StKTx23 were obtained successfully with a yield of approximately 1-2 mg/l culture. Electrophysiological experiments further showed that these two recombinant toxins showed good bioactivities, indicating that our method was effective in producing large amounts of functional disulfide-rich animal toxins. Interestingly, by analyzing the separated fractions of BmP01, StKTx23, and ImKTx1 using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, four new enterokinase secondary cleavage sites were found, consisting of the sequences "WEYR," "EDK," "QNAR," and "DNDK." To our knowledge, this is the first report of the presence of secondary cleavage sites for commercial enterokinase in animal toxins. These findings will help us use commercial enterokinase appropriately as a cleavage tool in the production of animal toxins.

  7. New strategy for specific activation of recombinant microbial pro-transglutaminase by introducing an enterokinase cleavage site.

    PubMed

    Wang, Kun; Wang, Bin; Yang, Hui-Lin; Pan, Li

    2013-03-01

    Recombinant microbial transglutaminase (rMTG) is usually expressed as a soluble zymogen (pro-rMTG) in heterologous expression systems but proteolytic activation of the inactive pro-rMTG is essential. Instead of screening proteases for activating pro-rMTG, we examined an alternative method by introducing a specific cleavage site of enterokinase between the pro-peptide and mature rMTG, generating three pro-rMTG variants (Pro-mrMTG, Pro-m-rMTG and mPro-rMTG). Pro-mrMTG and Pro-m-rMTG were activated by enterokinase without degrading mature rMTG. The activation productivity of Pro-m-rMTG by enterokinase reached 92 % after 22 h activation, while the activation productivity of Pro-rMTG activated by trypsin was 47 %. MALDI-MS analysis revealed that the pro-peptide including the cleavage site was specifically removed from Pro-m-rMTG after activation. This methodology has the potential to be applied in rMTG production by incorporating highly specific cleavage sites of other proteases.

  8. A Motivational Determinant of Facial Emotion Recognition: Regulatory Focus Affects Recognition of Emotions in Faces

    PubMed Central

    Sassenrath, Claudia; Sassenberg, Kai; Ray, Devin G.; Scheiter, Katharina; Jarodzka, Halszka

    2014-01-01

    Two studies examined an unexplored motivational determinant of facial emotion recognition: observer regulatory focus. It was predicted that a promotion focus would enhance facial emotion recognition relative to a prevention focus because the attentional strategies associated with promotion focus enhance performance on well-learned or innate tasks - such as facial emotion recognition. In Study 1, a promotion or a prevention focus was experimentally induced and better facial emotion recognition was observed in a promotion focus compared to a prevention focus. In Study 2, individual differences in chronic regulatory focus were assessed and attention allocation was measured using eye tracking during the facial emotion recognition task. Results indicated that the positive relation between a promotion focus and facial emotion recognition is mediated by shorter fixation duration on the face which reflects a pattern of attention allocation matched to the eager strategy in a promotion focus (i.e., striving to make hits). A prevention focus did not have an impact neither on perceptual processing nor on facial emotion recognition. Taken together, these findings demonstrate important mechanisms and consequences of observer motivational orientation for facial emotion recognition. PMID:25380247

  9. A motivational determinant of facial emotion recognition: regulatory focus affects recognition of emotions in faces.

    PubMed

    Sassenrath, Claudia; Sassenberg, Kai; Ray, Devin G; Scheiter, Katharina; Jarodzka, Halszka

    2014-01-01

    Two studies examined an unexplored motivational determinant of facial emotion recognition: observer regulatory focus. It was predicted that a promotion focus would enhance facial emotion recognition relative to a prevention focus because the attentional strategies associated with promotion focus enhance performance on well-learned or innate tasks - such as facial emotion recognition. In Study 1, a promotion or a prevention focus was experimentally induced and better facial emotion recognition was observed in a promotion focus compared to a prevention focus. In Study 2, individual differences in chronic regulatory focus were assessed and attention allocation was measured using eye tracking during the facial emotion recognition task. Results indicated that the positive relation between a promotion focus and facial emotion recognition is mediated by shorter fixation duration on the face which reflects a pattern of attention allocation matched to the eager strategy in a promotion focus (i.e., striving to make hits). A prevention focus did not have an impact neither on perceptual processing nor on facial emotion recognition. Taken together, these findings demonstrate important mechanisms and consequences of observer motivational orientation for facial emotion recognition.

  10. Characterization of clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses from wild birds possessing atypical hemagglutinin polybasic cleavage sites.

    PubMed

    Usui, Tatsufumi; Soda, Kosuke; Tomioka, Yukiko; Ito, Hiroshi; Yabuta, Toshiyo; Takakuwa, Hiroki; Otsuki, Koichi; Ito, Toshihiro; Yamaguchi, Tsuyoshi

    2017-02-01

    Since 2014, clade 2.3.4.4 H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have been distributed worldwide. These viruses, which were reported to be highly virulent in chickens by intravenous inoculation, have a consensus HPAI motif PLRERRRKR at the HA cleavage site. However, two-clade 2.3.4.4 H5N8 viruses which we isolated from wild migratory birds in late 2014 in Japan possessed atypical HA cleavage sequences. A swan isolate, Tottori/C6, had a novel polybasic cleavage sequence, PLGERRRKR, and another isolate from a dead mandarin duck, Gifu/01, had a heterogeneous mixture of consensus PLRERRRKR and variant PLRERRRRKR sequences. The polybasic HA cleavage site is the prime virulence determinant of AIVs. Therefore, in the present study, we examined the pathogenicity of these H5N8 isolates in chickens by intravenous inoculation. When 10(6) EID50 of these viruses were intravenously inoculated into chickens, the mean death time associated with Tottori/C6 was substantially longer (>6.1 days) than that associated with Gifu/01 (2.5 days). These viruses had comparable abilities to replicate in tissue culture cells in the presence and absence of exogenous trypsin, but the growth of Tottori/C6 was hampered. These results indicate that the novel cleavage motif of Tottori/C6 did not directly affect the infectivity of the virus, but Tottori/C6 caused attenuated pathogenicity in chickens because of hampered replication efficiency. It is important to test for the emergence of diversified HPAIVs, because introduction of HPAIVs with a lower virulence like Tottori/C6 might hinder early detection of affected birds in poultry farms.

  11. A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens

    PubMed Central

    Veits, Jutta; Tauscher, Kerstin; Ziller, Mario; Teifke, Jens P.; Stech, Jürgen; Mettenleiter, Thomas C.

    2015-01-01

    ABSTRACT In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding

  12. DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion

    PubMed Central

    Schwarz, Friedrich W.; van Aelst, Kara; Tóth, Júlia; Seidel, Ralf; Szczelkun, Mark D.

    2011-01-01

    DNA cleavage by the Type III Restriction–Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision. PMID:21724613

  13. Mapping sequence differences between thimet oligopeptidase and neurolysin implicates key residues in substrate recognition.

    PubMed

    Ray, Kallol; Hines, Christina S; Rodgers, David W

    2002-09-01

    The highly homologous endopeptidases thimet oligopeptidase and neurolysin are both restricted to short peptide substrates and share many of the same cleavage sites on bioactive and synthetic peptides. They sometimes target different sites on the same peptide, however, and defining the determinants of differential recognition will help us to understand how both enzymes specifically target a wide variety of cleavage site sequences. We have mapped the positions of the 224 surface residues that differ in sequence between the two enzymes onto the surface of the neurolysin crystal structure. Although the deep active site channel accounts for about one quarter of the total surface area, only 11% of the residue differences map to this region. Four isolated sequence changes (R470/E469, R491/M490, N496/H495, and T499/R498; neurolysin residues given first) are well positioned to affect recognition of substrate peptides, and differences in cleavage site specificity can be largely rationalized on the basis of these changes. We also mapped the positions of three cysteine residues believed to be responsible for multimerization of thimet oligopeptidase, a process that inactivates the enzyme. These residues are clustered on the outside of one channel wall, where multimerization via disulfide formation is unlikely to block the substrate-binding site. Finally, we mapped the regulatory phosphorylation site in thimet oligopeptidase to a location on the outside of the molecule well away from the active site, which indicates this modification has an indirect effect on activity.

  14. The Efficiency of Dentin Sialoprotein-Phosphophoryn Processing Is Affected by Mutations Both Flanking and Distant from the Cleavage Site*

    PubMed Central

    Yang, Robert T.; Lim, Glendale L.; Dong, Zhihong; Lee, Arthur M.; Yee, Colin T.; Fuller, Robert S.; Ritchie, Helena H.

    2013-01-01

    Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G447↓D448 cleavage site in DSP-PP240 had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P4 to P4′ blocked, impaired, or enhanced DSP-PP240 cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP240 had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP240 significantly modified the amount of PP240 product generated from DSP-PP240 precursor protein cleavage suggests that such mutation may affect the mineralization process. PMID:23297400

  15. In-line alignment and Mg2+ coordination at the cleavage site of the env22 twister ribozyme

    PubMed Central

    Ren, Aiming; Košutić, Marija; Rajashankar, Kanagalaghatta R.; Frener, Marina; Santner, Tobias; Westhof, Eric; Micura, Ronald; Patel, Dinshaw J.

    2015-01-01

    Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site-specific cleavage/ligation of phosphodiester backbones. We report on the 2.9-Å crystal structure of the env22 twister ribozyme, which adopts a compact tertiary fold stabilized by co-helical stacking, double-pseudoknot formation and long-range pairing interactions. The U-A cleavage site adopts a splayed-apart conformation with the modeled 2′-O of U positioned for in-line attack on the adjacent to-be-cleaved P-O5′ bond. Both an invariant guanosine and a Mg2+ are directly coordinated to the non-bridging phosphate oxygens at the U-A cleavage step, with the former positioned to contribute to catalysis and the latter to structural integrity. The impact of key mutations on cleavage activity identified an invariant guanosine that contributes to catalysis. Our structure of the in-line aligned env22 twister ribozyme is compared with two recently-reported twister ribozymes structures, which adopt similar global folds, but differ in conformational features around the cleavage site. PMID:25410397

  16. CaspNeuroD: a knowledgebase of predicted caspase cleavage sites in human proteins related to neurodegenerative diseases

    PubMed Central

    Kumar, Sonu; Cieplak, Piotr

    2016-01-01

    Background: A variety of neurodegenerative diseases (NDs) have been associated with deregulated caspase activation that leads to neuronal death. Caspases appear to be involved in the molecular pathology of NDs by directly cleaving important proteins. For instance, several proteins involved in Alzheimer’s disease, including β-amyloid precursor protein (APP) and presenilins, are known to be cleaved by caspases. Therefore, cell death pathway may play a central role in many neurological diseases, and targeting the important proteins that control the cell survival and death may potentially represent a therapeutic approach for chronic neurodegenerative disorders. Findings: We developed CaspNeuroD, a relational database of in silico predicted caspase cleavage sites in human proteins associated with NDs. The prediction has been done on collection of 249 human proteins reported in clinical studies of NDs using the recently published CaspDB Random Forest machine-learning model. This database could be used for identifying new caspase substrates and further our understanding of the caspase-mediated substrate cleavage in NDs. Conclusion: Our database provides information about potential caspase cleavage sites in a verified set of human proteins involved in NDs. It provides also information about the conservation of cleavage positions in corresponding orthologs, and information about the positions of single nucleotide polymorphisms and posttranslational modifications (PTMs) that may modulate the caspase cleavage efficiency. Database URL: caspdb.sanfordburnham.org/caspneurod.php . PMID:28025335

  17. Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide

    PubMed Central

    Quandte, Matthias; Cabartova, Zuzana; Bontjer, Ilja; Källgren, Carolina; Nilsson, IngMarie; Land, Aafke; von Heijne, Gunnar; Sanders, Rogier W

    2017-01-01

    Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV. PMID:28753126

  18. Protection of particular cleavage sites of restriction endonucleases by distamycin A and actinomycin D.

    PubMed Central

    Nosikov, V V; Braga, E A; Karlishev, A V; Zhuze, A L; Polyanovsky, O L

    1976-01-01

    It is shown here that distamycin A and actinomycin D can protect the recognition sites of endo R.EcoRI, EcoRII, HindII, HindIII, HpaI and HpaII from the attack of these restriction endonucleases. At proper distamycin concentrations only two endo R.EcoRI sites of phage lambda DNA are available for the restriction enzyme--sRI1 and sRI4. This phenomenon results in the appearance of larger DNA fragments comprising several consecutive fragments of endo R.EcoRI complete cleavage. The distamycin fragments isolated from the agarose gels can be subsequently cleaved by endo R.EcoRI with the yield of the fragments of complete digestion. We have compared the effect of distamycin A and actinomycin D on a number of restriction endonucleases having different nucleotide sequences in the recognition sites and established that antibiotic action depends on the nucleotide sequences of the recognition sites and their closest environment Images PMID:967694

  19. Recombinant Sendai viruses expressing fusion proteins with two furin cleavage sites mimic the syncytial and receptor-independent infection properties of respiratory syncytial virus.

    PubMed

    Rawling, Joanna; Cano, Olga; Garcin, Dominique; Kolakofsky, Daniel; Melero, José A

    2011-03-01

    Cell entry by paramyxoviruses requires fusion between viral and cellular membranes. Paramyxovirus infection also gives rise to the formation of multinuclear, fused cells (syncytia). Both types of fusion are mediated by the viral fusion (F) protein, which requires proteolytic processing at a basic cleavage site in order to be active for fusion. In common with most paramyxoviruses, fusion mediated by Sendai virus F protein (F(SeV)) requires coexpression of the homologous attachment (hemagglutinin-neuraminidase [HN]) protein, which binds to cell surface sialic acid receptors. In contrast, respiratory syncytial virus fusion protein (F(RSV)) is capable of fusing membranes in the absence of the viral attachment (G) protein. Moreover, F(RSV) is unique among paramyxovirus fusion proteins since F(RSV) possesses two multibasic cleavage sites, which are separated by an intervening region of 27 amino acids. We have previously shown that insertion of both F(RSV) cleavage sites in F(SeV) decreases dependency on the HN attachment protein for syncytium formation in transfected cells. We now describe recombinant Sendai viruses (rSeV) that express mutant F proteins containing one or both F(RSV) cleavage sites. All cleavage-site mutant viruses displayed reduced thermostability, with double-cleavage-site mutants exhibiting a hyperfusogenic phenotype in infected cells. Furthermore, insertion of both F(RSV) cleavage sites in F(SeV) reduced dependency on the interaction of HN with sialic acid for infection, thus mimicking the unique ability of RSV to fuse and infect cells in the absence of a separate attachment protein.

  20. The P4-P2′ Amino Acids Surrounding Human Norovirus Polyprotein Cleavage Sites Define the Core Sequence Regulating Self-Processing Order

    PubMed Central

    May, Jared; Viswanathan, Prasanth; Ng, Kenneth K.-S.; Medvedev, Alexei

    2014-01-01

    ABSTRACT Noroviruses (NoV) are members of the family Caliciviridae. The human NoV open reading frame 1 (ORF1) encodes a 200-kDa polyprotein which is cleaved by the viral 20-kDa 3C-like protease (Pro, NS6) into 6 nonstructural proteins that are necessary for viral replication. The NoV ORF1 polyprotein is processed in a specific order, with “early” sites (NS1/2-3 and NS3-4) being cleaved rapidly and three “late” sites (NS4-5, NS5-6, and NS6-7) processed subsequently and less efficiently. Previously, we demonstrated that the NoV polyprotein processing order is directly correlated with the efficiency of the enzyme, which is regulated by the primary amino acid sequences surrounding ORF1 cleavage sites. Using fluorescence resonance energy transfer (FRET) peptides representing the NS2-3 and NS6-7 ORF1 cleavage sites, we now demonstrate that the amino acids spanning positions P4 to P2′ (P4-P2′) surrounding each site comprise the core sequence controlling NoV protease enzyme efficiency. Furthermore, the NoV polyprotein self-processing order can be altered by interchanging this core sequence between NS2-3 and any of the three late sites in in vitro transcription-translation assays. We also demonstrate that the nature of the side chain at the P3 position for the NS1/2-3 (Nterm/NTPase) site confers significant influence on enzyme catalysis (kcat and kcat/Km), a feature overlooked in previous structural studies. Molecular modeling provides possible explanations for the P3 interactions with NoV protease. IMPORTANCE Noroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on health care systems. Proteolytic processing of the viral nonstructural polyprotein is required for norovirus replication. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full

  1. Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.

    PubMed Central

    Kufel, J; Kirsebom, L A

    1996-01-01

    We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process. Images Fig. 2 Fig. 3 Fig. 4 PMID:8650223

  2. Drug-resistant HIV-1 protease regains functional dynamics through cleavage site coevolution.

    PubMed

    Özer, Nevra; Özen, Ayşegül; Schiffer, Celia A; Haliloğlu, Türkan

    2015-02-01

    Drug resistance is caused by mutations that change the balance of recognition favoring substrate cleavage over inhibitor binding. Here, a structural dynamics perspective of the regained wild-type functioning in mutant HIV-1 proteases with coevolution of the natural substrates is provided. The collective dynamics of mutant structures of the protease bound to p1-p6 and NC-p1 substrates are assessed using the Anisotropic Network Model (ANM). The drug-induced protease mutations perturb the mechanistically crucial hinge axes that involve key sites for substrate binding and dimerization and mainly coordinate the intrinsic dynamics. Yet with substrate coevolution, while the wild-type dynamic behavior is restored in both p1-p6 ((LP) (1'F)p1-p6D30N/N88D) and NC-p1 ((AP) (2) (V)NC-p1V82A) bound proteases, the dynamic behavior of the NC-p1 bound protease variants (NC-p1V82A and (AP) (2) (V)NC-p1V82A) rather resemble those of the proteases bound to the other substrates, which is consistent with experimental studies. The orientational variations of residue fluctuations along the hinge axes in mutant structures justify the existence of coevolution in p1-p6 and NC-p1 substrates, that is, the dynamic behavior of hinge residues should contribute to the interdependent nature of substrate recognition. Overall, this study aids in the understanding of the structural dynamics basis of drug resistance and evolutionary optimization in the HIV-1 protease system.

  3. The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing*

    PubMed Central

    Textoris-Taube, Kathrin; Keller, Christin; Liepe, Juliane; Henklein, Petra; Sidney, John; Sette, Alessandro; Kloetzel, Peter M.; Mishto, Michele

    2015-01-01

    MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209–217tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201–230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209–217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8+ T cell stimulation in vitro similar to the wtgp100209–217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8+ T cell response also towards N-terminally extended versions of the minimal epitope. PMID:26507656

  4. Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants▿

    PubMed Central

    Oh, Hyung Suk; Pathak, Harsh B.; Goodfellow, Ian G.; Arnold, Jamie J.; Cameron, Craig E.

    2009-01-01

    A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes. PMID:19587035

  5. Cleavage site and Ectodomain of HA2 sub-unit sequence of three equine influenza virus isolated in Morocco.

    PubMed

    Boukharta, Mohamed; Zakham, Fathiah; Touil, Nadia; Elharrak, Mehdi; Ennaji, Moulay Mustapha

    2014-07-12

    The equine influenza (EI) is an infectious and contagious disease of the upper respiratory tract of horses. Two outbreaks were notified in Morocco during 1997 and 2004 respectively in Nador and Essaouira. The aims of the present study concern the amino acids sequences comparison with reference strain A/equine/Miami/1963(H3N8) of the HA2 subunit including the cleavage site of three equine influenza viruses (H3N8) isolated in Morocco: A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004 (H3N8) and A/equine/Essaouira/3/2004 (H3N8). The obtained results demonstrated that the substitutions were located at Ectodomain (ED) and transmembrane domain (TD), and they have only one arginine in cleavage site (HA1-PEKQI-R329-GI-HA2). In the Ectodomain, the mutation N/1542/T deleted the NGT glycosylation site at position 154 for both strains A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8). Except for mutation D/1602/Y of the A/equine/Nador/1/1997(H3N8) strain, the other mutations were involved in non conserved sites. While the transmembrane domain (TM) of the strain A/equine/Essaouira/3/2004(H3N8) exhibits a substitution at residue C/1992/F. For the A/equine/Nador/1/1997(H3N8) strain the HA2 shows a mutation at residue M/2072/L. Three Moroccan strains reveals a common substitution at the residue E/2112/Q located between transmembrane domain TM and the cytoplasmic domain (CD). The given nature virulence of three Moroccan strains, the identified and reported mutations certainly played a permissive role of infection viral process.

  6. SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula

    PubMed Central

    Wong, Emily S. W.; Hardy, Margaret C.; Wood, David; Bailey, Timothy; King, Glenn F.

    2013-01-01

    Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree) and developed an algorithm (SpiderP) for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM) framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor) from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP) is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html), a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from the Spider

  7. SVM-based prediction of propeptide cleavage sites in spider toxins identifies toxin innovation in an Australian tarantula.

    PubMed

    Wong, Emily S W; Hardy, Margaret C; Wood, David; Bailey, Timothy; King, Glenn F

    2013-01-01

    Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree) and developed an algorithm (SpiderP) for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM) framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor) from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP) is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html), a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from the Spider

  8. The mechanism behind the selection of two different cleavage sites in NAG-NAM polymers

    PubMed Central

    Mihelič, Marko; Vlahoviček-Kahlina, Kristina; Renko, Miha; Mesnage, Stephane; Doberšek, Andreja; Taler-Verčič, Ajda; Jakas, Andreja; Turk, Dušan

    2017-01-01

    Peptidoglycan is a giant molecule that forms the cell wall that surrounds bacterial cells. It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues connected by β-(1,4)-glycosidic bonds and cross-linked with short polypeptide chains. Owing to the increasing antibiotic resistance against drugs targeting peptidoglycan synthesis, studies of enzymes involved in the degradation of peptidoglycan, such as N-acetylglucos­aminidases, may expose new, valuable drug targets. The scientific challenge addressed here is how lysozymes, muramidases which are likely to be the most studied enzymes ever, and bacterial N-acetylglucosaminidases discriminate between two glycosidic bonds that are different in sequence yet chemically equivalent in the same NAG-NAM polymers. In spite of more than fifty years of structural studies of lysozyme, it is still not known how the enzyme selects the bond to be cleaved. Using macromolecular crystallography, chemical synthesis and molecular modelling, this study explains how these two groups of enzymes based on an equivalent structural core exhibit a difference in selectivity. The crystal structures of Staphylococcus aureus N-acetylglucosaminidase autolysin E (AtlE) alone and in complex with fragments of peptidoglycan revealed that N-acetylglucosaminidases and muramidases approach the substrate at alternate glycosidic bond positions from opposite sides. The recognition pocket for NAM residues in the active site of N-acetylglucosaminidases may make them a suitable drug target. PMID:28250957

  9. The mechanism behind the selection of two different cleavage sites in NAG-NAM polymers.

    PubMed

    Mihelič, Marko; Vlahoviček-Kahlina, Kristina; Renko, Miha; Mesnage, Stephane; Doberšek, Andreja; Taler-Verčič, Ajda; Jakas, Andreja; Turk, Dušan

    2017-03-01

    Peptidoglycan is a giant molecule that forms the cell wall that surrounds bacterial cells. It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues connected by β-(1,4)-glycosidic bonds and cross-linked with short polypeptide chains. Owing to the increasing antibiotic resistance against drugs targeting peptidoglycan synthesis, studies of enzymes involved in the degradation of peptidoglycan, such as N-acetylglucos-aminidases, may expose new, valuable drug targets. The scientific challenge addressed here is how lysozymes, muramidases which are likely to be the most studied enzymes ever, and bacterial N-acetylglucosaminidases discriminate between two glycosidic bonds that are different in sequence yet chemically equivalent in the same NAG-NAM polymers. In spite of more than fifty years of structural studies of lysozyme, it is still not known how the enzyme selects the bond to be cleaved. Using macromolecular crystallography, chemical synthesis and molecular modelling, this study explains how these two groups of enzymes based on an equivalent structural core exhibit a difference in selectivity. The crystal structures of Staphylococcus aureusN-acetylglucosaminidase autolysin E (AtlE) alone and in complex with fragments of peptidoglycan revealed that N-acetylglucosaminidases and muramidases approach the substrate at alternate glycosidic bond positions from opposite sides. The recognition pocket for NAM residues in the active site of N-acetylglucosaminidases may make them a suitable drug target.

  10. Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera).

    PubMed

    Snell, Terry W; Shearer, Tonya L; Smith, Hilary A; Kubanek, Julia; Gribble, Kristin E; Welch, David B Mark

    2009-09-09

    Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the

  11. Determination of candidate subjects for better recognition of faces

    NASA Astrophysics Data System (ADS)

    Wang, Xuansheng; Chen, Zhen; Teng, Zhongming

    2016-05-01

    In order to improve the accuracy of face recognition and to solve the problem of various poses, we present an improved collaborative representation classification (CRC) algorithm using original training samples and the corresponding mirror images. First, the mirror images are generated from the original training samples. Second, both original training samples and their mirror images are simultaneously used to represent the test sample via improved collaborative representation. Then, some classes which are "close" to the test sample are coarsely selected as candidate classes. At last, the candidate classes are used to represent the test sample again, and then the class most similar to the test sample can be determined finely. The experimental results show our proposed algorithm has more robustness than the original CRC algorithm and can effectively improve the accuracy of face recognition.

  12. A star pattern recognition algorithm for autonomous attitude determination

    NASA Technical Reports Server (NTRS)

    Van Bezooijen, R. W. H.

    1990-01-01

    The star-pattern recognition algorithm presented allows the advanced Full-sky Autonomous Star Tracker (FAST) device, such as the projected ASTROS II system of the Mariner Mark II planetary spacecraft, to reliably ascertain attitude about all three axes. An ASTROS II-based FAST, possessing an 11.5 x 11.5 deg field of view and 8-arcsec accuracy, can when integrated with an all-sky data base of 4100 guide stars determine its attitude in about 1 sec, with a success rate close to 100 percent. The present recognition algorithm can also be used for automating the acquisition of celestial targets by astronomy telescopes, autonomously updating the attitude of gyro-based attitude control systems, and automating ground-based attitude reconstruction.

  13. Preparation of recombinant thioredoxin fused N-terminal proCNP: Analysis of enterokinase cleavage products reveals new enterokinase cleavage sites.

    PubMed

    Liew, Oi Wah; Ching Chong, Jenny Pek; Yandle, Tim G; Brennan, Stephen O

    2005-06-01

    C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58% of total bacterial proteins. Greater than 90% of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-MS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited

  14. His-oriented peptide hydrolysis promoted by cis-[Pt(en)(H2O)2]2+: a new specific peptide cleavage site.

    PubMed

    Hong, Jin; Jiao, Yang; He, Weijiang; Guo, Zijian; Yu, Zhen; Zhang, Junfeng; Zhu, Longgen

    2010-09-06

    The new specific hydrolysis of histidine-containing peptides promoted by cis-[Pt(en)(H(2)O)(2)](2+) was investigated by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectrometry (NMR). MS determination demonstrated that cis-[Pt(en)(H(2)O)(2)](2+) anchors to AcGHG with the stoichiometry of either 1:1 or 2:1 (Pt/peptide), but only with 1:1 stoichoimetry to AcGHL. Cis-[Pt(en)(H(2)O)(2)](2+) is able to promote the cleavage of the first downstream peptide bond from histidine at 60 degrees C and pH 2.65, and Pt-anchored peptides are the essential intermediates for the promoted hydrolysis. Moreover, the larger amount of Pt(II) complex results in higher fragmental yield and higher hydrolysis rate. In the presence of 1 equiv of Pt(II) complex, (1)H NMR determination confirmed the apparent first-order kinetics of the Pt(II)-promoted hydrolysis and the hydrolysis rate for AcGHG and AcGHL is 0.20 day(-1) and 0.14 day(-1), respectively. Moreover, Pt(II) coordinating to histidine imidazole is the key step to form the Pt(II)-anchored peptides. The Pt(II)-activating the first His-downstream carbonyl group via synergic coordinating to His imidazole and carbonyl O atom has been proposed for the Pt(II)-promoted his-oriented peptide hydrolysis. The lower rate for AcGHL should be correlated to the steric hindrance of Leu side chain to the second Pt(II) coordinating to tripeptide. In addition, the newly confirmed specific His-oriented peptide cleavage site implies a new potential strategy for target cleavage of peptides or proteins.

  15. Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition

    PubMed Central

    Velmurugan, Punitha; Jonnalagadda, Raghava Rao; Unni Nair, Balachandran

    2015-01-01

    Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins. PMID:25973613

  16. A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

    PubMed Central

    Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

  17. Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA

    NASA Astrophysics Data System (ADS)

    Schmidt, Thomas P.; Perna, Anna M.; Fugmann, Tim; Böhm, Manja; Jan Hiss; Haller, Sarah; Götz, Camilla; Tegtmeyer, Nicole; Hoy, Benjamin; Rau, Tilman T.; Neri, Dario; Backert, Steffen; Schneider, Gisbert; Wessler, Silja

    2016-03-01

    The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.

  18. Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

    PubMed Central

    Hou, Yong; Li, Jianwei; Li, Yi; Dong, Zhaoming; Xia, Qingyou; Yuan, Y Adam

    2014-01-01

    In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level. PMID:24639361

  19. The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

    PubMed Central

    2011-01-01

    Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors. PMID:22151792

  20. Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition.

    PubMed

    Velmurugan, Punitha; Jonnalagadda, Raghava Rao; Nair, Balachandran Unni

    2015-01-01

    Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

  1. Modulation in kinetics of lactone ring hydrolysis of camptothecins upon interaction with topoisomerase I cleavage sites on DNA.

    PubMed

    Chourpa, I; Riou, J F; Millot, J M; Pommier, Y; Manfait, M

    1998-05-19

    The kinetics of hydrolysis of the alpha-hydroxylactone ring of anticancer agents belonging to the camptothecin (CPT) series has been followed using their fluorescence emission. Data obtained for CPT, CPT-11, and SN-38, either in their free form or in the presence of DNA and/or topoisomerase I (top1), have been compared. DNA was modeled using three types of double-strand oligonucleotides corresponding to top1 cleavage site enhanced in the presence of the drug (olg1), top1 site independent of CPT (olg2), and nonspecific synthetic oligonucleotide containing only AT and no GC base pairs (olg3). Cleavage assays indicated the absence of top1-mediated cleavage on olg3, both in the presence and in the absence of CPT. The kinetics data also showed ratio-dependent stabilization of the lactone forms of CPTs when in the presence of an excess of olg1 or olg2, but not of olg3. These observations correlate with the previously reported preferential binding of CPTs to guanines. Although lactone hydrolysis was not perturbed by top1 alone, this enzyme hindered lactone stabilization by specific oligonucleotides. After addition of top1 to CPT-olg1 or CPT-olg2 complexes, the lactone ring of the drug was destabilized. No lactone stabilization was observed when olg1 was added to CPT-top1 complexes or when olg1-top1 complexes were added to CPT.

  2. Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera)

    PubMed Central

    Snell, Terry W; Shearer, Tonya L; Smith, Hilary A; Kubanek, Julia; Gribble, Kristin E; Welch, David B Mark

    2009-01-01

    Background Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. Results A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions

  3. Sequence diversity and associated pathogenicity of the hemagglutinin cleavage site of H5N2 avian influenza viruses isolated from chickens in Taiwan during 2013–2015

    PubMed Central

    LI, Kuang-Po; CHANG, Poa-Chun; CHENG, Ming-Chu; TAN, Duen-Huey; CHEN, Li-Hsuan; LIU, Yu-Pin; LIN, Yu-Ju; TSAI, Hsiang-Jung; SHIEN, Jui-Hung

    2016-01-01

    The sequence at the hemagglutinin (HA) cleavage site (CS) plays a key role in determining the pathogenicity of avian influenza viruses. Three types of HA CS sequences, QREKR/GL, QRKKR/GL and QRRKR/GL, were previously reported in Taiwanese H5N2 viruses that were isolated from chickens from 2003 to 2013. However, no HA CS sequence was reported for viruses isolated after 2013. This article presents the HA CS sequences and pathogenicity of H5N2 viruses that were isolated from chickens in Taiwan during 2013–2015. Two novel HA CS sequences, QKEKR/GL and KREKREKR/GL, were found in the viruses isolated in 2013 and 2014, and pathogenicity tests showed that the viruses with these novel HA CS sequences are low and high pathogenic viruses, respectively. In contrast, the HA CS sequence QREKR/GL was found in all viruses that were isolated in 2015, and all of these viruses were low pathogenic viruses. After 10 passages in embryonated chicken eggs, a virus strain that was isolated in 2003 evolved into a viral quasispecies that contained at least four distinct types of HA CS sequences. These results highlight the potential of Taiwanese H5N2 viruses to change their pathogenicity and HA CS sequences via mutations. Furthermore, viruses with the HA CS sequence QREKR/GL were more prevalent than others in 2015. These findings are useful for understanding the mechanism of sequence changes at the HA CS and for refining H5N2 virus control measures in Taiwan. PMID:27725416

  4. Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

    PubMed Central

    Packman, L C; Perham, R N

    1987-01-01

    The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented. Images Fig. 1. Fig. 2. PMID:3297046

  5. Structural determinants for recognition and translocation by the anandamide transporter

    PubMed Central

    Piomelli, D.; Beltramo, M.; Glasnapp, S.; Lin, S. Y.; Goutopoulos, A.; Xie, Xiang-Qun; Makriyannis, A.

    1999-01-01

    The biological actions of anandamide (arachidonylethanolamide), an endogenous cannabinoid lipid, are terminated by a two-step inactivation process consisting of carrier-mediated uptake and intracellular hydrolysis. Anandamide uptake in neurons and astrocytes is mediated by a high-affinity, Na+-independent transporter that is selectively inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). In the present study, we examined the structural determinants governing recognition and translocation of substrates by the anandamide transporter constitutively expressed in a human astrocytoma cell line. Competition experiments with a select group of analogs suggest that substrate recognition by the transporter is favored by a polar nonionizable head group of defined stereochemical configuration containing a hydroxyl moiety at its distal end. The secondary carboxamide group interacts favorably with the transporter, but may be replaced with either a tertiary amide or an ester, suggesting that it may serve as hydrogen acceptor. Thus, 2-arachidonylglycerol, a putative endogenous cannabinoid ester, also may serve as a substrate for the transporter. Substrate recognition requires the presence of at least one cis double bond situated at the middle of the fatty acid carbon chain, indicating a preference for ligands whose hydrophobic tail can adopt a bent U-shaped conformation. On the other hand, uptake experiments with radioactively labeled substrates show that no fewer than four cis nonconjugated double bonds are required for optimal translocation across the cell membrane, suggesting that substrates are transported in a folded hairpin conformation. These results outline the general structural requisites for anandamide transport and may assist in the development of selective inhibitors with potential clinical applications. PMID:10318965

  6. Perceptual Organization as a Determinant of Visual Recognition Memory

    ERIC Educational Resources Information Center

    Wiseman, Sandor; Neisser, Ulric

    1974-01-01

    Ambiguous pictures that could be seen as faces or as meaningless patterns were the stimuli in two recognition-memory experiments. Recognition was far more accurate when the stimuli were seen as faces. (Editor)

  7. DNA bending is a determinant of calicheamicin target recognition.

    PubMed

    Salzberg, A A; Dedon, P C

    2000-06-27

    Calicheamicin is a hydrophobic enediyne antibiotic that binds noncovalently to DNA and causes sequence-selective oxidation of deoxyribose. While the drug makes several base contacts along the minor groove, the diversity of binding-site sequences and the effects of DNA conformation on calicheamicin-induced DNA cleavage suggest that sequence recognition per se is not the primary determinant of target selection. We now present evidence that calicheamicin bends its DNA targets. Using a gel mobility assay, we observed that polymers of oligonucleotide constructs containing AGGA and ACAA binding sites for calicheamicin did not possess intrinsic curvature. Binding of calicheamicin epsilon, the aromatized form of the parent calicheamicin gamma(1)(I), to oligonucleotide constructs containing binding sites in phase with the helical repeat caused a shift to smaller circle sizes in T4 ligase-mediated circle formation assays, with a much smaller shift observed with constructs containing out-of-phase binding sites. It was also observed that binding of calicheamicin epsilon to a 273 bp construct with phased binding sites caused an increase in the molar cyclization factor, J, from 8 x 10(-8) to 9 x 10(-6) M. These results are consistent with DNA bending as part of an induced-fit mechanism of DNA target recognition and with the hypothesis that the preferred targets of calicheamicin, the 3' ends of oligopurine tracts, are characterized by unique conformational properties.

  8. Linear models of ovine IgG1 and IgG2 subclasses and predicted pepsin cleavage sites.

    PubMed

    Jones, Russell G A; Martino, Angela

    2016-01-05

    Highly purified specific Fab antibody fragments derived from sheep have a long history of therapeutic use as safe and effective emergency medicines. In more recent years simple low-cost methods have been developed, which take advantage of the ability of pepsin under optimally controlled conditions to preferentially digest ovine IgG within the Fc region to produce F(ab')2 and easy to remove low MW Fc sub-fragments. Despite these developments no information is currently available on the pepsin digestion of ovine IgG at the amino acid level hindering the development of improved F(ab')2 processing methods. To gain knowledge of the fragments properties we have constructed linear models of ovine IgG1 and IgG2 subclasses, starting from the gamma-1 and gamma-2 chain amino acid sequences, which also incorporate the inter- and intra-chain disulphide bonds. Any potential pepsin cleavage site was initially predicted in silico, then high probability points identified for each of the molecules and mapped onto the individual models. A theoretical order of digestion was subsequently constructed, which appeared to agree with the experimental data, suggesting an accurate prediction model for ovine IgG1 and IgG2 subclasses. These findings lay the foundations for a more detailed analysis of pepsin cleavage fragments in the future. Additionally, the F(ab')2 generated following pepsin digestion were predicted to contain subclass unique C-terminal octapeptide neoepitopes, despite the high 89% sequence identity of the intact gamma-1 and gamma-2 chain constant regions. These neoepitopes have the potential to be utilised for identification purposes once confirmed experimentally.

  9. A catalytic metal ion interacts with the cleavage site G•U wobble in the HDV ribozyme†

    PubMed Central

    Chen, Jui-Hui; Gong, Bo; Bevilacqua, Philip C.; Carey, Paul R.; Golden, Barbara L.

    2009-01-01

    The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate a 2′,3′-cyclic phosphate and a 5′-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted > 2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335–13342.], while in crystal structures, it is well-positioned for proton transfer. However no crystallographic evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on rate and Mg2+-binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously-unobserved hydrated magnesium ion interacts with the N7 of the cleavage site G•U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data. PMID:19178151

  10. Peptidase specificity from the substrate cleavage collection in the MEROPS database and a tool to measure cleavage site conservation

    PubMed Central

    Rawlings, Neil D.

    2016-01-01

    One peptidase can usually be distinguished from another biochemically by its action on proteins, peptides and synthetic substrates. Since 1996, the MEROPS database (http://merops.sanger.ac.uk) has accumulated a collection of cleavages in substrates that now amounts to 66,615 cleavages. The total number of peptidases for which at least one cleavage is known is 1700 out of a total of 2457 different peptidases. This paper describes how the cleavages are obtained from the scientific literature, how they are annotated and how cleavages in peptides and proteins are cross-referenced to entries in the UniProt protein sequence database. The specificity profiles of 556 peptidases are shown for which ten or more substrate cleavages are known. However, it has been proposed that at least 40 cleavages in disparate proteins are required for specificity analysis to be meaningful, and only 163 peptidases (6.6%) fulfil this criterion. Also described are the various displays shown on the website to aid with the understanding of peptidase specificity, which are derived from the substrate cleavage collection. These displays include a logo, distribution matrix, and tables to summarize which amino acids or groups of amino acids are acceptable (or not acceptable) in each substrate binding pocket. For each protein substrate, there is a display to show how it is processed and degraded. Also described are tools on the website to help with the assessment of the physiological relevance of cleavages in a substrate. These tools rely on the hypothesis that a cleavage site that is conserved in orthologues is likely to be physiologically relevant, and alignments of substrate protein sequences are made utilizing the UniRef50 database, in which in each entry sequences are 50% or more identical. Conservation in this case means substitutions are permitted only if the amino acid is known to occupy the same substrate binding pocket from at least one other substrate cleaved by the same peptidase. PMID

  11. GFP is Efficiently Expressed by Wheat Streak Mosaic Virus Using a Range of Tritimovirus NIa Cleavage Sites and Forms Dense Aggregates in Cereal Hosts

    USDA-ARS?s Scientific Manuscript database

    Wheat streak mosaic virus (WSMV)-based transient expression vector was developed to express GFP as a marker protein. The GFP cistron was engineered between the P1 and HC-Pro cistrons in an infectious cDNA clone of WSMV. The cleavage sites, P3/6KI, 6KI/CI, NIa/NIb, or NIb/CP, from WSMV were fused to ...

  12. Effects of Recognition on Subsequent Recall: Comments on "Determinants of Recognition and Recall: Accessibility and Generation"

    ERIC Educational Resources Information Center

    Broadbent, Donald E.; Broadbent, Margaret H. P.

    1977-01-01

    Attempts have been made by Rabinowitz, Mandler, and Patterson (AA 527 084) to show that both recall and recognition involve the accessibility of individual words. Their recall tests preceded recognition tests, or vice versa, thus contaminating each other; a fresh experiment is presented to confirm that this is so. (Editor)

  13. Effects of Recognition on Subsequent Recall: Comments on "Determinants of Recognition and Recall: Accessibility and Generation"

    ERIC Educational Resources Information Center

    Broadbent, Donald E.; Broadbent, Margaret H. P.

    1977-01-01

    Attempts have been made by Rabinowitz, Mandler, and Patterson (AA 527 084) to show that both recall and recognition involve the accessibility of individual words. Their recall tests preceded recognition tests, or vice versa, thus contaminating each other; a fresh experiment is presented to confirm that this is so. (Editor)

  14. New Findings in Cleavage Sites Variability across Groups, Subtypes and Recombinants of Human Immunodeficiency Virus Type 1

    PubMed Central

    Torrecilla, Esther; Llácer Delicado, Teresa; Holguín, África

    2014-01-01

    Background Polymorphisms at cleavage sites (CS) can influence Gag and Pol proteins processing by the viral protease (PR), restore viral fitness and influence the virological outcome of specific antiretroviral drugs. However, data of HIV-1 variant-associated CS variability is scarce. Methods In this descriptive research, we examine the effect of HIV-1 variants on CS conservation using all 9,028 gag and 3,906 pol HIV-1 sequences deposited in GenBank, focusing on the 110 residues (10 per site) involved at 11 CS: P17/P24, P24/P2, P2/P7, P7/P1, P1/P6gag, NC/TFP, TFP/P6pol, P6pol/PR, PR/RTp51, RTp51/RTp66 and RTp66/IN. CS consensus amino acid sequences across HIV-1 groups (M, O, N, P), group M 9 subtypes and 51 circulating recombinant forms (CRF) were inferred from our alignments and compared to the HIV-1 consensus-of-consensuses sequence provided by GenBank. Results In all HIV-1 variants, the most conserved CS were PR/RTp51, RTp51/RTp66, P24/P2 and RTp66/IN and the least P2/P7 and P6pol/PR. Conservation was significantly lower in subtypes vs. recombinants in P2/P7 and TFP/P6pol and higher in P17/P24. We found a significantly higher conservation rate among Group M vs. non-M Groups HIV-1. The late processing sites at Gag (P7/P1) and GagPol precursors (PR/RTp51) presented a significantly higher conservation vs. the first CS (P2/P7) in the 4 HIV-1 groups. Here we show 52 highly conserved residues across HIV-1 variants in 11 CS and the amino acid consensus sequence in each HIV-1 group and HIV-1 group M variant for each 11 CS. Conclusions This is the first study to describe the CS conservation level across all HIV-1 variants and 11 sites in one of the largest available sequence HIV-1 dataset. These results could help other researchers for the future design of both novel antiretroviral agents acting as maturation inhibitors as well as for vaccine targeting CS. PMID:24516589

  15. RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA

    DOE PAGES

    Gordon, Gina C.; Cameron, Jeffrey C.; Pfleger, Brian F.

    2017-03-28

    needed to make predictions of transcript half-life and to design synthetic transcripts with optimal stability. RNase III does not have a conserved target sequence but instead recognizes RNA secondary structure. Prior to this study, only a few RNase III target sites in E. coli were known, so we used RNA sequencing to provide a more comprehensive list of cleavage sites and to examine the impact of RNase III on transcript degradation. Finally, with this added information on how RNase III participates in transcript regulation and recycling, a more complete picture of RNA turnover can be developed for E. coli. Similar approaches could be used to augment our understanding of RNA turnover in other bacteria.« less

  16. RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA.

    PubMed

    Gordon, Gina C; Cameron, Jeffrey C; Pfleger, Brian F

    2017-03-28

    predictions of transcript half-life and to design synthetic transcripts with optimal stability. RNase III does not have a conserved target sequence but instead recognizes RNA secondary structure. Prior to this study, only a few RNase III target sites in E. coli were known, so we used RNA sequencing to provide a more comprehensive list of cleavage sites and to examine the impact of RNase III on transcript degradation. With this added information on how RNase III participates in transcript regulation and recycling, a more complete picture of RNA turnover can be developed for E. coli Similar approaches could be used to augment our understanding of RNA turnover in other bacteria.

  17. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

    PubMed

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-02

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  18. Residues in Conserved Loops of Intramembrane Metalloprotease SpoIVFB Interact with Residues near the Cleavage Site in Pro-σK

    PubMed Central

    Zhang, Yang; Luethy, Paul M.

    2013-01-01

    Intramembrane metalloproteases (IMMPs) control critical biological processes by cleaving membrane-associated proteins within a transmembrane segment or at a site near the membrane surface. Phylogenetic analysis divides IMMPs into four groups. SpoIVFB is a group III IMMP that regulates Bacillus subtilis endospore formation by cleaving Pro-σK and releasing the active sigma factor from a membrane. To elucidate the enzyme-substrate interaction, single-cysteine versions of catalytically inactive SpoIVFB and C-terminally truncated Pro-σK(1-126) (which can be cleaved by active SpoIVFB) were coexpressed in Escherichia coli, and proximity was tested by disulfide cross-linking in vivo. As expected, the results provided evidence that catalytic residue Glu-44 of SpoIVFB is near the cleavage site in the substrate. Also near the cleavage site were two residues of SpoIVFB in predicted conserved loops; Pro-135 in a short loop and Val-70 in a longer loop. Pro-135 corresponds to Pro-399 of RseP, a group I IMMP, and Pro-399 was reported previously to interact with substrate near the cleavage site, suggesting a conserved interaction across IMMP subfamilies. Val-70 follows a newly recognized conserved motif, PXGG (X is a large hydrophobic residue), which is in a hydrophobic region predicted to be a membrane reentrant loop. Following the hydrophobic region is a negatively charged region that is conserved in IMMPs of groups I and III. At least two residues with a negatively charged side chain are required in this region for activity of SpoIVFB. The region exhibits other features in IMMPs of groups II and IV. Its possible roles, as well as that of the short loop, are discussed. New insights into IMMP-substrate interaction build toward understanding how IMMPs function and may facilitate manipulation of their activity. PMID:23995631

  19. Optimization of a multi-stage, multi-subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites.

    PubMed

    Spiegel, Holger; Schinkel, Helga; Kastilan, Robin; Dahm, Pia; Boes, Alexander; Scheuermayer, Matthias; Chudobová, Ivana; Maskus, Dominika; Fendel, Rolf; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-04-01

    We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.

  20. Visual Flight Feedback as a Determiner of Motor Response Recognition

    ERIC Educational Resources Information Center

    Newell, K. M.

    1975-01-01

    In this study, over a series of learning trials of projecting a ball a criterion distance, the hypotheses were tested and confirmed that withdrawal of visual feedback of the flight of the ball would produce a decrement in response recognition but not recall. (JS)

  1. Visual Flight Feedback as a Determiner of Motor Response Recognition

    ERIC Educational Resources Information Center

    Newell, K. M.

    1975-01-01

    In this study, over a series of learning trials of projecting a ball a criterion distance, the hypotheses were tested and confirmed that withdrawal of visual feedback of the flight of the ball would produce a decrement in response recognition but not recall. (JS)

  2. sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites.

    PubMed

    Xie, Shengsong; Shen, Bin; Zhang, Chaobao; Huang, Xingxu; Zhang, Yonglian

    2014-01-01

    Although the CRISPR/Cas9/sgRNA system efficiently cleaves intracellular DNA at desired target sites, major concerns remain on potential "off-target" cleavage that may occur throughout the whole genome. In order to improve CRISPR-Cas9 specificity for targeted genome editing and transcriptional control, we describe a bioinformatics tool "sgRNAcas9", which is a software package developed for fast design of CRISPR sgRNA with minimized off-target effects. This package consists of programs to perform a search for CRISPR target sites (protospacers) with user-defined parameters, predict genome-wide Cas9 potential off-target cleavage sites (POT), classify the POT into three categories, batch-design oligonucleotides for constructing 20-nt (nucleotides) or truncated sgRNA expression vectors, extract desired length nucleotide sequences flanking the on- or off-target cleavage sites for designing PCR primer pairs to validate the mutations by T7E1 cleavage assay. Importantly, by identifying potential off-target sites in silico, the sgRNAcas9 allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of sgRNA for genome editing applications. sgRNAcas9 software package is publicly available at BiooTools website (www.biootools.com) under the terms of the GNU General Public License.

  3. Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX

    PubMed Central

    Gaglia, Marta Maria; Rycroft, Chris H.; Glaunsinger, Britt A.

    2015-01-01

    Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity. PMID:26646420

  4. Endolysosomal Degradation of Allergenic Ole e 1-Like Proteins: Analysis of Proteolytic Cleavage Sites Revealing T Cell Epitope-Containing Peptides.

    PubMed

    Wildner, Sabrina; Elsässer, Brigitta; Stemeseder, Teresa; Briza, Peter; Soh, Wai Tuck; Villalba, Mayte; Lidholm, Jonas; Brandstetter, Hans; Gadermaier, Gabriele

    2017-08-16

    Knowledge of the susceptibility of proteins to endolysosomal proteases provides valuable information on immunogenicity. Though Ole e 1-like proteins are considered relevant allergens, little is known about their immunogenic properties and T cell epitopes. Thus, six representative molecules, i.e., Ole e 1, Fra e 1, Sal k 5, Che a 1, Phl p 11 and Pla l 1, were investigated. Endolysosomal degradation and peptide generation were simulated using microsomal fractions of JAWS II dendritic cells. Kinetics and peptide patterns were evaluated by gel electrophoresis and mass spectrometry. In silico MHC (major histocompatibility complex) class II binding prediction was performed with ProPred. Cleavage sites were assigned to the primary and secondary structure, and in silico docking experiments between the protease cathepsin S and Ole e 1 were performed. Different kinetics during endolysosomal degradation were observed while similar peptide profiles especially at the C-termini were detected. Typically, the identified peptide clusters comprised the previously-reported T cell epitopes of Ole e 1, consistent with an in silico analysis of the T cell epitopes. The results emphasize the importance of the fold on allergen processing, as also reflected by conserved cleavage sites located within the large flexible loop. In silico docking and mass spectrometry results suggest that one of the first Ole e 1 cleavages might occur at positions 107-108. Our results provided kinetic and structural information on endolysosomal processing of Ole e 1-like proteins.

  5. The Functional Maturation of A Disintegrin and Metalloproteinase (ADAM) 9, 10, and 17 Requires Processing at a Newly Identified Proprotein Convertase (PC) Cleavage Site*

    PubMed Central

    Wong, Eitan; Maretzky, Thorsten; Peleg, Yoav; Blobel, Carl P.; Sagi, Irit

    2015-01-01

    Proenzyme maturation is a general mechanism to control the activation of enzymes. Catalytically active members of the A Disintegrin And Metalloprotease (ADAM) family of membrane-anchored metalloproteases are synthesized as proenzymes, in which the latency is maintained by their autoinhibitory pro-domains. A proteolytic processing then transforms the proenzyme into a catalytically active form. The removal of the pro-domain of ADAMs is currently thought to depend on processing at a canonical consensus site for the proprotein convertase Furin (RXXR) between the pro- and the catalytic domain. Here, we demonstrate that this previously described canonical site is a secondary cleavage site to a prerequisite cleavage in a newly characterized upstream PC site embedded within the pro-domain sequence. The novel upstream regulatory site is important for the maturation of several ADAM proenzymes. Mutations in the upstream regulatory site of ADAM17, ADAM10, and ADAM9 do not prevent pro-domain processing between the pro- and metalloprotease domain, but nevertheless, cause significantly reduced catalytic activity. Thus, our results have uncovered a novel functionally relevant PC processing site in the N-terminal part of the pro-domain that is important for the activation of these ADAMs. These results suggest that the novel PC site is part of a general mechanism underlying proenzyme maturation of ADAMs that is independent of processing at the previously identified canonical Furin cleavage site. PMID:25795784

  6. Development of a novel anti-HIV-1 agent from within: Effect of chimeric Vpr-containing protease cleavage site residues on virus replication

    PubMed Central

    Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

    1997-01-01

    Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag–Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection. PMID:9096396

  7. The cleavage sites and localization of genes encoding the restriction endonucleases Eco1831I and EcoHI.

    PubMed

    Kravetz, A N; Zakharova, M V; Beletskaya, I V; Sineva, E V; Denjmuchametov, M M; Petrov, S I; Glatman, L I; Solonin, A S

    1993-07-15

    The restriction endonucleases Eco1831I and EcoHI cleave before the first 5'-cytosine in the recognition sequence 5'-decreases CCSGG--3'/3'--GGSCC increases-5' (where S = G or C), generate 5-base 5' cohesive ends, and are encoded by homologous plasmids that are restricted in McrA+ hosts. Thus, they differ in their cleavage specificity from that of the BcnI isoschizomer, which cleaves after the second 5' cytosine.

  8. Structural determinants of RNA recognition and cleavage by Dicer.

    PubMed

    MacRae, Ian J; Zhou, Kaihong; Doudna, Jennifer A

    2007-10-01

    A hallmark of RNA interference is the production of short double-stranded RNA (dsRNA) molecules 21-28 nucleotides in length by the specialized RNase III protein Dicer. Dicer enzymes uniquely generate RNA products of specific lengths by mechanisms that have not been fully elucidated. Here we show that the PAZ domain responsible for dsRNA end recognition confers this measuring ability through both its structural position and RNA-binding specificity. Point mutations define the dsRNA-binding surface and reveal a protein loop important for cleavage of substrates containing perfect or imperfect base pairing. On the basis of these results, we reengineered Dicer with a U1A RNA-binding domain in place of the PAZ domain to create an enzyme with altered end-recognition specificity and RNA product length. These results explain how Dicer functions as a molecular ruler and provide a structural basis for modifying its activity in cells.

  9. Active site determinants of substrate recognition by the metalloproteinases TACE and ADAM10

    PubMed Central

    Caescu, Cristina I.; Jeschke, Grace R.; Turk, Benjamin E.

    2009-01-01

    The metalloproteinases TACE (ADAM17) and ADAM10 are the primary enzymes responsible for catalyzing release of membrane anchored proteins from the cell surface in metazoan organisms. While the repertoire of protein substrates for these two proteases is partially overlapping, each one appears to target a subset of unique proteins in vivo. The mechanisms by which the two proteases achieve specificity for particular substrates are not completely understood. We have used peptide libraries to define the cleavage site selectivity of TACE and ADAM10. The two proteases have distinct primary sequence requirements at multiple positions surrounding the cleavage site in their substrates, which allowed us to generate peptide substrates that are highly specific for each of these proteases. The major difference between the two protease specificities maps to the P1′ position (immediately downstream of the cleavage site) of the substrate. At this position, TACE is selective for smaller aliphatic residues, while ADAM10 can accommodate aromatic amino acids. Using mutagenesis we identify three residues in the S1′ pockets of these enzymes that dramatically influence specificity for both peptide and protein substrates. Our results suggest that substrate selectivity of TACE and ADAM10 can be at least partly rationalized by specific features of their active sites. PMID:19715556

  10. Genetic determinants of self identity and social recognition in bacteria.

    PubMed

    Gibbs, Karine A; Urbanowski, Mark L; Greenberg, E Peter

    2008-07-11

    The bacterium Proteus mirabilis is capable of movement on solid surfaces by a type of motility called swarming. Boundaries form between swarming colonies of different P. mirabilis strains but not between colonies of a single strain. A fundamental requirement for boundary formation is the ability to discriminate between self and nonself. We have isolated mutants that form boundaries with their parent. The mutations map within a six-gene locus that we term ids for identification of self. Five of the genes in the ids locus are required for recognition of the parent strain as self. Three of the ids genes are interchangeable between strains, and two encode specific molecular identifiers.

  11. Determinants of novel object and location recognition during development.

    PubMed

    Jablonski, S A; Schreiber, W B; Westbrook, S R; Brennan, L E; Stanton, M E

    2013-11-01

    In the novel object recognition (OR) paradigm, rats are placed in an arena where they encounter two sample objects during a familiarization phase. A few minutes later, they are returned to the same arena and are presented with a familiar object and a novel object. The object location recognition (OL) variant involves the same familiarization procedure but during testing one of the familiar objects is placed in a novel location. Normal adult rats are able to perform both the OR and OL tasks, as indicated by enhanced exploration of the novel vs. the familiar test item. Rats with hippocampal lesions perform the OR but not OL task indicating a role of spatial memory in OL. Recently, these tasks have been used to study the ontogeny of spatial memory but the literature has yielded conflicting results. The current experiments add to this literature by: (1) behaviorally characterizing these paradigms in postnatal day (PD) 21, 26 and 31-day-old rats; (2) examining the role of NMDA systems in OR vs. OL; and (3) investigating the effects of neonatal alcohol exposure on both tasks. Results indicate that normal-developing rats are able to perform OR and OL by PD21, with greater novelty exploration in the OR task at each age. Second, memory acquisition in the OL but not OR task requires NMDA receptor function in juvenile rats [corrected]. Lastly, neonatal alcohol exposure does not disrupt performance in either task. Implications for the ontogeny of incidental spatial learning and its disruption by developmental alcohol exposure are discussed.

  12. Probing the Specificity Determinants of Amino Acid Recognition by Arginase

    SciTech Connect

    Shishova, E.; Di Costanzo, L; Emig, F; Ash, D; Christianson, D

    2009-01-01

    Arginase is a binuclear manganese metalloenzyme that serves as a therapeutic target for the treatment of asthma, erectile dysfunction, and atherosclerosis. In order to better understand the molecular basis of inhibitor affinity, we have employed site-directed mutagenesis, enzyme kinetics, and X-ray crystallography to probe the molecular recognition of the amino acid moiety (i.e., the ?-amino and ?-carboxylate groups) of substrate l-arginine and inhibitors in the active site of arginase I. Specifically, we focus on (1) a water-mediated hydrogen bond between the substrate ?-carboxylate and T135, (2) a direct hydrogen bond between the substrate ?-carboxylate and N130, and (3) a direct charged hydrogen bond between the substrate ?-amino group and D183. Amino acid substitutions for T135, N130, and D183 generally compromise substrate affinity as reflected by increased KM values but have less pronounced effects on catalytic function as reflected by minimal variations of kcat. As with substrate KM values, inhibitor Kd values increase for binding to enzyme mutants and suggest that the relative contribution of intermolecular interactions to amino acid affinity in the arginase active site is water-mediated hydrogen bond < direct hydrogen bond < direct charged hydrogen bond. Structural comparisons of arginase with the related binuclear manganese metalloenzymes agmatinase and proclavaminic acid amidinohydrolase suggest that the evolution of substrate recognition in the arginase fold occurs by mutation of residues contained in specificity loops flanking the mouth of the active site (especially loops 4 and 5), thereby allowing diverse guanidinium substrates to be accommodated for catalysis.

  13. Identification of the cleavage sites in the alpha6A integrin subunit: structural requirements for cleavage and functional analysis of the uncleaved alpha6Abeta1 integrin.

    PubMed Central

    Delwel, G O; Kuikman, I; van der Schors, R C; de Melker, A A; Sonnenberg, A

    1997-01-01

    The alpha6A and alpha6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the alpha6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of alpha6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the alpha6 precursor. Mutations were introduced in the codons for the R876KKR879, E883K884, R890K891 and R898K899 sequences, the potential proteolytic cleavage sites, and wild-type and mutant alpha6A cDNAs were transfected into K562 cells. The mutant alpha6A integrin subunits were expressed in association with endogenous beta1 at levels comparable to that of wild-type alpha6Abeta1. A single alpha6 polypeptide chain (150 kDa) was precipitated from transfectants expressing alpha6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded alpha6A subunits that were cleaved once into a heavy and a light chain, whereas alpha6A subunits with mutations in one of the two RK sequences were, like wild-type alpha6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of alpha6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two alpha6A light chains from platelets demonstrated that cleavage occurs after Arg879 and Lys884. Because alpha6(RKKG), alpha6(GKKR) and alpha6(RGGR) subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. alpha6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys884, were partly

  14. A strategy for fusion expression and preparation of functional glucagon-like peptide-1 (GLP-1) analogue by introducing an enterokinase cleavage site.

    PubMed

    Liu, Yang; Ren, Limei; Ge, Lingmiao; Cui, Qingxin; Cao, Xiaofang; Hou, Yuanyuan; Bai, Fang; Bai, Gang

    2014-08-01

    KGLP-1, a 31-amino acid glucagon-like peptide-1 (GLP-1) analogue, has a great therapeutic potential for anti-diabetes. In this work, a strategy for expression and purification of functional KGLP-1 peptide has been established. KGLP-1 cDNA was fused with glutathione S-transferase (GST), with an enterokinase cleavage site in the fusion junction. The recombinant fusion protein GST-KGLP-1 was affinity purified via the GST-tag, and then digested with enterokinase. The resulting GST part as well as the enzymes were eliminated by ultra-filtration followed by size exclusion chromatograph. The yield of purified KGLP-1 was approximately 12.1 mg/L, with purity of 96.18 %. The recombinant KGLP-1 was shown to have similar bioactivity as native GLP-1 when evaluated in a Chinese hamster ovary cell line expressing a GLP-1 receptor-egfp reporter gene.

  15. Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme.

    PubMed

    Klein, Daniel J; Wilkinson, Sara R; Been, Michael D; Ferré-D'Amaré, Adrian R

    2007-10-12

    The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.

  16. Nob1 binds the single-stranded cleavage site D at the 3'-end of 18S rRNA with its PIN domain.

    PubMed

    Lamanna, Allison C; Karbstein, Katrin

    2009-08-25

    Ribosome assembly is a hierarchical process that involves pre-rRNA folding, modification, and cleavage and assembly of ribosomal proteins. In eukaryotes, this process requires a macromolecular complex comprising over 200 proteins and RNAs. Whereas the rRNA modification machinery is well-characterized, rRNA cleavage to release mature rRNAs is poorly understood, and in yeast, only 2 of 8 endonucleases have been identified. The essential and conserved ribosome assembly factor Nob1 has been suggested to be the endonuclease responsible for generating the mature 3'-end of 18S rRNA by cleaving at site D. Here we provide evidence that recombinant Nob1 forms a tetramer that binds directly to pre-rRNA analogs containing cleavage site D. Analysis of Nob1's affinity to a series of RNA truncations, as well as Nob1-dependent protections of pre-rRNA in vitro and in vivo demonstrate that Nob1's binding site centers around the 3'-end of 18S rRNA, where our data also locate Nob1's suggested active site. Thus, Nob1 is poised for cleavage at the 3'-end of 18S rRNA. Together with prior data, these results strongly implicate Nob1 in cleavage at site D. In addition, our data provide evidence that the cleavage site at the 3'-end of 18S rRNA is single-stranded and not part of a duplex as commonly depicted. Using these results, we have built a model for Nob1's interaction with preribosomes.

  17. A peptide-based approach to evaluate the adaptability of influenza A virus to humans based on its hemagglutinin proteolytic cleavage site

    PubMed Central

    Straus, Marco R.; Whittaker, Gary R.

    2017-01-01

    Cleavage activation of the hemagglutinin (HA) protein by host proteases is a crucial step in the infection process of influenza A viruses (IAV). However, IAV exists in eighteen different HA subtypes in nature and their cleavage sites vary considerably. There is uncertainty regarding which specific proteases activate a given HA in the human respiratory tract. Understanding the relationship between different HA subtypes and human-specific proteases will be valuable in assessing the pandemic potential of circulating viruses. Here we utilized fluorogenic peptides mimicking the HA cleavage motif of representative IAV strains causing disease in humans or of zoonotic/pandemic potential and tested them with a range of proteases known to be present in the human respiratory tract. Our results show that peptides from the H1, H2 and H3 subtypes are cleaved efficiently by a wide range of proteases including trypsin, matriptase, human airway tryptase (HAT), kallikrein-related peptidases 5 (KLK5) and 12 (KLK12) and plasmin. Regarding IAVs currently of concern for human adaptation, cleavage site peptides from H10 viruses showed very limited cleavage by respiratory tract proteases. Peptide mimics from H6 viruses showed broader cleavage by respiratory tract proteases, while H5, H7 and H9 subtypes showed variable cleavage; particularly matriptase appeared to be a key protease capable of activating IAVs. We also tested HA substrate specificity of Factor Xa, a protease required for HA cleavage in chicken embryos and relevant for influenza virus production in eggs. Overall our data provide novel tool allowing the assessment of human adaptation of IAV HA subtypes. PMID:28358853

  18. Masking of a cathepsin G cleavage site in vivo contributes to the proteolytic resistance of major histocompatibility complex class II molecules

    PubMed Central

    Burster, Timo; Macmillan, Henriette; Hou, Tieying; Schilling, James; Truong, Phi; Boehm, Bernhard O; Zou, Fang; Lau, Kenneth; Strohman, Michael; Schaffert, Steven; Busch, Robert; Mellins, Elizabeth D

    2010-01-01

    The expression of major histocompatibility complex class II (MHC II) molecules is post-translationally regulated by endocytic protein turnover. Here, we identified the serine protease cathepsin G (CatG) as an MHC II-degrading protease by in vitro screening and examined its role in MHC II turnover in vivo. CatG, uniquely among endocytic proteases tested, initiated cleavage of detergent-solubilized native and recombinant soluble MHC II molecules. CatG cleaved human leukocyte antigen (HLA)-DR isolated from both HLA-DM-expressing and DM-null cells. Even following CatG cleavage, peptide binding was retained by pre-loaded, soluble recombinant HLA-DR. MHC II cleavage occurred on the loop between fx1 and fx2 of the membrane-proximal β2 domain. All allelic variants of HLA-DR tested and murine I-Ag7 class II molecules were susceptible, whereas murine I-Ek and HLA-DM were not, consistent with their altered sequence at the P1’ position of the CatG cleavage site. CatG effects were reduced on HLA-DR molecules with DRB mutations in the region implicated in interaction with HLA-DM. In contrast, addition of CatG to intact B-lymphoblastoid cell lines (B-LCLs) did not cause degradation of membrane-bound MHC II. Moreover, inhibition or genetic ablation of CatG in primary antigen-presenting cells did not cause accumulation of MHC II molecules. Thus, in vivo, the CatG cleavage site is sterically inaccessible or masked by associated molecules. A combination of intrinsic and context-dependent proteolytic resistance may allow peptide capture by MHC II molecules in harshly proteolytic endocytic compartments, as well as persistent antigen presentation in acute inflammatory settings with extracellular proteolysis. PMID:20331476

  19. Amylose recognition and ring-size determination of amylomaltase

    PubMed Central

    Roth, Christian; Weizenmann, Nicole; Bexten, Nicola; Saenger, Wolfram; Zimmermann, Wolfgang; Maier, Timm; Sträter, Norbert

    2017-01-01

    Starch is a major carbon and energy source throughout all kingdoms of life. It consists of two carbohydrate polymers, branched amylopectin and linear amylose, which are sparingly soluble in water. Hence, the enzymatic breakdown by glycoside hydrolases (GHs) is of great biological and societal importance. Amylomaltases (AMs) are GHs specialized in the hydrolysis of α-1,4–linked sugar chains such as amylose. They are able to catalyze an intramolecular transglycosylation of a bound sugar chain yielding polymeric sugar rings, the cycloamyloses (CAs), consisting of 20 to 100 glucose units. Despite a wealth of data on short oligosaccharide binding to GHs, no structural evidence is available for their interaction with polymeric substrates that better represent the natural polysaccharide. We have determined the crystal structure of Thermus aquaticus AM in complex with a 34-meric CA—one of the largest carbohydrates resolved by x-ray crystallography and a mimic of the natural polymeric amylose substrate. In total, 15 glucose residues interact with the protein in an extended crevice with a length of more than 40 Å. A modified succinimide, derived from aspartate, mediates protein-sugar interactions, suggesting a biological role for this nonstandard amino acid. The structure, together with functional assays, provides unique insights into the interaction of GHs with their polymeric substrate and reveals a molecular ruler mechanism for minimal ring-size determination of CA products. PMID:28097217

  20. Recognition and Specificity Determinants of the Human Cbx Chromodomains

    SciTech Connect

    Kaustov, Lilia; Ouyang, Hui; Amaya, Maria; Lemak, Alexander; Nady, Nataliya; Duan, Shili; Wasney, Gregory A.; Li, Zhihong; Vedadi, Masoud; Schapira, Matthieu; Min, Jinrong; Arrowsmith, Cheryl H.

    2011-08-17

    The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1-8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1-8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity.

  1. First genome report and analysis of chicken H7N9 influenza viruses with poly-basic amino acids insertion in the hemagglutinin cleavage site.

    PubMed

    Chen, Jidang; Zhang, Jipei; Zhu, Wanjun; Zhang, Yishan; Tan, Hualong; Liu, Minfang; Cai, Mingsheng; Shen, Jiaren; Ly, Hinh; Chen, Jianhong

    2017-08-30

    We report the full-length sequence of two chicken source influenza A (H7N9) viruses found in Guangdong live poultry market (LPM) during the most recent wave of human infections (from October 2016 to the present time). These viruses carry insertion of poly-basic amino acids (KGKRTAR/G) at the protease cleavage site of the HA protein, which were previously found in the highly pathogenic (HP) human influenza A (H7N9) [IAV(H7N9)] strains. Phylogenetic analysis of these two novel avian influenza viruses (AIVs) suggested that their genomes reassorted between the Yangtze River Delta (YRD) and Pearl River Delta (PRD) clades. Molecular clock analysis indicated that they emerged several months before the HP human strains. Collectively, our results suggest that IAV(H7N9) viruses evolve in chickens through antigenic drift to include a signature HP sequence in the HA gene, which highlights challenges in risk assessment and public health management of IAV(H7N9) infections at the human-animal interface.

  2. APASdb: a database describing alternative poly(A) sites and selection of heterogeneous cleavage sites downstream of poly(A) signals.

    PubMed

    You, Leiming; Wu, Jiexin; Feng, Yuchao; Fu, Yonggui; Guo, Yanan; Long, Liyuan; Zhang, Hui; Luan, Yijie; Tian, Peng; Chen, Liangfu; Huang, Guangrui; Huang, Shengfeng; Li, Yuxin; Li, Jie; Chen, Chengyong; Zhang, Yaqing; Chen, Shangwu; Xu, Anlong

    2015-01-01

    Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3'-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3'-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3'-untranslated regions (3'-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish.

  3. Systematic Design of Trypsin Cleavage Site Mutated Exendin4-Cysteine 1, an Orally Bioavailable Glucagon-Like Peptide-1 Receptor Agonist

    PubMed Central

    Sai, Wenbo; Tian, Hong; Yang, Kangmin; Tang, Daoqi; Bao, Jinxiao; Ge, Yang; Song, Xiaoda; Zhang, Yu; Luo, Cheng; Gao, Xiangdong; Yao, Wenbing

    2017-01-01

    Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1), an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes. PMID:28282854

  4. APASdb: a database describing alternative poly(A) sites and selection of heterogeneous cleavage sites downstream of poly(A) signals

    PubMed Central

    You, Leiming; Wu, Jiexin; Feng, Yuchao; Fu, Yonggui; Guo, Yanan; Long, Liyuan; Zhang, Hui; Luan, Yijie; Tian, Peng; Chen, Liangfu; Huang, Guangrui; Huang, Shengfeng; Li, Yuxin; Li, Jie; Chen, Chengyong; Zhang, Yaqing; Chen, Shangwu; Xu, Anlong

    2015-01-01

    Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3′-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3′-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3′-untranslated regions (3′-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish. PMID:25378337

  5. An albumin leader sequence coupled with a cleavage site modification enhances the yield of recombinant C-terminal Mullerian Inhibiting Substance

    PubMed Central

    Pépin, D.; Hoang, M.; Nicolaou, F.; Hendren, K.; Benedict, L.A.; Al-Moujahed, A.; Sosulski, A.; Marmalidou, A.; Vavvas, D.; Donahoe, P.K.

    2014-01-01

    Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. Furthermore, recent evidence suggests that MIS may effectively target a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin-cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here we show that, compared to wild type, the substitution of the MIS leader sequence to that of human serum albumin, combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased C-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing these alterations retains its capacity to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays. PMID:24729676

  6. Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin1

    PubMed Central

    Steere, Ashley N.; Roberts, Samantha E.; Byrne, Shaina L.; Chasteen, N. Dennis; Bobst, Cedric E.; Kaltashov, Igor A; Smith, Valerie C.; MacGillivray, Ross T. A.; Mason, Anne B.

    2010-01-01

    Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated FeChTF. The new bridge sequence of this construct, designated FeCTEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe. PMID:20064616

  7. Albumin Redhill (-1 Arg, 320 Ala yields Thr): A glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site

    SciTech Connect

    Brennan, S.O.; Myles, T.; Peach, R.J.; George, P.M. ); Donaldson, D. )

    1990-01-01

    Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind {sup 63}Ni{sup 2+} and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni{sup 2+} was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala {yields} Thr. This introduces as Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg {yields} Cys. The authors propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.

  8. Fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (SARS) coronavirus S protein neutralizes the virus in a rhesus macaque SARS model.

    PubMed

    Miyoshi-Akiyama, Tohru; Ishida, Isao; Fukushi, Masaya; Yamaguchi, Keina; Matsuoka, Yusuke; Ishihara, Takashi; Tsukahara, Masayoshi; Hatakeyama, Seisuke; Itoh, Norikazu; Morisawa, Aki; Yoshinaka, Yoshiyuki; Yamamoto, Naoki; Lianfeng, Zhang; Chuan, Qin; Kirikae, Teruo; Sasazuki, Takehiko

    2011-06-01

    There is still no effective method to prevent or treat severe acute respiratory syndrome (SARS), which is caused by SARS coronavirus (CoV). In the present study, we evaluated the efficacy of a fully human monoclonal antibody capable of neutralizing SARS-CoV in vitro in a Rhesus macaque model of SARS. The antibody 5H10 was obtained by vaccination of KM mice bearing human immunoglobulin genes with Escherichia coli-producing recombinant peptide containing the dominant epitope of the viral spike protein found in convalescent serum samples from patients with SARS. 5H10, which recognized the same epitope that is also a cleavage site critical for the entry of SARS-CoV into host cells, inhibited propagation of the virus and pathological changes found in Rhesus macaques infected with the virus through the nasal route. In addition, we analyzed the mode of action of 5H10, and the results suggested that 5H10 inhibited fusion between the virus envelope and host cell membrane. 5H10 has potential for use in prevention and treatment of SARS if it reemerges. This study represents a platform to produce fully human antibodies against emerging infectious diseases in a timely and safe manner.

  9. Immunotherapy against APP beta-secretase cleavage site improves cognitive function and reduces neuroinflammation in Tg2576 mice without a significant effect on brain abeta levels.

    PubMed

    Rakover, Idan; Arbel, Michal; Solomon, Beka

    2007-01-01

    Active and passive immunization methodologies against amyloid-beta (Abeta) are employed to clear and reduce cerebral Abetatowards treatment of Alzheimer's disease (AD) patients. The therapeutic potential of these antibodies in AD patients is limited because of adverse inflammatory reactions and cerebral hemorrhage, which are associated with the treatment. We propose a novel approach to inhibit Abeta production via antibodies against the beta-secretase cleavage site of the amyloid precursor protein (APP). Such an approach limits APP processing by beta-secretase, mainly through the endocytic pathway, and overcomes some of the limitations of BACE inhibition. Anti-APP beta-site antibodies, tested in a cellular model expressing wild-type APP, were found to bind full-length APP, internalize into the cells and interfere with BACE activity, inhibiting both intra- and extracellular Abeta peptide formation. We investigated the effect of anti-beta-site antibodies in an AD animal model regarding antibody efficacy, as well as possible adverse effects in the brain and periphery that may result from antibody treatment. Here, we show that long-term systemic administration of anti-APP beta-site antibodies to Tg2576 transgenic mice improved mouse cognitive functions associated with a reduction in both brain inflammation and the incidence of microhemorrhage. Furthermore, antibody treatment did not induce any peripheral autoimmunity responses. In spite of the beneficial effects observed in antibody-treated mice, brain Abeta levels were not altered as a result of antibody treatment. Copyright (c) 2007 S. Karger AG, Basel.

  10. Substrate recognition by ribonucleoprotein ribonuclease MRP.

    PubMed

    Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S

    2011-02-01

    The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

  11. Complex Consisting of β-Glucan and Antigenic Peptides with Cleavage Site for Glutathione and Aminopeptidases Induces Potent Cytotoxic T Lymphocytes.

    PubMed

    Mochizuki, Shinichi; Morishita, Hiromi; Sakurai, Kazuo

    2017-09-20

    The efficient induction of antigen-specific immune responses requires not only promotion of the uptake of antigens and adjuvant molecules into antigen-presenting cells but also control of their intracellular behavior. We previously demonstrated that the β-glucan schizophyllan (SPG) can form complexes with CpG oligonucleotides with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses. In this study, we prepared various conjugates composed of antigenic peptide (OVA257-264) and dA40 and made complexes with SPG. The conjugates with a disulfide bond between OVA257-264 and dA40 were easily cleaved by glutathione. The resultant peptides with a hydrophobic amino acid at the C-terminal end was recognized by puromycin-insensitive leucine aminopeptidase (PILS-AP), which trims antigenic peptide precursors and prepares peptides of eight or nine amino acids in length, which is the optimal length for binding to major histocompatibility complex (MHC)-I. The conjugate exposed to such enzymes induced a high antigen presentation level. The antigen presentation level was almost the same before and after the complexation with SPG. Immunization with a mixture of dA-OVA257-264/SPG and CpG-dA/SPG induced high antigen-specific cytotoxic T-lymphocyte activity at a much lower peptide dose than in previous studies. These results can be strongly ascribed to not only the cell-specific delivery by SPG but also the control of the intracellular behavior by the introduction of cleavage sites. Therefore, peptide-dA/SPG complexes could be used as potent vaccine antigens for the treatment of cancers and infectious diseases.

  12. A short double-stranded RNA motif of Peach latent mosaic viroid contains the initiation and the self-cleavage sites of both polarity strands.

    PubMed

    Delgado, Sonia; Martínez de Alba, Angel E; Hernández, Carmen; Flores, Ricardo

    2005-10-01

    The transcription initiation sites of viroid RNAs, despite their relevance for replication and in vivo folding, are poorly characterized. Here we have examined this question for Peach latent mosaic viroid (PLMVd), which belongs to the family of chloroplastic viroids with hammerhead ribozymes (Avsunviroidae), by adapting an RNA ligase-mediated rapid amplification of cDNA ends methodology developed for mapping the genuine capped 5' termini of eukaryotic messenger RNAs. To this aim, the characteristic free 5'-triphosphate group of chloroplastic primary transcripts from PLMVd-infected young fruits was previously capped in vitro with GTP and guanylyltransferase. PLMVd plus and minus initiation sites map at similar double-stranded motifs of 6 to 7 bp that also contain the conserved GUC triplet preceding the self-cleavage site in both polarity strands. Within the branched secondary structures predicted for the two PLMVd strands, this motif is located at the base of a similar long hairpin that presumably contains the promoters for a chloroplastic RNA polymerase. The transcription templates could be the circular viroid RNAs or their most abundant linear counterparts, assuming the involvement of an RNA polymerase able to jump over template discontinuities. Both PLMVd initiation sites were confirmed by applying the same methodology to two purified PLMVd subgenomic RNAs and by primer extension, and they therefore likely reflect the in vivo situation. The location of the PLMVd initiation sites provides a mechanistic view into how the nascent strands may fold and self-cleave during transcription. The approach described here may be extended to other chloroplastic RNA replicons and transcripts accumulating at low levels.

  13. Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin.

    PubMed

    Steere, Ashley N; Roberts, Samantha E; Byrne, Shaina L; Dennis Chasteen, N; Bobst, Cedric E; Kaltashov, Igor A; Smith, Valerie C; MacGillivray, Ross T A; Mason, Anne B

    2010-07-01

    Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated Fe(C)hTF. The new bridge sequence of this construct, designated Fe(C)TEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe. Copyright 2010 Elsevier Inc. All rights reserved.

  14. GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

    PubMed Central

    Sechi, Stefano; Frappaolo, Anna; Raffa, Grazia D.; Fuller, Margaret T.; Giansanti, Maria Grazia

    2014-01-01

    The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein has been described as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is also known as a potent oncogene, commonly amplified in several human tumors. However, the molecular pathways through which the oncoprotein GOLPH3 acts in malignant transformation are largely unknown. GOLPH3 has never been involved in cytokinesis. Here, we characterize the Drosophila melanogaster homologue of human GOLPH3 during cell division. We show that GOLPH3 accumulates at the cleavage furrow and is required for successful cytokinesis in Drosophila spermatocytes and larval neuroblasts. In premeiotic spermatocytes GOLPH3 protein is required for maintaining the organization of Golgi stacks. In dividing spermatocytes GOLPH3 is essential for both contractile ring and central spindle formation during cytokinesis. Wild type function of GOLPH3 enables maintenance of centralspindlin and Rho1 at cell equator and stabilization of Myosin II and Septin rings. We demonstrate that the molecular mechanism underlying GOLPH3 function in cytokinesis is strictly dependent on the ability of this protein to interact with PI(4)P. Mutations that abolish PI(4)P binding impair recruitment of GOLPH3 to both the Golgi and the cleavage furrow. Moreover telophase cells from mutants with defective GOLPH3-PI(4)P interaction fail to accumulate PI(4)P-and Rab11-associated secretory organelles at the cleavage site. Finally, we show that GOLPH3 protein interacts with components of both cytokinesis and membrane trafficking machineries in Drosophila cells. Based on these results we propose that GOLPH3 acts as a key molecule to coordinate phosphoinositide signaling with actomyosin dynamics and vesicle trafficking during cytokinesis. Because cytokinesis failures have been associated with premalignant disease and cancer, our studies suggest novel insight into molecular circuits involving the oncogene GOLPH3 in cytokinesis. PMID:24786584

  15. A Short Double-Stranded RNA Motif of Peach Latent Mosaic Viroid Contains the Initiation and the Self-Cleavage Sites of Both Polarity Strands†

    PubMed Central

    Delgado, Sonia; Martínez de Alba, Ángel E.; Hernández, Carmen; Flores, Ricardo

    2005-01-01

    The transcription initiation sites of viroid RNAs, despite their relevance for replication and in vivo folding, are poorly characterized. Here we have examined this question for Peach latent mosaic viroid (PLMVd), which belongs to the family of chloroplastic viroids with hammerhead ribozymes (Avsunviroidae), by adapting an RNA ligase-mediated rapid amplification of cDNA ends methodology developed for mapping the genuine capped 5′ termini of eukaryotic messenger RNAs. To this aim, the characteristic free 5′-triphosphate group of chloroplastic primary transcripts from PLMVd-infected young fruits was previously capped in vitro with GTP and guanylyltransferase. PLMVd plus and minus initiation sites map at similar double-stranded motifs of 6 to 7 bp that also contain the conserved GUC triplet preceding the self-cleavage site in both polarity strands. Within the branched secondary structures predicted for the two PLMVd strands, this motif is located at the base of a similar long hairpin that presumably contains the promoters for a chloroplastic RNA polymerase. The transcription templates could be the circular viroid RNAs or their most abundant linear counterparts, assuming the involvement of an RNA polymerase able to jump over template discontinuities. Both PLMVd initiation sites were confirmed by applying the same methodology to two purified PLMVd subgenomic RNAs and by primer extension, and they therefore likely reflect the in vivo situation. The location of the PLMVd initiation sites provides a mechanistic view into how the nascent strands may fold and self-cleave during transcription. The approach described here may be extended to other chloroplastic RNA replicons and transcripts accumulating at low levels. PMID:16188995

  16. Differential DNA sequence recognition is a determinant of specificity in homeotic gene action.

    PubMed Central

    Ekker, S C; von Kessler, D P; Beachy, P A

    1992-01-01

    The homeotic genes of Drosophila encode transcriptional regulatory proteins that specify distinct segment identities. Previous studies have implicated the homeodomain as a major determinant of biological specificity within these proteins, but have not established the physical basis of this specificity. We show here that the homeodomains encoded by the Ultrabithorax and Deformed homeotic genes bind optimally to distinct DNA sequences and have mapped the determinants responsible for differential recognition. We further show that relative transactivation by these two proteins in a simple in vivo system can differ by nearly two orders of magnitude. Such differences in DNA sequence recognition and target activation provide a biochemical basis for at least part of the biological specificity of homeotic gene action. Images PMID:1356765

  17. Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads

    PubMed Central

    Carbonell, Alberto; De la Peña, Marcos; Flores, Ricardo; Gago, Selma

    2006-01-01

    Eggplant latent viroid (ELVd) can form stable hammerhead structures in its (+) and (−) strands. These ribozymes have the longest helices I reported in natural hammerheads, with that of the ELVd (+) hammerhead being particularly stable (5/7 bp are G-C). Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (−) hammerhead. When the AUA trinucleotide preceding the self-cleavage site of the ELVd (+) hammerhead was substituted by GUA and GUC, as well as by AUC (essentially absent in natural hammerheads), the values of the self-cleavage rate constants at low magnesium of the purified hammerheads were: ELVd-(+)-AUC≈ELVd-(+)-GUC>ELVd-(+)-GUA> ELVd-(+)-AUA. However, the ELVd-(+)-AUC hammerhead was the catalytically less efficient during in vitro transcription, most likely because of the transient adoption of catalytically-inactive metastable structures. These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads. Comparisons with other natural hammerheads showed that the ELVd-(+)-GUC and ELVd-(+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium. PMID:17028097

  18. Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

    PubMed Central

    Burck, P J; Berg, D H; Luk, T P; Sassmannshausen, L M; Wakulchik, M; Smith, D P; Hsiung, H M; Becker, G W; Gibson, W; Villarreal, E C

    1994-01-01

    The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein

  19. The influence of socio-demographic indicators economic determinants and social recognition on sport participation in Germany.

    PubMed

    Hallmann, Kirstin; Breuer, Christoph

    2014-01-01

    This article analyses sport participation using a demographic-economic model which was extended by the construct 'social recognition'. Social recognition was integrated into the model on the understanding that it is the purpose of each individual to maximise his or her utility. A computer-assisted telephone interview survey was conducted in the city of Rheinberg, Germany, producing an overall sample of n=1934. Regression analyses were performed to estimate the impact of socio-demographic, economic determinants, and social recognition on sport participation. The results suggest that various socio-economic factors and social recognition are important determinants of sport participation on the one hand, and on sport frequency on the other. Social recognition plays a significant yet different role for both sport participation and sport frequency. While friends' involvement with sport influences one's sport participation, parents' involvement with sport influences one's sport frequency.

  20. Determinants of tRNA Recognition by the Radical SAM Enzyme RlmN

    PubMed Central

    Fitzsimmons, Christina M.; Fujimori, Danica Galonić

    2016-01-01

    RlmN, a bacterial radical SAM methylating enzyme, has the unusual ability to modify two distinct types of RNA: 23S rRNA and tRNA. In rRNA, RlmN installs a methyl group at the C2 position of A2503 of 23S rRNA, while in tRNA the modification occurs at nucleotide A37, immediately adjacent to the anticodon triplet. Intriguingly, only a subset of tRNAs that contain an adenosine at position 37 are substrates for RlmN, suggesting that the enzyme carefully probes the highly conserved tRNA fold and sequence features to identify its targets. Over the past several years, multiple studies have addressed rRNA modification by RlmN, while relatively few investigations have focused on the ability of this enzyme to modify tRNAs. In this study, we utilized in vitro transcribed tRNAs as model substrates to interrogate RNA recognition by RlmN. Using chimeras and point mutations, we probed how the structure and sequence of RNA influences methylation, identifying position 38 of tRNAs as a critical determinant of substrate recognition. We further demonstrate that, analogous to previous mechanistic studies with fragments of 23S rRNA, tRNA methylation requirements are consistent with radical SAM reactivity. Together, our findings provide detailed insight into tRNA recognition by a radical SAM methylating enzyme. PMID:27902775

  1. Molecular determinants of prokaryotic voltage-gated sodium channels for recognition of local anesthetics.

    PubMed

    Shimomura, Takushi; Irie, Katsumasa; Fujiyoshi, Yoshinori

    2016-08-01

    Local anesthetics (LAs) inhibit mammalian voltage-gated Na(+) channels (Navs) and are thus clinically important. LAs also inhibit prokaryotic Navs (BacNavs), which have a simpler structure than mammalian Navs. To elucidate the detailed mechanisms of LA inhibition to BacNavs, we used NavBh, a BacNav from Bacillus halodurans, to analyze the interactions of several LAs and quaternary ammoniums (QAs). Based on the chemical similarity of QA with the tertiary-alkylamine (TAA) group of LAs, QAs were used to determine the residues required for the recognition of TAA by NavBh. We confirmed that two residues, Thr220 and Phe227, are important for LA binding; a methyl group of Thr220 is important for recognizing both QAs and LAs, whereas Phe227 is involved in holding blockers at the binding site. In addition, we found that NavBh holds blockers in a closed state, consistent with the large inner cavity observed in the crystal structures of BacNavs. These findings reveal the inhibition mechanism of LAs in NavBh, where the methyl group of Thr220 provides the main receptor site for the TAA group and the bulky phenyl group of Phe227 holds the blockers inside the large inner cavity. These two residues correspond to the two LA recognition residues in mammalian Navs, which suggests the relevance of the LA recognition between BacNavs and mammalian Navs. © 2016 Federation of European Biochemical Societies.

  2. Neutrophil proteinase cathepsin G is proteolytically active on the human platelet glycoprotein Ib-IX receptor: characterization of the cleavage sites within the glycoprotein Ib alpha subunit.

    PubMed Central

    Pidard, D; Renesto, P; Berndt, M C; Rabhi, S; Clemetson, K J; Chignard, M

    1994-01-01

    The proteolytic activity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its

  3. Mutation of the f-protein cleavage site of avian paramyxovirus type 7 results in furin cleavage, fusion promotion, and increased replication in vitro but not increased replication, tissue tropism, or virulence in chickens.

    PubMed

    Xiao, Sa; Khattar, Sunil K; Subbiah, Madhuri; Collins, Peter L; Samal, Siba K

    2012-04-01

    We constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoprotein in tissue tropism and virulence. The AMPV-7 F protein has a single basic residue arginine (R) at position -1 in the F cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR↓FA) (underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR↓FI) or (RRKKR↓FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position -3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.

  4. On the Psychology of the Recognition Heuristic: Retrieval Primacy as a Key Determinant of Its Use

    ERIC Educational Resources Information Center

    Pachur, Thorsten; Hertwig, Ralph

    2006-01-01

    The recognition heuristic is a prime example of a boundedly rational mind tool that rests on an evolved capacity, recognition, and exploits environmental structures. When originally proposed, it was conjectured that no other probabilistic cue reverses the recognition-based inference (D. G. Goldstein & G. Gigerenzer, 2002). More recent studies…

  5. On the Psychology of the Recognition Heuristic: Retrieval Primacy as a Key Determinant of Its Use

    ERIC Educational Resources Information Center

    Pachur, Thorsten; Hertwig, Ralph

    2006-01-01

    The recognition heuristic is a prime example of a boundedly rational mind tool that rests on an evolved capacity, recognition, and exploits environmental structures. When originally proposed, it was conjectured that no other probabilistic cue reverses the recognition-based inference (D. G. Goldstein & G. Gigerenzer, 2002). More recent studies…

  6. Myc-binding-site recognition in the human genome is determined by chromatin context.

    PubMed

    Guccione, Ernesto; Martinato, Francesca; Finocchiaro, Giacomo; Luzi, Lucilla; Tizzoni, Laura; Dall' Olio, Valentina; Zardo, Giuseppe; Nervi, Clara; Bernard, Loris; Amati, Bruno

    2006-07-01

    Large-scale chromatin immunoprecipitation (ChIP) studies have been effective in unravelling the distribution of DNA-binding transcription factors along eukaryotic genomes, but specificity determinants remain elusive. Gene-regulatory regions display distinct histone variants and modifications (or marks). An attractive hypothesis is that these marks modulate protein recognition, but whether or not this applies to transcription factors remains unknown. Based on large-scale datasets and quantitative ChIP, we dissect the correlations between 35 histone marks and genomic binding by the transcription factor Myc. Our data reveal a relatively simple combinatorial organization of histone marks in human cells, with a few main groups of marks clustering on distinct promoter populations. A stretch of chromatin bearing high H3 K4/K79 methylation and H3 acetylation (or 'euchromatic island'), which is generally associated with a pre-engaged basal transcription machinery, is a strict pre-requisite for recognition of any target site by Myc (whether the consensus CACGTG or an alternative sequence). These data imply that tethering of a transcription factor to restricted chromatin domains is rate-limiting for sequence-specific DNA binding in vivo.

  7. Chiral recognition and determination of enantiomeric excess by mass spectrometry: A review.

    PubMed

    Yu, Xiangying; Yao, Zhong-Ping

    2017-05-22

    Chiral analysis is of great importance to fundamental and applied research in chemical, biological and pharmaceutical sciences. Due to the superiority of mass spectrometry (MS) over other analytical methods in terms of speed, specificity and sensitivity, chiral analysis by MS has attracted much interest in recent years. Chiral analysis by MS typically involves introduction of a chiral selector to form diastereomers with analyte enantiomers, and comparison of the behaviors of diastereomers in MS. Chiral differentiation can be achieved by comparing the relative abundances of diastereomers, the thermodynamic or kinetic constants of ion-molecule reactions of diastereomers in the gas phase, the dissociation of diastereomers in MS/MS, or the mobility of diastereomers in ion mobility mass spectrometry. In this review, chiral recognition and determination of enantiomeric excess by these chiral MS methods were summarized, and the prospects of chiral analysis by MS were discussed.

  8. In Vivo Recognition of Ovalbumin Expressed by Transgenic Leishmania Is Determined by Its Subcellular Localization1

    PubMed Central

    Prickett, Sara; Gray, Peter M.; Colpitts, Sara L.; Scott, Phillip; Kaye, Paul M.; Smith, Deborah F.

    2009-01-01

    The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity. PMID:16585577

  9. Structural determinants of imidazoacridinones facilitating antitumor activity are crucial for substrate recognition by ABCG2.

    PubMed

    Bram, Eran E; Adar, Yamit; Mesika, Nufar; Sabisz, Michal; Skladanowski, Andrzej; Assaraf, Yehuda G

    2009-05-01

    Symadex is the lead acridine compound of a novel class of imidazoacridinones (IAs) currently undergoing phase II clinical trials for the treatment of various cancers. Recently, we have shown that Symadex is extruded by ABCG2-overexpressing lung cancer A549/K1.5 cells, thereby resulting in a marked resistance to certain IAs. To identify the IA residues essential for substrate recognition by ABCG2, we here explored the ability of ABCG2 to extrude and confer resistance to a series of 23 IAs differing at defined residue(s) surrounding their common 10-azaanthracene structure. Taking advantage of the inherent fluorescent properties of IAs, ABCG2-dependent efflux and drug resistance were determined in A549/K1.5 cells using flow cytometry in the presence or absence of fumitremorgin C, a specific ABCG2 transport inhibitor. We find that a hydroxyl group at one of the R1, R2, or R3 positions in the proximal IA ring was essential for ABCG2-mediated efflux and consequent IA resistance. Moreover, elongation of the common distal aliphatic side chain attenuated ABCG2-dependent efflux, thereby resulting in the retention of parental cell sensitivity. Hence, the current study offers novel molecular insight into the structural determinants that facilitate ABCG2-mediated drug efflux and consequent drug resistance using a unique platform of fluorescent IAs. Moreover, these results establish that the IA determinants mediating cytotoxicity are precisely those that facilitate ABCG2-dependent drug efflux and IA resistance. The possible clinical implications for the future design of novel acridines that overcome ABCG2-dependent multidrug resistance are discussed.

  10. Molecular recognition of aromatic rings by flavin: electrostatics and dispersion determine ring positioning above isoalloxazine.

    PubMed

    Koziol, Lucas; Kumar, Neeraj; Wong, Sergio E; Lightstone, Felice C

    2013-12-05

    Aromatic stacking interactions between isoalloxazine (ISA) of flavin and three prototypical aromatics (benzene, pyridine, chlorobenzene) were investigated using electronic structure calculations with Monte Carlo simulated annealing. The Effective Fragment Potential (EFP) method was used to locate the low-energy equilibrium configurations for the three dimer systems. These structures were further characterized through DFT (M06-2X) and MP2 calculations. One equilibrium configuration exists for ISA-benzene; characterizing the stacked dimer surface revealed a steep, single-welled potential that funnels benzene directly between rings II and III, positioning a substituent hydrogen adjacent to the redox-active N5. ISA-pyridine and ISA-chlorobenzene minimum-energy structures contain the aromatic ring in very similar position to that in ISA-benzene. However, the added rotational degree of freedom leads to two distinct binding motifs, having approximately antiparallel or parallel dipole moment alignment with ISA. The existence of the latter binding configuration was unexpected but is explained by the shape of the ISA electrostatic potential. Dispersion is the primary noncovalent interaction driving the positioning of aromatic rings above ISA, while electrostatics determine the orientation in dipole-containing substituted benzenes. The interplay of these interactions can be used to tune molecular recognition properties of synthetic redox cofactors, including positioning desired functional groups adjacent to the redox-active N5.

  11. Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases.

    PubMed

    Niewoehner, Ole; Jinek, Martin; Doudna, Jennifer A

    2014-01-01

    In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site. To investigate the evolution of distinct mechanisms for the recognition of diverse CRISPR repeats by Cas6 enzymes, we determined crystal structures of two Thermus thermophilus Cas6 enzymes both alone and bound to substrate and product RNAs. These structures show how the scaffold common to all Cas6 endonucleases has evolved two binding sites with distinct modes of RNA recognition: one specific for a hairpin fold and the other for a single-stranded 5'-terminal segment preceding the hairpin. These findings explain how divergent Cas6 enzymes have emerged to mediate highly selective pre-CRISPR-derived RNA processing across diverse CRISPR systems.

  12. N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

    PubMed Central

    Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

    2015-01-01

    N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

  13. D-Isonucleotide (isoNA) incorporation around cleavage site of passenger strand promotes the vibration of Ago2-PAZ domain and enhances in vitro potency of siRNA.

    PubMed

    Huang, Ye; Tian, Miao; Zhang, Yichao; Sheng, Gang; Chen, Zhuo; Ma, Yuan; Chen, Yue; Peng, Yihong; Zhao, Yi-Lei; Wang, Yanli; Zhang, Lihe; Yang, Zhenjun

    2015-11-28

    It has been demonstrated that passenger strand cleavage is important for the activation of RNA-induced silencing complex (RISC), which is a crucial step for siRNA-mediated gene silencing. Herein, we report that isonucleotide (isoNA) modification around the cleavage site of the passenger strand would affect the in vitro potency of modified siRNAs by altering the motion pattern of the Ago2-PAZ domain. According to western blotting, q-PCR and antiviral test results, we proved that D-isonucleotide (isoNA) modification at the position 8 of the passenger strand (siMek1-S08D), which is adjacent to the cleavage site, markedly improved the in vitro potency of the modified siRNA, whereas siRNAs with D-isoNA incorporation at position 9 (siMek1-S09D) or L-isoNA incorporation at positions 8 and 9 (siMek1-S08L, siMek1-S09L) displayed lower activity compared to native siRNA. Kinetics evaluation of passenger strand cleavage induced by T. thermophilus Ago (Tt-Ago) showed that D-isoNA modification at position 8 of the passenger strand had no significant influence on the cleavage rate, but L-isoNA modification at position 8 slowed the cleavage rate markedly. Moreover, the results of molecular dynamics simulations showed that D-isoNA modification at position 8 affected the open-close motion of the PAZ domain in the Ago/siRNA complex, which may promote the loading of RISC and release of a passenger strand cleavage product, and consequently accelerate the activation of RISC and enhance silencing activity. However, D-isoNA modification at position 9 or L-isoNA modification at position 8 or 9 exerted opposite influences on the motion of the Ago-PAZ domain.

  14. N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites.

    PubMed

    Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong; Yao, Bin

    2015-12-04

    N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.

  15. Deformability in the cleavage site of primary microRNA is not sensed by the double-stranded RNA binding domains in the microprocessor component DGCR8.

    PubMed

    Quarles, Kaycee A; Chadalavada, Durga; Showalter, Scott A

    2015-06-01

    The prevalence of double-stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA-binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD-containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8-Core, consists of its two dsRBDs and a C-terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N-terminal dsRBD of DGCR8 in isolation nor the DGCR8-Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single-stranded segments flanking the stem. Extending DGCR8-Core to include an N-terminal heme-binding region does not change our conclusions. Thus, our data suggest that although the DGCR8-Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency.

  16. Semantic and visual determinants of face recognition in a prosopagnosic patient.

    PubMed

    Dixon, M J; Bub, D N; Arguin, M

    1998-05-01

    Prosopagnosia is the neuropathological inability to recognize familiar people by their faces. It can occur in isolation or can coincide with recognition deficits for other nonface objects. Often, patients whose prosopagnosia is accompanied by object recognition difficulties have more trouble identifying certain categories of objects relative to others. In previous research, we demonstrated that objects that shared multiple visual features and were semantically close posed severe recognition difficulties for a patient with temporal lobe damage. We now demonstrate that this patient's face recognition is constrained by these same parameters. The prosopagnosic patient ELM had difficulties pairing faces to names when the faces shared visual features and the names were semantically related (e.g., Tonya Harding, Nancy Kerrigan, and Josee Chouinard -three ice skaters). He made tenfold fewer errors when the exact same faces were associated with semantically unrelated people (e.g., singer Celine Dion, actress Betty Grable, and First Lady Hillary Clinton). We conclude that prosopagnosia and co-occurring category-specific recognition problems both stem from difficulties disambiguating the stored representations of objects that share multiple visual features and refer to semantically close identities or concepts.

  17. Size determines whether specialized expert processes are engaged for recognition of faces.

    PubMed

    Yang, Nan; Shafai, Fakhri; Oruc, Ipek

    2014-07-22

    Many influential models of face recognition postulate specialized expert processes that are engaged when viewing upright, own-race faces, as opposed to a general-purpose recognition route used for nonface objects and inverted or other-race faces. In contrast, others have argued that empirical differences do not stem from qualitatively distinct processing. We offer a potential resolution to this ongoing controversy. We hypothesize that faces engage specialized processes at large sizes only. To test this, we measured recognition efficiencies for a wide range of sizes. Upright face recognition efficiency increased with size. This was not due to better visibility of basic image features at large sizes. We ensured this by calculating efficiency relative to a specialized ideal observer unique to each individual that incorporated size-related changes in visibility and by measuring inverted efficiencies across the same range of face sizes. Inverted face recognition efficiencies did not change with size. A qualitative face inversion effect, defined as the ratio of relative upright and inverted efficiencies, showed a complete lack of inversion effects for small sizes up to 6°. In contrast, significant face inversion effects were found for all larger sizes. Size effects may stem from predominance of larger faces in the overall exposure to faces, which occur at closer viewing distances typical of social interaction. Our results offer a potential explanation for the contradictory findings in the literature regarding the special status of faces.

  18. An Efficient and Robust Singular Value Method for Star Pattern Recognition and Attitude Determination

    NASA Technical Reports Server (NTRS)

    Juang, Jer-Nan; Kim, Hye-Young; Junkins, John L.

    2003-01-01

    A new star pattern recognition method is developed using singular value decomposition of a measured unit column vector matrix in a measurement frame and the corresponding cataloged vector matrix in a reference frame. It is shown that singular values and right singular vectors are invariant with respect to coordinate transformation and robust under uncertainty. One advantage of singular value comparison is that a pairing process for individual measured and cataloged stars is not necessary, and the attitude estimation and pattern recognition process are not separated. An associated method for mission catalog design is introduced and simulation results are presented.

  19. Recognition and reporting of suspected adverse drug reactions by surveyed healthcare professionals in Uganda: key determinants

    PubMed Central

    Kiguba, Ronald; Karamagi, Charles; Waako, Paul; Ndagije, Helen B; Bird, Sheila M

    2014-01-01

    Objective To assess extent and determinants of past-month recognition of suspected adverse drug reactions (ADR) and past-year ADR reporting among healthcare professionals (HCPs) in Uganda. Setting Geographically diverse health facilities (public, private for-profit, private not-for-profit). Participants Of 2000 questionnaires distributed, 1345 were completed: return rate of 67%. Primary and secondary outcome measures Per cent HCPs who suspected ADR in the past month; reported ADR in the past year. Results Nurses were the majority (59%, 792/1345). Only half the respondents had heard about pharmacovigilance: 39% of nurses (295/763; 95% CI 35% to 42%), 70% otherwise (383/547; 95% CI 66% to 74%). One fifth (268/1289 or 21%; 95% CI 19% to 23%) had suspected an ADR in the previous 4 weeks, 111 of them were nurses; 15% (190/1296) had reported a suspected ADR in the past year, 103 of them were nurses. Past-month ADR suspicion was more likely by non-nurses (OR=1.7, 95% CI 1.16 to 2.40) and with medical research involvement (OR=1.5, 95% CI 1.05 to 2.15) but past-month receipt of patient ADR-complaint predominated (OR=19, 95% CI 14 to 28). Past-year ADR reporting was higher by hospital staff (OR=1.9, 95% CI 1.18 to 3.10), especially in medicine (OR=2.3, 95% CI 1.08 to 4.73); but lower from private for-profit health facilities (OR=0.5, 95% CI 0.28 to 0.77) and by older staff (OR=0.6, 95% CI 0.43 to 0.91); more likely by HCPs who had ever encountered a fatal ADR (OR=2.9, 95% CI 1.94 to 4.25), knew to whom to report (OR=1.7, 95% CI 1.18 to 2.46), or suggested how to improve ADR reporting (OR=1.6, 95% CI 1.04 to 2.49). Two attitudinal factors were important: diffidence and lethargy. Conclusions One in five HCPs suspected an ADR in the past-month and one in seven reported ADR in the previous year. Empowering patients could strengthen ADR detection and reporting in Africa. PMID:25421337

  20. Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

    PubMed Central

    Kolykhalov, A A; Agapov, E V; Rice, C M

    1994-01-01

    Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B

  1. Newcastle Disease Virus Establishes Persistent Infection in Tumor Cells In Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase.

    PubMed

    Rangaswamy, Udaya S; Wang, Weijia; Cheng, Xing; McTamney, Patrick; Carroll, Danielle; Jin, Hong

    2017-08-15

    Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses.IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in the

  2. In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain.

    PubMed

    Ursel, Christian; Fandrich, Uwe; Hoffmann, Anita; Sieg, Torsten; Ihling, Christian; Stubbs, Milton T

    2013-08-01

    Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.

  3. Development of a Pattern Recognition Methodology for Determining Operationally Optimal Heat Balance Instrumentation Calibration Schedules

    SciTech Connect

    Kurt Beran; John Christenson; Dragos Nica; Kenny Gross

    2002-12-15

    The goal of the project is to enable plant operators to detect with high sensitivity and reliability the onset of decalibration drifts in all of the instrumentation used as input to the reactor heat balance calculations. To achieve this objective, the collaborators developed and implemented at DBNPS an extension of the Multivariate State Estimation Technique (MSET) pattern recognition methodology pioneered by ANAL. The extension was implemented during the second phase of the project and fully achieved the project goal.

  4. Coiled coil miniprotein randomization on phage leads to charge pattern mimicry of the receptor recognition determinant of interleukin 5.

    PubMed

    Li, Chuanzhao; Plugariu, Carmela G; Bajgier, Joanna; White, John R; Liefer, Kristin M; Wu, Sheng-Jiun; Chaiken, Irwin

    2002-01-01

    Phage display was used to identify sequences that mimic structural determinants in interleukin5 (IL5) for IL5 receptor recognition. A coiled coil stem loop (CCSL) miniprotein scaffold library was constructed with its turn region randomized and panned for binding variants against human IL5 receptor alpha chain (IL5Ralpha). Competition enzyme-linked immunosorbent assays identified CCSL-phage selectants for which binding to IL5Ralpha was competed by IL5. The most frequently selected and IL5-competed CCSL-phage contain charged residues Arg and Glu in their turn sequences, in this regard resembling a beta strand sequence in the 'CD turn' region, of IL5, that has been proposed to present a key determinant for IL5 receptor alpha chain recognition. The most dominant CCSL-phage selectant sequence, PVEGRV, contains a negative/positive charge pattern similar to that seen in the original CD turn. To test the relatedness of CCSL-phage selectant sequences to the IL5 receptor recognition epitope, PVEGRV was grafted into the sequence 87--92 of a monomeric IL5. The resulting IL5 variant, [(87)PVEGRV(92)]GM1, was able to bind to IL5Ralpha in biosensor assays, to elicit TF-1 cell proliferation and to induce STAT5 phosphorylation in TF-1 cells. The results help discern sequence patterns in the IL5 CD turn region which are key in driving receptor recognition and demonstrate the utility of CCSL miniprotein scaffold phage display to identify local IL5 mimetic sequence arrangements that may ultimately lead to IL5 antagonists.

  5. Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop.

    PubMed Central

    Kettunen, R; Tyystjärvi, E; Aro, E M

    1996-01-01

    Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. this cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves. PMID:8756500

  6. Use of a recombinant fluorescent substrate with cleavage sites for all botulinum neurotoxins in high-throughput screening of natural product extracts for inhibitors of serotypes A, B, and E.

    PubMed

    Hines, Harry B; Kim, Alexander D; Stafford, Robert G; Badie, Shirin S; Brueggeman, Ernst E; Newman, David J; Schmidt, James J

    2008-02-01

    The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.

  7. Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

    SciTech Connect

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik; Whittaker, Gary R.

    2012-12-05

    A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

  8. Study to determine potential flight applications and human factors design guidelines for voice recognition and synthesis systems

    NASA Technical Reports Server (NTRS)

    White, R. W.; Parks, D. L.

    1985-01-01

    A study was conducted to determine potential commercial aircraft flight deck applications and implementation guidelines for voice recognition and synthesis. At first, a survey of voice recognition and synthesis technology was undertaken to develop a working knowledge base. Then, numerous potential aircraft and simulator flight deck voice applications were identified and each proposed application was rated on a number of criteria in order to achieve an overall payoff rating. The potential voice recognition applications fell into five general categories: programming, interrogation, data entry, switch and mode selection, and continuous/time-critical action control. The ratings of the first three categories showed the most promise of being beneficial to flight deck operations. Possible applications of voice synthesis systems were categorized as automatic or pilot selectable and many were rated as being potentially beneficial. In addition, voice system implementation guidelines and pertinent performance criteria are proposed. Finally, the findings of this study are compared with those made in a recent NASA study of a 1995 transport concept.

  9. Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element.

    PubMed Central

    Wang, P Z; Projan, S J; Henriquez, V; Novick, R P

    1993-01-01

    The leading strand replication origin of pT181 plasmids consists of two adjacent inverted repeat elements (IR-II and IR-III), which are involved in origin recognition by the initiator (Rep) protein. The conserved core element, IR-II, which contains the initiation nick site, is induced by Rep to form a cruciform structure, probably the primary substrate for the initiation of rolling circle replication. The divergent repeat, IR-III, constitutes the determinant of origin recognition specificity. We show here that the distal arm of IR-III is not required for sequence-specific recognition, whereas the proximal arm and central region are required. Since the initiator is dimeric, we presume that it binds symmetrically to IR-III. A unique type of DNA-protein interaction is proposed, in which the lack of sequence requirement for the distal arm is a consequence of binding to the adjacent IR-II, which thereby polarizes the stringency of binding to the two arms of IR-III. In addition, genetic evidence indicates that both the spacing and the phasing of IR-II to IR-III are crucial for function and that the central segment of IR-III may serve to position the two flanking half-sites for optimal interaction of Rep with IR-III. Images PMID:8428593

  10. A microflow chemiluminescence sensor for indirect determination of dibutyl phthalate by hydrolyzing based on biological recognition materials.

    PubMed

    Qiu, Huamin; Fan, Lulu; Li, Xiangjun; Li, Leilei; Sun, Min; Luo, Chuannan

    2013-03-05

    A microflow chemiluminescence (CL) sensor for determination of dibutyl phthalate (DBP) based on magnetic molecularly imprinted polymer (MMIP) as recognition element was fabricated. Briefly, a hydrophilic molecularly imprinted polymer layer was produced at the surface of Fe₃O₄@SiO₂ magnetic nanoparticles (MNPs) via combination of molecular imprinting and reversible stimuli responsive hydrogel. In this protocol, the initial step involved co-precipitation of Fe²⁺ and Fe³⁺ in an ammonia solution. Silica was then coated on the Fe₃O₄ nanoparticles using a sol-gel method to obtain silica shell magnetic nanoparticles. The MMIP was synthesized using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker and 2,2-azobisisobutyronitrile (AIBN) as initiator in chloroform. Then the synthesized MMIP and magnetic non-molecular imprinted polymers (MNIP) were employed as recognition by packing into lab-made straight shape tubes, connected in CL analyzer for establishing the novel sensor with a single channel syringe pump. And a mixer for hydrolyzing of DBP was followed. Based on this experiment principle, DBP was determined indirectly. And the MMIP showed satisfactory recognition capacity to DBP, resulting to the wide linear range of 3.84 × 10⁻⁸ to 2.08 × 10⁻⁵ M and the low detection limit of 2.09 × 10⁻⁹ M (3σ) for DBP. The relative standard deviation (RSD) for DBP (3.20 × 10⁻⁶ M) was 1.40% (n=11). Besides improving sensitivity and selectivity, the sensor was reusable. The proposed DBP-MMIP-CL sensor has been successfully applied to determine DBP in drink samples.

  11. The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

    PubMed Central

    Fung, Angela Wai Shan; Leung, Charles Chung Yun; Fahlman, Richard Peter

    2014-01-01

    Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNALeu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNALeu (CAG)—a G3:C70 base pair and a set of 4 nt (C72, A4:U69, C68)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed. PMID:24935875

  12. Timing of presentation and nature of stimuli determine retroactive interference with social recognition memory in mice.

    PubMed

    Perna, Judith Camats; Wotjak, Carsten T; Stork, Oliver; Engelmann, Mario

    2015-05-01

    The present study was designed to further investigate the nature of stimuli and the timing of their presentation, which can induce retroactive interference with social recognition memory in mice. In accordance with our previous observations, confrontation with an unfamiliar conspecific juvenile 3h and 6h, but not 22 h, after the initial learning session resulted in retroactive interference. The same effect was observed with the exposure to both enantiomers of the monomolecular odour carvone, and with a novel object. Exposure to a loud tone (12 KHz, 90 dB) caused retroactive interference at 6h, but not 3h and 22 h, after sampling. Our data show that retroactive interference of social recognition memory can be induced by exposing the experimental subjects to the defined stimuli presented <22 h after learning in their home cage. The distinct interference triggered by the tone presentation at 6h after sampling may be linked to the intrinsic aversiveness of the loud tone and suggests that at this time point memory consolidation is particularly sensitive to stress.

  13. Vγ2Vδ2 T Cell Receptor Recognition of Prenyl Pyrophosphates is Dependent on all Complementarity Determining Regions1

    PubMed Central

    Wang, Hong; Fang, Zhimei; Morita, Craig T.

    2010-01-01

    γδ T cells differ from αβ T cells in the antigens they recognize and their functions in immunity. While most αβ T cell receptors (TCR) recognize peptides presented by MHC class I or II, human γδ T cells expressing Vγ2Vδ2 TCRs recognize nonpeptide prenyl pyrophosphates. To define the molecular basis for this recognition, the effect of mutations in the TCR complementarity-determining regions (CDR) was assessed. Mutations in all CDR loops altered recognition and cover a large footprint. Unlike murine γδ TCR recognition of the MHC class Ib T22 protein, there was no CDR3δ motif required for recognition because only 1 residue is required. Instead, the length and sequence of CDR3γ was key. Although a potential prenyl pyrophosphate-binding site was defined by Lys109 in Jγ1.2 and Arg51 in CDR2δ, the area outlined by critical mutations is much larger. These results show that prenyl pyrophosphate recognition is primarily by germline-encoded regions of the γδ TCR, allowing a high proportion of Vγ2Vδ2 TCRs to respond. This underscores its parallels to innate immune receptors. Our results also provide strong evidence for the existence of an antigen-presenting molecule for prenyl pyrophosphates. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org. PMID:20483784

  14. Determining and improving the fault tolerance of multilayer perceptrons in a pattern-recognition application.

    PubMed

    Emmerson, M D; Damper, R I

    1993-01-01

    We investigate empirically the performance under damage conditions of single- and multilayer perceptrons (MLP's), with various numbers of hidden units, in a representative pattern-recognition task. While some degree of graceful degradation was observed, the single-layer perceptron was considerably less fault tolerant than any of the multilayer perceptrons, including one with fewer adjustable weights. Our initial hypothesis that fault tolerance would be significantly improved for multilayer nets with larger numbers of hidden units proved incorrect. Indeed, there appeared to be a liability to having excess hidden units. A simple technique (called augmentation) is described, which was successful in translating excess hidden units into improved fault tolerance. Finally, our results were supported by applying singular value decomposition (SVD) analysis to the MLP's internal representations.

  15. Recognition of protein-linked glycans as a determinant of peptidase activity.

    PubMed

    Noach, Ilit; Ficko-Blean, Elizabeth; Pluvinage, Benjamin; Stuart, Christopher; Jenkins, Meredith L; Brochu, Denis; Buenbrazo, Nakita; Wakarchuk, Warren; Burke, John E; Gilbert, Michel; Boraston, Alisdair B

    2017-01-31

    The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.

  16. Molecular surface recognition: determination of geometric fit between proteins and their ligands by correlation techniques.

    PubMed Central

    Katchalski-Katzir, E; Shariv, I; Eisenstein, M; Friesem, A A; Aflalo, C; Vakser, I A

    1992-01-01

    A geometric recognition algorithm was developed to identify molecular surface complementarity. It is based on a purely geometric approach and takes advantage of techniques applied in the field of pattern recognition. The algorithm involves an automated procedure including (i) a digital representation of the molecules (derived from atomic coordinates) by three-dimensional discrete functions that distinguishes between the surface and the interior; (ii) the calculation, using Fourier transformation, of a correlation function that assesses the degree of molecular surface overlap and penetration upon relative shifts of the molecules in three dimensions; and (iii) a scan of the relative orientations of the molecules in three dimensions. The algorithm provides a list of correlation values indicating the extent of geometric match between the surfaces of the molecules; each of these values is associated with six numbers describing the relative position (translation and rotation) of the molecules. The procedure is thus equivalent to a six-dimensional search but much faster by design, and the computation time is only moderately dependent on molecular size. The procedure was tested and validated by using five known complexes for which the correct relative position of the molecules in the respective adducts was successfully predicted. The molecular pairs were deoxyhemoglobin and methemoglobin, tRNA synthetase-tyrosinyl adenylate, aspartic proteinase-peptide inhibitor, and trypsin-trypsin inhibitor. A more realistic test was performed with the last two pairs by using the structures of uncomplexed aspartic proteinase and trypsin inhibitor, respectively. The results are indicative of the extent of conformational changes in the molecules tolerated by the algorithm. Images PMID:1549581

  17. Determinants of DNA mismatch recognition within the polymerase domain of the Klenow fragment.

    PubMed

    Thompson, Elizabeth H Z; Bailey, Michael F; van der Schans, Edwin J C; Joyce, Catherine M; Millar, David P

    2002-01-22

    The Klenow fragment of Escherichia coli DNA polymerase I catalyzes template-directed synthesis of DNA and uses a separate 3'-5' exonuclease activity to edit misincorporated bases. The polymerase and exonuclease activities are contained in separate structural domains. In this study, nine Klenow fragment derivatives containing mutations within the polymerase domain were examined for their interaction with model primer-template duplexes. The partitioning of the DNA primer terminus between the polymerase and 3'-5' exonuclease active sites of the mutant proteins was assessed by time-resolved fluorescence anisotropy, utilizing a dansyl fluorophore attached to the DNA. Mutation of N845 or R668 disrupted favorable interactions between the Klenow fragment and a duplex containing a matched terminal base pair but had little effect when the terminus was mismatched. Thus, N845 and R668 are required for recognition of correct terminal base pairs in the DNA substrate. Mutation of N675, R835, R836, or R841 resulted in tighter polymerase site binding of DNA, suggesting that the side chains of these residues induce strain in the DNA and/or protein backbone. A double mutant (N675A/R841A) showed an even greater polymerase site partitioning than was displayed by either single mutation, indicating that such strain is additive. In both groups of mutant proteins, the ability to discriminate between duplexes containing matched or mismatched base pairs was impaired. In contrast, mutation of K758 or Q849 had no effect on partitioning relative to wild type, regardless of DNA mismatch character. These results demonstrate that DNA mismatch recognition is dependent on specific amino acid residues within the polymerase domain and is not governed solely by thermodynamic differences between correct and mismatched base pairs. Moreover, this study suggests a mechanism whereby the Klenow fragment is able to recognize polymerase errors following a misincorporation event, leading to their eventual

  18. Cleavage of the NR2B subunit amino terminus of N-methyl-D-aspartate (NMDA) receptor by tissue plasminogen activator: identification of the cleavage site and characterization of ifenprodil and glycine affinities on truncated NMDA receptor.

    PubMed

    Ng, Kay-Siong; Leung, How-Wing; Wong, Peter T-H; Low, Chian-Ming

    2012-07-20

    Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicates tPA as an important neuromodulator of the N-methyl-d-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg(67)). This cleavage is tPA-specific, plasmin-independent, and removes a predicted ~4-kDa fragment (Arg(27)-Arg(67)) from the amino-terminal domain of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg(67) to Ala(67) impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg(27)-Arg(67)-truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln(29)-Arg(67) deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC(50) with no change in glutamate EC(50). Our results confirmed NR2B as a novel proteolytic substrate of tPA, where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.

  19. The human G1m1 allotype associates with CD4+ T-cell responsiveness to a highly conserved IgG1 constant region peptide and confers an asparaginyl endopeptidase cleavage site

    PubMed Central

    Stickler, M M; Reddy, A; Xiong, J M; Hinton, P R; DuBridge, R; Harding, F A

    2011-01-01

    The human G1m1 allotype comprises two amino acids, D12 and L14, in the CH3 domain of IGHG1. Although the G1m1 allotype is prevalent in human populations, ∼40% of Caucasiods are homozygous for the nG1m1 allotype corresponding to E12 and M14. Peptides derived from the G1m1 region were tested for their ability to induce CD4+ T-cell proliferative responses in vitro. A peptide immediately downstream from the G1m1 sequence was recognized by CD4+ T cells in a large percentage of donors (peptide CH315−29). CD4+ T-cell proliferative responses to CH315−29 were found at an increased frequency in nG1m1 homozygous donors. Homozygous nG1m1 donors possessing the HLA-DRB1*07 allele displayed the highest magnitudes of proliferation. CD4+ T cells from donors homozygous for nG1m1 proliferated to G1m1-carrying Fc-fragment proteins, whereas CD4+ T cells from G1m1 homozygous donors did not. The G1m1 sequence creates an enzymatic cleavage site for asparaginyl endopeptidase in vitro. Proteolytic activity at D12 may allow the presentation of the CH315−29 peptide, which in turn may result in the establishment of tolerance to this peptide in G1m1-positive donors. Homozygous nG1m1 patients may be more likely to develop CD4+ T-cell-mediated immune responses to therapeutic antibodies carrying the G1m1 allotype. PMID:21326320

  20. Pattern-Based Recognition for the Rapid Determination of Identity, Concentration, and Enantiomeric Excess of Subtly Different Threo Diols

    PubMed Central

    Shabbir, Shagufta H.; Joyce, Leo A.; da Cruz, Gabriella M.; Lynch, Vincent M.; Sorey, Steven; Anslyn, Eric V.

    2009-01-01

    A pattern based recognition approach was developed for the rapid determination of identity, concentration, and enantiomeric excess of chiral vicinal diols, specifically threo diols. A diverse enantioselective sensor array was generated using three chiral boronic acid receptors and three pH indicators. The optical response produced by the sensor array was analyzed by two pattern recognition algorithms: principal component analysis and artificial neural networks. Principle component analysis demonstrated good chemoselective and enantioselective separation of the analytes, and an artificial neural network was used to accurately determine the concentration and enantiomeric excess of five unknown samples with an average absolute error of ± 0.08 mM in concentration and 3.6% in enantiomeric excess. The speed of the analysis was enhanced by using a 96-well plate format, which portents to applications in high-throughput screening for asymmetric catalyst discovery. X-ray crystallography and 11B NMR was utilized to study the enantioselective nature of boronic acid host-2. PMID:19691315

  1. Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

    PubMed Central

    Dhiman, Monisha; Zago, Maria Paola; Nunez, Sonia; Amoroso, Alejandro; Rementeria, Hugo; Dousset, Pierre; Burgos, Federico Nunez; Garg, Nisha Jain

    2012-01-01

    Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD50 sensitivity to 30 µM 4-HNE and 100 µM H2O2 at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H2O2 resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We

  2. The serological recognition of the human MLC determinants using a modified cytotoxicity technique.

    PubMed

    van Rood, J J; van Leeuwen, A; Keuning, J J; van Oud Alblas, A B

    1975-04-01

    A new approach which presumably recognizes MLC determinants (or at least structures closely linked to MLC and different from the FOUR, AJ and LA series determinants) by serology is described. As the test can be performed within 10 h it could be used to match cadaveric kidney donors for these MLC determinants. Perhaps even more important is the fact that the antibodies detected by this technique could be used to study the immunochemistry of a class of determinants differing from the well known SD determinants.

  3. Determination of association constant of host-guest supramolecular complex (molecular recognition of carbamazepine, antiseizure drug, with calix(4)arene).

    PubMed

    Meenakshi, C; Jayabal, P; Ramakrishnan, V

    2015-12-05

    The thermodynamic property of the host-guest, inclusion complex formed between p-t-butyl calix(4)arene which is a supramolecule, and the antiseizure drug, carbamazepine was studied. p-t-Butyl calix(4)arene has been used as a host molecule and carbamazepine as a guest molecule. Optical absorption spectral studies were carried out to investigate the molecular recognition properties of p-t-butyl calix(4)arene with carbamazepine. The stochiometry of the host-guest complexes formed and the association constant were determined. An interesting 1:2 stochiometric host-guest complex was formed. Job's continuous method of variation and Benesi-Hildebrand expression were used for the determination of binding constant and the stochiometry of the host-guest complex formed. Molecular dimension of the host molecule plays a vital role in the formation of the host-guest stochiometric complexes.

  4. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    NASA Astrophysics Data System (ADS)

    Ebner, Andreas; Nikova, Dessy; Lange, Tobias; Häberle, Johannes; Falk, Sabine; Dübbers, Angelika; Bruns, Reimer; Hinterdorfer, Peter; Oberleithner, Hans; Schillers, Hermann

    2008-09-01

    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl-) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients.

  5. Environmental Determinants of Chronic Disease and Medical Approaches: Recognition, Avoidance, Supportive Therapy, and Detoxification

    PubMed Central

    Sears, Margaret E.; Genuis, Stephen J.

    2012-01-01

    The World Health Organization warns that chronic, noncommunicable diseases are rapidly becoming epidemic worldwide. Escalating rates of neurocognitive, metabolic, autoimmune and cardiovascular diseases cannot be ascribed only to genetics, lifestyle, and nutrition; early life and ongoing exposures, and bioaccumulated toxicants may also cause chronic disease. Contributors to ill health are summarized from multiple perspectives—biological effects of classes of toxicants, mechanisms of toxicity, and a synthesis of toxic contributors to major diseases. Healthcare practitioners have wide-ranging roles in addressing environmental factors in policy and public health and clinical practice. Public health initiatives include risk recognition and chemical assessment then exposure reduction, remediation, monitoring, and avoidance. The complex web of disease and environmental contributors is amenable to some straightforward clinical approaches addressing multiple toxicants. Widely applicable strategies include nutrition and supplements to counter toxic effects and to support metabolism; as well as exercise and sweating, and possibly medication to enhance excretion. Addressing environmental health and contributors to chronic disease has broad implications for society, with large potential benefits from improved health and productivity. PMID:22315626

  6. H9N2 avian influenza virus-derived natural reassortant H5N2 virus in swan containing the hemagglutinin segment from Eurasian H5 avian influenza virus with an in-frame deletion of four basic residues in the polybasic hemagglutinin cleavage site.

    PubMed

    Wang, Youling; Yuan, Xiaoyuan; Qi, Lihong; Zhang, Yuxia; Xu, Huaiying; Yang, Jinxing; Ai, Wu; Qi, Wenbao; Liao, Ming; Wang, Dan; Song, Minxun; Li, Feng

    2016-06-01

    We isolated a novel H5N2 avian influenza virus from swans in China. The virus was derived from a widespread H9N2 avian influenza virus but acquired the hemagglutinin gene from Eurasian H5 subtype with a naturally occurring in-frame deletion of four basic residues in the polybasic hemagglutinin cleavage site. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Structure-function analysis of Staphylococcus aureus amidase reveals the determinants of peptidoglycan recognition and cleavage.

    PubMed

    Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

    2014-04-18

    The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

  8. Alternative Selection of β-Site APP-Cleaving Enzyme 1 (BACE1) Cleavage Sites in Amyloid β-Protein Precursor (APP) Harboring Protective and Pathogenic Mutations within the Aβ Sequence.

    PubMed

    Kimura, Ayano; Hata, Saori; Suzuki, Toshiharu

    2016-11-11

    β-Site APP-cleaving enzyme 1 (BACE1) cleaves amyloid β-protein precursor (APP) at the bond between Met(671) and Asp(672) (β-site) to generate the carboxyl-terminal fragment (CTFβ/C99). BACE1 also cleaves APP at another bond between Thr(681) and Gln(682) (β'-site), yielding CTFβ'/C89. Cleavage of CTFβ/C99 by γ-secretase generates Aβ(1-XX), whereas cleavage of CTFβ'/C89 generates Aβ(11-XX). Thus, β'-site cleavage by BACE1 is amyloidolytic rather than amyloidogenic. β' cleavage of mouse APP is more common than the corresponding cleavage of human APP. We found that the H684R substitution within human Aβ, which replaces the histidine in the human protein with the arginine found at the corresponding position in mouse, facilitated β' cleavage irrespective of the species origin of BACE1, thereby significantly increasing the level of Aβ(11-XX) and decreasing the level of Aβ(1-XX). Thus, amino acid substitutions within the Aβ sequence influenced the selectivity of alternative β- or β'-site cleavage of APP by BACE1. In familial Alzheimer's disease (FAD), the APP gene harbors pathogenic variations such as the Swedish (K670N/M671L), Leuven (E682K), and A673V mutations, all of which decrease Aβ(11-40) generation, whereas the protective Icelandic mutation (A673T) increases generation of Aβ(11-40). Thus, A673T promotes β' cleavage of APP and protects subjects against AD. In addition, CTFβ/C99 was cleaved by excess BACE1 activity to generate CTFβ'/C89, followed by Aβ(11-40), even if APP harbored pathogenic mutations. The resultant Aβ(11-40) was more metabolically labile in vivo than Aβ(1-40). Our analysis suggests that some FAD mutations in APP are amyloidogenic and/or amyloidolytic via selection of alternative BACE1 cleavage sites.

  9. BCL::contact-low confidence fold recognition hits boost protein contact prediction and de novo structure determination.

    PubMed

    Karakaş, Mert; Woetzel, Nils; Meiler, Jens

    2010-02-01

    Knowledge of all residue-residue contacts within a protein allows determination of the protein fold. Accurate prediction of even a subset of long-range contacts (contacts between amino acids far apart in sequence) can be instrumental for determining tertiary structure. Here we present BCL::Contact, a novel contact prediction method that utilizes artificial neural networks (ANNs) and specializes in the prediction of medium to long-range contacts. BCL::Contact comes in two modes: sequence-based and structure-based. The sequence-based mode uses only sequence information and has individual ANNs specialized for helix-helix, helix-strand, strand-helix, strand-strand, and sheet-sheet contacts. The structure-based mode combines results from 32-fold recognition methods with sequence information to a consensus prediction. The two methods were presented in the 6(th) and 7(th) Critical Assessment of Techniques for Protein Structure Prediction (CASP) experiments. The present work focuses on elucidating the impact of fold recognition results onto contact prediction via a direct comparison of both methods on a joined benchmark set of proteins. The sequence-based mode predicted contacts with 42% accuracy (7% false positive rate), while the structure-based mode achieved 45% accuracy (2% false positive rate). Predictions by both modes of BCL::Contact were supplied as input to the protein tertiary structure prediction program Rosetta for a benchmark of 17 proteins with no close sequence homologs in the protein data bank (PDB). Rosetta created higher accuracy models, signified by an improvement of 1.3 A on average root mean square deviation (RMSD), when driven by the predicted contacts. Further, filtering Rosetta models by agreement with the predicted contacts enriches for native-like fold topologies.

  10. Determination of the influence of substrate concentration on enzyme selectivity using whey protein Isolate and Bacillus licheniformis protease.

    PubMed

    Butré, Claire I; Sforza, Stefano; Gruppen, Harry; Wierenga, Peter A

    2014-10-22

    Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each cleavage site in the protein. It was determined from the identification and quantification of the peptides present in the hydrolysates. Solutions of 0.1-10% (w/v) whey protein isolate (WPI) were hydrolyzed by Bacillus licheniformis protease at constant enzyme-to-substrate ratio. The cleavage sites were divided into five groups, from very high (>10%) to very low selectivity (<0.1%). The selectivity toward cleavage sites after Glu 62 and 134 was 2 times higher at 10% (w/v) WPI than at the lower protein concentrations. This finding shows that both the rate of hydrolysis and the enzyme selectivity were influenced by the substrate concentration.

  11. Gender-Marked Determiners Help Dutch Learners' Word Recognition when Gender Information Itself Does Not

    ERIC Educational Resources Information Center

    van Heugten, Marieke; Johnson, Elizabeth K.

    2011-01-01

    Dutch, unlike English, contains two gender-marked forms of the definite article. Does the presence of multiple definite article forms lead Dutch learners to be delayed relative to English learners in the acquisition of their determiner system? Using the Preferential Looking Procedure, we found that Dutch-learning children aged 1 ; 7 to 2 ; 0 use…

  12. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

    SciTech Connect

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A.

    2011-08-16

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

  13. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease▿

    PubMed Central

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A.

    2011-01-01

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance. PMID:21507982

  14. Novel Protein–Protein Contacts Facilitate mRNA 3'-Processing Signal Recognition by Rna15 and Hrp1

    SciTech Connect

    Leeper, Thomas C.; Qu, Xiangping; Lu, Connie; Moore, Claire; Varani, Gabriele

    2010-08-01

    Precise 3'-end processing of mRNA is essential for correct gene expression, yet in yeast, 3'-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3'-processing and are recognized by the RNA-binding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein–protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3'-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.

  15. Novel Protein-Protein Contacts Facilitate mRNA 3'-Processing Signal Recognition by Rna15 and Hrp1.

    SciTech Connect

    Leeper, Thomas C; Qu, Xiangping; Lu, Connie; Moore, Claire; Varani, Gabriele

    2010-06-19

    Precise 3'-end processing of mRNA is essential for correct gene expression, yet in yeast, 3'-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3'-processing and are recognized by the RNAbinding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein–protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3'-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.

  16. Mutagenic dissection of the sequence determinants of protein folding, recognition, and machine function.

    PubMed

    Sauer, Robert T

    2013-11-01

    Understanding the relationship between the amino-acid sequence of a protein and its ability to fold and to function is one of the major challenges of protein science. Here, cases are reviewed in which mutagenesis, biochemistry, structure determination, protein engineering, and single-molecule biophysics have illuminated the sequence determinants of folding, binding specificity, and biological function for DNA-binding proteins and ATP-fueled machines that forcibly unfold native proteins as a prelude to degradation. In addition to structure-function relationships, these studies provide information about folding intermediates, mutations that accelerate folding, slow unfolding, and stabilize proteins against denaturation, show how new binding specificities and folds can evolve, and reveal strategies that proteolytic machines use to recognize, unfold, and degrade thousands of distinct substrates. © 2013 The Protein Society.

  17. Multiple modes of peptide recognition by the PTB domain of the cell fate determinant Numb

    PubMed Central

    Zwahlen, Catherine; Li, Shun-Cheng; Kay, Lewis E.; Pawson, Tony; Forman-Kay, Julie D.

    2000-01-01

    The phosphotyrosine-binding (PTB) domain of the cell fate determinant Numb is involved in the formation of multiple protein complexes in vivo and can bind a diverse array of peptide sequences in vitro. To investigate the structural basis for the promiscuous nature of this protein module, we have determined its solution structure by NMR in a complex with a peptide containing an NMSF sequence derived from the Numb-associated kinase (Nak). The Nak peptide was found to adopt a significantly different structure from that of a GPpY sequence-containing peptide previously determined. In contrast to the helical turn adopted by the GPpY peptide, the Nak peptide forms a β–turn at the NMSF site followed by another turn near the C–terminus. The Numb PTB domain appears to recognize peptides that differ in both primary and secondary structures by engaging various amounts of the binding surface of the protein. Our results suggest a mechanism through which a single PTB domain might interact with multiple distinct target proteins to control a complex biological process such as asymmetric cell division. PMID:10747019

  18. Nanosilica-based molecularly imprinted polymer nanoshell for specific recognition and determination of rhodamine B in red wine and beverages.

    PubMed

    Long, Zerong; Xu, Weiwei; Lu, Yi; Qiu, Hongdeng

    2016-09-01

    A new and facile rhodamine B (RhB)-imprinted polymer nanoshell coating for SiO2 nanoparticles was readily prepared by a combination of silica gel modification and molecular surface imprinting. The RhB-imprinted polymers (RhB-MIPs) were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and UV-vis spectroscopy; the binding properties and selectivity of these MIPs were investigated in detail. The uniformly imprinted nanoparticles displayed a rather thin shell thickness (23nm) with highly effective recognition sites, showing homogenous distribution and monolayer adsorption. The maximum MIP adsorption capacity (Qm) was as high as 45.2mgg(-1), with an adsorption equilibrium time of about 15min at ambient temperature. Dynamic rebinding experiments showed that chemical adsorption is crucial for RhB binding to RhB-MIPs. The adsorption isotherm for RhB-MIPs binding could also be described by the Langmuir equation at different temperatures and pH values. Increasing temperature led to an enhanced Qm, a decreased dissociation constant (K'd), and a more negative free energy (ΔG), indicating that adsorption is favored at higher temperatures. Moreover, the adsorption capacity of RhB was remarkably affected by pH. At pH>7, the adsorption of RhB was driven by hydrogen bonding interactions, while at pH<7 electrostatic forces were dominant. Additionally, the MIPs also showed specific recognition of RhB from the standard mixture solution containing five structurally analogs. This method was also successfully employed to determine RhB content in red wine and beverages using three levels of spiking, with recoveries in the range of 91.6-93.1% and relative standard deviations lower than 4.1%. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants.

    PubMed Central

    Klock, G; Unger, B; Gatz, C; Hillen, W; Altenbuchner, J; Schmid, K; Schmitt, R

    1985-01-01

    Homologous and heterologous repressor-operator interactions among four different classes of tetracycline resistance determinants have been compared. These are represented by RP1/Tn1721 (class A), R222/Tn10 (class B), pSC101/pBR322 (class C), and RA1 (class D). By the use of the purified repressor proteins of class A (TetRA) and class B (TetRB), operator sequences of all four classes are recognized by both with an identical stoichiometry of four repressor subunits per control sequence, but with different affinities. In vitro transcription has been used to demonstrate regulatory activities of TetRA and TetRB upon all four classes of tet genes. Tetracycline acted as an inducer. A functional relationship among the tet regulatory systems was also shown in vivo by complementation of a class A tetR'-galK fusion mutant with the tetR genes of classes A, B, and C. Repression of tetRA-linked galactokinase was ca. 80% in the presence of tetRA or tetRC, and ca. 50% in the presence of tetRB. Taken together, these results demonstrate heterologous repressor-operator interaction, suggesting close relationships among the four classes of Tcr determinants. Images PMID:3881391

  20. Lck availability during thymic selection determines the recognition specificity of the T cell repertoire

    PubMed Central

    Van Laethem, François; Tikhonova, Anastasia N.; Pobezinsky, Leonid A.; Tai, Xuguang; Kimura, Motoko Y.; Le Saout, Cecile; Guinter, Terry I.; Adams, Anthony; Sharrow, Susan O.; Bernhardt, Günter; Feigenbaum, Lionel; Singer, Alfred

    2013-01-01

    Summary Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αβ T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αβTCR that are restricted by major histocompatibility complex (MHC) encoded determinants. Here, we report that Lck proteins that were coreceptor-associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor-free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αβTCR depending on whether Lck is coreceptor-associated or coreceptor-free. We conclude that the intracellular state of Lck determines the specificity of thymic selection, and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αβTCR repertoire. PMID:24034254

  1. Research progress in radiation detectors, pattern recognition programs, and radiation damage determination in DNA

    NASA Technical Reports Server (NTRS)

    Baily, N. A.

    1973-01-01

    The radiological implications of statistical variations in energy deposition by ionizing radiation were investigated in the conduct of the following experiments: (1) study of the production of secondary particles generated by the passage of the primary radiation through bone and muscle; (2) the study of the ratio of nonreparable to reparable damage in DNA as a function of different energy deposition patterns generated by X rays versus heavy fast charged particles; (3) the use of electronic radiography systems for direct fluoroscopic tomography and for the synthesis of multiple planes and; (4) the determination of the characteristics of systems response to split fields having different contrast levels, and of minimum detectable contrast levels between the halves under realistic clinical situations.

  2. Echocardiographic evaluation of left ventricular diastolic function in cats: Hemodynamic determinants and pattern recognition.

    PubMed

    Schober, Karsten E; Chetboul, Valérie

    2015-12-01

    Left ventricular (LV) diastolic dysfunction is highly prevalent in cats and is a functional hallmark of feline cardiomyopathy. The majority of cats with hypertrophic, restrictive, and dilated cardiomyopathy have echocardiographic evidence of abnormal LV filling, even during the occult (preclinical) phase. Moderate and severe diastolic dysfunction is an indicator of advanced myocardial disease, is associated with clinical signs including exercise intolerance and congestive heart failure, affects outcome, and influences therapeutic decisions. Therefore, identification and quantification of LV diastolic dysfunction are clinically important. Surrogate measures of diastolic function determined by transthoracic two-dimensional, M-mode, and Doppler echocardiographic (DE) methods have been used widely for such purpose. Major functional characteristics of LV diastole, including global function, relaxation and untwist, chamber compliance, filling volume, and the resultant filling pressures can be semi-quantified by echocardiographic methods, and variables retrieved from transmitral flow, pulmonary vein flow, and tissue Doppler recordings are most frequently used. Although there is still a critical lack of well-designed studies in the field, knowledge has steadily accumulated over the past 20 years, reference ranges of diastolic echocardiographic variables have been determined, epidemiological studies have been conducted, and new treatments of diastolic dysfunction in cats have been evaluated. This report will give the reader a summary of the current status in the field of feline diastology with focus on the noninvasive diagnostic methods and interpretation of echocardiographic surrogate measures of LV diastolic function. Lastly, a grading system using a composite of left atrial size and various DE variables potentially useful in the functional classification of LV diastole in cats is introduced.

  3. Air segmented amplitude modulated multiplexed flow analysis with software-based phase recognition: determination of phosphate ion.

    PubMed

    Ogusu, Takeshi; Uchimoto, Katsuya; Takeuchi, Masaki; Tanaka, Hideji

    2014-01-01

    Amplitude modulated multiplexed flow analysis (AMMFA) has been improved by introducing air segmentation and software-based phase recognition. Sample solutions, the flow rates of which are respectively varied at different frequencies, are merged. Air is introduced to the merged liquid stream in order to limit the dispersion of analytes within each liquid segment separated by air bubbles. The stream is led to a detector with no physical deaeration. Air signals are distinguished from liquid signals through the analysis of detector output signals, and are suppressed down to the level of liquid signals. Resulting signals are smoothed based on moving average computation. Thus processed signals are analyzed by fast Fourier transform. The analytes in the samples are respectively determined from the amplitudes of the corresponding wave components obtained. The developed system has been applied to the simultaneous determinations of phosphate ions in water samples by a Malachite Green method. The linearity of the analytical curve (0.0-31.0 μmol dm(-3)) is good (r(2)>0.999) and the detection limit (3.3 σ) at the modulation period of 30s is 0.52 μmol dm(-3). Good recoveries around 100% have been obtained for phosphate ions spiked into real water samples. © 2013 Elsevier B.V. All rights reserved.

  4. Visual determination of aliphatic diamines based on host-guest recognition of calix[4]arene derivatives capped gold nanoparticles.

    PubMed

    Chen, Yangyang; Zhang, Jiangjiang; Gao, Yanmin; Lee, Jaebeom; Chen, Hongxia; Yin, Yongmei

    2015-10-15

    Since amine compounds have been widespread pollutants in nature and they are extensively used in pharmaceutical industries and dye manufacturing, it is highly desirable to develop simple, effective and naked-eye available analytical methods for such aliphatic diamines determination. Calixarenes as macrocycles have drawn intensive interests for fields such as biomedicine, supramolecular chemistry and smart materials. Here, instead of the normal complicated modification strategy, a facile and efficient method for one-pot synthesis of calix[4]arene crown ether (CCE4) capped gold nanoparticles (AuNPs) is proposed. The as-prepared CCE4-AuNPs are not only high water dispersity and stability even after storage for 3 months, but also capable of host-guest recognition of diamines in aqueous systems. Size-selective encapsulation of amine group between CCE4 and diamines carry out the aggregation of CCE4-AuNPs. The determination of diamines such as hexamethylenediamine or spermine can be realized by the UV-vis absorbance change and visual color difference.

  5. Rationalization and Design of the Complementarity Determining Region Sequences in an Antibody-Antigen Recognition Interface

    PubMed Central

    Chen, Ing-Chien; Lee, Yu-Ching; Chen, Jun-Bo; Tsai, Keng-Chang; Chen, Ching-Tai; Chang, Jeng-Yih; Yang, Ei-Wen; Hsu, Po-Chiang; Jian, Jhih-Wei; Hsu, Hung-Ju; Chang, Hung-Ju; Hsu, Wen-Lian; Huang, Kai-Fa; Ma, Alex Che; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes. PMID:22457753

  6. A bimetallic nanocomposite modified genosensor for recognition and determination of thalassemia gene.

    PubMed

    Hamidi-Asl, Ezat; Raoof, Jahan Bakhsh; Naghizadeh, Nahid; Akhavan-Niaki, Haleh; Ojani, Reza; Banihashemi, Ali

    2016-10-01

    The main roles of DNA in the cells are to maintain and properly express genetic information. It is important to have analytical methods capable of fast and sensitive detection of DNA damage. DNA hybridization sensors are well suited for diagnostics and other purposes, including determination of bacteria and viruses. Beta thalassemias (βth) are due to mutations in the β-globin gene. In this study, an electrochemical biosensor which detects the sequences related to the β-globin gene issued from real samples amplified by polymerase chain reaction (PCR) is described for the first time. The biosensor relies on the immobilization of 20-mer single stranded oligonucleotide (probe) related to βth sequence on the carbon paste electrode (CPE) modified by 15% silver (Ag) and platinum (Pt) nanoparticles to prepare the bimetallic nanocomposite electrode and hybridization of this oligonucleotide with its complementary sequence (target). The extent of hybridization between the probe and target sequences was shown by using linear sweep voltammetry (LSV) with methylene blue (MB) as hybridization indicator. The selectivity of sensor was investigated using PCR samples containing non-complementary oligonucleotides. The detection limit of biosensor was calculated about 470.0pg/μL. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. RNA recognition by the Caenorhabditis elegans oocyte maturation determinant OMA-1.

    PubMed

    Kaymak, Ebru; Ryder, Sean P

    2013-10-18

    Maternally supplied mRNAs encode proteins that pattern early embryos in many species. In the nematode Caenorhabditis elegans, a suite of RNA-binding proteins regulates expression of maternal mRNAs during oogenesis, the oocyte to embryo transition, and early embryogenesis. To understand how these RNA-binding proteins contribute to development, it is necessary to determine how they select specific mRNA targets for regulation. OMA-1 and OMA-2 are redundant proteins required for oocyte maturation--an essential part of meiosis that prepares oocytes for fertilization. Both proteins have CCCH type tandem zinc finger RNA-binding domains. Here, we define the RNA binding specificity of OMA-1 and demonstrate that OMA-1/2 are required to repress the expression of a glp-1 3'-UTR reporter in developing oocytes. OMA-1 binds with high affinity to a conserved region of the glp-1 3'-UTR previously shown to interact with POS-1 and GLD-1, RNA-binding proteins required for glp-1 reporter repression in the posterior of fertilized embryos. Our results reveal that OMA-1 is a sequence-specific RNA-binding protein required to repress expression of maternal transcripts during oogenesis and suggest that interplay between OMA-1 and other factors for overlapping binding sites helps to coordinate the transition from oocyte to embryo.

  8. Evaluating a Computer Flash-Card Sight-Word Recognition Intervention with Self-Determined Response Intervals in Elementary Students with Intellectual Disability

    ERIC Educational Resources Information Center

    Cazzell, Samantha; Skinner, Christopher H.; Ciancio, Dennis; Aspiranti, Kathleen; Watson, Tiffany; Taylor, Kala; McCurdy, Merilee; Skinner, Amy

    2017-01-01

    A concurrent multiple-baseline across-tasks design was used to evaluate the effectiveness of a computer flash-card sight-word recognition intervention with elementary-school students with intellectual disability. This intervention allowed the participants to self-determine each response interval and resulted in both participants acquiring…

  9. Determining detection, recognition, and identification ranges of thermal cameras on the basis of laboratory measurements and TTP model

    NASA Astrophysics Data System (ADS)

    Bareła, J.; Kastek, M.; Firmanty, K.; Trzaskawka, P.; Dulski, R.

    2012-06-01

    TTP (Targeting Task Performance) model is widely used for the estimation of theoretical performance of observation devices. It is used, for example, in the NVTERM software and makes it possible to determine the detection, recognition and identification ranges for a standard target types on the basis of known technical parameters of analyzed device. Many theoretical analysis concerning TTP model can be found, as well as a few experimental, field test results. However the usability of the TTP model for the calculation of range parameters on the basis of laboratory test results has not been widely analyzed. The paper presents an attempt to apply TTP model for the estimation of range parameters of thermal cameras using laboratory measurement results of camera properties. The test stand consists of an IR collimator, a standard IR source, a set of test targets and a computer with data acquisition card. The method used for the measurement of aforementioned characteristics will be described and the algorithms used to finally estimate the range parameters of a tested thermal camera using TTP model.

  10. Crystal structure of pentapeptide-independent chemotaxis receptor methyltransferase (CheR) reveals idiosyncratic structural determinants for receptor recognition.

    PubMed

    Batra, Monu; Sharma, Rajesh; Malik, Anjali; Dhindwal, Sonali; Kumar, Pravindra; Tomar, Shailly

    2016-12-01

    Chemotactic methyltransferase, CheR catalyse methylation of specific glutamate residues in the cytoplasmic domain of methyl-accepting chemotactic protein receptors (MCPRs). The methylation of MCPRs is essential for the chemical sensing and chemotactic bacterial mobility towards favorable chemicals or away from unfavorable ones. In this study, crystal structure of B. subtilis CheR (BsCheR) in complex with S-adenosyl-l-homocysteine (SAH) has been determined to 1.8Å resolution. This is the first report of crystal structure belonging to the pentapeptide-independent CheR (PICheR) class. Till date, only one crystal structure of CheR from S. typhimurium (StCheR) belonging to pentapeptide-dependent CheR (PDCheR) class is available. Structural analysis of BsCheR reveals a helix-X-helix motif (HXH) with Asp53 as the linker residue in the N-terminal domain. The key structural features of the PDCheR β-subdomain involved in the formation of a tight complex with the pentapeptide binding motif in MCPRs were found to be absent in the structure of BsCheR. Additionally, isothermal titration calorimetry (ITC) experiments were performed to investigate S-adenosyl-(l)-methionine (SAM) binding affinity and KD was determined to be 0.32mM. The structure of BsCheR reveals that mostly residues of the large C-terminal domain contribute to SAH binding, with contributions of few residues from the linker region and the N-terminal domain. Structural investigations and sequence analysis carried out in this study provide critical insights into the distinct receptor recognition mechanism of the PDCheR and PICheR methyltransferase classes.

  11. Flow injection chemiluminescence sensor using core-shell molecularly imprinted polymers as recognition element for determination of dapsone.

    PubMed

    Lu, Fuguang; Yang, Jinlong; Sun, Min; Fan, Lulu; Qiu, Huamin; Li, Xiangjun; Luo, Chuannan

    2012-07-01

    This paper reports the preparation of dapsone (DDS) imprinted polymer layer-coated silica submicron particles (SiO(2)) combined with chemiluminescence (CL) toward analysis of tracing DDS in practical samples. To induce the selective occurrence of surface polymerization, the amino groups were first grafted at the surface of SiO(2) by the (3-aminopropyl)triethoxysilane (APTES). The molecularly imprinted polymers (MIP) were coated at the surface of modified SiO(2) by the graft copolymerization. After the removal of templates, recognition sites of DDS were exposed in the polymer layers. The DDS-imprinted products were characterized by FT-IR, SEM, TEM, dynamic adsorption, and static adsorption tests. The proximity between the thickness of MIP layer and the spatial size of DDS indicated that the imprinted sites almost situated at the surface of MIP, leading to rapid adsorption saturation within 90 min. The apparent maximum binding amount of MIP toward DDS was evaluated as 14.98 mg·g(-1), which was much higher than that of non-molecularly imprinted polymers. The CL sensor provided a wide linear range for DDS within 1.0 × 10(-6) to 1.0 × 10(-4) mol·L(-1) with a detection limit of 5.27 × 10(-7) mol·L(-1) and the relative standard deviation of 1.8 % (n = 11) by determinations of 5.0 × 10(-6) mol·L(-1) DDS. This method was applied to determine DDS in urine samples and satisfactory results were obtained.

  12. Proposed study to determine potential flight applications and human factors design guidelines of voice recognition/synthesis systems

    NASA Technical Reports Server (NTRS)

    Bergeron, H. P.

    1983-01-01

    An effort to evaluate the human factors aspects and potential of voice recognition/synthesis techniques and the application of present and near-future (5 years) voice recognition/synthesis systems as a pilot/aircraft cockpit interface capability in an operational environment is discussed. The analysis will emphasize applications for single pilot instrument flight rules operations but will also include applications for other categories of aircraft with various levels of complexity.

  13. Evaluating a computer flash-card sight-word recognition intervention with self-determined response intervals in elementary students with intellectual disability.

    PubMed

    Cazzell, Samantha; Skinner, Christopher H; Ciancio, Dennis; Aspiranti, Kathleen; Watson, Tiffany; Taylor, Kala; McCurdy, Merilee; Skinner, Amy

    2017-09-01

    A concurrent multiple-baseline across-tasks design was used to evaluate the effectiveness of a computer flash-card sight-word recognition intervention with elementary-school students with intellectual disability. This intervention allowed the participants to self-determine each response interval and resulted in both participants acquiring previously unknown words across all word sets. Discussion focuses on the need to evaluate and compare computer flash-card sight-word recognition interventions with fixed and self-determined response intervals across students and dependent variables, including rates of inappropriate behavior and self-determination in students with intellectual disability. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  14. Global analysis of Drosophila Cys₂-His₂ zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants.

    PubMed

    Enuameh, Metewo Selase; Asriyan, Yuna; Richards, Adam; Christensen, Ryan G; Hall, Victoria L; Kazemian, Majid; Zhu, Cong; Pham, Hannah; Cheng, Qiong; Blatti, Charles; Brasefield, Jessie A; Basciotta, Matthew D; Ou, Jianhong; McNulty, Joseph C; Zhu, Lihua J; Celniker, Susan E; Sinha, Saurabh; Stormo, Gary D; Brodsky, Michael H; Wolfe, Scot A

    2013-06-01

    Cys2-His2 zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes.

  15. Calicheamicin-DNA complexes: warhead alignment and saccharide recognition of the minor groove.

    PubMed

    Ikemoto, N; Kumar, R A; Ling, T T; Ellestad, G A; Danishefsky, S J; Patel, D J

    1995-11-07

    The solution structures of calicheamicin gamma 1I, its cycloaromatized analog (calicheamicin epsilon), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin gamma 1I such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin gamma 1I is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.

  16. Trend recognition and failure prediction of the attitude determination and control system of the Space Station Freedom

    NASA Astrophysics Data System (ADS)

    Nelson, Kyle S.; Hadden, George D.

    An approach to automated trend recognition and failure prediction in the health parameter data of spacecraft is described. The approach, State-Based Feature Recognition (SBER), combines intelligent data filtering with state machines to detect the presence of features (trends and impending failures) in the health parameter data of spacecraft. SBFR, when implemented in a space-based or ground-based monitoring system, can increase spacecraft autonomy and decrease technician workload. An implemented, prototype Space Station Freedom (SSF) Maintenance and Diagnostic System (SSFMDS) that demonstrates the applicability of SBFR to trend detection and failure prediction will be described. SBFR allows features to be tracked, using specialized state machines, as they develop in a time-independent manner, allowing both short term and long term features to be detected. Each state machine operates independently of the other machines, making simultaneous feature tracking possible.

  17. Structure of the propeptide of prothrombin containing the. gamma. -carboxylation recognition site determined by two-dimensional NMR spectroscopy

    SciTech Connect

    Sanford, D.G.; Sudmeier, J.L.; Bachovchin, W.W.; Kanagy, C.; Furie, B.C.; Furie, B. )

    1991-10-15

    The propeptides of the vitamin K dependent blood clotting and regulatory proteins contain a {gamma}-carboxylation recognition site that directs precursor forms of these proteins for posttranslational {gamma}-carboxylation. Peptides corresponding to the propeptide of prothrombin were synthesized and examined by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). CD spectra indicate that these peptides have little or no secondary structure in aqueous solutions but that the addition of trifluoroethanol induces or stabilizes a structure containing {alpha}-helical character. The maximum helical content occurs at 35-40% trifluoroethanol. This trifluoroethanol-stabilized structure was solved by two-dimensional NMR spectroscopy. The NMR results demonstrate that residues {minus}13 to {minus}3 form an amphipathic {alpha}-helix. NMR spectra indicate that a similar structure is present at 5C, in the absence of trifluoroethanol. Of the residues previously implicated in defining the {gamma}-carboxylation recognition site, four residues ({minus}18, {minus}17, {minus}16, and {minus}15) are adjacent to the helical region and one residue ({minus}10) is located within the helix. The potential role of the amphipathic {alpha}-helix in the {gamma}-carboxylation recognition site is discussed.

  18. Recognition sequences of restriction endonucleases and methylases--a review.

    PubMed

    Kessler, C; Neumaier, P S; Wolf, W

    1985-01-01

    The properties and sources of all known endonucleases and methylases acting site-specifically on DNA are listed. The enzymes are crossindexed (Table I), classified according to homologies within their recognition sequences (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328 and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (Table III), the structure of the restriction fragment ends (Table IV), and the sensitivity to different kinds of DNA methylation (Table V). Table VI classifies the methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises those restriction endonucleases, which are known to be inhibited by the modified nucleotides. Furthermore, this review includes a restriction map of bacteriophage lambda DNA based on sequence data. Table VII lists the exact nucleotide positions of the cleavage sites, the length of the generated fragments ordered according to size, and the effects of the Escherichia coli dam- and dcmI-coded methylases M X Eco dam and M X Eco dcmI on the particular recognition sites.

  19. Large domain motions in Ago protein controlled by the guide DNA-strand seed region determine the Ago-DNA-mRNA complex recognition process.

    PubMed

    Xia, Zhen; Huynh, Tien; Ren, Pengyu; Zhou, Ruhong

    2013-01-01

    The recognition mechanism and cleavage activity of argonaute (Ago), miRNA, and mRNA complexes are the core processes to the small non-coding RNA world. The 5' nucleation at the 'seed' region (position 2-8) of miRNA was believed to play a significant role in guiding the recognition of target mRNAs to the given miRNA family. In this paper, we have performed all-atom molecular dynamics simulations of the related and recently revealed Ago-DNA:mRNA ternary complexes to study the dynamics of the guide-target recognition and the effect of mutations by introducing "damaging" C·C mismatches at different positions in the seed region of the DNA-RNA duplex. Our simulations show that the A-form-like helix duplex gradually distorts as the number of seed mismatches increases and the complex can survive no more than two such mismatches. Severe distortions of the guide-target heteroduplex are observed in the ruinous 4-sites mismatch mutant, which give rise to a bending motion of the PAZ domain along the L1/L2 "hinge-like" connection segment, resulting in the opening of the nucleic-acid-binding channel. These long-range interactions between the seed region and PAZ domain, moderated by the L1/L2 segments, reveal the central role of the seed region in the guide-target strands recognition: it not only determines the guide-target heteroduplex's nucleation and propagation, but also regulates the dynamic motions of Ago domains around the nucleic-acid-binding channel.

  20. Investigation and evaluation of a method for determination of ethanol with the SIRE Biosensor P100, using alcohol dehydrogenase as recognition element.

    PubMed

    Svensson, Katrin; Bülow, Leif; Kriz, Dario; Krook, Margareta

    2005-11-15

    A new method for rapid determination of ethanol was developed, using alcohol dehydrogenase as recognition element for the SIRE (sensors based on injection of the recognition element) Biosensor, which is an amperometric biosensor. The method was simple, fast, accurate, specific and cost-effective. The recognition element solution used was stable at least for 24 h in room temperature, and at least one month when lyophilised. The optimal potential versus the silver wire electrode, the optimal pH of the buffer and the optimal temperature of the water bath was determined to be +950 mV, 8.1 and 308 K, respectively. The optimal concentrations of alcohol dehydrogenase, BSA and NAD(+) were determined to be 200 U/ml, 20 mg/ml and 15 mM, respectively. The total analysis time was between 50 s and 4 min per analysis, depending on the concentration range. The linear range was 0-12.5 mM. The detection limit was less than 0.1 mM. The repeatability (%R.S.D.) was 3-5% (n=10). The reproducibility was 5-8% (n=5). Methanol gave no signal at all, but higher alcohols, such as propanol, pentanol and hexanol, gave significant signals, decreasing with increasing length of the carbon chain. The price for one measurement was calculated to be 0.052 euro. The results from measurements with the biosensor were compared to those from an established analysis kit for ethanol. The results correlated well (R(2)=0.9874). The concentration of ethanol in different alcoholic beverages was investigated and correlated well with the concentrations given by the manufacturers.

  1. Molecular Determinants of Antibiotic Recognition and Resistance by Aminoglycoside Phosphotransferase (3′)-IIIa: A Calorimetric and Mutational Analysis

    PubMed Central

    Kaul, Malvika; Barbieri, Christopher M.; Srinivasan, Annankoil R.; Pilch, Daniel S.

    2007-01-01

    Summary The growing threat from the emergence of multidrug resistant pathogens highlight a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3′)-IIIa (an antibiotic phosphorylating enzyme that produces resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with 15N NMR-derived pKa and calorimetrically-derived binding-linked drug protonation data, identify the 1-, 3-, and 2′-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3′)-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3′)-IIIa-induced resistance. PMID:17418235

  2. Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis.

    PubMed

    Kaul, Malvika; Barbieri, Christopher M; Srinivasan, Annankoil R; Pilch, Daniel S

    2007-05-25

    The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.

  3. Nicotine, cotinine, and myosmine determination using polymer films of tailor-designed zinc porphyrins as recognition units for piezoelectric microgravimetry chemosensors.

    PubMed

    Noworyta, Krzysztof; Kutner, Wlodzimierz; Wijesinghe, Channa A; Srour, Serge G; D'Souza, Francis

    2012-03-06

    Two electropolymerizable zinc porphyrins with receptor sites tailor-designed for selective recognition of the nicotine, cotinine, or myosmine alkaloids were synthesized. These were 5-(2-phenoxyacetamide)-10,15,20-tris(triphenylamino)porphyrinato zinc(II) 1 and 5-(2,5-phenylene-bis(oxy)diacetamide)-10,15,20-tris(triphenylamino)porphyrinato zinc(II) 2 featuring one and two pendant amide side "pincers", respectively, and three triphenylamine substituents at the meso positions of the porphyrin macrocycles capable of electrochemical polymerization. Thin polymerfilms of these porphyrins served for recognition and the piezoelectric microgravimetry (PM) for analytical signal transduction of a new chemical sensor devised for determination of these alkaloids. The films were deposited by potentiodynamic electropolymerization on the 10 MHz quartz resonators of the electrochemical quartz crystal microbalance (EQCM) without affecting the electronic structure of the porphyrin macrocycles. Under favorable flow injection analysis (FIA) conditions, the alkaloid analytes were determined at the concentration level of 0.1 mM with high sensitivity and selectivity. Affinity toward the analytes of the polymer of 2 was higher than that of 1 due to the higher binding ability offered by two pendant pincers of the former. Because of the selective receptors and PM applied under FIA conditions, the developed procedure offered an alternative to the time-consuming and relatively expensive high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and gas chromatography mass spectrometry (GC-MS) methods of detection and quantification of these alkaloids.

  4. Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

    PubMed

    Buch, Michael H C; Liaci, A Manuel; O'Hara, Samantha D; Garcea, Robert L; Neu, Ursula; Stehle, Thilo

    2015-10-01

    Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid

  5. Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity

    PubMed Central

    O’Hara, Samantha D.; Garcea, Robert L.; Neu, Ursula; Stehle, Thilo

    2015-01-01

    Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid

  6. Fingerprint Recognition

    DTIC Science & Technology

    2006-06-01

    their central lines. The rule- based algorithm developed for character recognition by Ahmed and Ward (2002) can be applied to a fingerprint image...REFERENCES Ahmed, M., & Ward, R. (2002). A rotation invariant rule- based thinning algorithm for character recognition . IEEE Transactions on Pattern...various steps present in a fingerprint recognition system. The study develops a working algorithm to extract fingerprint minutiae from an input

  7. Structural Determinants of Substrate Recognition in the HAD Superfamily Member D-glycero-D-manno-Heptose-1,7-bisphosphate Phosphatase (GmhB)

    SciTech Connect

    Nguyen, H.; Wang, L; Huang, H; Peisach, E; Dunaway-Mariano, D; Allen, K

    2010-01-01

    The haloalkanoic acid dehalogenase (HAD) enzyme superfamily is the largest family of phosphohydrolases. In HAD members, the structural elements that provide the binding interactions that support substrate specificity are separated from those that orchestrate catalysis. For most HAD phosphatases, a cap domain functions in substrate recognition. However, for the HAD phosphatases that lack a cap domain, an alternate strategy for substrate selection must be operative. One such HAD phosphatase, GmhB of the HisB subfamily, was selected for structure-function analysis. Herein, the X-ray crystallographic structures of Escherichia coli GmhB in the apo form (1.6 {angstrom} resolution), in a complex with Mg{sup 2+} and orthophosphate (1.8 {angstrom} resolution), and in a complex with Mg{sup 2+} and D-glycero-D-manno-heptose 1{beta},7-bisphosphate (2.2 {angstrom} resolution) were determined, in addition to the structure of Bordetella bronchiseptica GmhB bound to Mg{sup 2+} and orthophosphate (1.7 {angstrom} resolution). The structures show that in place of a cap domain, the GmhB catalytic site is elaborated by three peptide inserts or loops that pack to form a concave, semicircular surface around the substrate leaving group. Structure-guided kinetic analysis of site-directed mutants was conducted in parallel with a bioinformatics study of sequence diversification within the HisB subfamily to identify loop residues that serve as substrate recognition elements and that distinguish GmhB from its subfamily counterpart, the histidinol-phosphate phosphatase domain of HisB. We show that GmhB and the histidinol-phosphate phosphatase domain use the same design of three substrate recognition loops inserted into the cap domain yet, through selective residue usage on the loops, have achieved unique substrate specificity and thus novel biochemical function.

  8. Structural determinants of substrate recognition in the HAD superfamily member D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) .

    PubMed

    Nguyen, Henry H; Wang, Liangbing; Huang, Hua; Peisach, Ezra; Dunaway-Mariano, Debra; Allen, Karen N

    2010-02-16

    The haloalkanoic acid dehalogenase (HAD) enzyme superfamily is the largest family of phosphohydrolases. In HAD members, the structural elements that provide the binding interactions that support substrate specificity are separated from those that orchestrate catalysis. For most HAD phosphatases, a cap domain functions in substrate recognition. However, for the HAD phosphatases that lack a cap domain, an alternate strategy for substrate selection must be operative. One such HAD phosphatase, GmhB of the HisB subfamily, was selected for structure-function analysis. Herein, the X-ray crystallographic structures of Escherichia coli GmhB in the apo form (1.6 A resolution), in a complex with Mg(2+) and orthophosphate (1.8 A resolution), and in a complex with Mg(2+) and d-glycero-d-manno-heptose 1beta,7-bisphosphate (2.2 A resolution) were determined, in addition to the structure of Bordetella bronchiseptica GmhB bound to Mg(2+) and orthophosphate (1.7 A resolution). The structures show that in place of a cap domain, the GmhB catalytic site is elaborated by three peptide inserts or loops that pack to form a concave, semicircular surface around the substrate leaving group. Structure-guided kinetic analysis of site-directed mutants was conducted in parallel with a bioinformatics study of sequence diversification within the HisB subfamily to identify loop residues that serve as substrate recognition elements and that distinguish GmhB from its subfamily counterpart, the histidinol-phosphate phosphatase domain of HisB. We show that GmhB and the histidinol-phosphate phosphatase domain use the same design of three substrate recognition loops inserted into the cap domain yet, through selective residue usage on the loops, have achieved unique substrate specificity and thus novel biochemical function.

  9. A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation

    PubMed Central

    Liu, Xin; Zhang, Chen-Song; Lu, Chang; Lin, Sheng-Cai; Wu, Jia-Wei; Wang, Zhi-Xin

    2016-01-01

    Mitogen-activated protein kinases (MAPKs), important in a large array of signalling pathways, are tightly controlled by a cascade of protein kinases and by MAPK phosphatases (MKPs). MAPK signalling efficiency and specificity is modulated by protein–protein interactions between individual MAPKs and the docking motifs in cognate binding partners. Two types of docking interactions have been identified: D-motif-mediated interaction and FXF-docking interaction. Here we report the crystal structure of JNK1 bound to the catalytic domain of MKP7 at 2.4-Å resolution, providing high-resolution structural insight into the FXF-docking interaction. The 285FNFL288 segment in MKP7 directly binds to a hydrophobic site on JNK1 that is near the MAPK insertion and helix αG. Biochemical studies further reveal that this highly conserved structural motif is present in all members of the MKP family, and the interaction mode is universal and critical for the MKP-MAPK recognition and biological function. PMID:26988444

  10. The structure and specificity of the type III secretion system effector NleC suggest a DNA mimicry mechanism of substrate recognition.

    PubMed

    Turco, Michelle Marian; Sousa, Marcelo Carlos

    2014-08-12

    Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn(2+) proteases beyond its conserved HExxH Zn(2+)-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation.

  11. Unraveling a Hotspot for TCR Recognition on HLA-A2: Evidence Against the Existence of Peptide-independent TCR Binding Determinants

    SciTech Connect

    Gagnon, Susan J.; Borbulevych, Oleg Y.; Davis-Harrison, Rebecca L.; Baxter, Tiffany K.; Clemens, John R.; Armstrong, Kathryn M.; Turner, Richard V.; Damirjian, Marale; Biddison, William E.; Baker, Brian M.

    2010-07-19

    T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 {alpha}1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.

  12. A Combined NMR and Computational Approach to Determine the RGDechi-hCit-αv β3 Integrin Recognition Mode in Isolated Cell Membranes.

    PubMed

    Farina, Biancamaria; de Paola, Ivan; Russo, Luigi; Capasso, Domenica; Liguoro, Annamaria; Gatto, Annarita Del; Saviano, Michele; Pedone, Paolo V; Di Gaetano, Sonia; Malgieri, Gaetano; Zaccaro, Laura; Fattorusso, Roberto

    2016-01-11

    The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, αv β3 and αv β5 integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively αv β3 integrin both in vitro and in vivo. High-resolution molecular details of the selective αv β3 recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into αv β3 molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for αv β5 integrin.

  13. Chemical cross-linking of HIV-1 Env for direct TLR7/8 ligand conjugation compromises recognition of conserved antigenic determinants

    PubMed Central

    Feng, Yu; Forsell, Mattias N. E.; Flynn, Barbara; Adams, William; Loré, Karin; Seder, Robert; Wyatt, Richard T.; Karlsson Hedestam, Gunilla B.

    2013-01-01

    Covalent conjugation of immune-stimulatory compounds to protein antigens is a potential means to self-adjuvant non-replicating subunit vaccines. Previously, it was demonstrated that covalent coupling of a Toll-like receptor (TLR) ligand to the exterior HIV-1 envelope glycoprotein, gp120, enhanced its immunogenicity. However, the consequences of chemical conjugation to gp120 on broadly neutralizing antibody (bNAb) epitopes were so far not examined. Here, we conjugated a TLR7/8 ligand to lysine residues on gp120 using NHS-PEO8-maleimide linkers and investigated if this affected Ab recognition of the CD4 binding site (CD4bs), a highly conserved target for bNAbs. We demonstrate that the recognition of the CD4bs was reduced following coupling, especially at a higher coupling ratio. These results have implications for the coupling of ligands to vaccine antigens where elicitation of humoral immune responses to specific neutralizing determinants is desired. PMID:24074567

  14. Critical determinants for substrate recognition and catalysis in the M. tuberculosis class II AP-endonuclease/3'-5' exonuclease III.

    PubMed

    Khanam, Taran; Shukla, Ankita; Rai, Niyati; Ramachandran, Ravishankar

    2015-05-01

    The Mycobacterium tuberculosis AP-endonuclease/3'-5' exodeoxyribonuclease (MtbXthA) is an important player in DNA base excision repair (BER). We demonstrate that the enzyme has robust apurinic/apyrimidinic (AP) endonuclease activity, 3'-5' exonuclease, phosphatase, and phosphodiesterase activities. The enzyme functions as an AP-endonuclease at high ionic environments, while the 3'-5'-exonuclease activity is predominant at low ionic environments. Our molecular modelling and mutational experiments show that E57 and D251 are critical for catalysis. Although nicked DNA and gapped DNA are fair substrates of MtbXthA, the gap-size did not affect the excision activity and furthermore, a substrate with a recessed 3'-end is preferred. To understand the determinants of abasic-site recognition, we examined the possible roles of (i) the base opposite the abasic site, (ii) the abasic ribose ring itself, (iii) local distortions in the AP-site, and (iv) conserved residues located near the active site. Our experiments demonstrate that the first three determinants do not play a role in MtbXthA, and in fact the enzyme exhibits robust endonucleolytic activity against single-stranded AP DNA also. Regarding the fourth determinant, it is known that the catalytic-site of AP endonucleases is surrounded by conserved aromatic residues and intriguingly, the exact residues that are directly involved in abasic site recognition vary with the individual proteins. We therefore, used a combination of mutational analysis, kinetic assays, and structure-based modelling, to identify that Y237, supported by Y137, mediates the formation of the MtbXthA-AP-DNA complex and AP-site incision.

  15. Palindromic oligonucleotides containing 7-deaza-2'-deoxyguanosine: solid-phase synthesis of d[(p)GG*AATTCC] octamers and recognition by the endodeoxyribonuclease EcoRI.

    PubMed Central

    Seela, F; Driller, H

    1986-01-01

    Octadeoxynucleotides with the sequence d[(p)GG*AATTCC] have been prepared by solid-phase synthesis employing regular and base-modified phosphoramidites. These oligomers which contain an isosterically altered recognition sequence of the endodeoxyribonuclease Eco RI form duplexes under appropriate salt conditions. Since G* can represent 7-deaza-2'-deoxyguanosine the oligomers were used as probes to study their cleavage by the endodeoxyribonuclease Eco RI. The enzymatic hydrolysis of the modified octamer was strongly decreased compared to the regular DNA-fragment. This shows that guanine N-7 located at the cleavage site is important for the recognition process by the enzyme. The residual enzymatic activity is discussed on the basis of reduced specificity towards the recognition fragment. The fact that this cleavage occurs already under regular conditions indicates that the process described here bases on an intrinsic property of the oligomer and is different from the star activity. PMID:3008089

  16. Latent ClpX-recognition signals ensure LexA destruction after DNA damage

    PubMed Central

    Neher, Saskia B.; Flynn, Julia M.; Sauer, Robert T.; Baker, Tania A.

    2003-01-01

    The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein. Intact LexA is stable to intracellular degradation but its auto-cleavage fragments are degraded rapidly. Here, both fragments of LexA are shown to be substrates for the ClpXP protease. ClpXP recognizes these fragments using sequence motifs that flank the auto-cleavage site but are dormant in intact LexA. Furthermore, ClpXP degradation of the LexA-DNA-binding fragment is important to cell survival after DNA damage. These results demonstrate how one protein-processing event can activate latent protease recognition signals, triggering a cascade of protein turnover in response to environmental stress. PMID:12730132

  17. Molecular recognition in a propazine-imprinted polymer and its application to the determination of triazines in environmental samples.

    PubMed

    Turiel, E; Martin-Esteban, A; Fernández, P; Pérez-Conde, C; Cámara, C

    2001-11-01

    An analytical methodology for the determination of triazines in environmental samples incorporating a molecularly imprinted solid-phase extraction (MISPE) process using a propazine-imprinted polymer was developed. Two different polymers were prepared using acetonitrile or toluene as porogen, and their optimum loading, washing, and elution conditions were established. Although both polymers were able to recognize several chlorotriazines (propazine, atrazine, simazine, desethylatrazine, and desisopropylatrazine), the polymer prepared in toluene showed the best performance and was also capable of recognizing a methylthiotriazine (prometryn). A binding study carried out in this polymer demonstrated that it possesses heterogeneous binding sites with different binding abilities. From this study, it was also concluded that desethylatrazine and desisopropylatrazine displace the other triazines at high concentrations, including the template molecule. The accuracy and selectivity of the MISPE process developed was verified using a certified reference material for drinking water containing atrazine and simazine among other commonly used pesticides. Finally, the MISPE procedure was successfully applied to the cleanup of drinking and groundwater, soil, and corn sample extracts, and the triazines were determined by micellar electrokinetic chromatography.

  18. Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

    PubMed Central

    Kaufmann, Kristian W.; Dawson, Eric S.; Henry, L. Keith; Field, Julie R.; Blakely, Randy D.; Meiler, Jens

    2009-01-01

    To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuTAa) structure reported by Yamashita et al. (Nature 2005;437:215–223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuTAa is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to cHitically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuTAa structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 Å of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected. PMID:18704946

  19. Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies.

    PubMed

    Kaufmann, Kristian W; Dawson, Eric S; Henry, L Keith; Field, Julie R; Blakely, Randy D; Meiler, Jens

    2009-02-15

    To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuT(Aa)) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT(Aa) is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to critically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT(Aa) structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 A of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected.

  20. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  1. Cleavage sites within the poliovirus capsid protein precursors

    SciTech Connect

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.

  2. von Willebrand factor storage requires intact prosequence cleavage site.

    PubMed

    Journet, A M; Saffaripour, S; Cramer, E M; Tenza, D; Wagner, D D

    1993-02-01

    Large multimers of the adhesive glycoprotein von Willebrand factor (vWf) are stored in endothelial cells in rod-shaped granules called Weibel-Palade bodies, while small multimers are secreted constitutively. Expression of pro-vWf in other cells with a regulated pathway of secretion, results in formation of vWf-containing storage granules that have a morphology similar to Weibel-Palade bodies. vWf expressed without its prosequence is not stored. To evaluate the importance of prosequence cleavage in vWf storage, the Arg at position -1, known to be necessary for cleavage, was mutated to Gly. Transfection of this cleavage mutant into two cell lines with a regulated pathway of secretion (RIN 5F and AtT-20 cells) led to the formation of large multimers. However, treatment of the cell lysates by the enzyme endoglycosidase H (Endo-H) did not reveal significant amounts of intracellular Endo-H-resistant vWf, which indicates the absence of a pool of stored processed vWf. In addition, no Weibel-Palade body-like structure was detected in these cells by immunofluorescence labeling with anti-vWf antiserum. Electron microscopy and immunocytochemistry of RIN 5F cells expressing the pro-vWf mutant confirmed the absence of Weibel-Palade body-like structures. In addition, anti-vWf-linked gold particles were found in the ER, occasionally in rounded granules and particularly in lysosomal structures which were abundant. We conclude that the formation of large aggregates is not sufficient to induce efficient vWf storage, and that the lack of cleavage of the prosequence may direct the mutant pro-vWf molecule to a degradative pathway. Therefore, the prosequence cleavage is a requirement for vWf storage.

  3. Recognition memory for faces: when familiarity supports associative recognition judgments.

    PubMed

    Yonelinas, A P; Kroll, N E; Dobbins, I G; Soltani, M

    1999-12-01

    Recognition memory for single items can be dissociated from recognition memory for the associations between items. For example, recognition tests for single words produce curvilinear receiver operating characteristics (ROCs), but associative recognition tests for word pairs produce linear ROCs. These dissociations are consistent with dual-process theories of recognition and suggest that associative recognition relies on recollection but that item recognition relies on a combination of recollection and assessments of familiarity. In the present study, we examined associative recognition ROCs for facial stimuli by manipulating the central and external features, in order to determine whether linear ROCs would be observed for stimuli other than arbitrary word pairs. When the faces were presented upright, familiarity estimates were significantly above zero, and the associative ROCs were curvilinear, suggesting that familiarity contributed to associative judgments. However, presenting the faces upside down effectively eliminated the contribution of familiarity to associative recognition, and the ROCs were linear. The results suggest that familiarity can support associative recognition judgments, if the associated components are encoded as a coherent gestalt, as in upright faces.

  4. Genetic specificity of face recognition.

    PubMed

    Shakeshaft, Nicholas G; Plomin, Robert

    2015-10-13

    Specific cognitive abilities in diverse domains are typically found to be highly heritable and substantially correlated with general cognitive ability (g), both phenotypically and genetically. Recent twin studies have found the ability to memorize and recognize faces to be an exception, being similarly heritable but phenotypically substantially uncorrelated both with g and with general object recognition. However, the genetic relationships between face recognition and other abilities (the extent to which they share a common genetic etiology) cannot be determined from phenotypic associations. In this, to our knowledge, first study of the genetic associations between face recognition and other domains, 2,000 18- and 19-year-old United Kingdom twins completed tests assessing their face recognition, object recognition, and general cognitive abilities. Results confirmed the substantial heritability of face recognition (61%), and multivariate genetic analyses found that most of this genetic influence is unique and not shared with other cognitive abilities.

  5. Genetic specificity of face recognition

    PubMed Central

    Shakeshaft, Nicholas G.; Plomin, Robert

    2015-01-01

    Specific cognitive abilities in diverse domains are typically found to be highly heritable and substantially correlated with general cognitive ability (g), both phenotypically and genetically. Recent twin studies have found the ability to memorize and recognize faces to be an exception, being similarly heritable but phenotypically substantially uncorrelated both with g and with general object recognition. However, the genetic relationships between face recognition and other abilities (the extent to which they share a common genetic etiology) cannot be determined from phenotypic associations. In this, to our knowledge, first study of the genetic associations between face recognition and other domains, 2,000 18- and 19-year-old United Kingdom twins completed tests assessing their face recognition, object recognition, and general cognitive abilities. Results confirmed the substantial heritability of face recognition (61%), and multivariate genetic analyses found that most of this genetic influence is unique and not shared with other cognitive abilities. PMID:26417086

  6. Conjoint Recognition.

    ERIC Educational Resources Information Center

    Brainerd, C. J.; Reyna, V. F.; Mojardin, A. H.

    1999-01-01

    Reviews some limiting properties of the process-dissociation model as it applies to the study of dual-process conceptions of memory. A second-generation model (conjoint recognition) is proposed to address these limitations and supply additional capabilities. Worked applications to data are provided. (Author/GCP)

  7. Androgenic control of male-typical behavior, morphology and sex recognition is independent of the mode of sex determination: A case study on Lichtenfelder's gecko (Eublepharidae: Goniurosaurus lichtenfelderi).

    PubMed

    Golinski, Alison; Kubička, Lukáš; John-Alder, Henry; Kratochvíl, Lukáš

    2015-06-01

    Previous work on lizards has shown that many sexually dimorphic traits depend on testosterone (T), but the details of this control can vary among species. Here, we tested the role of T on the expression of morphological, physiological, and behavioral traits in Lichtenfelder's gecko (Goniurosaurus lichtenfelderi), from the lizard family Eublepharidae notable for interspecific variation in sexually dimorphic traits and the mode of sex determination. Experiments included three groups of males (intact control, surgically castrated, castrated with T replacement) and two groups of females (intact control, T supplemented). In males, castration caused reductions in 1) the size of hemipenes, 2) offensive aggression, 3) male sexual behavior in a neutral arena, 4) activity of precloacal glands, and 5) loss of male chemical cues for sex recognition. These reductions were not observed in castrated males with T replacement. Interestingly, castrated males performed sexual behavior in their home cages, which shows that the effect of T depends on the environmental context. Notably, tail vibration, previously reported as a courtship behavior in other eublepharids, is displayed by males of G. lichtenfelderi during interactions with conspecifics of both sexes, suggesting an evolutionary shift in the meaning of this signal. In females, T induced growth of hemipenes and male-typical courtship but did not induce precloacal pore activity, aggression, or mounting. In comparison to previous reports on Eublepharis macularius, our results indicate that effects of T do not depend on the mode of sex determination. Further, our results extend our understanding of the complexity of control of male traits and illustrate how lability in the effects of T can be a general mechanism causing evolutionary changes in the components of suites of functionally correlated traits. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Pocket 4 of the HLA-DR(alpha,beta 1*0401) molecule is a major determinant of T cells recognition of peptide

    PubMed Central

    1995-01-01

    To investigate the functional roles of individual HLA-DR residues in T cell recognition, transfectants expressing wild-type or mutant DR(alpha,beta 1*0401) molecules with single amino acid substitutions at 14 polymorphic positions of the DR beta 1*0401 chain or 19 positions of the DR alpha chain were used as antigen-presenting cells for five T cell clones specific for the influenza hemagglutinin peptide, HA307-19. Of the six polymorphic positions in the DR beta floor that were examined, mutations at only two positions eliminated T cell recognition: positions 13 (four clones) and 28 (one clone). In contrast, individual mutations at DR beta positions 70, 71, 78, and 86 on the alpha helix eliminated recognition by each of the clones, and mutations at positions 74 and 67 eliminated recognition by four and two clones, respectively. Most of the DR alpha mutations had minimal or no effect on most of the clones, although one clone was very sensitive to changes in the DR alpha chain, with loss of recognition in response to 10 mutants. Mutants that abrogated recognition by all of the clones were assessed for peptide binding, and only the beta 86 mutation drastically decreased peptide binding. Single amino acid substitutions at polymorphic positions in the central part of the DR beta alpha helix disrupted T cell recognition much more frequently than substitutions in the floor, suggesting that DR beta residues on the alpha helix make relatively greater contributions than those in the floor to the ability of the DR(alpha,beta 1*0401) molecule to present HA307-19. The data indicate that DR beta residues 13, 70, 71, 74, and 78, which are located in pocket 4 of the peptide binding site in the crystal structure of the DR1 molecule, exert a major and disproportionate influence on the outcome of T cell recognition, compared with other polymorphic residues. PMID:7869051

  9. Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Brunzelle, Joseph; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-03-27

    Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

  10. Graphene sheet-starch platform based on the groove recognition for the sensitive and highly selective determination of iodide in seafood samples.

    PubMed

    Liu, Shou-Qing; Hu, Feng-Tian; Liu, Cheng-Bao; Chen, Feng; Wu, Zheng-Ying; Liang, Zuo-Qin; Xu, Nan; Chen, Zhi-Gang

    2013-09-15

    The functionalization of graphene nanosheets was realized using a simple starch mixture to achieve a highly selective recognition of iodide, thereby surmounting the complicated reactions possibly leading to low yield during functionalization. The groove recognition for starch to iodide, a novel recognition model, was established. The starch-to-graphene nanosheet mass ratio of 3:2 produced an optimal current signal. The recognition and measurement procedures were conducted in different cells, respectively. These procedures improved the selectivity and sensitivity, and overcame the possibility of interference from coexisting ions. Under optimal conditions, the graphene sheet-starch electrode was immersed in a recognition cell at pH 2.0 for 10min, afterward, in a measurement cell at pH 1.0 for quantitative analysis, resulting in the highest current signals obtained. The quantitative electrochemical measurements yielded a mean value of 214.6mg/kg in actual samples of commercially available seafood sample, whereas the spectrophotometric measurements produced a mean value of 226.7mg/kg. If the spectrophotometric value for the seafood sample is accurate, the percentage error for the electrochemical method is only 5.3%. Therefore, the electrochemical method is reliable for qualitative iodide measurements. The groove recognition was highlighted to elucidate the specific selectivity.

  11. Conjoint recognition.

    PubMed

    Brainerd, C J; Reyna, V F; Mojardin, A H

    1999-01-01

    The process-dissociation model has stimulated important advances in the study of dual-process conceptions of memory. The authors review some limiting properties of that model and consider the degree of support for its parent theory (the recollection-familiarity distinction). A 2nd-generation model (conjoint recognition) is proposed that addresses these limitations and supplies additional capabilities, such as goodness-of-fit tests, the ability to measure dual processes for false-memory responses, and statistical procedures for testing within- and between-conditions hypotheses about its parameters. The conjoint-recognition model also implements an alternative theoretical interpretation (the identity-similarity distinction of fuzzy-trace theory). Worked applications to data are provided.

  12. Andes virus recognition of human and Syrian hamster beta3 integrins is determined by an L33P substitution in the PSI domain.

    PubMed

    Matthys, Valery S; Gorbunova, Elena E; Gavrilovskaya, Irina N; Mackow, Erich R

    2010-01-01

    Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human alpha(v)beta(3) integrins are receptors for several pathogenic hantaviruses, and the function of alpha(v)beta(3) integrins on endothelial cells suggests a role for alpha(v)beta(3) in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity alpha(v)beta(3) integrin ligand vitronectin and by antibodies to alpha(v)beta(3) integrins. Further, antibodies to the beta(3) integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster beta(3) subunits, but not murine or bovine beta(3), inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with beta(3) subunits through PSI domain residues conserved in both Syrian hamster and human beta(3) integrins. Sequencing the Syrian hamster beta(3) integrin PSI domain revealed eight differences between Syrian hamster and human beta(3) integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine beta(3) integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human beta(3) PSI domain to contain the L33P substitution present in bovine beta(3) integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human beta(3) permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine beta(3) integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster alpha(v)beta(3) integrins are key receptors for ANDV and that specific residues within the

  13. Structural Insights into Substrate Recognition by Clostridium difficile Sortase

    PubMed Central

    Yin, Jui-Chieh; Fei, Chun-Hsien; Lo, Yen-Chen; Hsiao, Yu-Yuan; Chang, Jyun-Cyuan; Nix, Jay C.; Chang, Yuan-Yu; Yang, Lee-Wei; Huang, I-Hsiu; Wang, Shuying

    2016-01-01

    Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26–PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB. PMID:27921010

  14. Determination of the Processing Sites of an Arabidopsis 2S Albumin and Characterization of the Complete Gene Family.

    PubMed

    Krebbers, E; Herdies, L; De Clercq, A; Seurinck, J; Leemans, J; Van Damme, J; Segura, M; Gheysen, G; Van Montagu, M; Vandekerckhove, J

    1988-08-01

    The most abundant isoform of the 2S albumin present in seeds of Arabidopsis thaliana has been sequenced and the corresponding gene isolated. Examination of the protein and DNA sequences allows the determination of the exact proteolytic cleavage sites during posttranslational processing. Like other 2S albumins, that of Arabidopsis is made as a prepropeptide. After removal of the signal peptide, the propeptide is cleaved at four other points, giving two subunits linked by a disulfide bridge(s). Comparison of these cleavage sites with those of 2S albumins of Brassica napus and Bertholletia excelsa suggests that while individual cleavage sites between species are conserved, the four processing sites within a species are not similar, suggesting that up to four different proteases may be involved in processing 2S albumins. The Arabidopsis 2S albumin gene was used to isolate the entire gene family. There are four genes, tightly linked in a tandem array. None of the genes contains an intron. Comparison of the predicted protein sequences shows that only one of the genes can encode the isoform determined by protein analysis to be the most abundant, and therefore this gene is certain to be expressed. It is possible that some or all of the other three genes are also active.

  15. Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

    PubMed Central

    Enuameh, Metewo Selase; Asriyan, Yuna; Richards, Adam; Christensen, Ryan G.; Hall, Victoria L.; Kazemian, Majid; Zhu, Cong; Pham, Hannah; Cheng, Qiong; Blatti, Charles; Brasefield, Jessie A.; Basciotta, Matthew D.; Ou, Jianhong; McNulty, Joseph C.; Zhu, Lihua J.; Celniker, Susan E.; Sinha, Saurabh; Stormo, Gary D.; Brodsky, Michael H.; Wolfe, Scot A.

    2013-01-01

    Cys2-His2 zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes. PMID:23471540

  16. The chemoenzymatic synthesis of clofarabine and related 2'-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases.

    PubMed

    Fateev, Ilja V; Antonov, Konstantin V; Konstantinova, Irina D; Muravyova, Tatyana I; Seela, Frank; Esipov, Roman S; Miroshnikov, Anatoly I; Mikhailopulo, Igor A

    2014-01-01

    Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (1, clofarabine) were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, (2F)Ara-1P) via three step conversion of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (9) into the phosphate 12a without isolation of intermediary products. Condensation of 12a with 2-chloroadenine catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of clofarabine in 67% yield. The reaction was also studied with a number of purine bases (2-aminoadenine and hypoxanthine), their analogues (5-aza-7-deazaguanine and 8-aza-7-deazahypoxanthine) and thymine. The results were compared with those of a similar reaction with α-D-arabinofuranose-1-phosphate (13a, Ara-1P). Differences of the reactivity of various substrates were analyzed by ab initio calculations in terms of the electronic structure (natural purines vs analogues) and stereochemical features ((2F)Ara-1P vs Ara-1P) of the studied compounds to determine the substrate recognition by E. coli nucleoside phosphorylases. The second approach starts with the cascade one-pot enzymatic transformation of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a, followed by its condensation with 2-chloroadenine thereby affording clofarabine in ca. 48% yield in 24 h. The following recombinant E. coli enzymes catalyze the sequential conversion of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a: ribokinase (2-deoxy-2-fluoro-D-arabinofuranose-5-phosphate), phosphopentomutase (PPN; no 1,6-diphosphates of D-hexoses as co-factors required) (12a), and finally PNP. The substrate activities of D-arabinose, D-ribose and D-xylose in the similar cascade syntheses of the relevant 2-chloroadenine nucleosides were studied and compared with the activities of 2-deoxy-2-fluoro-D-arabinose. As expected, D-ribose exhibited the best substrate activity

  17. Highly conserved regions within the spike proteins of human coronaviruses 229E and NL63 determine recognition of their respective cellular receptors.

    PubMed

    Hofmann, Heike; Simmons, Graham; Rennekamp, Andrew J; Chaipan, Chawaree; Gramberg, Thomas; Heck, Elke; Geier, Martina; Wegele, Anja; Marzi, Andrea; Bates, Paul; Pöhlmann, Stefan

    2006-09-01

    We have recently demonstrated that the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor angiotensin converting enzyme 2 (ACE2) also mediates cellular entry of the newly discovered human coronavirus (hCoV) NL63. Here, we show that expression of DC-SIGN augments NL63 spike (S)-protein-driven infection of susceptible cells, while only expression of ACE2 but not DC-SIGN is sufficient for entry into nonpermissive cells, indicating that ACE2 fulfills the criteria of a bona fide hCoV-NL63 receptor. As for SARS-CoV, murine ACE2 is used less efficiently by NL63-S for entry than human ACE2. In contrast, several amino acid exchanges in human ACE2 which diminish SARS-S-driven entry do not interfere with NL63-S-mediated infection, suggesting that SARS-S and NL63-S might engage human ACE2 differentially. Moreover, we observed that NL63-S-driven entry was less dependent on a low-pH environment and activity of endosomal proteases compared to infection mediated by SARS-S, further suggesting differences in hCoV-NL63 and SARS-CoV cellular entry. NL63-S does not exhibit significant homology to SARS-S but is highly related to the S-protein of hCoV-229E, which enters target cells by engaging CD13. Employing mutagenic analyses, we found that the N-terminal unique domain in NL63-S, which is absent in 229E-S, does not confer binding to ACE2. In contrast, the highly homologous C-terminal parts of the NL63-S1 and 229E-S1 subunits in conjunction with distinct amino acids in the central regions of these proteins confer recognition of ACE2 and CD13, respectively. Therefore, despite the high homology of these sequences, they likely form sufficiently distinct surfaces, thus determining receptor specificity.

  18. Recognition intent and visual word recognition.

    PubMed

    Wang, Man-Ying; Ching, Chi-Le

    2009-03-01

    This study adopted a change detection task to investigate whether and how recognition intent affects the construction of orthographic representation in visual word recognition. Chinese readers (Experiment 1-1) and nonreaders (Experiment 1-2) detected color changes in radical components of Chinese characters. Explicit recognition demand was imposed in Experiment 2 by an additional recognition task. When the recognition was implicit, a bias favoring the radical location informative of character identity was found in Chinese readers (Experiment 1-1), but not nonreaders (Experiment 1-2). With explicit recognition demands, the effect of radical location interacted with radical function and word frequency (Experiment 2). An estimate of identification performance under implicit recognition was derived in Experiment 3. These findings reflect the joint influence of recognition intent and orthographic regularity in shaping readers' orthographic representation. The implication for the role of visual attention in word recognition was also discussed.

  19. Individual recognition between mother and infant bats (Myotis)

    NASA Technical Reports Server (NTRS)

    Turner, D.; Shaughnessy, A.; Gould, E.

    1972-01-01

    The recognition process and the basis for that recognition, in brown bats, between mother and infant are analyzed. Two parameters, ultrasonic communication and olfactory stimuli, are investigated. The test animals were not allowed any visual contact. It was concluded that individual recognition between mother and infant occurred. However, it could not be determined if the recognition was based on ultrasonic signals or olfactory stimuli.

  20. Recognition Tunneling

    PubMed Central

    Lindsay, Stuart; He, Jin; Sankey, Otto; Hapala, Prokop; Jelinek, Pavel; Zhang, Peiming; Chang, Shuai; Huang, Shuo

    2010-01-01

    Single molecules in a tunnel junction can now be interrogated reliably using chemically-functionalized electrodes. Monitoring stochastic bonding fluctuations between a ligand bound to one electrode and its target bound to a second electrode (“tethered molecule-pair” configuration) gives insight into the nature of the intermolecular bonding at a single molecule-pair level, and defines the requirements for reproducible tunneling data. Simulations show that there is an instability in the tunnel gap at large currents, and this results in a multiplicity of contacts with a corresponding spread in the measured currents. At small currents (i.e. large gaps) the gap is stable, and functionalizing a pair of electrodes with recognition reagents (the “free analyte” configuration) can generate a distinct tunneling signal when an analyte molecule is trapped in the gap. This opens up a new interface between chemistry and electronics with immediate implications for rapid sequencing of single DNA molecules. PMID:20522930

  1. Pattern Recognition Control Design

    NASA Technical Reports Server (NTRS)

    Gambone, Elisabeth A.

    2018-01-01

    Spacecraft control algorithms must know the expected vehicle response to any command to the available control effectors, such as reaction thrusters or torque devices. Spacecraft control system design approaches have traditionally relied on the estimated vehicle mass properties to determine the desired force and moment, as well as knowledge of the effector performance to efficiently control the spacecraft. A pattern recognition approach was used to investigate the relationship between the control effector commands and spacecraft responses. Instead of supplying the approximated vehicle properties and the thruster performance characteristics, a database of information relating the thruster ring commands and the desired vehicle response was used for closed-loop control. A Monte Carlo simulation data set of the spacecraft dynamic response to effector commands was analyzed to establish the influence a command has on the behavior of the spacecraft. A tool developed at NASA Johnson Space Center to analyze flight dynamics Monte Carlo data sets through pattern recognition methods was used to perform this analysis. Once a comprehensive data set relating spacecraft responses with commands was established, it was used in place of traditional control methods and gains set. This pattern recognition approach was compared with traditional control algorithms to determine the potential benefits and uses.

  2. Pattern Recognition Control Design

    NASA Technical Reports Server (NTRS)

    Gambone, Elisabeth

    2016-01-01

    Spacecraft control algorithms must know the expected spacecraft response to any command to the available control effectors, such as reaction thrusters or torque devices. Spacecraft control system design approaches have traditionally relied on the estimated vehicle mass properties to determine the desired force and moment, as well as knowledge of the effector performance to efficiently control the spacecraft. A pattern recognition approach can be used to investigate the relationship between the control effector commands and the spacecraft responses. Instead of supplying the approximated vehicle properties and the effector performance characteristics, a database of information relating the effector commands and the desired vehicle response can be used for closed-loop control. A Monte Carlo simulation data set of the spacecraft dynamic response to effector commands can be analyzed to establish the influence a command has on the behavior of the spacecraft. A tool developed at NASA Johnson Space Center (Ref. 1) to analyze flight dynamics Monte Carlo data sets through pattern recognition methods can be used to perform this analysis. Once a comprehensive data set relating spacecraft responses with commands is established, it can be used in place of traditional control laws and gains set. This pattern recognition approach can be compared with traditional control algorithms to determine the potential benefits and uses.

  3. Recognition of Teaching Excellence*

    PubMed Central

    Piascik, Peggy; Medina, Melissa; Pittenger, Amy; Rose, Renee; Creekmore, Freddy; Soltis, Robert; Bouldin, Alicia; Schwarz, Lindsay; Scott, Steven

    2010-01-01

    The 2008-2009 Task Force for the Recognition of Teaching Excellence was charged by the AACP Council of Faculties Leadership to examine teaching excellence by collecting best practices from colleges and schools of pharmacy, evaluating the literature to identify evidence-based criteria for excellent teaching, and recommending appropriate means to acknowledge and reward teaching excellence. This report defines teaching excellence and discusses a variety of ways to assess it, including student, alumni, peer, and self-assessment. The task force identifies important considerations that colleges and schools must address when establishing teaching recognition programs including the purpose, criteria, number and mix of awards, frequency, type of award, and method of nominating and determining awardees. The report concludes with recommendations for the academy to consider when establishing and revising teaching award programs. PMID:21301598

  4. Recognition of teaching excellence.

    PubMed

    Hammer, Dana; Piascik, Peggy; Medina, Melissa; Pittenger, Amy; Rose, Renee; Creekmore, Freddy; Soltis, Robert; Bouldin, Alicia; Schwarz, Lindsay; Scott, Steven

    2010-11-10

    The 2008-2009 Task Force for the Recognition of Teaching Excellence was charged by the AACP Council of Faculties Leadership to examine teaching excellence by collecting best practices from colleges and schools of pharmacy, evaluating the literature to identify evidence-based criteria for excellent teaching, and recommending appropriate means to acknowledge and reward teaching excellence. This report defines teaching excellence and discusses a variety of ways to assess it, including student, alumni, peer, and self-assessment. The task force identifies important considerations that colleges and schools must address when establishing teaching recognition programs including the purpose, criteria, number and mix of awards, frequency, type of award, and method of nominating and determining awardees. The report concludes with recommendations for the academy to consider when establishing and revising teaching award programs.

  5. Phrasal recognition.

    PubMed

    Farhadi, Ali; Sadeghi, Mohammad Amin

    2013-12-01

    In this paper, we introduce visual phrases, complex visual composites like "a person riding a horse." Visual phrases often display significantly reduced visual complexity compared to their component objects because the appearance of those objects can change profoundly when they participate in relations. We introduce a dataset suitable for phrasal recognition that uses familiar PASCAL object categories, and demonstrate significant experimental gains resulting from exploiting visual phrases. We show that a visual phrase detector significantly outperforms a baseline which detects component objects and reasons about relations, even though visual phrase training sets tend to be smaller than those for objects. We argue that any multiclass detection system must decode detector outputs to produce final results; this is usually done with nonmaximum suppression. We describe a novel decoding procedure that can account accurately for local context without solving difficult inference problems. We show this decoding procedure outperforms the state of the art. Finally, we show that decoding a combination of phrasal and object detectors produces real improvements in detector results.

  6. Proteolytic Characteristics of Cathepsin D Related to the Recognition and Cleavage of Its Target Proteins

    PubMed Central

    Sun, Huiying; Lou, Xiaomin; Shan, Qiang; Zhang, Ju; Zhu, Xu; Zhang, Jia; Wang, Yang; Xie, Yingying; Xu, Ningzhi; Liu, Siqi

    2013-01-01

    Cathepsin D (CD) plays an important role in both biological and pathological processes, although the cleavage characteristics and substrate selection of CD have yet to be fully explored. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the CD cleavage sites in bovine serum albumin (BSA). We found that the hydrophobic residues at P1 were not only a preferential factor for CD cleavage but that the hydrophobicity at P1’ also contributed to CD recognition. The concept of hydrophobic scores of neighbors (HSN) was proposed to describe the hydrophobic microenvironment of CD recognition sites. The survey of CD cleavage characteristics in several proteins suggested that the HSN was a sensitive indicator for judging the favorable sites in peptides for CD cleavage, with HSN values of 0.5–1.0 representing a likely threshold. Ovalbumin (OVA), a protein resistant to CD cleavage in its native state, was easily cleaved by CD after denaturation, and the features of the cleaved peptides were quite similar to those found in BSA, where a higher HSN value indicated greater cleavability. We further conducted two-dimensional gel electrophoresis (2DE) to find more proteins that were insensitive to CD cleavage in CD-knockdown cells. Based on an analysis of secondary and three-dimensional structures, we postulated that intact proteins with a structure consisting of all α-helices would be relatively accessible to CD cleavage. PMID:23840360

  7. Base-pair opening dynamics of primary miR156a using NMR elucidates structural determinants important for its processing level and leaf number phenotype in Arabidopsis.

    PubMed

    Kim, Wanhui; Kim, Hee-Eun; Lee, Ae-Ree; Jun, A Rim; Jung, Myeong Gyo; Ahn, Ji Hoon; Lee, Joon-Hwa

    2017-01-25

    MicroRNAs originate from primary transcripts containing hairpin structures. The levels of mature miR156 influence the leaf number prior to flowering in the life cycle of plants. To understand the molecular mechanism of biogenesis of primary miR156a (pri-miR156a) to mature miR156, a base-pair opening dynamics study was performed using model RNAs mimicking the cleavage site of wild type and B5 bulge-stabilizing mutant pri-miR156a constructs. We also determined the mature miR156 levels and measured leaf numbers at flowering of plants overexpressing the wild type and mutant constructs. Our results suggest that the stabilities and/or opening dynamics of the C15·G98 and U16·A97 base-pairs at the cleavage site are essential for formation of the active conformation and for efficient processing of pri-miR156a, and that mutations of the B5 bulge can modulate mature miR156 levels as well as miR156-driven leaf number phenotypes via changes in the base-pair stability of the cleavage site. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Base-pair opening dynamics of primary miR156a using NMR elucidates structural determinants important for its processing level and leaf number phenotype in Arabidopsis

    PubMed Central

    Kim, Wanhui; Kim, Hee-Eun; Lee, Ae-Ree; Jun, A Rim; Jung, Myeong Gyo; Ahn, Ji Hoon; Lee, Joon-Hwa

    2017-01-01

    MicroRNAs originate from primary transcripts containing hairpin structures. The levels of mature miR156 influence the leaf number prior to flowering in the life cycle of plants. To understand the molecular mechanism of biogenesis of primary miR156a (pri-miR156a) to mature miR156, a base-pair opening dynamics study was performed using model RNAs mimicking the cleavage site of wild type and B5 bulge-stabilizing mutant pri-miR156a constructs. We also determined the mature miR156 levels and measured leaf numbers at flowering of plants overexpressing the wild type and mutant constructs. Our results suggest that the stabilities and/or opening dynamics of the C15·G98 and U16·A97 base-pairs at the cleavage site are essential for formation of the active conformation and for efficient processing of pri-miR156a, and that mutations of the B5 bulge can modulate mature miR156 levels as well as miR156-driven leaf number phenotypes via changes in the base-pair stability of the cleavage site. PMID:27574118

  9. Infrared target recognition

    NASA Astrophysics Data System (ADS)

    Singstock, Brian D.

    1991-12-01

    In this thesis, three approaches were used for Automatic Target Recognition (ATR). These approaches were shape, moment and Fourier generated features, Karhunen-Loeve Transform (KLT) generated features and Discrete Cosine Transform (DCT) generated features. The KLT approach was modelled after the face recognition research by Suarez, AFIT, and Turk and Pentland, MIT. A KLT is taken of a reduced covariance matrix, composed all three classes of targets, and the resulting eigenimages are used to reconstruct the original images. The reconstruction coefficients for each original image are found by taking the dot product of the original image with each eigenimage. These reconstruction coefficients were implemented as features into a three layer backprop with momentum network. Using the hold one-cut-out technique of testing data, the net could correctly differentiate the targets 100 percent of the time. Using standard features, the correct classification rate was 99.33 percent. The DCT was also taken of each image, and 16 low frequency Fourier components were kept as features. These recognition rates were compared to FFT results where each set contained the top five feature, as determined by a saliency test. The results proved that the DCT and the FFT were equivalent concerning classification of targets.

  10. Structural requirements for recognition of the HLA-Dw14 class II epitope: A key HLA determinant associated with rheumatoid arthritis

    SciTech Connect

    Hiraiwa, Akikazu; Yamanaka, Katsuo; Kwok, W.W.; Nepom, G.T. ); Mickelson, E.M.; Masewicz, S.; Hansen, J.A. ); Radka, S.F. )

    1990-10-01

    Although HLA genes have been shown to be associated with certain diseases, the basis for this association is unknown. Recent studies, however, have documented patterns of nucleotide sequence variation among some HLA genes associated with a particular disease. For rheumatoid arthritis, HLA genes in most patients have a shared nucleotide sequence encoding a key structural element of an HLA class II polypeptide; this sequence element is critical for the interaction of the HLA molecule with antigenic peptides and with responding T cells, suggestive of a direct role for this sequence element in disease susceptibility. The authors describe the serological and cellular immunologic characteristics encoded by this rheumatoid arthritis-associated sequence element. Site-directed mutagenesis of the DRB1 gene was used to define amino acids critical for antibody and T-cell recognition of this structural element, focusing on residues that distinguish the rheumatoid arthritis-associated alleles Dw4 and Dw14 from a closely related allele, Dw10, not associated with disease. Both the gain and loss of rheumatoid arthritis-associated epitopes were highly dependent on three residues within a discrete domain of the HLA-DR molecule. Recognition was most strongly influenced by the following amino acids (in order): 70 > 71 > 67. Some alloreactive T-cell clones were also influenced by amino acid variation in portions of the DR molecule lying outside the shared sequence element.

  11. Determination of multiple elements in samples of the medicinal plant Marsdenia tenacissima and estimation of geographic origin via pattern recognition techniques.

    PubMed

    Li, Chao; Guo, Qiao-sheng; Yang, Sheng-chao; Zheng, Kai-yan; Li, Wang-ping; Meng, Zhen-gui; Xu, Xiang-zeng

    2015-01-01

    Multi-element analysis of the medicinal plant Marsdenia tenacissima was used to develop a reliable method of tracing the geographical source of the samples. The concentrations of 27 elements in 128 samples from 4 provinces in China were analyzed by inductively coupled plasma-atomic emission spectroscopy. Pattern recognition techniques, viz. principal component analysis (PCA), cluster analysis (CA), stepwise linear discriminant analysis (SLDA) and k-nearest neighbor analysis (KNN), were used for this purpose. It was verified that 21 elements in the M. tenacissima samples from different regions showed significant differences (P < 0.05). The PCA explained 87.36 % of the variance with the first seven principal component variables, and a score plot produced from the largest three principal components showed that the source area of most samples could be correctly distinguished. The CA showed that samples were separated into three clusters. The SLDA produced an overall correct classification rate of 87.5 % and a cross-validation rate of 85.2 %. The KNN analysis performed ideally, with an average identification rate of 100 % for the training set and 93.33 % for the test set. These results laid the foundation for the application of multi-element analysis combined with pattern recognition techniques for tracing the geographical origin of samples of medicinal plants.

  12. A novel and sensitive method for recognition and indirect determination of Al III in biological fluid based on the quenching of resonance Rayleigh scattering intensities of "Al III-EV-DNA" complexing system

    NASA Astrophysics Data System (ADS)

    Long, Xiufen; Li, Desheng; Wang, Na; Zhang, Caihua; Cao, Qing; Xiancong, Tao; Bi, Shuping

    2008-01-01

    A novel method for recognition and indirect determination of Al III by using biological molecules has been established based on the quenching of RRS intensity. In the weak acidic medium, the reaction of ethyl violet (EV) and DNA would result in great enhancement of RRS intensity. However, the presence of Al III would lead to the decrease of the RRS intensity owing to the competition coordination of Al with DNA. The decreased intensity of RRS is directly proportional to the concentration of Al III in the range of (0.1-2.5) × 10 -6 and (0.30-4.5) × 10 -5 M, respectively. The method has high sensitivity and its detection limit (3 σ) is 3.6 × 10 -8 M. The characteristics of RRS spectra of the system, the optimum conditions of the reaction, and the reaction mechanism have been investigated. The method can recognize Al III selectively owing to its strong binding to the phosphate backbone of DNA, and has been applied to the determination of Al III concentration in synthetic biological samples with satisfactory results. Therefore, the proposed method is promising as an effective means for selective recognition and sensitive determination in situ of Al III. Furthermore, this study would contribute to further understanding of the biological significance of Al neurotoxicity.

  13. Visual recognition memory across contexts.

    PubMed

    Jones, Emily J H; Pascalis, Olivier; Eacott, Madeline J; Herbert, Jane S

    2011-01-01

    In two experiments, we investigated the development of representational flexibility in visual recognition memory during infancy using the Visual Paired Comparison (VPC) task. In Experiment 1, 6- and 9-month-old infants exhibited recognition when familiarization and test occurred in the same room, but showed no evidence of recognition when familiarization and test occurred in different rooms. In contrast, 12- and 18-month-old infants exhibited recognition irrespective of testing room. Thus, flexibility across a change of room was observed at a younger age than flexibility across a change of background that has previously been seen with the VPC procedure (Robinson & Pascalis, 2004). To determine if limitations in representational flexibility across a change of background could be overcome by experiences during encoding, in Experiment 2, 6-, 9-, 12- and 18-month-old infants were familiarized with a picture on multiple backgrounds. At all ages, infants showed recognition across a change in background at test. These findings indicate that dissociating an item from its context during encoding may be an important factor in understanding the representational flexibility of visual recognition memory in infancy. Developmental changes in representational flexibility are likely driven by changes in the functional maturity of the hippocampal formation, and experience. © 2010 Blackwell Publishing Ltd.

  14. Information Security: Securing Smart Cards With IRIS Recognition

    DTIC Science & Technology

    2001-03-01

    recognition code was examined. It was necessary to determine the time necessary to break the iris recognition code should the smart card be...are excessive. Additionally, smart card technology was examined to determine if existing technology could store the necessary iris recognition

  15. Pattern recognition system and procedures

    NASA Technical Reports Server (NTRS)

    Nelson, G. D.; Serreyn, D. V.

    1972-01-01

    The ratio transformation technique is used to determine effective features as function of time in remote multiple sensing of crops and soils. The selection of quantizer parameters for a two-class recognition problem under the criteria of minimizing the probability of errors is also discussed.

  16. Recognition of the T stem-loop of a pre-tRNA substrate by the ribozyme from Bacillus subtilis ribonuclease P.

    PubMed

    Loria, A; Pan, T

    1997-05-27

    The ribozyme from bacterial ribonuclease P (denoted P RNA) specifically recognizes the coaxially stacked T stem-loop and the acceptor stem of a tRNA substrate. This recognition is mediated primarily through tertiary interactions. At least four 2'-OH groups in the T stem-loop region have been implicated as direct contacts with Bacillus subtilis P RNA [Pan, T., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12510]. Effects of six single 2'-OH --> 2'-H substitutions and two base mutants of the G19-C56 tertiary interaction in tRNA on substrate binding (Kd) and the chemical step of the reaction (k2) have been determined using a tRNA(Phe) substrate containing a 2'-deoxy residue at the cleavage site. Our results show that at least five functional groups in the T stem-loop of tRNA directly participate in P RNA binding. They include the 2'-OH groups of residues 54, 56, 61, and 62 and possibly the 4-amino group of the conserved C56. The 2'-OHs of residues 54, 61, and 62 are positioned within the same minor groove due to stacking of the reverse Hoogsteen U54-A58 pair on the G53-C61 Watson-Crick pair in the T stem. This groove is extended to the 4-amino group of C56 through the tertiary structure of tRNA. We use the term "tertiary groove" to describe alignment of functional groups through tertiary folding of an RNA. The binding also includes the 2'-OH of nucleotide C56 which is not located in this tertiary groove. Assuming additivity, these five interactions can contribute 7.4 kcal/mol or 10(5)-fold in binding but only -0.5 kcal/mol or approximately 2-fold in chemistry at 37 degrees C. The P RNA binding site for the T stem-loop includes at least the previously identified A230 as well as the A130 in B. subtilis P RNA. The Kd and k2 data from the A130G mutant of B. subtilis P RNA suggest that A130 may be proximal to residue 56 in tRNA. These results show how the highly structured T stem-loop region in a pre-tRNA substrate is bound by the B. subtilis P RNA. This is among the

  17. A comparison of iron extraction methods for the determination of degree of pyritisation and the recognition of iron-limited pyrite formation

    NASA Technical Reports Server (NTRS)

    Raiswell, R.; Canfield, D. E.; Berner, R. A.

    1994-01-01

    Measurements of degree of pyritisation require an estimate of sediment iron which is capable of reaction with dissolved sulphide to form pyrite, either directly or indirectly via iron monosulphide precursors. Three dissolution techniques (buffered dithionite, cold 1 M HCl, boiling 12 M HCl) were examined for their capacity to extract iron from a variety of iron minerals, and iron-bearing sediments, as a function of different extraction times and different grain sizes. All the iron oxides studied are quantitatively extracted by dithionite and boiling HCl (but not by cold HCl). Both HCl techniques extract more iron from silicates than does dithionite but probably about the same amounts as are potentially capable of sulphidation. Modern sediment studies indicate that most sedimentary pyrite is formed rapidly from iron oxides, with smaller amounts formed more slowly from iron silicates (if sufficient geologic time is available). It is therefore recommended that the degree of pyritisation be defined with respect to the dithionite-extractable (mainly iron oxide) pool and/or the boiling HCl-extractable pool (which includes some silicate iron) for the recognition of iron-limited pyritisation.

  18. The Hidden Conformation of Lewis x, a Human Histo-Blood Group Antigen, Is a Determinant for Recognition by Pathogen Lectins.

    PubMed

    Topin, Jérémie; Lelimousin, Mickaël; Arnaud, Julie; Audfray, Aymeric; Pérez, Serge; Varrot, Annabelle; Imberty, Anne

    2016-07-15

    Histo-blood group epitopes are fucosylated branched oligosaccharides with well-defined conformations in solution that are recognized by receptors, such as lectins from pathogens. We report here the results of a series of experimental and computational endeavors revealing the unusual distortion of histo-blood group antigens by bacterial and fungal lectins. The Lewis x trisaccharide adopts a rigid closed conformation in solution, while crystallography and molecular dynamics reveal several higher energy open conformations when bound to the Ralstonia solanacearum lectin, which is in agreement with thermodynamic and kinetic measurements. Extensive molecular dynamics simulations confirm rare transient Le(x) openings in solution, frequently assisted by distortion of the central N-acetyl-glucosamine ring. Additional directed molecular dynamic trajectories revealed the role of a conserved tryptophan residue in guiding the fucose into the binding site. Our findings show that conformational adaptation of oligosaccharides is of paramount importance in cell recognition and should be considered when designing anti-infective glyco-compounds.

  19. Structure and DNA-Binding Sites of the SWI1 AT-rich Interaction Domain (ARID) Suggest Determinants for Sequence-Specific DNA Recognition

    SciTech Connect

    Kim, Suhkmann; Zhang, Ziming; Upchurch, Sean; Isern, Nancy G.; Chen, Yuan

    2004-04-16

    2 ARID is a homologous family of DNA-binding domains that occur in DNA binding proteins from a wide variety of species, ranging from yeast to nematodes, insects, mammals and plants. SWI1, a member of the SWI/SNF protein complex that is involved in chromatin remodeling during transcription, contains the ARID motif. The ARID domain of human SWI1 (also known as p270) does not select for a specific DNA sequence from a random sequence pool. The lack of sequence specificity shown by the SWI1 ARID domain stands in contrast to the other characterized ARID domains, which recognize specific AT-rich sequences. We have solved the three-dimensional structure of human SWI1 ARID using solution NMR methods. In addition, we have characterized non-specific DNA-binding by the SWI1 ARID domain. Results from this study indicate that a flexible long internal loop in ARID motif is likely to be important for sequence specific DNA-recognition. The structure of human SWI1 ARID domain also represents a distinct structural subfamily. Studies of ARID indicate that boundary of the DNA binding structural and functional domains can extend beyond the sequence homologous region in a homologous family of proteins. Structural studies of homologous domains such as ARID family of DNA-binding domains should provide information to better predict the boundary of structural and functional domains in structural genomic studies. Key Words: ARID, SWI1, NMR, structural genomics, protein-DNA interaction.

  20. Machine Recognition vs Human Recognition of Voices

    DTIC Science & Technology

    2012-05-01

    good as seen in NIST Speaker Recognition Evaluations, performance can still suffer when the environmental conditions, emotions , or recording quality...recognized. The accuracy of speaker recognition for disyllables was 87%. For monosyllables, it was 81%, consonant- vowel excerpts were 63%, and... vowel excerpts were 56%. Thus, they demonstrated that the identification performance decreased as the number of phonemes decreased. In [2], the

  1. Exploring a recognition-induced recognition decrement

    PubMed Central

    Dopkins, Stephen; Ngo, Catherine Trinh; Sargent, Jesse

    2007-01-01

    Four experiments explored a recognition decrement that is associated with the recognition of a word from a short list. The stimulus material for demonstrating the phenomenon was a list of words of different syntactic types. A word from the list was recognized less well following a decision that a word of the same type had occurred in the list than following a decision that such a word had not occurred in the list. A recognition decrement did not occur for a word of a given type following a positive recognition decision to a word of a different type. A recognition decrement did not occur when the list consisted exclusively of nouns. It was concluded that the phenomenon may reflect a criterion shift but probably does not reflect a list strength effect, suppression, or familiarity attribution consequent to a perceived discrepancy between actual and expected fluency. PMID:17063915

  2. Building Group Recognition.

    ERIC Educational Resources Information Center

    Chartier, George

    1994-01-01

    Discusses the value of name recognition for theater companies. Describes steps toward identity and recognition, analyzing the group, the mission statement, symbolic logic, designing and identity, developing a communications plan, and meaningful activities. (SR)

  3. Speech Recognition by Computer.

    ERIC Educational Resources Information Center

    Levinson, Stephen E.; Liberman, Mark Y.

    1981-01-01

    Speech recognition by computers is discussed, including methods of recognizing isolated words and procedures for analyzing connected speech. Describes Bell Laboratories' speech recognition system which attempts to combine major elements of human communication into a single operating unit. (DS)

  4. Speech recognition and understanding

    SciTech Connect

    Vintsyuk, T.K.

    1983-05-01

    This article discusses the automatic processing of speech signals with the aim of finding a sequence of works (speech recognition) or a concept (speech understanding) being transmitted by the speech signal. The goal of the research is to develop an automatic typewriter that will automatically edit and type text under voice control. A dynamic programming method is proposed in which all possible class signals are stored, after which the presented signal is compared to all the stored signals during the recognition phase. Topics considered include element-by-element recognition of words of speech, learning speech recognition, phoneme-by-phoneme speech recognition, the recognition of connected speech, understanding connected speech, and prospects for designing speech recognition and understanding systems. An application of the composition dynamic programming method for the solution of basic problems in the recognition and understanding of speech is presented.

  5. Early development of visual recognition.

    PubMed

    Plebe, Alessio; Domenella, Rosaria Grazia

    2006-01-01

    The most important ability of the human vision is object recognition, yet it is exactly the less understood aspect of the vision system. Computational models have been helpful in progressing towards an explanation of this obscure cognitive ability, and today it is possible to conceive more refined models, thanks to the new availability of neuroscientific data about the human visual cortex. This work proposes a model of the development of the object recognition capability, under a different perspective with respect to the most common approaches, with a precise theoretical epistemology. It is assumed that the main processing functions involved in recognition are not genetically determined and hardwired in the neural circuits, but are the result of interactions between epigenetic influences and the basic neural plasticity mechanisms. The model is organized in modules related with the main visual biological areas, and is implemented mainly using the LISSOM architecture, a recent self-organizing algorithm closely reflecting the essential behavior of cortical circuits.

  6. Computational analysis of siRNA recognition by the Ago2 PAZ domain and identification of the determinants of RNA-induced gene silencing.

    PubMed

    Kandeel, Mahmoud; Kitade, Yukio

    2013-01-01

    RNA interference (RNAi) is a highly specialized process of protein-siRNA interaction that results in the regulation of gene expression and cleavage of target mRNA. The PAZ domain of the Argonaute proteins binds to the 3' end of siRNA, and during RNAi the attaching end of the siRNA switches between binding and release from its binding pocket. This biphasic interaction of the 3' end of siRNA with the PAZ domain is essential for RNAi activity; however, it remains unclear whether stronger or weaker binding with PAZ domain will facilitate or hinder the overall RNAi process. Here we report the correlation between the binding of modified siRNA 3' overhang analogues and their in vivo RNAi efficacy. We found that higher RNAi efficacy was associated with the parameters of lower Ki value, lower total intermolecular energy, lower free energy, higher hydrogen bonding, smaller total surface of interaction and fewer van der Waals interactions. Electrostatic interaction was a minor contributor to compounds recognition, underscoring the presence of phosphate groups in the modified analogues. Thus, compounds with lower binding affinity are associated with better gene silencing. Lower binding strength along with the smaller interaction surface, higher hydrogen bonding and fewer van der Waals interactions were among the markers for favorable RNAi activity. Within the measured parameters, the interaction surface, van der Waals interactions and inhibition constant showed a statistically significant correlation with measured RNAi efficacy. The considerations provided in this report will be helpful in the design of new compounds with better gene silencing ability.

  7. Molecular dynamics investigations of ozone on an ab initio potential energy surface with the utilization of pattern-recognition neural network for accurate determination of product formation.

    PubMed

    Le, Hung M; Dinh, Thach S; Le, Hieu V

    2011-10-13

    The singlet-triplet transformation and molecular dissociation of ozone (O(3)) gas is investigated by performing quasi-classical molecular dynamics (MD) simulations on an ab initio potential energy surface (PES) with visible and near-infrared excitations. MP4(SDQ) level of theory with the 6-311g(2d,2p) basis set is executed for three different electronic spin states (singlet, triplet, and quintet). In order to simplify the potential energy function, an approximation is adopted by ignoring the spin-orbit coupling and allowing the molecule to switch favorably and instantaneously to the spin state that is more energetically stable (lowest in energy among the three spin states). This assumption has previously been utilized to study the SiO(2) system as reported by Agrawal et al. (J. Chem. Phys. 2006, 124 (13), 134306). The use of such assumption in this study probably makes the upper limits of computed rate coefficients the true rate coefficients. The global PES for ozone is constructed by fitting 5906 ab initio data points using a 60-neuron two-layer feed-forward neural network. The mean-absolute error and root-mean-squared error of this fit are 0.0446 eV (1.03 kcal/mol) and 0.0756 eV (1.74 kcal/mol), respectively, which reveal very good fitting accuracy. The parameter coefficients of the global PES are reported in this paper. In order to identify the spin state with high confidence, we propose the use of a pattern-recognition neural network, which is trained to predict the spin state of a given configuration (with a prediction accuracy being 95.6% on a set of testing data points). To enhance the prediction effectiveness, a buffer series of five points are validated to confirm the spin state during the MD process to gain better confidence. Quasi-classical MD simulations from 1.2 to 2.4 eV of total internal energy (including zero-point energy) result in rate coefficients of singlet-triplet transformation in the range of 0.027 ps(-1) to 1.21 ps(-1). Also, we find very

  8. Determination of the recognition site for adenine-specific methylase of Shigella sonnei 47 by hydazinolysis of DNA, followed by separation of the purine oligonucleotides by thin-layer chromatography on DEAE-cellulose

    SciTech Connect

    Lopatina, N.G.; Kirnos, M.D.; Suchkov, S.V.; Vanyushin, B.F.; Nikol'skaya, I.I.; Debov, S.S.

    1985-09-20

    A method has been developed for the separation of oligopurine units according to length and composition by two-dimensional thin-layer chromatography on plates with DEAE-cellulose, permitting a comparative analysis of the content of various purine isopliths in DNA of different origin. In the case of the analysis of methylated DNA, the method permits a comparison of the substrate specificity of various enzymes of methylation of the adenine residues in DNA. In conjunction with enzymatic treatment of labeled methylated isopliths, the method permits determination of the methylatable sequence and in a number of cases an ascertainment of the recognition site for adenine-specific methylase as a whole. The proposed method was used to establish the fact that the methylase Ssol recognizes the sequence 5'...G-A-A-T-T-C...3' and methylates the adenine residue closest to its 5'-end.

  9. Global identification of target recognition and cleavage by the Microprocessor in human ES cells.

    PubMed

    Seong, Youngmo; Lim, Do-Hwan; Kim, Augustine; Seo, Jae Hong; Lee, Young Sik; Song, Hoseok; Kwon, Young-Soo

    2014-11-10

    The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3' overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.

  10. Face recognition for uncontrolled environments

    NASA Astrophysics Data System (ADS)

    Podilchuk, Christine; Hulbert, William; Flachsbart, Ralph; Barinov, Lev

    2010-04-01

    A new face recognition algorithm has been proposed which is robust to variations in pose, expression, illumination and occlusions such as sunglasses. The algorithm is motivated by the Edit Distance used to determine the similarity between strings of one dimensional data such as DNA and text. The key to this approach is how to extend the concept of an Edit Distance on one-dimensional data to two-dimensional image data. The algorithm is based on mapping one image into another and using the characteristics of the mapping to determine a two-dimensional Pictorial-Edit Distance or P-Edit Distance. We show how the properties of the mapping are similar to insertion, deletion and substitution errors defined in an Edit Distance. This algorithm is particularly well suited for face recognition in uncontrolled environments such as stand-off and other surveillance applications. We will describe an entire system designed for face recognition at a distance including face detection, pose estimation, multi-sample fusion of video frames and identification. Here we describe how the algorithm is used for face recognition at a distance, present some initial results and describe future research directions.(

  11. Image Recognition Based on Biometric Pattern Recognition

    NASA Astrophysics Data System (ADS)

    Sun, Shuliang; Chen, Zhong; Liu, Chenglian; Guo, Yongning; Lin, Xueyun

    2011-09-01

    A new method, biomimetric pattern recognition, is mentioned to recognize images. At first, the image is pretreatment and feature extraction, then a high vector is got. A biomimetric pattern recognition model is designed. The judgment function is used to discriminate the classification of the samples. It is showed that the method is effective for little samples by experiment. It would be useful in many fields in future.

  12. On three dimensional object recognition and pose-determination: An abstraction based approach. Ph.D. Thesis - Michigan Univ. Final Report

    NASA Technical Reports Server (NTRS)

    Quek, Kok How Francis

    1990-01-01

    A method of computing reliable Gaussian and mean curvature sign-map descriptors from the polynomial approximation of surfaces was demonstrated. Such descriptors which are invariant under perspective variation are suitable for hypothesis generation. A means for determining the pose of constructed geometric forms whose algebraic surface descriptors are nonlinear in terms of their orienting parameters was developed. This was done by means of linear functions which are capable of approximating nonlinear forms and determining their parameters. It was shown that biquadratic surfaces are suitable companion linear forms for cylindrical approximation and parameter estimation. The estimates provided the initial parametric approximations necessary for a nonlinear regression stage to fine tune the estimates by fitting the actual nonlinear form to the data. A hypothesis-based split-merge algorithm for extraction and pose determination of cylinders and planes which merge smoothly into other surfaces was developed. It was shown that all split-merge algorithms are hypothesis-based. A finite-state algorithm for the extraction of the boundaries of run-length regions was developed. The computation takes advantage of the run list topology and boundary direction constraints implicit in the run-length encoding.

  13. Optical Pattern Recognition

    NASA Astrophysics Data System (ADS)

    Yu, Francis T. S.; Jutamulia, Suganda

    2008-10-01

    Contributors; Preface; 1. Pattern recognition with optics Francis T. S. Yu and Don A. Gregory; 2. Hybrid neural networks for nonlinear pattern recognition Taiwei Lu; 3. Wavelets, optics, and pattern recognition Yao Li and Yunglong Sheng; 4. Applications of the fractional Fourier transform to optical pattern recognition David Mendlovic, Zeev Zalesky and Haldum M. Oxaktas; 5. Optical implementation of mathematical morphology Tien-Hsin Chao; 6. Nonlinear optical correlators with improved discrimination capability for object location and recognition Leonid P. Yaroslavsky; 7. Distortion-invariant quadratic filters Gregory Gheen; 8. Composite filter synthesis as applied to pattern recognition Shizhou Yin and Guowen Lu; 9. Iterative procedures in electro-optical pattern recognition Joseph Shamir; 10. Optoelectronic hybrid system for three-dimensional object pattern recognition Guoguang Mu, Mingzhe Lu and Ying Sun; 11. Applications of photrefractive devices in optical pattern recognition Ziangyang Yang; 12. Optical pattern recognition with microlasers Eung-Gi Paek; 13. Optical properties and applications of bacteriorhodopsin Q. Wang Song and Yu-He Zhang; 14. Liquid-crystal spatial light modulators Aris Tanone and Suganda Jutamulia; 15. Representations of fully complex functions on real-time spatial light modulators Robert W. Cohn and Laurence G. Hassbrook; Index.

  14. Recognition of Epstein-Barr virus (EBV)-infected cells by T cell colonies from a human chimera: restriction by allogeneic determinants.

    PubMed Central

    Plotnicky, H; Touraine, J L

    1993-01-01

    The anti-EBV T cell response was studied in a severe combined immunodeficiency patient (PS) who received two transplants of fetal liver cells. His peripheral blood mononuclear cells (PBMC) were incubated with EBV and cultured during 15 days. Eleven colonies were derived from the T lymphocytes causing the regression of the infected cell foci: nine were constituted with CD3+ CD4+ CD8- lymphocytes and two with CD3+ CD4- CD8+ cells. HLA typing of six colonies showed that two of them derived from the first transplant and four from the second one. The colonies killed the cells of the lymphoblastoid line (LCL) derived from the recipient (PS-LCL), but failed to kill the LCL matched with the transplants. With only one exception, they all lysed also the LCL derived from the mother or from the father, but they were ineffective on the EBV-negative lymphoblasts. Two colonies recognized determinants which did not appear to be HLA antigens, although they were shared by PS and by one of his parents, two (CD4- CD8+) reacted against the LCL which shared HLA-A3 or -A33 with PS-LCL, and four (CD4+ CD8-) lysed the LCL sharing HLA-A3, -A33 or -DR5 with PS-LCL, among which only one was demonstrated to interact directly with host HLA-class I determinants. These data indicate that T lymphocytes differentiating in contact with histo-incompatible determinants may express the capability to recognize viral antigens and to lyse virus-infected cells in the context of allogeneic MHC or non-MHC molecules. PMID:7504600

  15. Study on Information Fusion Based Check Recognition System

    NASA Astrophysics Data System (ADS)

    Wang, Dong

    Automatic check recognition techniques play an important role in financial systems, especially in risk management. This paper presents a novel check recognition system based on multi-cue information fusion theory. For Chinese bank check, the amount can be independently determined by legal amount, courtesy amount, or E13B code. The check recognition algorithm consists of four steps: preprocessing, check layout analysis, segmentation and recognition, and information fusion. For layout analysis, an adaptive template matching algorithm is presented to locate the target recognition regions on the check. The hidden markov model is used to segment and recognize legal amount. Courtesy and E13B code are recognized by artificial neural network method, respectively. Finally, D-S evidence theory is then introduced to fuse above three recognition results for better recognition performance. Experimental results demonstrate that the system can robustly recognize checks and the information fusion based algorithm improves the recognition rate by 5~10 percent.

  16. Kin Recognition in Bacteria.

    PubMed

    Wall, Daniel

    2016-09-08

    The ability of bacteria to recognize kin provides a means to form social groups. In turn these groups can lead to cooperative behaviors that surpass the ability of the individual. Kin recognition involves specific biochemical interactions between a receptor(s) and an identification molecule(s). Recognition specificity, ensuring that nonkin are excluded and kin are included, is critical and depends on the number of loci and polymorphisms involved. After recognition and biochemical perception, the common ensuing cooperative behaviors include biofilm formation, quorum responses, development, and swarming motility. Although kin recognition is a fundamental mechanism through which cells might interact, microbiologists are only beginning to explore the topic. This review considers both molecular and theoretical aspects of bacterial kin recognition. Consideration is also given to bacterial diversity, genetic relatedness, kin selection theory, and mechanisms of recognition.

  17. Multimodal eye recognition

    NASA Astrophysics Data System (ADS)

    Zhou, Zhi; Du, Yingzi; Thomas, N. L.; Delp, Edward J., III

    2010-04-01

    Multimodal biometrics use more than one means of biometric identification to achieve higher recognition accuracy, since sometimes a unimodal biometric is not good enough used to do identification and classification. In this paper, we proposed a multimodal eye recognition system, which can obtain both iris and sclera patterns from one color eye image. Gabor filter and 1-D Log-Gabor filter algorithms have been applied as the iris recognition algorithms. In sclera recognition, we introduced automatic sclera segmentation, sclera pattern enhancement, sclera pattern template generation, and sclera pattern matching. We applied kernelbased matching score fusion to improve the performance of the eye recognition system. The experimental results show that the proposed eye recognition method can achieve better performance compared to unimodal biometric identification, and the accuracy of our proposed kernel-based matching score fusion method is higher than two classic linear matching score fusion methods: Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA).

  18. Molecular determinants of MAO selectivity in a series of indolylmethylamine derivatives: biological activities, 3D-QSAR/CoMFA analysis, and computational simulation of ligand recognition.

    PubMed

    Morón, J A; Campillo, M; Perez, V; Unzeta, M; Pardo, L

    2000-05-04

    A series of indolylmethylamine derivatives were assayed toward MAO-A and MAO-B inhibition. The K(i) values of these compounds are in the range from 0.8 to >10(6) nM for MAO-A or from 0.75 to 476000 nM for MAO-B. The most selective MAO-A or MAO-B inhibitors elicit a ratio of K(i) in the order of 1500 or 1000, respectively. Comparison of MAO-A and MAO-B CoMFA models showed that both the steric and electrostatic properties at the 5 position of the indole ring are determinant for MAO selectivity. Computational simulations of the complex between this part of the ligand and Phe-208 of MAO-A or Ile-199 of MAO-B, experimentally identified as responsible for substrate selectivity, allowed us to further characterize the nature of these enzyme-inhibitor interactions.

  19. Hydrogen-bonding recognition-induced aggregation of gold nanoparticles for the determination of the migration of melamine monomers using dynamic light scattering.

    PubMed

    Wu, Long; Chen, Kun; Lu, Zhicheng; Li, Tingting; Shao, Kang; Shao, Feng; Han, Heyou

    2014-10-03

    The migration of melamine monomers from food contact materials has aroused particular attention since the 2008 melamine-tainted milk scandal in China. However, the determination of melamine monomer's migratory quantity (MMMQ) has remained an open question because of the complex sample pretreatment and the low sensitivity. Based on the hydrogen bonding interaction between DNA thymine and melamine, this paper described a simple and rapid method focusing on the measurement of MMMQ from melamine tableware by gold nanoparticles (GNPs) and dynamic light scattering (DLS). With the presence of probe DNA (p-DNA), the GNPs were stable in NaCl solution (0.06 M), whereas they became aggregated when the p-DNA hybridized with melamine. The change in the hydrodynamic diameter of GNPs could be detected by DLS technology. Under the optimal conditions, the average diameter increased linearly with the concentration of melamine over the range from 5.0 to 320.0 μg L(-1), and showed a detection limit of 2.0 μg L(-1) (3σ/slope). The MMMQ was investigated within a range from 6.00×10(-4) to 2.58×10(-1) mg dm(-2) (n≥3) in four different food simulants at different temperatures and time points. The results suggest that the DLS method has great potential in the analysis of the migration of melamine monomers. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Reconstruction of LPS Transfer Cascade Reveals Structural Determinants within LBP, CD14, and TLR4-MD2 for Efficient LPS Recognition and Transfer.

    PubMed

    Ryu, Je-Kyung; Kim, Soo Jin; Rah, Sang-Hyun; Kang, Ji In; Jung, Hi Eun; Lee, Dongsun; Lee, Heung Kyu; Lee, Jie-Oh; Park, Beom Seok; Yoon, Tae-Young; Kim, Ho Min

    2017-01-17

    Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, binds Toll-like receptor 4 (TLR4)-MD2 complex and activates innate immune responses. LPS transfer to TLR4-MD2 is catalyzed by both LPS binding protein (LBP) and CD14. To define the sequential molecular interactions underlying this transfer, we reconstituted in vitro the entire LPS transfer process from LPS micelles to TLR4-MD2. Using electron microscopy and single-molecule approaches, we characterized the dynamic intermediate complexes for LPS transfer: LBP-LPS micelles, CD14-LBP-LPS micelle, and CD14-LPS-TLR4-MD2 complex. A single LBP molecule bound longitudinally to LPS micelles catalyzed multi-rounds of LPS transfer to CD14s that rapidly dissociated from LPB-LPS complex upon LPS transfer via electrostatic interactions. Subsequently, the single LPS molecule bound to CD14 was transferred to TLR4-MD2 in a TLR4-dependent manner. The definition of the structural determinants of the LPS transfer cascade to TLR4 may enable the development of targeted therapeutics for intervention in LPS-induced sepsis.

  1. Antigen Recognition By Variable Lymphocyte Receptors

    SciTech Connect

    Han, B.W.; Herrin, B.R.; Cooper, M.D.; Wilson, I.A.

    2009-05-18

    Variable lymphocyte receptors (VLRs) rather than antibodies play the primary role in recognition of antigens in the adaptive immune system of jawless vertebrates. Combinatorial assembly of leucine-rich repeat (LRR) gene segments achieves the required repertoire for antigen recognition. We have determined a crystal structure for a VLR-antigen complex, VLR RBC36 in complex with the H-antigen trisaccharide from human blood type O erythrocytes, at 1.67 angstrom resolution. RBC36 binds the H-trisaccharide on the concave surface of the LRR modules of the solenoid structure where three key hydrophilic residues, multiple van der Waals interactions, and the highly variable insert of the carboxyl-terminal LRR module determine antigen recognition and specificity. The concave surface assembled from the most highly variable regions of the LRRs, along with diversity in the sequence and length of the highly variable insert, can account for the recognition of diverse antigens by VLRs.

  2. Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1.

    PubMed

    Xu, Huifang; Raynal, Nicolas; Stathopoulos, Stavros; Myllyharju, Johanna; Farndale, Richard W; Leitinger, Birgit

    2011-01-01

    The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.

  3. Collagen binding specificity of the discoidin domain receptors: Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

    PubMed Central

    Xu, Huifang; Raynal, Nicolas; Stathopoulos, Stavros; Myllyharju, Johanna; Farndale, Richard W.; Leitinger, Birgit

    2011-01-01

    The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I–III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor. PMID:21044884

  4. Moreland Recognition Program.

    ERIC Educational Resources Information Center

    Moreland Elementary School District, San Jose, CA.

    THE FOLLOWING IS THE FULL TEXT OF THIS DOCUMENT: Recognition for special effort and achievement has been noted as a component of effective schools. Schools in the Moreland School District have effectively improved standards of discipline and achievement by providing forty-six different ways for children to receive positive recognition. Good…

  5. Moreland Recognition Program.

    ERIC Educational Resources Information Center

    Moreland Elementary School District, San Jose, CA.

    THE FOLLOWING IS THE FULL TEXT OF THIS DOCUMENT: Recognition for special effort and achievement has been noted as a component of effective schools. Schools in the Moreland School District have effectively improved standards of discipline and achievement by providing forty-six different ways for children to receive positive recognition. Good…

  6. Visual object recognition.

    PubMed

    Logothetis, N K; Sheinberg, D L

    1996-01-01

    Visual object recognition is of fundamental importance to most animals. The diversity of tasks that any biological recognition system must solve suggests that object recognition is not a single, general purpose process. In this review, we consider evidence from the fields of psychology, neuropsychology, and neurophysiology, all of which supports the idea that there are multiple systems for recognition. Data from normal adults, infants, animals, and brain damaged patients reveal a major distinction between the classification of objects at a basic category level and the identification of individual objects from a homogeneous object class. An additional distinction between object representations used for visual perception and those used for visually guided movements provides further support for a multiplicity of visual recognition systems. Recent evidence from psychophysical and neurophysiological studies indicates that one system may represent objects by combinations of multiple views, or aspects, and another may represent objects by structural primitives and their spatial interrelationships.

  7. 8 CFR 292.2 - Organizations qualified for recognition; requests for recognition; withdrawal of recognition...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Organizations qualified for recognition; requests for recognition; withdrawal of recognition; accreditation of representatives; roster. 292.2... REPRESENTATION AND APPEARANCES § 292.2 Organizations qualified for recognition; requests for recognition...

  8. Molecular Recognition and Ligand Association

    NASA Astrophysics Data System (ADS)

    Baron, Riccardo; McCammon, J. Andrew

    2013-04-01

    We review recent developments in our understanding of molecular recognition and ligand association, focusing on two major viewpoints: (a) studies that highlight new physical insight into the molecular recognition process and the driving forces determining thermodynamic signatures of binding and (b) recent methodological advances in applications to protein-ligand binding. In particular, we highlight the challenges posed by compensating enthalpic and entropic terms, competing solute and solvent contributions, and the relevance of complex configurational ensembles comprising multiple protein, ligand, and solvent intermediate states. As more complete physics is taken into account, computational approaches increase their ability to complement experimental measurements, by providing a microscopic, dynamic view of ensemble-averaged experimental observables. Physics-based approaches are increasingly expanding their power in pharmacology applications.

  9. Automated smartphone audiometry: Validation of a word recognition test app.

    PubMed

    Dewyer, Nicholas A; Jiradejvong, Patpong; Henderson Sabes, Jennifer; Limb, Charles J

    2017-05-23

    Develop and validate an automated smartphone word recognition test. Cross-sectional case-control diagnostic test comparison. An automated word recognition test was developed as an app for a smartphone with earphones. English-speaking adults with recent audiograms and various levels of hearing loss were recruited from an audiology clinic and were administered the smartphone word recognition test. Word recognition scores determined by the smartphone app and the gold standard speech audiometry test performed by an audiologist were compared. Test scores for 37 ears were analyzed. Word recognition scores determined by the smartphone app and audiologist testing were in agreement, with 86% of the data points within a clinically acceptable margin of error and a linear correlation value between test scores of 0.89. The WordRec automated smartphone app accurately determines word recognition scores. 3b Laryngoscope, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  10. Star Pattern Recognition and Spacecraft Attitude Determination.

    DTIC Science & Technology

    1981-05-01

    PsO)) I Constants for orbit calculation. 790 DeO=OT(’e6O,eO)1 80 Re6=3QR(DCT(FeB, PeG )) 810 T-O- I Set reference time. 820 - 830 REDIII W1(.:3) 840...34;@’ Beta~old) Beta(new) Delta( Deta )" 11798 FOR [=1 TO 4 11886 Bvn( I) =I:vn( I>+Del x( I) 11816 PRINT JElING ’X3(MD.DDDDDDD,XXXX)"Bs(I),Bvn( I),Delx(I

  11. Protease recognition sites in Bet v 1a are cryptic, explaining its slow processing relevant to its allergenicity

    PubMed Central

    Freier, Regina; Dall, Elfriede; Brandstetter, Hans

    2015-01-01

    Despite a high similarity with homologous protein families, only few proteins trigger an allergic immune response with characteristic TH2 polarization. This puzzling observation is illustrated by the major birch pollen allergen Bet v 1a and its hypoallergenic protein isoforms, e.g., Bet v 1d. Given the key role of proteolytic processing in antigen presentation and T cell polarization, we investigated the recognition of Bet v 1 isoforms by the relevant protease cathepsin S. We found that at moderately acidic pH values Bet v 1a bound to cathepsin S with significantly lower affinity and was more slowly cleaved than its hypoallergenic isoform Bet v 1d. Only at pH values ≤4.5 the known proteolytic cleavage sites in Bet v 1a became accessible, resulting in a strong increase in affinity towards cathepsin S. Antigen processing and class II MHC loading occurs at moderately acidic compartments where processing of Bet v 1a and Bet v 1d differs distinctly. This difference translates into low and high density class II MHC loading and subsequently in TH2 and TH1 polarization, respectively. PMID:26235974

  12. Protease recognition sites in Bet v 1a are cryptic, explaining its slow processing relevant to its allergenicity.

    PubMed

    Freier, Regina; Dall, Elfriede; Brandstetter, Hans

    2015-08-03

    Despite a high similarity with homologous protein families, only few proteins trigger an allergic immune response with characteristic TH2 polarization. This puzzling observation is illustrated by the major birch pollen allergen Bet v 1a and its hypoallergenic protein isoforms, e.g., Bet v 1d. Given the key role of proteolytic processing in antigen presentation and T cell polarization, we investigated the recognition of Bet v 1 isoforms by the relevant protease cathepsin S. We found that at moderately acidic pH values Bet v 1a bound to cathepsin S with significantly lower affinity and was more slowly cleaved than its hypoallergenic isoform Bet v 1d. Only at pH values ≤ 4.5 the known proteolytic cleavage sites in Bet v 1a became accessible, resulting in a strong increase in affinity towards cathepsin S. Antigen processing and class II MHC loading occurs at moderately acidic compartments where processing of Bet v 1a and Bet v 1d differs distinctly. This difference translates into low and high density class II MHC loading and subsequently in TH2 and TH1 polarization, respectively.

  13. Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent on S-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE11[W][OPEN

    PubMed Central

    Qi, Dong; Dubiella, Ullrich; Kim, Sang Hee; Sloss, D. Isaiah; Dowen, Robert H.; Dixon, Jack E.; Innes, Roger W.

    2014-01-01

    The recognition of pathogen effector proteins by plants is typically mediated by intracellular receptors belonging to the nucleotide-binding leucine-rich repeat (NLR) family. NLR proteins often detect pathogen effector proteins indirectly by detecting modification of their targets. How NLR proteins detect such modifications is poorly understood. To address these questions, we have been investigating the Arabidopsis (Arabidopsis thaliana) NLR protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5), which detects the Pseudomonas syringae effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB). AvrPphB is a cysteine protease that specifically targets a subfamily of receptor-like cytoplasmic kinases, including the Arabidopsis protein kinase AVRPPHB Susceptible1 (PBS1). RPS5 is activated by the cleavage of PBS1 at the apex of its activation loop. Here, we show that RPS5 activation requires that PBS1 be localized to the plasma membrane and that plasma membrane localization of PBS1 is mediated by amino-terminal S-acylation. We also describe the development of a high-throughput screen for mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles, two of which blocked cleavage by AvrPphB. Lastly, we show that RPS5 distinguishes among closely related kinases by the amino acid sequence (SEMPH) within an exposed loop in the C-terminal one-third of PBS1. The SEMPH loop is located on the opposite side of PBS1 from the AvrPphB cleavage site, suggesting that RPS5 associates with the SEMPH loop while leaving the AvrPphB cleavage site exposed. These findings provide support for a model of NLR activation in which NLR proteins form a preactivation complex with effector targets and then sense a conformational change in the target induced by effector modification. PMID:24225654

  14. Visual recognition of permuted words

    NASA Astrophysics Data System (ADS)

    Rashid, Sheikh Faisal; Shafait, Faisal; Breuel, Thomas M.

    2010-02-01

    In current study we examine how letter permutation affects in visual recognition of words for two orthographically dissimilar languages, Urdu and German. We present the hypothesis that recognition or reading of permuted and non-permuted words are two distinct mental level processes, and that people use different strategies in handling permuted words as compared to normal words. A comparison between reading behavior of people in these languages is also presented. We present our study in context of dual route theories of reading and it is observed that the dual-route theory is consistent with explanation of our hypothesis of distinction in underlying cognitive behavior for reading permuted and non-permuted words. We conducted three experiments in lexical decision tasks to analyze how reading is degraded or affected by letter permutation. We performed analysis of variance (ANOVA), distribution free rank test, and t-test to determine the significance differences in response time latencies for two classes of data. Results showed that the recognition accuracy for permuted words is decreased 31% in case of Urdu and 11% in case of German language. We also found a considerable difference in reading behavior for cursive and alphabetic languages and it is observed that reading of Urdu is comparatively slower than reading of German due to characteristics of cursive script.

  15. Modeling protein recognition of carbohydrates.

    PubMed

    Laederach, Alain; Reilly, Peter J

    2005-09-01

    We have a limited understanding of the details of molecular recognition of carbohydrates by proteins, which is critical to a multitude of biological processes. Furthermore, carbohydrate-modifying proteins such as glycosyl hydrolases and phosphorylases are of growing importance as potential drug targets. Interactions between proteins and carbohydrates have complex thermodynamics, and in general the specific positioning of only a few hydroxyl groups determines their binding affinities. A thorough understanding of both carbohydrate and protein structures is thus essential to predict these interactions. An atomic-level view of carbohydrate recognition through structures of carbohydrate-active enzymes complexed with transition-state inhibitors reveals some of the distinctive molecular features unique to protein-carbohydrate complexes. However, the inherent flexibility of carbohydrates and their often water-mediated hydrogen bonding to proteins makes simulation of their complexes difficult. Nonetheless, recent developments such as the parameterization of specific force fields and docking scoring functions have greatly improved our ability to predict protein-carbohydrate interactions. We review protein-carbohydrate complexes having defined molecular requirements for specific carbohydrate recognition by proteins, providing an overview of the different computational techniques available to model them. Copyright 2005 Wiley-Liss, Inc.

  16. Pattern recognition technique

    NASA Technical Reports Server (NTRS)

    Hong, J. P.

    1971-01-01

    Technique operates regardless of pattern rotation, translation or magnification and successfully detects out-of-register patterns. It improves accuracy and reduces cost of various optical character recognition devices and page readers and provides data input to computer.

  17. Mechanistic insights into ectodomain shedding: susceptibility of CADM1 adhesion molecule is determined by alternative splicing and O-glycosylation

    PubMed Central

    Shirakabe, Kyoko; Omura, Takuya; Shibagaki, Yoshio; Mihara, Emiko; Homma, Keiichi; Kato, Yukinari; Yoshimura, Akihiko; Murakami, Yoshinori; Takagi, Junichi; Hattori, Seisuke; Ogawa, Yoshihiro

    2017-01-01

    Ectodomain shedding (shedding) is a post-translational modification, which liberates the extracellular domain of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Because shedding alters characteristics of cells in a rapid and irreversible manner, it should be strictly regulated. However, the molecular mechanisms determining membrane protein susceptibility to shedding (shedding susceptibility) are largely unknown. Here we report that alternative splicing can give rise to both shedding-susceptible and shedding-resistant CADM1 (cell adhesion molecule 1) variant proteins. We further show that O-glycans adjacent to the shedding cleavage site interfere with CADM1 shedding, and the only 33-bp alternative exon confers shedding susceptibility to CADM1 by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. These results demonstrate that shedding susceptibility of membrane protein can be determined at two different levels of its biosynthesis pathway, alternative splicing and O-glycosylation. PMID:28393893

  18. Recognition of the HLA class II-peptide complex by T-cell receptor: reversal of major histocompatibility complex restriction of a T-cell clone by a point mutation in the peptide determinant.

    PubMed

    Rothbard, J B; Busch, R; Lechler, R; Trowsdale, J; Lamb, J R

    1989-06-12

    Recognition of the HLA DR-peptide complex by an influenza haemagglutinin-specific T-cell clone was examined by assaying a variety of peptide analogues for their ability to be recognized. Consistent with earlier experiments arguing that the peptide blinds the restriction element in a helical conformation, acetylation of the amino terminus and amidation of the carboxy terminus of the natural determinant (residues 307-319) resulted in a peptide that exhibited both greater propensity to form a helix, as judged by circular dichroism, and the ability to stimulate the clone at concentrations approximately two orders of magnitude lower than the native sequence. The peptide was modelled into the potential antigen-combining site of HLA class II based on the ability of analogues containing point mutations to stimulate the T-cell clone. The working model was initially tested by examining the ability of Epstein-Barr-transformed B-cell lines expressing in different DR4 subtypes to present the native haemagglutinin sequence and analogues to the clone. The different alleles could be categorized as high, intermediate, or low responders based on the resulting proliferation. DR4 dw15 was a high-responding allele, dw4, 13, and 14 were intermediate-responding alleles, whereas dw10 was a low responder. Mutation of Gln to Arg at 312 in the haemagglutinin sequence converted the high and intermediate responders to non-responders, while turning the low-responding allele into an intermediate responder. Potential explanations for these effects are discussed in the context of the model of the complex between peptide and the major histocompatibility complex.

  19. Object recognition by artificial cortical maps.

    PubMed

    Plebe, Alessio; Domenella, Rosaria Grazia

    2007-09-01

    Object recognition is one of the most important functions of the human visual system, yet one of the least understood, this despite the fact that vision is certainly the most studied function of the brain. We understand relatively well how several processes in the cortical visual areas that support recognition capabilities take place, such as orientation discrimination and color constancy. This paper proposes a model of the development of object recognition capability, based on two main theoretical principles. The first is that recognition does not imply any sort of geometrical reconstruction, it is instead fully driven by the two dimensional view captured by the retina. The second assumption is that all the processing functions involved in recognition are not genetically determined or hardwired in neural circuits, but are the result of interactions between epigenetic influences and basic neural plasticity mechanisms. The model is organized in modules roughly related to the main visual biological areas, and is implemented mainly using the LISSOM architecture, a recent neural self-organizing map model that simulates the effects of intercortical lateral connections. This paper shows how recognition capabilities, similar to those found in brain ventral visual areas, can develop spontaneously by exposure to natural images in an artificial cortical model.

  20. 8 CFR 1292.2 - Organizations qualified for recognition; requests for recognition; withdrawal of recognition...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Organizations qualified for recognition; requests for recognition; withdrawal of recognition; accreditation of representatives; roster. 1292.2... IMMIGRATION REGULATIONS REPRESENTATION AND APPEARANCES § 1292.2 Organizations qualified for recognition...

  1. Sequence divergence and loss-of-function phenotypes of S locus F-box brothers genes are consistent with non-self recognition by multiple pollen determinants in self-incompatibility of Japanese pear (Pyrus pyrifolia).

    PubMed

    Kakui, Hiroyuki; Kato, Masaki; Ushijima, Koichiro; Kitaguchi, Miyoko; Kato, Shu; Sassa, Hidenori

    2011-12-01

    The S-RNase-based gametophytic self-incompatibility (SI) of Rosaceae, Solanaceae, and Plantaginaceae is controlled by at least two tightly linked genes located at the complex S locus; the highly polymorphic S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen. Self-incompatibility in Prunus (Rosaceae) is considered to represent a 'self recognition by a single factor' system, because loss-of-function of SFB is associated with self-compatibility, and allelic divergence of SFB is high and comparable to that of S-RNase. In contrast, Petunia (Solanaceae) exhibits 'non-self recognition by multiple factors'. However, the distribution of 'self recognition' and 'non-self recognition' SI systems in different taxa is not clear. In addition, in 'non-self recognition' systems, a loss-of-function phenotype of pollen S is unknown. Here we analyze the divergence of SFBB genes, the multiple pollen S candidates, of a rosaceous plant Japanese pear (Pyrus pyrifolia) and show that intrahaplotypic divergence is high and comparable to the allelic diversity of S-RNase while interhaplotypic divergence is very low. Next, we analyzed loss-of-function of the SFBB1 type gene. Genetic analysis showed that pollen with the mutant haplotype S(4sm) lacking SFBB1-S(4) is rejected by pistils with an otherwise compatible S(1) while it is accepted by other non-self pistils. We found that the S(5) haplotype encodes a truncated SFBB1 protein, even though S(5) pollen is accepted normally by pistils with S(1) and other non-self haplotypes. These findings suggest that Japanese pear has a 'non-self recognition by multiple factors' SI system, although it is a species of Rosaceae to which Prunus also belongs.

  2. Probabilistic Open Set Recognition

    NASA Astrophysics Data System (ADS)

    Jain, Lalit Prithviraj

    Real-world tasks in computer vision, pattern recognition and machine learning often touch upon the open set recognition problem: multi-class recognition with incomplete knowledge of the world and many unknown inputs. An obvious way to approach such problems is to develop a recognition system that thresholds probabilities to reject unknown classes. Traditional rejection techniques are not about the unknown; they are about the uncertain boundary and rejection around that boundary. Thus traditional techniques only represent the "known unknowns". However, a proper open set recognition algorithm is needed to reduce the risk from the "unknown unknowns". This dissertation examines this concept and finds existing probabilistic multi-class recognition approaches are ineffective for true open set recognition. We hypothesize the cause is due to weak adhoc assumptions combined with closed-world assumptions made by existing calibration techniques. Intuitively, if we could accurately model just the positive data for any known class without overfitting, we could reject the large set of unknown classes even under this assumption of incomplete class knowledge. For this, we formulate the problem as one of modeling positive training data by invoking statistical extreme value theory (EVT) near the decision boundary of positive data with respect to negative data. We provide a new algorithm called the PI-SVM for estimating the unnormalized posterior probability of class inclusion. This dissertation also introduces a new open set recognition model called Compact Abating Probability (CAP), where the probability of class membership decreases in value (abates) as points move from known data toward open space. We show that CAP models improve open set recognition for multiple algorithms. Leveraging the CAP formulation, we go on to describe the novel Weibull-calibrated SVM (W-SVM) algorithm, which combines the useful properties of statistical EVT for score calibration with one-class and binary

  3. Toward hyperspectral face recognition

    NASA Astrophysics Data System (ADS)

    Robila, Stefan A.

    2008-02-01

    Face recognition continues to meet significant challenges in reaching accurate results and still remains one of the activities where humans outperform technology. An attractive approach in improving face identification is provided by the fusion of multiple imaging sources such as visible and infrared images. Hyperspectral data, i.e. images collected over hundreds of narrow contiguous light spectrum intervals constitute a natural choice for expanding face recognition image fusion, especially since it may provide information beyond the normal visible range, thus exceeding the normal human sensing. In this paper we investigate the efficiency of hyperspectral face recognition through an in house experiment that collected data in over 120 bands within the visible and near infrared range. The imagery was produced using an off the shelf sensor in both indoors and outdoors with the subjects being photographed from various angles. Further processing included spectra collection and feature extraction. Human matching performance based on spectral properties is discussed.

  4. Macrophage pattern recognition receptors in immunity, homeostasis and self tolerance.

    PubMed

    Mukhopadhyay, Subhankar; Plüddemann, Annette; Gordon, Siamon

    2009-01-01

    Macrophages, a major component of innate immune defence, express a large repertoire of different classes of pattern recognition receptors and other surface antigens which determine the immunologic and homeostatic potential of these versatile cells. In the light of present knowledge ofmacrophage surface antigens, we discuss self versus nonself recognition, microbicidal effector functions and self tolerance in the innate immune system.

  5. Neonatal Recognition Processes and Attachment: The Masking Experiment.

    ERIC Educational Resources Information Center

    Cassel, Thomas Z. K.; Sander, Louis W.

    This research project was designed to determine whether 1-week-old neonates would indicate biological recognition of their mothers. Biological recognition is defined as the particular configuration of sensory, kinesthetic, and motor cues and the temporal patterning of these cues which characterizes infants' exchange processes with their…

  6. Recognition of emotions in autism: a formal meta-analysis.

    PubMed

    Uljarevic, Mirko; Hamilton, Antonia

    2013-07-01

    Determining the integrity of emotion recognition in autistic spectrum disorder is important to our theoretical understanding of autism and to teaching social skills. Previous studies have reported both positive and negative results. Here, we take a formal meta-analytic approach, bringing together data from 48 papers testing over 980 participants with autism. Results show there is an emotion recognition difficulty in autism, with a mean effect size of 0.80 which reduces to 0.41 when a correction for publication bias is applied. Recognition of happiness was only marginally impaired in autism, but recognition of fear was marginally worse than recognition of happiness. This meta-analysis provides an opportunity to survey the state of emotion recognition research in autism and to outline potential future directions.

  7. Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-[alpha], and Amylin by Human Insulin-Degrading Enzyme

    SciTech Connect

    Guo, Qing; Manolopoulou, Marika; Bian, Yao; Schilling, Alexander B.; Tang, Wei-Jen

    2010-02-11

    Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-{beta}, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-{alpha} (TGF-{alpha}) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-{alpha}, amylin, reduced amylin, and amyloid-{beta} by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-{alpha} at 2.3 {angstrom} and IDE-amylin at 2.9 {angstrom}. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.

  8. [Prosopagnosia and facial expression recognition].

    PubMed

    Koyama, Shinichi

    2014-04-01

    This paper reviews clinical neuropsychological studies that have indicated that the recognition of a person's identity and the recognition of facial expressions are processed by different cortical and subcortical areas of the brain. The fusiform gyrus, especially the right fusiform gyrus, plays an important role in the recognition of identity. The superior temporal sulcus, amygdala, and medial frontal cortex play important roles in facial-expression recognition. Both facial recognition and facial-expression recognition are highly intellectual processes that involve several regions of the brain.

  9. Risk Recognition and Intimate Partner Violence

    ERIC Educational Resources Information Center

    Witte, Tricia H.; Kendra, Rachel

    2010-01-01

    The objective of this study was to determine whether female victims of physical forms of intimate partner violence (IPV) displayed deficits in risk recognition, or the ability to detect danger, in physically violent dating encounters. A total of 182 women watched a video depicting a psychologically and physically aggressive encounter between…

  10. Risk Recognition and Intimate Partner Violence

    ERIC Educational Resources Information Center

    Witte, Tricia H.; Kendra, Rachel

    2010-01-01

    The objective of this study was to determine whether female victims of physical forms of intimate partner violence (IPV) displayed deficits in risk recognition, or the ability to detect danger, in physically violent dating encounters. A total of 182 women watched a video depicting a psychologically and physically aggressive encounter between…

  11. Letter Recognition Difficulties: Their Real Nature.

    ERIC Educational Resources Information Center

    Cohn, Marvin

    This paper examines the letter recognition difficulties of 322 primary grade students with decoding problems. Each student was asked to name all of the lower case letters, which were presented in non-alphabetical order. Each response was accurately recorded to determine which letters were taken for others. All responses were then tallied, and an…

  12. Optimizing the Reliability of Speech Recognition Scores.

    ERIC Educational Resources Information Center

    Gelfand, Stanley A.

    1998-01-01

    This study developed a testing approach to increase sample size to approximately 450 scorable items, previously determined as necessary to optimize the reliability of a speech recognition test. The approach involves an interactive computer program which presents words in 50 three-word groups. The approach was evaluated with 100 persons with normal…

  13. Pattern recognition systems and procedures

    NASA Technical Reports Server (NTRS)

    Nelson, G. D.; Serreyn, D. V.

    1972-01-01

    The objectives of the pattern recognition tasks are to develop (1) a man-machine interactive data processing system; and (2) procedures to determine effective features as a function of time for crops and soils. The signal analysis and dissemination equipment, SADE, is being developed as a man-machine interactive data processing system. SADE will provide imagery and multi-channel analog tape inputs for digitation and a color display of the data. SADE is an essential tool to aid in the investigation to determine useful features as a function of time for crops and soils. Four related studies are: (1) reliability of the multivariate Gaussian assumption; (2) usefulness of transforming features with regard to the classifier probability of error; (3) advantage of selecting quantizer parameters to minimize the classifier probability of error; and (4) advantage of using contextual data. The study of transformation of variables (features), especially those experimental studies which can be completed with the SADE system, will be done.

  14. Segmental Rescoring in Text Recognition

    DTIC Science & Technology

    2014-02-04

    ttm № tes/m, m* tmvr mowm* a Smyrna Of l δrtA£ACf02S’ A w m - y i p m AmiKSiS € f № ) C № № m .. sg6#?«rA fiθN ; Atφ h Sft№’·’Spxn mm m fim f№b t&m&mm...applying a Hidden Markov Model (HMM) recognition approach. Generating the plurality text hypotheses for the image forming includes generating a first...image. Applying segmental analysis to a segmentation determined by a first OCR engine, such as a segmentation determined by a Hidden Markov Model (HMM

  15. Recognition of caudal regression syndrome.

    PubMed

    Boulas, Mari M

    2009-04-01

    Caudal regression syndrome, also referred to as caudal dysplasia and sacral agenesis syndrome, is a rare congenital malformation characterized by varying degrees of developmental failure early in gestation. It involves the lower extremities, the lumbar and coccygeal vertebrae, and corresponding segments of the spinal cord. This is a rare disorder, and true pathogenesis is unclear. The etiology is thought to be related to maternal diabetes, genetic predisposition, and vascular hypoperfusion, but no true causative factor has been determined. Fetal diagnostic tools allow for early recognition of the syndrome, and careful examination of the newborn is essential to determine the extent of the disorder. Associated organ system dysfunction depends on the severity of the disease. Related defects are structural, and systematic problems including respiratory, cardiac, gastrointestinal, urinary, orthopedic, and neurologic can be present in varying degrees of severity and in different combinations. A multidisciplinary approach to management is crucial. Because the primary pathology is irreversible, treatment is only supportive.

  16. View Invariant Gait Recognition

    NASA Astrophysics Data System (ADS)

    Seely, Richard D.; Goffredo, Michela; Carter, John N.; Nixon, Mark S.

    Recognition by gait is of particular interest since it is the biometric that is available at the lowest resolution, or when other biometrics are (intentionally) obscured. Gait as a biometric has now shown increasing recognition capability. There are many approaches and these show that recognition can achieve excellent performance on current large databases. The majority of these approaches are planar 2D, largely since the early large databases featured subjects walking in a plane normal to the camera view. To extend deployment capability, we need viewpoint invariant gait biometrics. We describe approaches where viewpoint invariance is achieved by 3D approaches or in 2D. In the first group, the identification relies on parameters extracted from the 3D body deformation during walking. These methods use several video cameras and the 3D reconstruction is achieved after a camera calibration process. On the other hand, the 2D gait biometric approaches use a single camera, usually positioned perpendicular to the subject’s walking direction. Because in real surveillance scenarios a system that operates in an unconstrained environment is necessary, many of the recent gait analysis approaches are orientated toward view-invariant gait recognition.

  17. Automated Optical Target Recognition.

    DTIC Science & Technology

    1994-12-01

    A multi-resolution signal processing approach to object recognition is presented using an optical correlator for generating a wavelet transform . The...This report presents an overview of continuous and discrete wavelet transforms. Both digital and optical implementations of the discrete wavelet ... transform are discussed. Examples of typical wavelet basis functions are compared and the constraints imposed by optical implementations are discussed

  18. Teaching Word Recognition Skills.

    ERIC Educational Resources Information Center

    Dawson, Mildred A., Comp.

    A series of articles with the chief emphasis on phonics as a means of analyzing words is presented. Various articles pertain to elementary, secondary, and college level instruction. The first of the five parts into which the volume is divided is comprised of a single article which gives an excellent overview of the field of word recognition. Part…

  19. Pattern recognition in bioinformatics.

    PubMed

    de Ridder, Dick; de Ridder, Jeroen; Reinders, Marcel J T

    2013-09-01

    Pattern recognition is concerned with the development of systems that learn to solve a given problem using a set of example instances, each represented by a number of features. These problems include clustering, the grouping of similar instances; classification, the task of assigning a discrete label to a given instance; and dimensionality reduction, combining or selecting features to arrive at a more useful representation. The use of statistical pattern recognition algorithms in bioinformatics is pervasive. Classification and clustering are often applied to high-throughput measurement data arising from microarray, mass spectrometry and next-generation sequencing experiments for selecting markers, predicting phenotype and grouping objects or genes. Less explicitly, classification is at the core of a wide range of tools such as predictors of genes, protein function, functional or genetic interactions, etc., and used extensively in systems biology. A course on pattern recognition (or machine learning) should therefore be at the core of any bioinformatics education program. In this review, we discuss the main elements of a pattern recognition course, based on material developed for courses taught at the BSc, MSc and PhD levels to an audience of bioinformaticians, computer scientists and life scientists. We pay attention to common problems and pitfalls encountered in applications and in interpretation of the results obtained.

  20. Geophysical Signal Recognition,

    DTIC Science & Technology

    1981-01-01

    quite helpful in the magnetosphere. Detecting a particular in earthquake prediction . However pattern recog- micropulsation event can provide a diagnosis...bio- In su..a.iry, application of pattern recognition to medical signals, progress in geophysical signal earthquake prediction is in its infancy

  1. Optical Character Recognition.

    ERIC Educational Resources Information Center

    Converso, L.; Hocek, S.

    1990-01-01

    This paper describes computer-based optical character recognition (OCR) systems, focusing on their components (the computer, the scanner, the OCR, and the output device); how the systems work; and features to consider in selecting a system. A list of 26 questions to ask to evaluate systems for potential purchase is included. (JDD)

  2. Units of Word Recognition.

    ERIC Educational Resources Information Center

    Santa, Carol M.; And Others

    Both psychologists and reading specialists have been interested in whether words are processed letter by letter or in larger units. A reaction time paradigm was used to evaluate these options with interest focused on potential units of word recognition which might be functional within single syllable words. The basic paradigm involved presenting…

  3. Intralist Cueing of Recognition

    ERIC Educational Resources Information Center

    Slamecka, Norman J.

    1975-01-01

    Two experiments tested for effects of intralist cues upon recognition probability. Categorized and random lists were each tested, with targets appearing with zero, one or three intralist cues. Experiments showed substantial effects of trials and list type, but not of intralist context. (CHK)

  4. Optical Character Recognition.

    ERIC Educational Resources Information Center

    Converso, L.; Hocek, S.

    1990-01-01

    This paper describes computer-based optical character recognition (OCR) systems, focusing on their components (the computer, the scanner, the OCR, and the output device); how the systems work; and features to consider in selecting a system. A list of 26 questions to ask to evaluate systems for potential purchase is included. (JDD)

  5. Recognition for Employed Inventors.

    ERIC Educational Resources Information Center

    Sanders, Howard J.

    1980-01-01

    Presents arguments for monetary rewards and other forms of recognition by employers for inventions of employed inventors, particularly as the concept applies to stimulating innovativeness in America. Discusses the controversy of federally mandated compensation for employed inventors. The efforts of the American Chemical Society along these lines…

  6. Automatic object recognition

    NASA Technical Reports Server (NTRS)

    Ranganath, H. S.; Mcingvale, Pat; Sage, Heinz

    1988-01-01

    Geometric and intensity features are very useful in object recognition. An intensity feature is a measure of contrast between object pixels and background pixels. Geometric features provide shape and size information. A model based approach is presented for computing geometric features. Knowledge about objects and imaging system is used to estimate orientation of objects with respect to the line of sight.

  7. Recognition Memory for Pseudowords

    ERIC Educational Resources Information Center

    Greene, Robert L.

    2004-01-01

    Participants are more likely to give positive responses on a recognition test to pseudowords (pronounceable nonwords) than words. A series of experiments suggests that this difference reflects the greater overall familiarity of pseudowords than of words. Pseudowords receive higher ratings of similarity to a studied list than do words. Pseudowords…

  8. Automatic aircraft recognition

    NASA Astrophysics Data System (ADS)

    Hmam, Hatem; Kim, Jijoong

    2002-08-01

    Automatic aircraft recognition is very complex because of clutter, shadows, clouds, self-occlusion and degraded imaging conditions. This paper presents an aircraft recognition system, which assumes from the start that the image is possibly degraded, and implements a number of strategies to overcome edge fragmentation and distortion. The current vision system employs a bottom up approach, where recognition begins by locating image primitives (e.g., lines and corners), which are then combined in an incremental fashion into larger sets of line groupings using knowledge about aircraft, as viewed from a generic viewpoint. Knowledge about aircraft is represented in the form of whole/part shape description and the connectedness property, and is embedded in production rules, which primarily aim at finding instances of the aircraft parts in the image and checking the connectedness property between the parts. Once a match is found, a confidence score is assigned and as evidence in support of an aircraft interpretation is accumulated, the score is increased proportionally. Finally a selection of the resulting image interpretations with the highest scores, is subjected to competition tests, and only non-ambiguous interpretations are allowed to survive. Experimental results demonstrating the effectiveness of the current recognition system are given.

  9. School IPM Recognition and Certification

    EPA Pesticide Factsheets

    Schools and school districts can get support and recognition for implementation of school IPM. EPA is developing a program to provide recognition for school districts that are working towards or have achieved a level of success with school IPM programs.

  10. Anticipatory coarticulation facilitates word recognition in toddlers.

    PubMed

    Mahr, Tristan; McMillan, Brianna T M; Saffran, Jenny R; Ellis Weismer, Susan; Edwards, Jan

    2015-09-01

    Children learn from their environments and their caregivers. To capitalize on learning opportunities, young children have to recognize familiar words efficiently by integrating contextual cues across word boundaries. Previous research has shown that adults can use phonetic cues from anticipatory coarticulation during word recognition. We asked whether 18-24 month-olds (n=29) used coarticulatory cues on the word "the" when recognizing the following noun. We performed a looking-while-listening eyetracking experiment to examine word recognition in neutral vs. facilitating coarticulatory conditions. Participants looked to the target image significantly sooner when the determiner contained facilitating coarticulatory cues. These results provide the first evidence that novice word-learners can take advantage of anticipatory sub-phonemic cues during word recognition.

  11. Recognition memory: material, processes, and substrates.

    PubMed

    Brown, Malcolm W; Warburton, E Clea; Aggleton, John P

    2010-11-01

    The proposal that a system centering on the perirhinal cortex is responsible for familiarity discrimination, particularly for single items, whereas a system centering on the hippocampus is responsible for recollective and more complex associational aspects of recognition memory is reviewed in the light of recent findings. In particular, the proposal is reviewed in relation to recent animal work with rats and results from human clinical studies. Notably, progress has been made in determining potential neural memory substrate mechanisms within the perirhinal cortex in rats. Recent findings have emphasized the importance of specifying the type of material, the type of test, and the strategy used by subjects to solve recognition memory tests if substrates are to be accurately inferred. It is to be expected that the default condition is that both the hippocampal and perirhinal systems will contribute to recognition memory performance. Indeed, rat lesion experiments provide examples of where cooperation between both systems is essential. Nevertheless, there remain examples of the independent operation of the hippocampal and perirhinal systems. Overall, it is concluded that most, though not all, of the recent findings are in support of the proposal. However, there is also evidence that the systems involved in recognition memory need to include structures outside the medial temporal lobe: there are significant but as yet only partially defined roles for the prefrontal cortex and sensory association cortices in recognition memory processes. © 2010 Wiley-Liss, Inc.

  12. Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection

    USDA-ARS?s Scientific Manuscript database

    Staphylococcus aureus is a Gram-positive pathogen relevant for both human and animal health. With multi-drug resistant S. aureus strains becoming increasingly prevalent, alternative therapeutics are urgently needed. Bacteriophage endolysins (peptidoglycan hydrolases, PGH) are capable of killing Gra...

  13. Support vector machines for prediction of protein signal sequences and their cleavage sites.

    PubMed

    Cai, Yu-Dong; Lin, Shuo-liang; Chou, Kuo-Chen

    2003-01-01

    Given a nascent protein sequence, how can one predict its signal peptide or "Zipcode" sequence? This is an important problem for scientists to use signal peptides as a vehicle to find new drugs or to reprogram cells for gene therapy (see, e.g. K.C. Chou, Current Protein and Peptide Science 2002;3:615-22). In this paper, support vector machines (SVMs), a new machine learning method, is applied to approach this problem. The overall rate of correct prediction for 1939 secretary proteins and 1440 nonsecretary proteins was over 91%. It has not escaped our attention that the new method may also serve as a useful tool for further investigating many unclear details regarding the molecular mechanism of the ZIP code protein-sorting system in cells. Copyright 2002 Elsevier Science Inc.

  14. Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

    SciTech Connect

    Selmer, B.L.; Hanecak, R.; Anderson, C.W.; Wimmer, E.

    1981-01-01

    Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.

  15. Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26.

    PubMed

    Park, Hyun I; Turk, Benjamin E; Gerkema, Ferry E; Cantley, Lewis C; Sang, Qing-Xiang Amy

    2002-09-20

    Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is a novel epithelial and cancer-specific metalloproteinase. Peptide libraries were used to profile the substrate specificity of MMP-26 from the P4-P4' sites. The optimal cleavage motifs for MMP-26 were Lys-Pro-Ile/Leu-Ser(P1)-Leu/Met(P1')-Ile/Thr-Ser/Ala-Ser. The strongest preference was observed at the P1' and P2 sites where hydrophobic residues were favored. Proline was preferred at P3, and Serine was preferred at P1. The overall specificity was similar to that of other MMPs with the exception that more flexibility was observed at P1, P2', and P3'. Accordingly, synthetic inhibitors of gelatinases and collagenases inhibited MMP-26 with similar efficacy. A pair of stereoisomers had only a 40-fold difference in K(i)(app) values against MMP-26 compared with a 250-fold difference against neutrophil collagenase, indicating that MMP-26 is less stereoselective for its inhibitors. MMP-26 autodigested itself during the folding process. Two of the major autolytic sites were Leu(49)-Thr(50) and Ala(75)-Leu(76), which still left the cysteine switch sequence (PHC(82)GVPD) intact. This suggests that Cys(82) may not play a role in the latency of the zymogen. Interestingly, inhibitor titration studies revealed that only approximately 5% of the total MMP-26 molecules was catalytically active, indicating that the thiol groups of Cys(82) in the active molecules may be dissociated or removed from the active site zinc ions. MMP-26 cleaved Phe(352)-Leu(353) and Pro(357)-Met(358) in the reactive loop of alpha(1)-proteinase inhibitor and His(140)-Val(141) in insulin-like growth factor-binding protein-1, probably rendering these substrates inactive. Among the fluorescent peptide substrates analyzed, Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH(2) displayed the highest specificity constant (30,000/molar second) with MMP-26. This report proposes a working model for the future studies of pro-MMP-26 activation, the design of inhibitors, and the identification of optimal physiological and pathological substrates of MMP-26 in vivo.

  16. International Recognition of Vocational Qualifications.

    ERIC Educational Resources Information Center

    Imrie, Bradford W.

    Certain issues are relevant to the international recognition of vocational qualifications: (1) the assumption that each country does or should value vocational education and training; (2) the quality of the national system and the implications for international recognition of qualifications, including recognition of the accrediting and awarding…

  17. Speech Recognition: A General Overview.

    ERIC Educational Resources Information Center

    de Sopena, Luis

    Speech recognition is one of five main areas in the field of speech processing. Difficulties in speech recognition include variability in sound within and across speakers, in channel, in background noise, and of speech production. Speech recognition can be used in a variety of situations: to perform query operations and phone call transfers; for…

  18. Word Recognition in Auditory Cortex

    ERIC Educational Resources Information Center

    DeWitt, Iain D. J.

    2013-01-01

    Although spoken word recognition is more fundamental to human communication than text recognition, knowledge of word-processing in auditory cortex is comparatively impoverished. This dissertation synthesizes current models of auditory cortex, models of cortical pattern recognition, models of single-word reading, results in phonetics and results in…

  19. Supporting Quality Teachers with Recognition

    ERIC Educational Resources Information Center

    Andrews, Hans A.

    2011-01-01

    Value has been found in providing recognition and awards programs for excellent teachers. Research has also found a major lack of these programs in both the USA and in Australia. Teachers receiving recognition and awards for their teaching have praised recognition programs as providing motivation for them to continue high-level instruction.…

  20. Visual Recognition Memory across Contexts

    ERIC Educational Resources Information Center

    Jones, Emily J. H.; Pascalis, Olivier; Eacott, Madeline J.; Herbert, Jane S.

    2011-01-01

    In two experiments, we investigated the development of representational flexibility in visual recognition memory during infancy using the Visual Paired Comparison (VPC) task. In Experiment 1, 6- and 9-month-old infants exhibited recognition when familiarization and test occurred in the same room, but showed no evidence of recognition when…

  1. Word Recognition in Auditory Cortex

    ERIC Educational Resources Information Center

    DeWitt, Iain D. J.

    2013-01-01

    Although spoken word recognition is more fundamental to human communication than text recognition, knowledge of word-processing in auditory cortex is comparatively impoverished. This dissertation synthesizes current models of auditory cortex, models of cortical pattern recognition, models of single-word reading, results in phonetics and results in…

  2. Superficial Priming in Episodic Recognition

    ERIC Educational Resources Information Center

    Dopkins, Stephen; Sargent, Jesse; Ngo, Catherine T.

    2010-01-01

    We explored the effect of superficial priming in episodic recognition and found it to be different from the effect of semantic priming in episodic recognition. Participants made recognition judgments to pairs of items, with each pair consisting of a prime item and a test item. Correct positive responses to the test item were impeded if the prime…

  3. Visual Recognition Memory across Contexts

    ERIC Educational Resources Information Center

    Jones, Emily J. H.; Pascalis, Olivier; Eacott, Madeline J.; Herbert, Jane S.

    2011-01-01

    In two experiments, we investigated the development of representational flexibility in visual recognition memory during infancy using the Visual Paired Comparison (VPC) task. In Experiment 1, 6- and 9-month-old infants exhibited recognition when familiarization and test occurred in the same room, but showed no evidence of recognition when…

  4. Automatic testing of speech recognition.

    PubMed

    Francart, Tom; Moonen, Marc; Wouters, Jan

    2009-02-01

    Speech reception tests are commonly administered by manually scoring the oral response of the subject. This requires a test supervisor to be continuously present. To avoid this, a subject can type the response, after which it can be scored automatically. However, spelling errors may then be counted as recognition errors, influencing the test results. We demonstrate an autocorrection approach based on two scoring algorithms to cope with spelling errors. The first algorithm deals with sentences and is based on word scores. The second algorithm deals with single words and is based on phoneme scores. Both algorithms were evaluated with a corpus of typed answers based on three different Dutch speech materials. The percentage of differences between automatic and manual scoring was determined, in addition to the mean difference in speech recognition threshold. The sentence correction algorithm performed at a higher accuracy than commonly obtained with these speech materials. The word correction algorithm performed better than the human operator. Both algorithms can be used in practice and allow speech reception tests with open set speech materials over the internet.

  5. When true recognition suppresses false recognition: evidence from amnesic patients.

    PubMed

    Schacter, D L; Verfaellie, M; Anes, M D; Racine, C

    1998-11-01

    False recognition occurs when people mistakenly claim that a novel item is familiar. After studying lists of semantically related words, healthy controls show extraordinarily high levels of false recognition to nonstudied lures that are semantic associates of study list words. In previous experiments, we found that both Korsakoff and non-Korsakoff amnesic patients show reduced levels of false recognition to semantic associates, implying that the medial temporal/diencephalic structures that are damaged in amnesic patients are involved in the encoding and/or retrieval of information that underlies false recognition. These data contrast with earlier results indicating greater false recognition in Korsakoff amnesics than in control subjects. The present experiment tests the hypothesis that greater or lesser false recognition of semantic associates in amnesic patients, relative to normal controls, can be demonstrated by creating conditions that are more or less conducive to allowing true recognition to suppress false recognition. With repeated presentation and testing of lists of semantic associates, control subjects and both Korsakoff and non-Korsakoff amnesics showed increasing levels of true recognition across trials. However, control subjects exhibited decreasing levels of false recognition across trials, whereas Korsakoff amnesic patients showed increases across trials and non-Korsakoff amnesics showed a fluctuating pattern. Consideration of signal detection analyses and differences between the two types of amnesic patients provides insight into how mechanisms of veridical episodic memory can be used to suppress false recognition.

  6. Prosody and Spoken Word Recognition in Early and Late Spanish-English Bilingual Individuals

    ERIC Educational Resources Information Center

    Boutsen, Frank R.; Dvorak, Justin D.; Deweber, Derick D.

    2017-01-01

    Purpose: This study was conducted to compare the influence of word properties on gated single-word recognition in monolingual and bilingual individuals under conditions of native and nonnative accent and to determine whether word-form prosody facilitates recognition in bilingual individuals. Method: Word recognition was assessed in monolingual and…

  7. Age of Acquisition and Sensitivity to Gender in Spanish Word Recognition

    ERIC Educational Resources Information Center

    Foote, Rebecca

    2014-01-01

    Speakers of gender-agreement languages use gender-marked elements of the noun phrase in spoken-word recognition: A congruent marking on a determiner or adjective facilitates the recognition of a subsequent noun, while an incongruent marking inhibits its recognition. However, while monolinguals and early language learners evidence this…

  8. Age of Acquisition and Sensitivity to Gender in Spanish Word Recognition

    ERIC Educational Resources Information Center

    Foote, Rebecca

    2014-01-01

    Speakers of gender-agreement languages use gender-marked elements of the noun phrase in spoken-word recognition: A congruent marking on a determiner or adjective facilitates the recognition of a subsequent noun, while an incongruent marking inhibits its recognition. However, while monolinguals and early language learners evidence this…

  9. Image recognition: Visual grouping, recognition, and learning

    PubMed Central

    Buhmann, Joachim M.; Malik, Jitendra; Perona, Pietro

    1999-01-01

    Vision extracts useful information from images. Reconstructing the three-dimensional structure of our environment and recognizing the objects that populate it are among the most important functions of our visual system. Computer vision researchers study the computational principles of vision and aim at designing algorithms that reproduce these functions. Vision is difficult: the same scene may give rise to very different images depending on illumination and viewpoint. Typically, an astronomical number of hypotheses exist that in principle have to be analyzed to infer a correct scene description. Moreover, image information might be extracted at different levels of spatial and logical resolution dependent on the image processing task. Knowledge of the world allows the visual system to limit the amount of ambiguity and to greatly simplify visual computations. We discuss how simple properties of the world are captured by the Gestalt rules of grouping, how the visual system may learn and organize models of objects for recognition, and how one may control the complexity of the description that the visual system computes. PMID:10588681

  10. Image recognition: visual grouping, recognition, and learning.

    PubMed

    Buhmann, J M; Malik, J; Perona, P

    1999-12-07

    Vision extracts useful information from images. Reconstructing the three-dimensional structure of our environment and recognizing the objects that populate it are among the most important functions of our visual system. Computer vision researchers study the computational principles of vision and aim at designing algorithms that reproduce these functions. Vision is difficult: the same scene may give rise to very different images depending on illumination and viewpoint. Typically, an astronomical number of hypotheses exist that in principle have to be analyzed to infer a correct scene description. Moreover, image information might be extracted at different levels of spatial and logical resolution dependent on the image processing task. Knowledge of the world allows the visual system to limit the amount of ambiguity and to greatly simplify visual computations. We discuss how simple properties of the world are captured by the Gestalt rules of grouping, how the visual system may learn and organize models of objects for recognition, and how one may control the complexity of the description that the visual system computes.

  11. Exploring the DNA-recognition potential of homeodomains.

    PubMed

    Chu, Stephanie W; Noyes, Marcus B; Christensen, Ryan G; Pierce, Brian G; Zhu, Lihua J; Weng, Zhiping; Stormo, Gary D; Wolfe, Scot A

    2012-10-01

    The recognition potential of most families of DNA-binding domains (DBDs) remains relatively unexplored. Homeodomains (HDs), like many other families of DBDs, display limited diversity in their preferred recognition sequences. To explore the recognition potential of HDs, we utilized a bacterial selection system to isolate HD variants, from a randomized library, that are compatible with each of the 64 possible 3' triplet sites (i.e., TAANNN). The majority of these selections yielded sets of HDs with overrepresented residues at specific recognition positions, implying the selection of specific binders. The DNA-binding specificity of 151 representative HD variants was subsequently characterized, identifying HDs that preferentially recognize 44 of these target sites. Many of these variants contain novel combinations of specificity determinants that are uncommon or absent in extant HDs. These novel determinants, when grafted into different HD backbones, produce a corresponding alteration in specificity. This information was used to create more explicit HD recognition models, which can inform the prediction of transcriptional regulatory networks for extant HDs or the engineering of HDs with novel DNA-recognition potential. The diversity of recovered HD recognition sequences raises important questions about the fitness barrier that restricts the evolution of alternate recognition modalities in natural systems.

  12. Optical Pattern Recognition

    NASA Astrophysics Data System (ADS)

    Yu, Francis T. S.; Jutamulia, Suganda

    1998-06-01

    This book provides a comprehensive review of optical pattern recognition, covering theoretical aspects as well as details of practical implementations and signal processing techniques. The first chapter is devoted to pattern recognition performed with optical correlators. Later chapters discuss new approaches based on neural networks, wavelet transforms, and the fractional Fourier transform. The book also covers nonlinear filter methods and optical-electronic hybrid systems. The final part deals with the devices and materials employed in modern systems, such as photorefractive crystals, microlasers, and liquid crystal spatial light modulators. The volume gives many examples of working systems that integrate optics, electronics, and computers, and it covers a range of new developments from mathematical theories to novel optical materials. It will be of great interest to graduate students and researchers in optical engineering and machine vision.

  13. Audio-visual gender recognition

    NASA Astrophysics Data System (ADS)

    Liu, Ming; Xu, Xun; Huang, Thomas S.

    2007-11-01

    Combining different modalities for pattern recognition task is a very promising field. Basically, human always fuse information from different modalities to recognize object and perform inference, etc. Audio-Visual gender recognition is one of the most common task in human social communication. Human can identify the gender by facial appearance, by speech and also by body gait. Indeed, human gender recognition is a multi-modal data acquisition and processing procedure. However, computational multimodal gender recognition has not been extensively investigated in the literature. In this paper, speech and facial image are fused to perform a mutli-modal gender recognition for exploring the improvement of combining different modalities.

  14. Advanced Pattern Recognition.

    DTIC Science & Technology

    1983-05-01

    classification via statistical pattern recognition; image preprocessing, enhancement, and filtering; image warping , resampling, and point positioning; and...obj_region training files *•* Edit Programs »*» mode_filter ( mdf ) - mode filtering of a classified image (noise cleaning) edge_thin - thin... mdf 5 5 comments: experimental Method to segregate Water, Urban, Vegetation urban edges method_type: edge measurements: avg 3/ep_smooth 2

  15. Recognition by Prototypes

    DTIC Science & Technology

    1992-12-01

    as many other cues, such as color , texture, motion. views ii cf each model Mi, is composed of the k eigen- and context, and objects are categorized in...such as color and texture. [15] Grirnson W.E.L. and Lozano-P~rez T., 1984. Model-based recognition and localization from Acknowledgement sparse data...but not to see. A case study of visual agnosia . gie. Lawrence Erlbaum Associates, Pub., London. [2] Bajcsy R. and Solina F., 1987. Three dimensional

  16. Homology recognition funnel

    NASA Astrophysics Data System (ADS)

    Lee, Dominic; Kornyshev, Alexei A.

    2009-10-01

    The recognition of homologous sequences of DNA before strand exchange is considered to be the most puzzling stage of homologous recombination. A mechanism for two homologous dsDNAs to recognize each other from a distance in electrolytic solution without unzipping had been proposed in an earlier paper [A. A. Kornyshev and S. Leikin, Phys. Rev. Lett. 86, 366 (2001)]. In that work, the difference in the electrostatic interaction energy between homologous duplexes and between nonhomologous duplexes, termed the recognition energy, has been calculated. That calculation was later extended in a series of papers to account for torsional elasticity of the molecules. A recent paper [A. A. Kornyshev and A. Wynveen, Proc. Natl. Acad. Sci. U.S.A. 106, 4683 (2009)] investigated the form of the potential well that homologous DNA molecules may feel when sliding along each other. A simple formula for the shape of the well was obtained. However, this latter study was performed under the approximation that the sliding molecules are torsionally rigid. Following on from this work, in the present article we investigate the effect of torsional flexibility of the molecules on the shape of the well. A variational approach to this problem results in a transcendental equation that is easily solved numerically. Its solutions show that at large interaxial separations the recognition well becomes wider and shallower, whereas at closer distances further unexpected features arise related to an abrupt change in the mean azimuthal alignment of the molecules. The energy surface as a function of interaxial separation and the axial shift defines what we call the recognition funnel. We show that it depends dramatically on the patterns of adsorption of counterions on DNA.

  17. Metamorphopsia and letter recognition

    PubMed Central

    Wiecek, Emily; Dakin, Steven C.; Bex, Peter

    2014-01-01

    Acuity is the most commonly used measure of visual function, and reductions in acuity are associated with most eye diseases. Metamorphopsia—a perceived distortion of visual space—is another common symptom of visual impairment and is currently assessed qualitatively using Amsler (1953) charts. In order to quantify the impact of metamorphopsia on acuity, we measured the effect of physical spatial distortion on letter recognition. Following earlier work showing that letter recognition is tuned to specific spatial frequency (SF) channels, we hypothesized that the effect of distortion might depend on the spatial scale of visual distortion just as it depends on the spatial scale of masking noise. Six normally sighted observers completed a 26 alternate forced choice (AFC) Sloan letter identification task at five different viewing distances, and the letters underwent different levels of spatial distortion. Distortion was controlled using spatially band-pass filtered noise that spatially remapped pixel locations. Noise was varied over five spatial frequencies and five magnitudes. Performance was modeled with logistic regression and worsened linearly with increasing distortion magnitude and decreasing letter size. We found that retinal SF affects distortion at midrange frequencies and can be explained with the tuning of a basic contrast sensitivity function, while object-centered distortion SF follows a similar pattern of letter object recognition sensitivity and is tuned to approximately three cycles per letter (CPL). The interaction between letter size and distortion makes acuity an unreliable outcome for metamorphopsia assessment. PMID:25453116

  18. Metamorphopsia and letter recognition.

    PubMed

    Wiecek, Emily; Dakin, Steven C; Bex, Peter

    2014-12-01

    Acuity is the most commonly used measure of visual function, and reductions in acuity are associated with most eye diseases. Metamorphopsia--a perceived distortion of visual space--is another common symptom of visual impairment and is currently assessed qualitatively using Amsler (1953) charts. In order to quantify the impact of metamorphopsia on acuity, we measured the effect of physical spatial distortion on letter recognition. Following earlier work showing that letter recognition is tuned to specific spatial frequency (SF) channels, we hypothesized that the effect of distortion might depend on the spatial scale of visual distortion just as it depends on the spatial scale of masking noise. Six normally sighted observers completed a 26 alternate forced choice (AFC) Sloan letter identification task at five different viewing distances, and the letters underwent different levels of spatial distortion. Distortion was controlled using spatially band-pass filtered noise that spatially remapped pixel locations. Noise was varied over five spatial frequencies and five magnitudes. Performance was modeled with logistic regression and worsened linearly with increasing distortion magnitude and decreasing letter size. We found that retinal SF affects distortion at midrange frequencies and can be explained with the tuning of a basic contrast sensitivity function, while object-centered distortion SF follows a similar pattern of letter object recognition sensitivity and is tuned to approximately three cycles per letter (CPL). The interaction between letter size and distortion makes acuity an unreliable outcome for metamorphopsia assessment.

  19. Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

    PubMed

    Xie, H; Zhang, B; Matsumoto, Y; Li, Q; Notkins, A L; Lan, M S

    1997-10-01

    IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.

  20. Dissociating viewpoint costs in mental rotation and object recognition.

    PubMed

    Hayward, William G; Zhou, Guomei; Gauthier, Isabel; Harris, Irina M

    2006-10-01

    In a mental rotation task, participants must determine whether two stimuli match when one undergoes a rotation in 3-D space relative to the other. The key evidence for mental rotation is the finding of a linear increase in response times as objects are rotated farther apart. This signature increase in response times is also found in recognition of rotated objects, which has led many theorists to postulate mental rotation as a key transformational procedure in object recognition. We compared mental rotation and object recognition in tasks that used the same stimuli and presentation conditions and found that, whereas mental rotation costs increased relatively linearly with rotation, object recognition costs increased only over small rotations. Taken in conjunction with a recent brain imaging study, this dissociation in behavioral performance suggests that object recognition is based on matching of image features rather than on 3-D mental transformations.

  1. Human motion recognition based on features and models selected HMM

    NASA Astrophysics Data System (ADS)

    Lu, Haixiang; Zhou, Hongjun

    2015-03-01

    This paper research on the motion recognition based on HMM with Kinect. Kinect provides skeletal data consist of 3D body joints with its lower price and convenience. In this work, several methods are used to determine the optimal subset of features among Cartesian coordinates, distance to hip center, velocity, angle and angular velocity, in order to improve the recognition rate. K-means is used for vector quantization and HMM is used as recognition method. HMM is an effective signal processing method which contains time calibration, provides a learning mechanism and recognition ability. Cluster numbers of K-means, structure and state numbers of HMM are optimized as well. The proposed methods are applied to the MSR Action3D dataset. Results show that the proposed methods obtain better recognition accuracy than the state of the art methods.

  2. NMR-based analysis of aminoglycoside recognition by the resistance enzyme ANT(4'): the pattern of OH/NH3(+) substitution determines the preferred antibiotic binding mode and is critical for drug inactivation.

    PubMed

    Revuelta, Julia; Vacas, Tatiana; Torrado, Mario; Corzana, Francisco; Gonzalez, Carlos; Jiménez-Barbero, Jesús; Menendez, Margarita; Bastida, Agatha; Asensio, Juan Luis

    2008-04-16

    The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic inactivation of the drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside

  3. Determination of polychlorinated biphenyl levels in the serum of residents and in the homogenates of seafood from the New Bedford, Massachusetts Area: A comparison of exposure sources through pattern recognition techniques

    SciTech Connect

    Burse, V.W.; Groce, D.F.; Caudill, S.P.; Korver, M.P.; Phillips, D.L.

    1994-01-01

    Gas chromatographic patterns of polychlorinated biophenyls (PCBs) found in the serum of New Bedford, MA residents with high serum PCBs were compared to patterns found in lobsters and bluefish taken from local waters, and goats fed selected technical Aroclors (e.g., Aroclors 1016, 1242, 1254, or 1260) using Jaccard measures of similarity and Principal Component Analysis. Pattern in humans were silimar to patterns in lobsters and both were more similar to those in the goat fed Aroclor 1254 as demonstrated by both pattern recognition techniques. However, patterns observed in humans, lobsters and bluefish all exhibited some presence of PCBs more characteristic of Aroclors 1016 and/or 1242 or 1260.

  4. Sequence determinants for DNA packaging specificity in the S. aureus pathogenicity island SaPI1.

    PubMed

    Bento, Joana C; Lane, Kristin D; Read, Erik K; Cerca, Nuno; Christie, Gail E

    2014-01-01

    The SaPIs and their relatives are a family of genomic islands that exploit helper phages for high frequency horizontal transfer. One of the mechanisms used by SaPIs to accomplish this molecular piracy is the redirection of the helper phage DNA packaging machinery. SaPIs encode a small terminase subunit that can be substituted for that of the phage. In this study we have determined the initial packaging cleavage sites for helper phage 80α, which uses the phage-encoded small terminase subunit, and for SaPI1, which uses the SaPI-encoded small terminase subunit. We have identified a 19nt SaPI1 sequence that is necessary and sufficient to allow high frequency 80α transduction of a plasmid by a terminase carrying the SaPI1-encoded small subunit. We also show that the hybrid enzyme with the SaPI1 small terminase subunit is capable of generalized transduction.

  5. Smart pattern recognition

    NASA Astrophysics Data System (ADS)

    Alfalou, A.; Brosseau, C.; Alam, M. S.

    2013-03-01

    The purpose of this paper is to test correlation methods for pattern recognition applications. A broad overview of the main correlation architectures is first given. Many correlation data are compared with those obtained from standard pattern recognition methods. We used our simulations to predict improved decisional performance from correlation methods. More specifically, we are focused on the POF filter and composite filter family. We present an optimized composite correlation filter, called asymmetric segmented phase-only filter (ASPOF) for mobile target recognition applications. The main objective is to find a compromise between the number of references to be merged in the correlation filter and the time needed for making a decision. We suggest an all-numerical implementation of a VanderLugt (VLC) type composite filter. The aim of this all-numerical implementation is to take advantage of the benefits of the correlation methods and make the correlator easily reconfigurable for various scenarios. The use of numerical implementation of the optical Fourier transform improves the decisional performance of the correlator. Further, it renders the correlator less sensitive to the saturation phenomenon caused by the increased number of references used for fabricating the composite filter. Different tests are presented making use of the peak-to-correlation energy criterion and ROC curves. These tests confirm the validity ofour technique. Elderly fall detection and underwater mine detection are two applications which are considered for illustrating the benefits of our approach. The present work is motivated by the need for detailed discussions of the choice of the correlation architecture for these specific applications, pre-processing in the input plane and post processing in the output plane techniques for such analysis.

  6. Laptop Computer - Based Facial Recognition System Assessment

    SciTech Connect

    R. A. Cain; G. B. Singleton

    2001-03-01

    The objective of this project was to assess the performance of the leading commercial-off-the-shelf (COTS) facial recognition software package when used as a laptop application. We performed the assessment to determine the system's usefulness for enrolling facial images in a database from remote locations and conducting real-time searches against a database of previously enrolled images. The assessment involved creating a database of 40 images and conducting 2 series of tests to determine the product's ability to recognize and match subject faces under varying conditions. This report describes the test results and includes a description of the factors affecting the results. After an extensive market survey, we selected Visionics' FaceIt{reg_sign} software package for evaluation and a review of the Facial Recognition Vendor Test 2000 (FRVT 2000). This test was co-sponsored by the US Department of Defense (DOD) Counterdrug Technology Development Program Office, the National Institute of Justice, and the Defense Advanced Research Projects Agency (DARPA). Administered in May-June 2000, the FRVT 2000 assessed the capabilities of facial recognition systems that were currently available for purchase on the US market. Our selection of this Visionics product does not indicate that it is the ''best'' facial recognition software package for all uses. It was the most appropriate package based on the specific applications and requirements for this specific application. In this assessment, the system configuration was evaluated for effectiveness in identifying individuals by searching for facial images captured from video displays against those stored in a facial image database. An additional criterion was that the system be capable of operating discretely. For this application, an operational facial recognition system would consist of one central computer hosting the master image database with multiple standalone systems configured with duplicates of the master operating in

  7. Contributions of area Te2 to rat recognition memory

    PubMed Central

    Ho, Jonathan Weng-Thim; Narduzzo, Katherine Elizabeth; Outram, Alexandra; Tinsley, Christopher John; Henley, Jeremy Martin; Warburton, Elizabeth Clea; Brown, Malcolm Watson

    2011-01-01

    Ablations and local intracerebral infusions were used to determine the role of rat temporal association cortex (area Te2) in object recognition memory, so that this role might be compared with that of the adjacent perirhinal cortex (PRH). Bilateral lesions of Te2 impaired recognition memory measured by preferential exploration of a novel rather than a familiar object at delays ≥20 min but not after a 5-min delay. Local infusion bilaterally into Te2 of (1) CNQX to block AMPA/kainate receptors or (2) lidocaine to block axonal transmission or (3) AP5, an NMDA receptor antagonist, impaired recognition memory after a 24-h but not a 20-min delay. In PRH all these manipulations impair recognition memory after a 20-min as well as a 24-h delay. UBP302, a GluK1 kainate receptor antagonist, impaired recognition memory after a 24-h but not a 20-min delay, contrasting with its action in PRH where it impairs only shorter-term (20 min) recognition memory. Also in contrast to PRH, infusion of the muscarinic receptor antagonist scopolamine was without effect. The Te2 impairments could not readily be ascribed to perceptual deficits. Hence, Te2 is essential for object recognition memory at delays >5 or 20 min. Thus, at long delays both area Te2 and PRH are necessary for object recognition memory. PMID:21700715

  8. Disorders of visual recognition.

    PubMed

    De Renzi, E

    2000-01-01

    Agnosias are disorders of recognition, specific to one sensory channel, that affect either the perceptual analysis of the stimulus or the recognition of its meaning. In the visual modality, objects, faces, and colors can be separately disrupted. Apperceptive object agnosia refers to failure to achieve a structured description of the shape of the object. Associative agnosia refers to inability to attribute a meaning to a correctly perceived stimulus. It must be differentiated from optic aphasia, in which the object is recognized but cannot be named in the visual modality. Associative agnosia and optic aphasia are associated with left occipitotemporal damage, and they differ more quantitatively than qualitatively. The inability to recognize familiar faces (prosopagnosia) can appear in isolation and be, in some cases, associated with a lesion confined to the occipitotemporal region of the right hemisphere. These findings are supportive of the idea that faces have a separate representation in the brain. Disorders of color cognition can affect color categorization, color-name association, and color-object association. They are linked to left hemisphere damage. The ability to recognize objects presented in the visual modality is a hierarchical process in which several cortical areas, corresponding to about 30% of the cortical mantle, participate. Their selective lesion results in a gamut of disorders whose identification provides the experienced neurologist with clues to the locus of damage and contributes to the understanding of the cognitive architecture underpinning recognition. They can result either in the inability to detect any change occurring in the visual field or in the impairment of further stages of the recognition process, from the analysis of the perceptual properties of the stimulus (form, color, motion, depth, etc.) to the achievement of its structural description and, eventually, the attribution of a meaning. In this paper, I focus on the diagnostic and

  9. Automatic Speech Recognition

    NASA Astrophysics Data System (ADS)

    Potamianos, Gerasimos; Lamel, Lori; Wölfel, Matthias; Huang, Jing; Marcheret, Etienne; Barras, Claude; Zhu, Xuan; McDonough, John; Hernando, Javier; Macho, Dusan; Nadeu, Climent

    Automatic speech recognition (ASR) is a critical component for CHIL services. For example, it provides the input to higher-level technologies, such as summarization and question answering, as discussed in Chapter 8. In the spirit of ubiquitous computing, the goal of ASR in CHIL is to achieve a high performance using far-field sensors (networks of microphone arrays and distributed far-field microphones). However, close-talking microphones are also of interest, as they are used to benchmark ASR system development by providing a best-case acoustic channel scenario to compare against.

  10. [Comparative studies of face recognition].

    PubMed

    Kawai, Nobuyuki

    2012-07-01

    Every human being is proficient in face recognition. However, the reason for and the manner in which humans have attained such an ability remain unknown. These questions can be best answered-through comparative studies of face recognition in non-human animals. Studies in both primates and non-primates show that not only primates, but also non-primates possess the ability to extract information from their conspecifics and from human experimenters. Neural specialization for face recognition is shared with mammals in distant taxa, suggesting that face recognition evolved earlier than the emergence of mammals. A recent study indicated that a social insect, the golden paper wasp, can distinguish their conspecific faces, whereas a closely related species, which has a less complex social lifestyle with just one queen ruling a nest of underlings, did not show strong face recognition for their conspecifics. Social complexity and the need to differentiate between one another likely led humans to evolve their face recognition abilities.

  11. The program complex for vocal recognition

    NASA Astrophysics Data System (ADS)

    Konev, Anton; Kostyuchenko, Evgeny; Yakimuk, Alexey

    2017-01-01

    This article discusses the possibility of applying the algorithm of determining the pitch frequency for the note recognition problems. Preliminary study of programs-analogues were carried out for programs with function “recognition of the music”. The software package based on the algorithm for pitch frequency calculation was implemented and tested. It was shown that the algorithm allows recognizing the notes in the vocal performance of the user. A single musical instrument, a set of musical instruments, and a human voice humming a tune can be the sound source. The input file is initially presented in the .wav format or is recorded in this format from a microphone. Processing is performed by sequentially determining the pitch frequency and conversion of its values to the note. According to test results, modification of algorithms used in the complex was planned.

  12. Image recognition and consistency of response

    NASA Astrophysics Data System (ADS)

    Haygood, Tamara M.; Ryan, John; Liu, Qing Mary A.; Bassett, Roland; Brennan, Patrick C.

    2012-02-01

    Purpose: To investigate the connection between conscious recognition of an image previously encountered in an experimental setting and consistency of response to the experimental question.
    Materials and Methods: Twenty-four radiologists viewed 40 frontal chest radiographs and gave their opinion as to the position of a central venous catheter. One-to-three days later they again viewed 40 frontal chest radiographs and again gave their opinion as to the position of the central venous catheter. Half of the radiographs in the second set were repeated images from the first set and half were new. The radiologists were asked of each image whether it had been included in the first set. For this study, we are evaluating only the 20 repeated images. We used the Kruskal-Wallis test and Fisher's exact test to determine the relationship between conscious recognition of a previously interpreted image and consistency in interpretation of the image.
    Results. There was no significant correlation between recognition of the image and consistency in response regarding the position of the central venous catheter. In fact, there was a trend in the opposite direction, with radiologists being slightly more likely to give a consistent response with respect to images they did not recognize than with respect to those they did recognize.
    Conclusion: Radiologists' recognition of previously-encountered images in an observer-performance study does not noticeably color their interpretation on the second encounter.

  13. Sparsity Motivated Automated Target Recognition

    DTIC Science & Technology

    2010-09-29

    been suggested for tasks such as face and iris recognition . In this project, we evaluated the effectiveness of such methods for automatic target...Sparsity-based methods have recently been suggested for tasks such as face and iris recognition . In this project, we evaluated the effectiveness of...have recently been suggested for tasks such as face and iris recognition . In this project, we evaluated the effectiveness of such methods for

  14. Complex cell prototype representation for face recognition.

    PubMed

    Prssoa, L; Leitao, A P

    1999-01-01

    In this paper we propose a new face recognition system based on a biologically inspired filtering method. Our work differs from previous proposals in: 1) the multistage filtering method employed; 2) the pyramid structure used, and most importantly; 3) the prototype construction scheme to determine the models stored in memory. The method is much simpler than previous proposals and relatively inexpensive computationally, while attaining error rates as low as 5%, very close to the best reported results.

  15. New FASB standard addresses revenue recognition considerations.

    PubMed

    McKee, Thomas E

    2015-12-01

    Healthcare organizations are expected to apply the following steps in revenue recognition under the new standard issued in May 2014 by the Financial Accounting Standards Board: Identify the customer contract. Identify the performance obligations in the contract. Determine the transaction price. Allocate the transaction price to the performance obligations in the contract. Recognize revenue when--or in some circumstances, as--the entity satisfies the performance obligation.

  16. A Fuzzy Aproach For Facial Emotion Recognition

    NASA Astrophysics Data System (ADS)

    Gîlcă, Gheorghe; Bîzdoacă, Nicu-George

    2015-09-01

    This article deals with an emotion recognition system based on the fuzzy sets. Human faces are detected in images with the Viola - Jones algorithm and for its tracking in video sequences we used the Camshift algorithm. The detected human faces are transferred to the decisional fuzzy system, which is based on the variable fuzzyfication measurements of the face: eyebrow, eyelid and mouth. The system can easily determine the emotional state of a person.

  17. Retina vascular network recognition

    NASA Astrophysics Data System (ADS)

    Tascini, Guido; Passerini, Giorgio; Puliti, Paolo; Zingaretti, Primo

    1993-09-01

    The analysis of morphological and structural modifications of the retina vascular network is an interesting investigation method in the study of diabetes and hypertension. Normally this analysis is carried out by qualitative evaluations, according to standardized criteria, though medical research attaches great importance to quantitative analysis of vessel color, shape and dimensions. The paper describes a system which automatically segments and recognizes the ocular fundus circulation and micro circulation network, and extracts a set of features related to morphometric aspects of vessels. For this class of images the classical segmentation methods seem weak. We propose a computer vision system in which segmentation and recognition phases are strictly connected. The system is hierarchically organized in four modules. Firstly the Image Enhancement Module (IEM) operates a set of custom image enhancements to remove blur and to prepare data for subsequent segmentation and recognition processes. Secondly the Papilla Border Analysis Module (PBAM) automatically recognizes number, position and local diameter of blood vessels departing from optical papilla. Then the Vessel Tracking Module (VTM) analyses vessels comparing the results of body and edge tracking and detects branches and crossings. Finally the Feature Extraction Module evaluates PBAM and VTM output data and extracts some numerical indexes. Used algorithms appear to be robust and have been successfully tested on various ocular fundus images.

  18. Radically enhanced molecular recognition

    NASA Astrophysics Data System (ADS)

    Trabolsi, Ali; Khashab, Niveen; Fahrenbach, Albert C.; Friedman, Douglas C.; Colvin, Michael T.; Cotí, Karla K.; Benítez, Diego; Tkatchouk, Ekaterina; Olsen, John-Carl; Belowich, Matthew E.; Carmielli, Raanan; Khatib, Hussam A.; Goddard, William A.; Wasielewski, Michael R.; Stoddart, J. Fraser

    2010-01-01

    The tendency for viologen radical cations to dimerize has been harnessed to establish a recognition motif based on their ability to form extremely strong inclusion complexes with cyclobis(paraquat-p-phenylene) in its diradical dicationic redox state. This previously unreported complex involving three bipyridinium cation radicals increases the versatility of host-guest chemistry, extending its practice beyond the traditional reliance on neutral and charged guests and hosts. In particular, transporting the concept of radical dimerization into the field of mechanically interlocked molecules introduces a higher level of control within molecular switches and machines. Herein, we report that bistable and tristable [2]rotaxanes can be switched by altering electrochemical potentials. In a tristable [2]rotaxane composed of a cyclobis(paraquat-p-phenylene) ring and a dumbbell with tetrathiafulvalene, dioxynaphthalene and bipyridinium recognition sites, the position of the ring can be switched. On oxidation, it moves from the tetrathiafulvalene to the dioxynaphthalene, and on reduction, to the bipyridinium radical cation, provided the ring is also reduced simultaneously to the diradical dication.

  19. Complex Event Recognition Architecture

    NASA Technical Reports Server (NTRS)

    Fitzgerald, William A.; Firby, R. James

    2009-01-01

    Complex Event Recognition Architecture (CERA) is the name of a computational architecture, and software that implements the architecture, for recognizing complex event patterns that may be spread across multiple streams of input data. One of the main components of CERA is an intuitive event pattern language that simplifies what would otherwise be the complex, difficult tasks of creating logical descriptions of combinations of temporal events and defining rules for combining information from different sources over time. In this language, recognition patterns are defined in simple, declarative statements that combine point events from given input streams with those from other streams, using conjunction, disjunction, and negation. Patterns can be built on one another recursively to describe very rich, temporally extended combinations of events. Thereafter, a run-time matching algorithm in CERA efficiently matches these patterns against input data and signals when patterns are recognized. CERA can be used to monitor complex systems and to signal operators or initiate corrective actions when anomalous conditions are recognized. CERA can be run as a stand-alone monitoring system, or it can be integrated into a larger system to automatically trigger responses to changing environments or problematic situations.

  20. Sudden Event Recognition: A Survey

    PubMed Central

    Suriani, Nor Surayahani; Hussain, Aini; Zulkifley, Mohd Asyraf

    2013-01-01

    Event recognition is one of the most active research areas in video surveillance fields. Advancement in event recognition systems mainly aims to provide convenience, safety and an efficient lifestyle for humanity. A precise, accurate and robust approach is necessary to enable event recognition systems to respond to sudden changes in various uncontrolled environments, such as the case of an emergency, physical threat and a fire or bomb alert. The performance of sudden event recognition systems depends heavily on the accuracy of low level processing, like detection, recognition, tracking and machine learning algorithms. This survey aims to detect and characterize a sudden event, which is a subset of an abnormal event in several video surveillance applications. This paper discusses the following in detail: (1) the importance of a sudden event over a general anomalous event; (2) frameworks used in sudden event recognition; (3) the requirements and comparative studies of a sudden event recognition system and (4) various decision-making approaches for sudden event recognition. The advantages and drawbacks of using 3D images from multiple cameras for real-time application are also discussed. The paper concludes with suggestions for future research directions in sudden event recognition. PMID:23921828

  1. Multisensory encoding improves auditory recognition.

    PubMed

    Moran, Zachary D; Bachman, Peter; Pham, Phillip; Cho, Seong Hah; Cannon, Tyrone D; Shams, Ladan

    2013-01-01

    Recent studies have challenged the long-held belief that recognition is unfailingly degraded by contextual differences between study and test items. In these studies, recognition of pictures presented in silence was better when during study or initial exposure the images were accompanied by a semantically congruent sound rather than silence. In the present study, we sought to examine the generalization of this phenomenon to auditory recognition and found a significant improvement in the recognition of auditory items when coupled with a congruent picture. We discuss these findings within the framework of the redintegration hypothesis of memory retrieval as well as Bayesian inference and learning.

  2. Sudden event recognition: a survey.

    PubMed

    Suriani, Nor Surayahani; Hussain, Aini; Zulkifley, Mohd Asyraf

    2013-08-05

    Event recognition is one of the most active research areas in video surveillance fields. Advancement in event recognition systems mainly aims to provide convenience, safety and an efficient lifestyle for humanity. A precise, accurate and robust approach is necessary to enable event recognition systems to respond to sudden changes in various uncontrolled environments, such as the case of an emergency, physical threat and a fire or bomb alert. The performance of sudden event recognition systems depends heavily on the accuracy of low level processing, like detection, recognition, tracking and machine learning algorithms. This survey aims to detect and characterize a sudden event, which is a subset of an abnormal event in several video surveillance applications. This paper discusses the following in detail: (1) the importance of a sudden event over a general anomalous event; (2) frameworks used in sudden event recognition; (3) the requirements and comparative studies of a sudden event recognition system and (4) various decision-making approaches for sudden event recognition. The advantages and drawbacks of using 3D images from multiple cameras for real-time application are also discussed. The paper concludes with suggestions for future research directions in sudden event recognition.

  3. The neural correlates of visual self-recognition.

    PubMed

    Devue, Christel; Brédart, Serge

    2011-03-01

    This paper presents a review of studies that were aimed at determining which brain regions are recruited during visual self-recognition, with a particular focus on self-face recognition. A complex bilateral network, involving frontal, parietal and occipital areas, appears to be associated with self-face recognition, with a particularly high implication of the right hemisphere. Results indicate that it remains difficult to determine which specific cognitive operation is reflected by each recruited brain area, in part due to the variability of used control stimuli and experimental tasks. A synthesis of the interpretations provided by previous studies is presented. The relevance of using self-recognition as an indicator of self-awareness is discussed. We argue that a major aim of future research in the field should be to identify more clearly the cognitive operations induced by the perception of the self-face, and search for dissociations between neural correlates and cognitive components.

  4. Defining protein electrostatic recognition processes

    NASA Astrophysics Data System (ADS)

    Getzoff, Elizabeth D.; Roberts, Victoria A.

    The objective is to elucidate the nature of electrostatic forces controlling protein recognition processes by using a tightly coupled computational and interactive computer graphics approach. The TURNIP program was developed to determine the most favorable precollision orientations for two molecules by systematic search of all orientations and evaluation of the resulting electrostatic interactions. TURNIP was applied to the transient interaction between two electron transfer metalloproteins, plastocyanin and cytochrome c. The results suggest that the productive electron-transfer complex involves interaction of the positive region of cytochrome c with the negative patch of plastocyanin, consistent with experimental data. Application of TURNIP to the formation of the stable complex between the HyHEL-5 antibody and its protein antigen lysozyme showed that long-distance electrostatic forces guide lysozyme toward the HyHEL-5 binding site, but do not fine tune its orientation. Determination of docked antigen/antibody complexes requires including steric as well as electrostatic interactions, as was done for the U10 mutant of the anti-phosphorylcholine antibody S107. The graphics program Flex, a convenient desktop workstation program for visualizing molecular dynamics and normal mode motions, was enhanced. Flex now has a user interface and was rewritten to use standard graphics libraries, so as to run on most desktop workstations.

  5. The Legal Recognition of Sign Languages

    ERIC Educational Resources Information Center

    De Meulder, Maartje

    2015-01-01

    This article provides an analytical overview of the different types of explicit legal recognition of sign languages. Five categories are distinguished: constitutional recognition, recognition by means of general language legislation, recognition by means of a sign language law or act, recognition by means of a sign language law or act including…

  6. Teaching and the Dialectic of Recognition

    ERIC Educational Resources Information Center

    Huttunen, Rauno; Heikkinen, Hannu L. T.

    2004-01-01

    In this article, the processes of recognition within education are discussed. Frequently, recognition is reduced to polite behaviour or etiquette. Another narrow view of recognition is, behaviouristically speaking, to regard it as mere feedback. We claim that authentic recognition is a different matter. Receiving recognition, as Charles Taylor has…

  7. The Legal Recognition of Sign Languages

    ERIC Educational Resources Information Center

    De Meulder, Maartje

    2015-01-01

    This article provides an analytical overview of the different types of explicit legal recognition of sign languages. Five categories are distinguished: constitutional recognition, recognition by means of general language legislation, recognition by means of a sign language law or act, recognition by means of a sign language law or act including…

  8. Amygdala damage impairs emotion recognition from music.

    PubMed

    Gosselin, Nathalie; Peretz, Isabelle; Johnsen, Erica; Adolphs, Ralph

    2007-01-28

    The role of the amygdala in recognition of danger is well established for visual stimuli such as faces. A similar role in another class of emotionally potent stimuli -- music -- has been recently suggested by the study of epileptic patients with unilateral resection of the anteromedian part of the temporal lobe [Gosselin, N., Peretz, I., Noulhiane, M., Hasboun, D., Beckett, C., & Baulac, M., et al. (2005). Impaired recognition of scary music following unilateral temporal lobe excision. Brain, 128(Pt 3), 628-640]. The goal of the present study was to assess the specific role of the amygdala in the recognition of fear from music. To this aim, we investigated a rare subject, S.M., who has complete bilateral damage relatively restricted to the amygdala and not encompassing other sectors of the temporal lobe. In Experiment 1, S.M. and four matched controls were asked to rate the intensity of fear, peacefulness, happiness, and sadness from computer-generated instrumental music purposely created to express those emotions. Subjects also rated the arousal and valence of each musical stimulus. An error detection task assessed basic auditory perceptual function. S.M. performed normally in this perceptual task, but was selectively impaired in the recognition of scary and sad music. In contrast, her recognition of happy music was normal. Furthermore, S.M. judged the scary music to be less arousing and the peaceful music less relaxing than did the controls. Overall, the pattern of impairment in S.M. is similar to that previously reported in patients with unilateral anteromedial temporal lobe damage. S.M.'s impaired emotional judgments occur in the face of otherwise intact processing of musical features that are emotionally determinant. The use of tempo and mode cues in distinguishing happy from sad music was also spared in S.M. Thus, the amygdala appears to be necessary for emotional processing of music rather than the perceptual processing itself.

  9. Chemical recognition of gases and gas mixtures with terahertz waves

    NASA Astrophysics Data System (ADS)

    Jacobsen, R. H.; Mittleman, D. M.; Nuss, M. C.

    1996-12-01

    A time-domain chemical-recognition system for classifying gases and analyzing gas mixtures is presented. We analyze the free induction decay exhibited by gases excited by far-infrared (terahertz) pulses in the time domain, using digital signal-processing techniques. A simple geometric picture is used for the classification of the waveforms measured for unknown gas species. We demonstrate how the recognition system can be used to determine the partial pressures of an ammonia-water gas mixture.

  10. Chemical recognition software

    SciTech Connect

    Wagner, J.S.; Trahan, M.W.; Nelson, W.E.; Hargis, P.H. Jr.; Tisone, G.C.

    1994-06-01

    We have developed a capability to make real time concentration measurements of individual chemicals in a complex mixture using a multispectral laser remote sensing system. Our chemical recognition and analysis software consists of three parts: (1) a rigorous multivariate analysis package for quantitative concentration and uncertainty estimates, (2) a genetic optimizer which customizes and tailors the multivariate algorithm for a particular application, and (3) an intelligent neural net chemical filter which pre-selects from the chemical database to find the appropriate candidate chemicals for quantitative analyses by the multivariate algorithms, as well as providing a quick-look concentration estimate and consistency check. Detailed simulations using both laboratory fluorescence data and computer synthesized spectra indicate that our software can make accurate concentration estimates from complex multicomponent mixtures, even when the mixture is noisy and contaminated with unknowns.

  11. Chemical recognition software

    SciTech Connect

    Wagner, J.S.; Trahan, M.W.; Nelson, W.E.; Hargis, P.J. Jr.; Tisone, G.C.

    1994-12-01

    We have developed a capability to make real time concentration measurements of individual chemicals in a complex mixture using a multispectral laser remote sensing system. Our chemical recognition and analysis software consists of three parts: (1) a rigorous multivariate analysis package for quantitative concentration and uncertainty estimates, (2) a genetic optimizer which customizes and tailors the multivariate algorithm for a particular application, and (3) an intelligent neural net chemical filter which pre-selects from the chemical database to find the appropriate candidate chemicals for quantitative analyses by the multivariate algorithms, as well as providing a quick-look concentration estimate and consistency check. Detailed simulations using both laboratory fluorescence data and computer synthesized spectra indicate that our software can make accurate concentration estimates from complex multicomponent mixtures. even when the mixture is noisy and contaminated with unknowns.

  12. Early recognition of speech

    PubMed Central

    Remez, Robert E; Thomas, Emily F

    2013-01-01

    Classic research on the perception of speech sought to identify minimal acoustic correlates of each consonant and vowel. In explaining perception, this view designated momentary components of an acoustic spectrum as cues to the recognition of elementary phonemes. This conceptualization of speech perception is untenable given the findings of phonetic sensitivity to modulation independent of the acoustic and auditory form of the carrier. The empirical key is provided by studies of the perceptual organization of speech, a low-level integrative function that finds and follows the sensory effects of speech amid concurrent events. These projects have shown that the perceptual organization of speech is keyed to modulation; fast; unlearned; nonsymbolic; indifferent to short-term auditory properties; and organization requires attention. The ineluctably multisensory nature of speech perception also imposes conditions that distinguish language among cognitive systems. WIREs Cogn Sci 2013, 4:213–223. doi: 10.1002/wcs.1213 PMID:23926454

  13. Recognition Using Hybrid Classifiers.

    PubMed

    Osadchy, Margarita; Keren, Daniel; Raviv, Dolev

    2016-04-01

    A canonical problem in computer vision is category recognition (e.g., find all instances of human faces, cars etc., in an image). Typically, the input for training a binary classifier is a relatively small sample of positive examples, and a huge sample of negative examples, which can be very diverse, consisting of images from a large number of categories. The difficulty of the problem sharply increases with the dimension and size of the negative example set. We propose to alleviate this problem by applying a "hybrid" classifier, which replaces the negative samples by a prior, and then finds a hyperplane which separates the positive samples from this prior. The method is extended to kernel space and to an ensemble-based approach. The resulting binary classifiers achieve an identical or better classification rate than SVM, while requiring far smaller memory and lower computational complexity to train and apply.

  14. Automatic speech recognition

    NASA Astrophysics Data System (ADS)

    Espy-Wilson, Carol

    2005-04-01

    Great strides have been made in the development of automatic speech recognition (ASR) technology over the past thirty years. Most of this effort has been centered around the extension and improvement of Hidden Markov Model (HMM) approaches to ASR. Current commercially-available and industry systems based on HMMs can perform well for certain situational tasks that restrict variability such as phone dialing or limited voice commands. However, the holy grail of ASR systems is performance comparable to humans-in other words, the ability to automatically transcribe unrestricted conversational speech spoken by an infinite number of speakers under varying acoustic environments. This goal is far from being reached. Key to the success of ASR is effective modeling of variability in the speech signal. This tutorial will review the basics of ASR and the various ways in which our current knowledge of speech production, speech perception and prosody can be exploited to improve robustness at every level of the system.

  15. Computer image processing and recognition

    NASA Technical Reports Server (NTRS)

    Hall, E. L.

    1979-01-01

    A systematic introduction to the concepts and techniques of computer image processing and recognition is presented. Consideration is given to such topics as image formation and perception; computer representation of images; image enhancement and restoration; reconstruction from projections; digital television, encoding, and data compression; scene understanding; scene matching and recognition; and processing techniques for linear systems.

  16. Methods of Teaching Speech Recognition

    ERIC Educational Resources Information Center

    Rader, Martha H.; Bailey, Glenn A.

    2010-01-01

    Objective: This article introduces the history and development of speech recognition, addresses its role in the business curriculum, outlines related national and state standards, describes instructional strategies, and discusses the assessment of student achievement in speech recognition classes. Methods: Research methods included a synthesis of…

  17. Automatic Recognition of Deaf Speech.

    ERIC Educational Resources Information Center

    Abdelhamied, Kadry; And Others

    1990-01-01

    This paper describes a speech perception system for automatic recognition of deaf speech. Using a 2-step segmentation approach for 468 utterances by 2 hearing-impaired men and 2 normal-hearing men, rates as high as 93.01 percent and 81.81 percent recognition were obtained in recognizing from deaf speech isolated words and connected speech,…

  18. Coordinate Transformations in Object Recognition

    ERIC Educational Resources Information Center

    Graf, Markus

    2006-01-01

    A basic problem of visual perception is how human beings recognize objects after spatial transformations. Three central classes of findings have to be accounted for: (a) Recognition performance varies systematically with orientation, size, and position; (b) recognition latencies are sequentially additive, suggesting analogue transformation…

  19. Children's Recognition of Cartoon Voices.

    ERIC Educational Resources Information Center

    Spence, Melanie J.; Rollins, Pamela R.; Jerger, Susan

    2002-01-01

    A study examined developmental changes in talker recognition skills by assessing 72 children's (ages 3-5) recognition of 20 cartoon characters' voices. Four- and 5-year-old children recognized more of the voices than did 3-year-olds. All children were more accurate at recognizing more familiar characters than less familiar characters. (Contains…

  20. Computer image processing and recognition

    NASA Technical Reports Server (NTRS)

    Hall, E. L.

    1979-01-01

    A systematic introduction to the concepts and techniques of computer image processing and recognition is presented. Consideration is given to such topics as image formation and perception; computer representation of images; image enhancement and restoration; reconstruction from projections; digital television, encoding, and data compression; scene understanding; scene matching and recognition; and processing techniques for linear systems.

  1. Quantum-Limited Image Recognition

    DTIC Science & Technology

    1989-12-01

    J. S. Bomba ,’Alpha-numeric character recognition using local operations,’ Fall Joint Comput. Conf., 218-224 (1959). 53. D. Barnea and H. Silverman...for Chapter 6 1. J. S. Bomba ,’Alpha-numeric character recognition using local operations,’ Fall Joint Comput. Conf., 218-224 (1959). 2. D. Bamea and H

  2. Conjoint recognition and phantom recollection.

    PubMed

    Brainerd, C J; Wright, R; Reyna, V F; Mojardin, A H

    2001-03-01

    A new methodology for measuring illusory conscious experience of the "presentation" of unstudied material (phantom recollection) is evaluated that extracts measurements directly from recognition responses, rather than indirectly from introspective reports. Application of this methodology in the Deese-Roediger-McDermott (DRM) paradigm (Experiments 1 and 2) and in a more conventional paradigm (Experiment 3) showed that 2 processes (phantom recollection and familiarity) contribute to false recognition of semantically related distractors. Phantom recollection was the larger contributor to false recognition of critical distractors in the DRM paradigm, but surprisingly, it was also the larger contributor to false recognition of other types of distractors. Variability in false recognition was tied to variability in phantom recollection. Experimental control of phantom recollection was achieved with manipulations that were motivated by fuzzy-trace theory's hypothesis that the phenomenon is gist-based.

  3. Determining bacteriophage endopeptidase activity using either fluorophore-quencher labeled peptides combined with liquid chromatography-mass spectrometry (LC-MS) or Förster resonance energy transfer (FRET) assays

    PubMed Central

    Molina, Henrik; Fischetti, Vincent A.

    2017-01-01

    The necessity of identifying novel methods to combat infections caused by antibiotic resistant bacteria is increasing each year. Recent advancements in the development of peptidoglycan hydrolases (e.g. lysins) from bacterial viruses (bacteriophages) have revealed the efficiency of this class of enzymes in treating serious bacterial infections. Though promising results have been obtained regarding the lethal action of lysin on bacterial pathogens both in vitro and in vivo, an often-overlooked factor in these studies is precisely identifying their peptidoglycan cleavage site. This knowledge would be useful for following the activity of the enzyme during development, without the need for whole-organism lytic assays. However, more importantly, it would enable the selection of lysins with different cleavage activities that would act synergistically for enhanced efficacy. Here, we have developed two new methods to accurately identify the cleavage site of lysins using liquid chromatography mass spectrometry (LC-MS) on peptidoglycan-like fluorophore-quencher modified synthetic peptides, as well as determining the enzymatic action and kinetics of the enzymes on modified peptides in a Förster resonance energy transfer (FRET) assay. These methods should facilitate progress within the lysin field, accelerating the development of therapeutic lysins to combat antibiotic resistant bacterial infections. PMID:28296948

  4. Recognition memory impairments caused by false recognition of novel objects.

    PubMed

    Yeung, Lok-Kin; Ryan, Jennifer D; Cowell, Rosemary A; Barense, Morgan D

    2013-11-01

    A fundamental assumption underlying most current theories of amnesia is that memory impairments arise because previously studied information either is lost rapidly or is made inaccessible (i.e., the old information appears to be new). Recent studies in rodents have challenged this view, suggesting instead that under conditions of high interference, recognition memory impairments following medial temporal lobe damage arise because novel information appears as though it has been previously seen. Here, we developed a new object recognition memory paradigm that distinguished whether object recognition memory impairments were driven by previously viewed objects being treated as if they were novel or by novel objects falsely recognized as though they were previously seen. In this indirect, eyetracking-based passive viewing task, older adults at risk for mild cognitive impairment showed false recognition to high-interference novel items (with a significant degree of feature overlap with previously studied items) but normal novelty responses to low-interference novel items (with a lower degree of feature overlap). The indirect nature of the task minimized the effects of response bias and other memory-based decision processes, suggesting that these factors cannot solely account for false recognition. These findings support the counterintuitive notion that recognition memory impairments in this memory-impaired population are not characterized by forgetting but rather are driven by the failure to differentiate perceptually similar objects, leading to the false recognition of novel objects as having been seen before.

  5. Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

    PubMed Central

    Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

    2013-01-01

    Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

  6. Speech Recognition: How Do We Teach It?

    ERIC Educational Resources Information Center

    Barksdale, Karl

    2002-01-01

    States that growing use of speech recognition software has made voice writing an essential computer skill. Describes how to present the topic, develop basic speech recognition skills, and teach speech recognition outlining, writing, proofreading, and editing. (Contains 14 references.) (SK)

  7. Face recognition from a moving platform via sparse representation

    NASA Astrophysics Data System (ADS)

    Hsu, Ming Kai; Hsu, Charles; Lee, Ting N.; Szu, Harold

    2012-06-01

    A video-based surveillance system for passengers includes face detection, face tracking and face recognition. In general, the final recognition result of the video-based surveillance system is usually determined by the cumulative recognition results. Under this strategy, the correctness of face tracking plays an important role for the system recognition rate. For face tracking, the challenges of face tracking on a moving platform are that the space and time information used for conventional face tracking algorithms may be lost. Consequently, conventional face tracking algorithms can barely handle the face tracking on a moving platform. In this paper, we have verified the state-of-the-art technologies for face detection, face tracking and face recognition on a moving platform. In the mean time, we also proposed a new strategy for face tracking on a moving platform or face tracking under very low frame rate. The steps of the new strategy for face detection are: (1) classification the detected faces over a certain period instead of every frame (2) Tracking of each passenger is equivalent to reconstruct the time order of certain period for each passenger. If the cumulative recognition results are the only part needed for the surveillance system, step 2 can be skipped. In addition, if the additional information from the passengers is required, such as path tracking, lip read, gesture recognition, etc, time order reconstruction in step 2 can offer the information required.

  8. Face and body recognition show similar improvement during childhood.

    PubMed

    Bank, Samantha; Rhodes, Gillian; Read, Ainsley; Jeffery, Linda

    2015-09-01

    Adults are proficient in extracting identity cues from faces. This proficiency develops slowly during childhood, with performance not reaching adult levels until adolescence. Bodies are similar to faces in that they convey identity cues and rely on specialized perceptual mechanisms. However, it is currently unclear whether body recognition mirrors the slow development of face recognition during childhood. Recent evidence suggests that body recognition develops faster than face recognition. Here we measured body and face recognition in 6- and 10-year-old children and adults to determine whether these two skills show different amounts of improvement during childhood. We found no evidence that they do. Face and body recognition showed similar improvement with age, and children, like adults, were better at recognizing faces than bodies. These results suggest that the mechanisms of face and body memory mature at a similar rate or that improvement of more general cognitive and perceptual skills underlies improvement of both face and body recognition. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Probabilistic view clustering in object recognition

    NASA Astrophysics Data System (ADS)

    Camps, Octavia I.; Christoffel, Douglas W.; Pathak, Anjali

    1992-11-01

    To recognize objects and to determine their poses in a scene we need to find correspondences between the features extracted from the image and those of the object models. Models are commonly represented by describing a few characteristic views of the object representing groups of views with similar properties. Most feature-based matching schemes assume that all the features that are potentially visible in a view will appear with equal probability, and the resulting matching algorithms have to allow for 'errors' without really understanding what they mean. PREMIO is an object recognition system that uses CAD models of 3D objects and knowledge of surface reflectance properties, light sources, sensor characteristics, and feature detector algorithms to estimate the probability of the features being detectable and correctly matched. The purpose of this paper is to describe the predictions generated by PREMIO, how they are combined into a single probabilistic model, and illustrative examples showing its use in object recognition.

  10. Photonics: From target recognition to lesion detection

    NASA Technical Reports Server (NTRS)

    Henry, E. Michael

    1994-01-01

    Since 1989, Martin Marietta has invested in the development of an innovative concept for robust real-time pattern recognition for any two-dimensioanal sensor. This concept has been tested in simulation, and in laboratory and field hardware, for a number of DOD and commercial uses from automatic target recognition to manufacturing inspection. We have now joined Rose Health Care Systems in developing its use for medical diagnostics. The concept is based on determining regions of interest by using optical Fourier bandpassing as a scene segmentation technique, enhancing those regions using wavelet filters, passing the enhanced regions to a neural network for analysis and initial pattern identification, and following this initial identification with confirmation by optical correlation. The optical scene segmentation and pattern confirmation are performed by the same optical module. The neural network is a recursive error minimization network with a small number of connections and nodes that rapidly converges to a global minimum.

  11. Building Hierarchical Representations for Oracle Character and Sketch Recognition.

    PubMed

    Jun Guo; Changhu Wang; Roman-Rangel, Edgar; Hongyang Chao; Yong Rui

    2016-01-01

    In this paper, we study oracle character recognition and general sketch recognition. First, a data set of oracle characters, which are the oldest hieroglyphs in China yet remain a part of modern Chinese characters, is collected for analysis. Second, typical visual representations in shape- and sketch-related works are evaluated. We analyze the problems suffered when addressing these representations and determine several representation design criteria. Based on the analysis, we propose a novel hierarchical representation that combines a Gabor-related low-level representation and a sparse-encoder-related mid-level representation. Extensive experiments show the effectiveness of the proposed representation in both oracle character recognition and general sketch recognition. The proposed representation is also complementary to convolutional neural network (CNN)-based models. We introduce a solution to combine the proposed representation with CNN-based models, and achieve better performances over both approaches. This solution has beaten humans at recognizing general sketches.

  12. Pattern recognition in spectra

    NASA Astrophysics Data System (ADS)

    Gebran, M.; Paletou, F.

    2017-06-01

    We present a new automated procedure that simultaneously derives the effective temperature Teff, surface gravity log g, metallicity [Fe/H], and equatorial projected rotational velocity ve sin i for stars. The procedure is inspired by the well-known PCA-based inversion of spectropolarimetric full-Stokes solar data, which was used both for Zeeman and Hanle effects. The efficiency and accuracy of this procedure have been proven for FGK, A, and late type dwarf stars of K and M spectral types. Learning databases are generated from the Elodie stellar spectra library using observed spectra for which fundamental parameters were already evaluated or with synthetic data. The synthetic spectra are calculated using ATLAS9 model atmospheres. This technique helped us to detect many peculiar stars such as Am, Ap, HgMn, SiEuCr and binaries. This fast and efficient technique could be used every time a pattern recognition is needed. One important application is the understanding of the physical properties of planetary surfaces by comparing aboard instrument data to synthetic ones.

  13. Recognition of speech spectrograms.

    PubMed

    Greene, B G; Pisoni, D B; Carrell, T D

    1984-07-01

    The performance of eight naive observers in learning to identify speech spectrograms was studied over a 2-month period. Single tokens from a 50-word phonetically balanced (PB) list were recorded by several talkers and displayed on a Spectraphonics Speech Spectrographic Display system. Identification testing occurred immediately after daily training sessions. After approximately 20 h of training, naive subjects correctly identified the 50 PB words from a single talker over 95% of the time. Generalization tests with the same words were then carried out with different tokens from the original talker, new tokens from another male talker, a female talker, and finally, a synthetic talker. The generalization results for these talkers showed recognition performance at 91%, 76%, 76%, and 48%, respectively. Finally, generalization tests with a novel set of PB words produced by the original talker were also carried out to examine in detail the perceptual strategies and visual features that subjects abstracted from the training set. Our results demonstrate that even without formal training in phonetics or acoustics naive observers can learn to identify visual displays of speech at very high levels of accuracy. Analysis of subjects' performance in a verbal protocol task demonstrated that they rely on salient visual correlates of many phonetic features in speech.

  14. Protospacer recognition motifs

    PubMed Central

    Shah, Shiraz A.; Erdmann, Susanne; Mojica, Francisco J.M.; Garrett, Roger A.

    2013-01-01

    Protospacer adjacent motifs (PAMs) were originally characterized for CRISPR-Cas systems that were classified on the basis of their CRISPR repeat sequences. A few short 2–5 bp sequences were identified adjacent to one end of the protospacers. Experimental and bioinformatical results linked the motif to the excision of protospacers and their insertion into CRISPR loci. Subsequently, evidence accumulated from different virus- and plasmid-targeting assays, suggesting that these motifs were also recognized during DNA interference, at least for the recently classified type I and type II CRISPR-based systems. The two processes, spacer acquisition and protospacer interference, employ different molecular mechanisms, and there is increasing evidence to suggest that the sequence motifs that are recognized, while overlapping, are unlikely to be identical. In this article, we consider the properties of PAM sequences and summarize the evidence for their dual functional roles. It is proposed to use the terms protospacer associated motif (PAM) for the conserved DNA sequence and to employ spacer acqusition motif (SAM) and target interference motif (TIM), respectively, for acquisition and interference recognition sites. PMID:23403393

  15. Macromolecular recognition: Recognition of polymer side chains by cyclodextrin

    NASA Astrophysics Data System (ADS)

    Hashidzume, Akihito; Harada, Akira

    2015-12-01

    The interaction of cyclodextrins (CD) with water soluble polymers possessing guest residues has been investigated as model systems in biological molecular recognition. The selectivity of interaction of CD with polymer-carrying guest residues is controlled by polymer chains, i.e., the steric effect of polymer main chain, the conformational effect of polymer main chain, and multi-site interaction. Macroscopic assemblies have been also realized based on molecular recognition using polyacrylamide-based gels possessing CD and guest residues.

  16. 21 CFR 26.9 - Equivalence determination.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... RECOGNITION OF PHARMACEUTICAL GOOD MANUFACTURING PRACTICE REPORTS, MEDICAL DEVICE QUALITY SYSTEM AUDIT REPORTS... Specific Sector Provisions for Pharmaceutical Good Manufacturing Practices § 26.9 Equivalence determination...

  17. 21 CFR 26.9 - Equivalence determination.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... RECOGNITION OF PHARMACEUTICAL GOOD MANUFACTURING PRACTICE REPORTS, MEDICAL DEVICE QUALITY SYSTEM AUDIT REPORTS... Specific Sector Provisions for Pharmaceutical Good Manufacturing Practices § 26.9 Equivalence determination...

  18. 21 CFR 26.9 - Equivalence determination.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... RECOGNITION OF PHARMACEUTICAL GOOD MANUFACTURING PRACTICE REPORTS, MEDICAL DEVICE QUALITY SYSTEM AUDIT REPORTS... Specific Sector Provisions for Pharmaceutical Good Manufacturing Practices § 26.9 Equivalence determination...

  19. 21 CFR 26.9 - Equivalence determination.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... RECOGNITION OF PHARMACEUTICAL GOOD MANUFACTURING PRACTICE REPORTS, MEDICAL DEVICE QUALITY SYSTEM AUDIT REPORTS... Specific Sector Provisions for Pharmaceutical Good Manufacturing Practices § 26.9 Equivalence determination...

  20. Structural Target Analysis And Recognition System

    NASA Astrophysics Data System (ADS)

    Lee, Harry C.

    1984-06-01

    The structural target analysis and recognition system (STARS) is a pyramid and syntactical based vision system that uniquely classifies targets, using their viewable internal structure. Being a totally structural approach, STARS uses a resolution sequence to develop a hierarchical pyramid organized segmentation and formal language to perform the recognition function. Global structure of the target is derived by the segment connectivity of the inter-resolution levels, while local structure is based on the local relationship of segments at a single level. The relationships of both the global and local structures form a resolution syntax tree (RST). Two targets are said to be structurally similar if they have similar RSTs. The matching process of the RSTs proceeds from the root to the leaves of the tree. The depth to which the match progresses before failure or completion determines the degree of patch in a resolution sense. RSTs from various views of a target are grouped together to form a formal language. The underlying grammar is transformed into a stochastic grammar so as to accommodate segmentation and environmental variations. Recognition metrics are a function of the resolution structure and posterior probability at each resolution level. Because of the inherent resolution sequence, STARS can accommodate both candidate and reference targets from various resolutions.

  1. Gait recognition and walking exercise intensity estimation.

    PubMed

    Lin, Bor-Shing; Liu, Yu-Ting; Yu, Chu; Jan, Gene Eu; Hsiao, Bo-Tang

    2014-04-04

    Cardiovascular patients consult doctors for advice regarding regular exercise, whereas obese patients must self-manage their weight. Because a system for permanently monitoring and tracking patients' exercise intensities and workouts is necessary, a system for recognizing gait and estimating walking exercise intensity was proposed. For gait recognition analysis, αβ filters were used to improve the recognition of athletic attitude. Furthermore, empirical mode decomposition (EMD) was used to filter the noise of patients' attitude to acquire the Fourier transform energy spectrum. Linear discriminant analysis was then applied to this energy spectrum for training and recognition. When the gait or motion was recognized, the walking exercise intensity was estimated. In addition, this study addressed the correlation between inertia and exercise intensity by using the residual function of the EMD and quadratic approximation to filter the effect of the baseline drift integral of the acceleration sensor. The increase in the determination coefficient of the regression equation from 0.55 to 0.81 proved that the accuracy of the method for estimating walking exercise intensity proposed by Kurihara was improved in this study.

  2. Pattern recognition using asymmetric attractor neural networks

    SciTech Connect

    Jin Tao; Zhao Hong

    2005-12-15

    The asymmetric attractor neural networks designed by the Monte Carlo- (MC-) adaptation rule are shown to be promising candidates for pattern recognition. In such a neural network with relatively low symmetry, when the members of a set of template patterns are stored as fixed-point attractors, their attraction basins are shown to be isolated islands embedded in a ''chaotic sea.'' The sizes of these islands can be controlled by a single parameter. We show that these properties can be used for effective pattern recognition and rejection. In our method, the pattern to be identified is attracted to a template pattern or a chaotic attractor. If the difference between the pattern to be identified and the template pattern is smaller than a predescribed threshold, the pattern is attracted to the template pattern automatically and thus is identified as belonging to this template pattern. Otherwise, it wanders in a chaotic attractor for ever and thus is rejected as an unknown pattern. The maximum sizes of these islands allowed by this kind of neural networks are determined by a modified MC-adaptation rule which are shown to be able to dramatically enlarge the sizes of the islands. We illustrate the use of our method for pattern recognition and rejection with an example of recognizing a set of Chinese characters.

  3. Stimulus Recognition and Associative Coding

    ERIC Educational Resources Information Center

    Runquist, Willard N.; Evans, Annabel

    1972-01-01

    Purpose of this experiment was to investigate the relationship between stimulus recognition and various learning conditions which were designed to affect both stimulus encoding and associative learning in a paired-associate task. (Authors)

  4. Gesture recognition on smart cameras

    NASA Astrophysics Data System (ADS)

    Dziri, Aziz; Chevobbe, Stephane; Darouich, Mehdi

    2013-02-01

    Gesture recognition is a feature in human-machine interaction that allows more natural interaction without the use of complex devices. For this reason, several methods of gesture recognition have been developed in recent years. However, most real time methods are designed to operate on a Personal Computer with high computing resources and memory. In this paper, we analyze relevant methods found in the literature in order to investigate the ability of smart camera to execute gesture recognition algorithms. We elaborate two hand gesture recognition pipelines. The first method is based on invariant moments extraction and the second on finger tips detection. The hand detection method used for both pipeline is based on skin color segmentation. The results obtained show that the un-optimized versions of invariant moments method and finger tips detection method can reach 10 fps on embedded processor and use about 200 kB of memory.

  5. Molecular recognition of bilayer vesicles.

    PubMed

    Voskuhl, Jens; Ravoo, Bart Jan

    2009-02-01

    Vesicles have been a versatile topic of research in chemistry ever since the discovery that, besides phospholipids, synthetic amphiphiles can also form molecular bilayers enclosing a small aqueous compartment. Non-covalent interactions of receptors and ligands or hosts and guests at vesicle surfaces resemble recognition processes at biological membranes, including cell recognition, adhesion and fusion. Molecular recognition at membranes is often mediated by a multivalent instead of a monovalent interaction. This tutorial review describes the basics as well as the latest developments in biomimetic supramolecular chemistry of bilayer vesicles. We describe how molecular recognition can mediate the interaction between vesicles, and how the biomimetic supramolecular chemistry of vesicles furthers our understanding of biological membranes.

  6. Emotion recognition from physiological signals.

    PubMed

    Gouizi, K; Bereksi Reguig, F; Maaoui, C

    2011-01-01

    Emotion recognition is one of the great challenges in human-human and human-computer interaction. Accurate emotion recognition would allow computers to recognize human emotions and therefore react accordingly. In this paper, an approach for emotion recognition based on physiological signals is proposed. Six basic emotions: joy, sadness, fear, disgust, neutrality and amusement are analysed using physiological signals. These emotions are induced through the presentation of International Affecting Picture System (IAPS) pictures to the subjects. The physiological signals of interest in this analysis are: electromyogram signal (EMG), respiratory volume (RV), skin temperature (SKT), skin conductance (SKC), blood volume pulse (BVP) and heart rate (HR). These are selected to extract characteristic parameters, which will be used for classifying the emotions. The SVM (support vector machine) technique is used for classifying these parameters. The experimental results show that the proposed methodology provides in general a recognition rate of 85% for different emotional states.

  7. Effective indexing for face recognition

    NASA Astrophysics Data System (ADS)

    Sochenkov, I.; Sochenkova, A.; Vokhmintsev, A.; Makovetskii, A.; Melnikov, A.

    2016-09-01

    Face recognition is one of the most important tasks in computer vision and pattern recognition. Face recognition is useful for security systems to provide safety. In some situations it is necessary to identify the person among many others. In this case this work presents new approach in data indexing, which provides fast retrieval in big image collections. Data indexing in this research consists of five steps. First, we detect the area containing face, second we align face, and then we detect areas containing eyes and eyebrows, nose, mouth. After that we find key points of each area using different descriptors and finally index these descriptors with help of quantization procedure. The experimental analysis of this method is performed. This paper shows that performing method has results at the level of state-of-the-art face recognition methods, but it is also gives results fast that is important for the systems that provide safety.

  8. The neuroecology of competitor recognition.

    PubMed

    Grether, Gregory F

    2011-11-01

    Territorial animals can be expected to distinguish among the types of competitors and noncompetitors that they encounter on a regular basis, including prospective mates and rivals of their own species, but they may not correctly classify individuals of other species. Closely related species often have similar phenotypes and this can cause confusion when formerly allopatric populations first come into contact. Errors in recognizing competitors can have important ecological and evolutionary effects. I review what is known about the mechanisms of competitor recognition in animals generally, focusing on cases in which the targets of recognition include other species. Case studies include damselflies, ants, skinks, salamanders, reef fishes, and birds. In general, recognition systems consist of a phenotypic cue (e.g., chemical, color, song), a neural template against which cues are compared, a motor response (e.g., aggression), and sensory integration circuits for context dependency of the response (if any). Little is known about how competitor recognition systems work at the neural level, but inferences about specificity of cues and about sensory integration can be drawn from the responses of territory residents to simulated intruders. Competitor recognition often involves multiple cues in the same, or different, sensory modalities. The same cues and templates are often, but not always, used for intraspecific and interspecific recognition. Experiments have shown that imprinting on local cues is common, which may enable templates to track evolved changes in cues automatically. The dependence of aggression and tolerance on context is important even in the simplest systems. Species in which mechanisms of competitor recognition are best known offer untapped opportunities to examine how competitor-recognition systems evolve (e.g., by comparing allopatric and sympatric populations). Cues that are gene products (peptides, proteins) may provide insights into rates of evolution

  9. Computer Recognition of Facial Profiles

    DTIC Science & Technology

    1974-08-01

    effective in identifying those feature vectors which are of most importance in the recognition process . Thus the training procedure generally produces...ga Ente#lodI- i COMPUTER RECOGNITTON OF FACIAL PROFILES iU A Thesis i Presented in Partial Fulfillment of the Requirements for the Degree Master of... thesis , the suggestion that the state of the art in pattern recognition was sufficient to enable a machine capable of recognizing human faces to be built

  10. Thermal to Visible Face Recognition

    DTIC Science & Technology

    2012-04-01

    recognition has been an active area of research for the past two decades due its wide range of applications in law enforcement and verification...an ideal modality for nighttime tasks, but the large disparateness between the thermal IR and visible spectrums results in a wide modality gap that...CONCLUSION AND FUTURE WORK In this study, we investigated the thermal-to-visible face recognition problem, which has a wide modality gap. We showed

  11. [Neurological disease and facial recognition].

    PubMed

    Kawamura, Mitsuru; Sugimoto, Azusa; Kobayakawa, Mutsutaka; Tsuruya, Natsuko

    2012-07-01

    To discuss the neurological basis of facial recognition, we present our case reports of impaired recognition and a review of previous literature. First, we present a case of infarction and discuss prosopagnosia, which has had a large impact on face recognition research. From a study of patient symptoms, we assume that prosopagnosia may be caused by unilateral right occipitotemporal lesion and right cerebral dominance of facial recognition. Further, circumscribed lesion and degenerative disease may also cause progressive prosopagnosia. Apperceptive prosopagnosia is observed in patients with posterior cortical atrophy (PCA), pathologically considered as Alzheimer's disease, and associative prosopagnosia in frontotemporal lobar degeneration (FTLD). Second, we discuss face recognition as part of communication. Patients with Parkinson disease show social cognitive impairments, such as difficulty in facial expression recognition and deficits in theory of mind as detected by the reading the mind in the eyes test. Pathological and functional imaging studies indicate that social cognitive impairment in Parkinson disease is possibly related to damages in the amygdalae and surrounding limbic system. The social cognitive deficits can be observed in the early stages of Parkinson disease, and even in the prodromal stage, for example, patients with rapid eye movement (REM) sleep behavior disorder (RBD) show impairment in facial expression recognition. Further, patients with myotonic dystrophy type 1 (DM 1), which is a multisystem disease that mainly affects the muscles, show social cognitive impairment similar to that of Parkinson disease. Our previous study showed that facial expression recognition impairment of DM 1 patients is associated with lesion in the amygdalae and insulae. Our study results indicate that behaviors and personality traits in DM 1 patients, which are revealed by social cognitive impairment, are attributable to dysfunction of the limbic system.

  12. A two-stage exon recognition model based on synergetic neural network.

    PubMed

    Huang, Zhehuang; Chen, Yidong

    2014-01-01

    Exon recognition is a fundamental task in bioinformatics to identify the exons of DNA sequence. Currently, exon recognition algorithms based on digital signal processing techniques have been widely used. Unfortunately, these methods require many calculations, resulting in low recognition efficiency. In order to overcome this limitation, a two-stage exon recognition model is proposed and implemented in this paper. There are three main works. Firstly, we use synergetic neural network to rapidly determine initial exon intervals. Secondly, adaptive sliding window is used to accurately discriminate the final exon intervals. Finally, parameter optimization based on artificial fish swarm algorithm is used to determine different species thresholds and corresponding adjustment parameters of adaptive windows. Experimental results show that the proposed model has better performance for exon recognition and provides a practical solution and a promising future for other recognition tasks.

  13. Applications of chaotic neurodynamics in pattern recognition

    NASA Astrophysics Data System (ADS)

    Baird, Bill; Freeman, Walter J.; Eeckman, Frank H.; Yao, Yong

    1991-08-01

    Network algorithms and architectures for pattern recognition derived from neural models of the olfactory system are reviewed. These span a range from highly abstract to physiologically detailed, and employ the kind of dynamical complexity observed in olfactory cortex, ranging from oscillation to chaos. A simple architecture and algorithm for analytically guaranteed associative memory storage of analog patterns, continuous sequences, and chaotic attractors in the same network is described. A matrix inversion determines network weights, given prototype patterns to be stored. There are N units of capacity in an N node network with 3N2 weights. It costs one unit per static attractor, two per Fourier component of each sequence, and three to four per chaotic attractor. There are no spurious attractors, and for sequences there is a Liapunov function in a special coordinate system which governs the approach of transient states to stored trajectories. Unsupervised or supervised incremental learning algorithms for pattern classification, such as competitive learning or bootstrap Widrow-Hoff can easily be implemented. The architecture can be ''folded'' into a recurrent network with higher order weights that can be used as a model of cortex that stores oscillatory and chaotic attractors by a Hebb rule. Network performance is demonstrated by application to the problem of real-time handwritten digit recognition. An effective system with on-line learning has been written by Eeckman and Baird for the Macintosh. It utilizes static, oscillatory, and/or chaotic attractors of two kinds--Lorenze attractors, or attractors resulting from chaotically interacting oscillatory modes. The successful application to an industrial pattern recognition problem of a network architecture of considerable physiological and dynamical complexity, developed by Freeman and Yao, is described. The data sets of the problem come in three classes of difficulty, and performance of the biological network is

  14. Invariant object recognition based on extended fragments.

    PubMed

    Bart, Evgeniy; Hegdé, Jay

    2012-01-01

    Visual appearance of natural objects is profoundly affected by viewing conditions such as viewpoint and illumination. Human subjects can nevertheless compensate well for variations in these viewing conditions. The strategies that the visual system uses to accomplish this are largely unclear. Previous computational studies have suggested that in principle, certain types of object fragments (rather than whole objects) can be used for invariant recognition. However, whether the human visual system is actually capable of using this strategy remains unknown. Here, we show that human observers can achieve illumination invariance by using object fragments that carry the relevant information. To determine this, we have used novel, but naturalistic, 3-D visual objects called "digital embryos." Using novel instances of whole embryos, not fragments, we trained subjects to recognize individual embryos across illuminations. We then tested the illumination-invariant object recognition performance of subjects using fragments. We found that the performance was strongly correlated with the mutual information (MI) of the fragments, provided that MI value took variations in illumination into consideration. This correlation was not attributable to any systematic differences in task difficulty between different fragments. These results reveal two important principles of invariant object recognition. First, the subjects can achieve invariance at least in part by compensating for the changes in the appearance of small local features, rather than of whole objects. Second, the subjects do not always rely on generic or pre-existing invariance of features (i.e., features whose appearance remains largely unchanged by variations in illumination), and are capable of using learning to compensate for appearance changes when necessary. These psychophysical results closely fit the predictions of earlier computational studies of fragment-based invariant object recognition.

  15. Chinese character recognition using simulated phosphene maps.

    PubMed

    Zhao, Ying; Lu, Yanyu; Zhou, Chuanqing; Chen, Yao; Ren, Qiushi; Chai, Xinyu

    2011-05-01

    A visual prosthetic device may produce phosphene maps in which individual phosphene characteristics can be altered. This study was an investigation of the ability of normally sighted subjects to recognize Chinese characters (CCs) after altering simulated phosphene maps. Thirty volunteers with normal or corrected visual acuity of 20/20 were recruited. CC recognition accuracy and response time were investigated while one parameter was changed (distortion, pixel dropout percentage, pixel size variability, or pixel gray level) or different combinations of three parameters were used. Five hundred CCs consisting of 1 to 16 strokes were used for the character sets. CC recognition accuracy and response times respectively decreased and increased when distortion, dropout, and pixel size variability increased. Gray levels did not significantly affect the results, except when eight levels were used. To maintain an 80% accuracy rate, there should be a distortion index (k) of no more than 0.2 (irregularity), a pixel dropout of 20%, and a pixel size range of 1 to 16 mm (7-112 min arc). Only a combination of a k=0.1 distortion index, a dropout of 10%, and a pixel size range of 1.33 to 12 mm (9.3-84 min arc) achieved a goal of ≥80% accuracy. Distortion, dropout percentage, and pixel size variability have a significant impact on pixelated CC recognition. Although at present the visual ability of prosthesis users is limited, it should be possible to extend this to CC recognition and reading in the future. The results will help visual prosthesis researchers determine the effects of altering phosphene maps and improve outcomes for patients.

  16. 77 FR 64799 - Notice of Submission for OMB Review; Office of Postsecondary Education; Secretary's Recognition...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-23

    ...The information collected is required to determine if an accrediting agency complies with the Secretary of Education's Criteria for Recognition and is used to allow the Secretary to make determinations on extending and/or continuing...

  17. Holistic processing predicts face recognition.

    PubMed

    Richler, Jennifer J; Cheung, Olivia S; Gauthier, Isabel

    2011-04-01

    The concept of holistic processing is a cornerstone of face-recognition research. In the study reported here, we demonstrated that holistic processing predicts face-recognition abilities on the Cambridge Face Memory Test and on a perceptual face-identification task. Our findings validate a large body of work that relies on the assumption that holistic processing is related to face recognition. These findings also reconcile the study of face recognition with the perceptual-expertise work it inspired; such work links holistic processing of objects with people's ability to individuate them. Our results differ from those of a recent study showing no link between holistic processing and face recognition. This discrepancy can be attributed to the use in prior research of a popular but flawed measure of holistic processing. Our findings salvage the central role of holistic processing in face recognition and cast doubt on a subset of the face-perception literature that relies on a problematic measure of holistic processing.

  18. Sampling design for face recognition

    NASA Astrophysics Data System (ADS)

    Yan, Yanjun; Osadciw, Lisa A.

    2006-04-01

    A face recognition system consists of two integrated parts: One is the face recognition algorithm, the other is the selected classifier and derived features by the algorithm from a data set. The face recognition algorithm definitely plays a central role, but this paper does not aim at evaluating the algorithm, but deriving the best features for this algorithm from a specific database through sampling design of the training set, which directs how the sample should be collected and dictates the sample space. Sampling design can help exert the full potential of the face recognition algorithm without overhaul. Conventional statistical analysis usually assume some distribution to draw the inference, but the design-based inference does not assume any distribution of the data and it does not assume the independency between the sample observations. The simulations illustrates that the systematic sampling scheme performs better than the simple random sampling scheme, and the systematic sampling is comparable to using all available training images in recognition performance. Meanwhile the sampling schemes can save the system resources and alleviate the overfitting problem. However, the post stratification by sex is not shown to be significant in improving the recognition performance.

  19. Voice Congruency Facilitates Word Recognition

    PubMed Central

    Campeanu, Sandra; Craik, Fergus I. M.; Alain, Claude

    2013-01-01

    Behavioral studies of spoken word memory have shown that context congruency facilitates both word and source recognition, though the level at which context exerts its influence remains equivocal. We measured event-related potentials (ERPs) while participants performed both types of recognition task with words spoken in four voices. Two voice parameters (i.e., gender and accent) varied between speakers, with the possibility that none, one or two of these parameters was congruent between study and test. Results indicated that reinstating the study voice at test facilitated both word and source recognition, compared to similar or no context congruency at test. Behavioral effects were paralleled by two ERP modulations. First, in the word recognition test, the left parietal old/new effect showed a positive deflection reflective of context congruency between study and test words. Namely, the same speaker condition provided the most positive deflection of all correctly identified old words. In the source recognition test, a right frontal positivity was found for the same speaker condition compared to the different speaker conditions, regardless of response success. Taken together, the results of this study suggest that the benefit of context congruency is reflected behaviorally and in ERP modulations traditionally associated with recognition memory. PMID:23527021

  20. Voice congruency facilitates word recognition.

    PubMed

    Campeanu, Sandra; Craik, Fergus I M; Alain, Claude

    2013-01-01

    Behavioral studies of spoken word memory have shown that context congruency facilitates both word and source recognition, though the level at which context exerts its influence remains equivocal. We measured event-related potentials (ERPs) while participants performed both types of recognition task with words spoken in four voices. Two voice parameters (i.e., gender and accent) varied between speakers, with the possibility that none, one or two of these parameters was congruent between study and test. Results indicated that reinstating the study voice at test facilitated both word and source recognition, compared to similar or no context congruency at test. Behavioral effects were paralleled by two ERP modulations. First, in the word recognition test, the left parietal old/new effect showed a positive deflection reflective of context congruency between study and test words. Namely, the same speaker condition provided the most positive deflection of all correctly identified old words. In the source recognition test, a right frontal positivity was found for the same speaker condition compared to the different speaker conditions, regardless of response success. Taken together, the results of this study suggest that the benefit of context congruency is reflected behaviorally and in ERP modulations traditionally associated with recognition memory.

  1. Perceptual Plasticity for Auditory Object Recognition

    PubMed Central

    Heald, Shannon L. M.; Van Hedger, Stephen C.; Nusbaum, Howard C.

    2017-01-01

    In our auditory environment, we rarely experience the exact acoustic waveform twice. This is especially true for communicative signals that have meaning for listeners. In speech and music, the acoustic signal changes as a function of the talker (or instrument), speaking (or playing) rate, and room acoustics, to name a few factors. Yet, despite this acoustic variability, we are able to recognize a sentence or melody as the same across various kinds of acoustic inputs and determine meaning based on listening goals, expectations, context, and experience. The recognition process relates acoustic signals to prior experience despite variability in signal-relevant and signal-irrelevant acoustic properties, some of which could be considered as “noise” in service of a recognition goal. However, some acoustic variability, if systematic, is lawful and can be exploited by listeners to aid in recognition. Perceivable changes in systematic variability can herald a need for listeners to reorganize perception and reorient their attention to more immediately signal-relevant cues. This view is not incorporated currently in many extant theories of auditory perception, which traditionally reduce psychological or neural representations of perceptual objects and the processes that act on them to static entities. While this reduction is likely done for the sake of empirical tractability, such a reduction may seriously distort the perceptual process to be modeled. We argue that perceptual representations, as well as the processes underlying perception, are dynamically determined by an interaction between the uncertainty of the auditory signal and constraints of context. This suggests that the process of auditory recognition is highly context-dependent in that the identity of a given auditory object may be intrinsically tied to its preceding context. To argue for the flexible neural and psychological updating of sound-to-meaning mappings across speech and music, we draw upon examples

  2. Recognition Failure: Another Case of Retrieval Failure

    ERIC Educational Resources Information Center

    Rabinowitz, Jan; And Others

    1977-01-01

    A theoretical explanation of the phenomenon of recognition failure and a presentation of seven experiments investigating performance. Recognition failure is reduced when a more stringent recognition criterion is used, essentially eliminated when the proper access test is used and significantly reduced when variability in recognition performance is…

  3. Pattern recognition characterizations of micromechanical and morphological materials states via analytical quantitative ultrasonics

    NASA Technical Reports Server (NTRS)

    Williams, J. H., Jr.; Lee, S. S.

    1986-01-01

    One potential approach to the quantitative acquisition of discriminatory information that can isolate a single structural state is pattern recognition. The pattern recognition characterizations of micromechanical and morphological materials states via analytical quantiative ultrasonics are outlined. The concepts, terminology, and techniques of statistical pattern recognition are reviewed. Feature extraction and classification and states of the structure can be determined via a program of ultrasonic data generation.

  4. Document Form and Character Recognition using SVM

    NASA Astrophysics Data System (ADS)

    Park, Sang-Sung; Shin, Young-Geun; Jung, Won-Kyo; Ahn, Dong-Kyu; Jang, Dong-Sik

    2009-08-01

    Because of development of computer and information communication, EDI (Electronic Data Interchange) has been developing. There is OCR (Optical Character Recognition) of Pattern recognition technology for EDI. OCR contributed to changing many manual in the past into automation. But for the more perfect database of document, much manual is needed for excluding unnecessary recognition. To resolve this problem, we propose document form based character recognition method in this study. Proposed method is divided into document form recognition part and character recognition part. Especially, in character recognition, change character into binarization by using SVM algorithm and extract more correct feature value.

  5. Alzheimer’s disease-associated mutations increase amyloid precursor protein resistance to γ-secretase cleavage and the Aβ42/Aβ40 ratio

    PubMed Central

    Xu, Ting-Hai; Yan, Yan; Kang, Yanyong; Jiang, Yi; Melcher, Karsten; Xu, H Eric

    2016-01-01

    Mutations in the amyloid precursor protein (APP) gene and the aberrant cleavage of APP by γ-secretase are associated with Alzheimer’s disease (AD). Here we have developed a simple and sensitive cell-based assay to detect APP cleavage by γ-secretase. Unexpectedly, most familial AD (FAD)-linked APP mutations make APP partially resistant to γ-secretase. Mutations that alter residues N terminal to the γ-secretase cleavage site Aβ42 have subtle effects on cleavage efficiency and cleavage-site selectivity. In contrast, mutations that alter residues C terminal to the Aβ42 site reduce cleavage efficiency and dramatically shift cleavage-site specificity toward the aggregation-prone Aβ42. Moreover, mutations that remove positive charge at residue 53 greatly reduce the APP cleavage by γ-secretase. These results suggest a model of γ-secretase substrate recognition, in which the APP region C terminal to the Aβ42 site and the positively charged residue at position 53 are the primary determinants for substrate binding and cleavage-site selectivity. We further demonstrate that this model can be extended to γ-secretase processing of notch receptors, a family of highly conserved cell-surface signaling proteins. PMID:27625790

  6. Pattern recognition monitoring of PEM fuel cell

    DOEpatents

    Meltser, M.A.

    1999-08-31

    The CO-concentration in the H{sub 2} feed stream to a PEM fuel cell stack is monitored by measuring current and voltage behavior patterns from an auxiliary cell attached to the end of the stack. The auxiliary cell is connected to the same oxygen and hydrogen feed manifolds that supply the stack, and discharges through a constant load. Pattern recognition software compares the current and voltage patterns from the auxiliary cell to current and voltage signature determined from a reference cell similar to the auxiliary cell and operated under controlled conditions over a wide range of CO-concentrations in the H{sub 2} fuel stream. 4 figs.

  7. Statistical pattern recognition algorithms for autofluorescence imaging

    NASA Astrophysics Data System (ADS)

    Kulas, Zbigniew; Bereś-Pawlik, Elżbieta; Wierzbicki, Jarosław

    2009-02-01

    In cancer diagnostics the most important problems are the early identification and estimation of the tumor growth and spread in order to determine the area to be operated. The aim of the work was to design of statistical algorithms helping doctors to objectively estimate pathologically changed areas and to assess the disease advancement. In the research, algorithms for classifying endoscopic autofluorescence images of larynx and intestine were used. The results show that the statistical pattern recognition offers new possibilities for endoscopic diagnostics and can be of a tremendous help in assessing the area of the pathological changes.

  8. Pattern recognition monitoring of PEM fuel cell

    DOEpatents

    Meltser, Mark Alexander

    1999-01-01

    The CO-concentration in the H.sub.2 feed stream to a PEM fuel cell stack is monitored by measuring current and voltage behavior patterns from an auxiliary cell attached to the end of the stack. The auxiliary cell is connected to the same oxygen and hydrogen feed manifolds that supply the stack, and discharges through a constant load. Pattern recognition software compares the current and voltage patterns from the auxiliary cell to current and voltage signature determined from a reference cell similar to the auxiliary cell and operated under controlled conditions over a wide range of CO-concentrations in the H.sub.2 fuel stream.

  9. Visual landmark recognition for autonomous robot navigation

    NASA Astrophysics Data System (ADS)

    Cicerone, M.; Stella, Ettore; Caponetti, Laura; Distante, Arcangelo

    1997-09-01

    Self-location is the capability of a mobile robot to determine its position in the environment referring to absolute landmarks. The possibility to use natural visual landmarks for self-location augments the autonomy and the flexibility of mobile vehicles. In this paper the use of junctions, detected in real images, as landmarks is proposed. The use of visual cues means that problems regarding variations of perspective and scale must be resolved. We propose to formulate the junction recognition as a graph matching problem and resolved using standard methods. Experimental results are shown on real contexts.

  10. Recognition memory: neuronal substrates of the judgement of prior occurrence.

    PubMed

    Brown, M W; Xiang, J Z

    1998-06-01

    Recognition memory relies on two processes: (i) identification and (ii) judgement concerning prior occurrence. A system centred on perirhinal cortex appears to be responsible for judgement of prior occurrence based on discrimination of the familiarity of stimuli or their recency of occurrence; in contrast, a hippocampal system probably supplies information concerning the episodic, contextual aspects of recognition memory. This review chiefly concerns the perirhinal system and, in particular, neurones that signal the prior occurrence of stimuli by a decrease in response. Details concerning such decremental responses are given and it is argued that such responses in perirhinal cortex are adequate for and central to discrimination of stimulus familiarity and recency in a wide range of situations. Information is given of similar types of neuronal responses in anatomically related brain regions and what may be deduced about the operation of the recognition memory system. The possibility is discussed that the neuronal responses that signal information concerning the recent occurrence of stimuli may contribute to repetition priming as well as recognition memory. Other described changes in the activity of individual neurones such as response enhancements, or sustained (delay) activity may allow solution of specialised forms of recognition memory tasks where relatively short-term working memory is adequate. Implications of the multi-faceted nature of recognition memory for the interpretation of results are emphasised. Unsolved problems and avenues for future experimentation, including determining the nature of possible underlying synaptic plastic changes, are discussed.

  11. Structural determinants of miR156a precursor processing in temperature-responsive flowering in Arabidopsis

    PubMed Central

    Kim, Wanhui; Kim, Hee-Eun; Jun, A Rim; Jung, Myeong Gyo; Jin, Suhyun; Lee, Joon-Hwa; Ahn, Ji Hoon

    2016-01-01

    MicroRNAs originate from primary transcripts (pri-miRNAs) containing hairpin structures. Plant pri-miRNAs have highly variable structures and little is known about the information encoded in their secondary structures. Arabidopsis miR156 is an ambient temperature-responsive miRNA and plays an important role in regulating flowering time. To identify the structural determinants for miR156 processing, we analyzed the effects of mutations introduced in the upper stem of pri-miR156a on its temperature-dependent processing and flowering time. The levels of pri-miR156a and mature miR156 were opposite at different temperatures. Mutations in the upper stem, especially the region closer to the miR156a/miR156a* duplex, reduced miR156 processing at 23 °C and 16 °C and caused a less severe phenotype compared with the un-mutated construct. Mutation in the second stem near the first cleavage site of pri-miR156a affected miR156 processing at 23 °C, but not at 16 °C. This was also seen in pri-miR172a, another ambient temperature-responsive miRNA. Replacement of the upper stem of pri-miR156a with that of pri-miR172a severely affected miR156 processing and flowering time. These results suggested that the upper stem of pri-miR156a is important for miR156 processing at different temperatures. In particular, the second stem adjacent to the first cleavage site plays a role in the regulation of ambient temperature-responsive flowering. PMID:27335452

  12. Two new restriction endonucleases DraII and DraIII from Deinococcus radiophilus.

    PubMed Central

    Grosskopf, R; Wolf, W; Kessler, C

    1985-01-01

    In addition to recently characterized DraI (1), two new Type II restriction endonucleases, DraII and DraIII, with novel site-specificities were isolated and purified from Deinococcus radiophilus ATCC 27603. DraII and DraIII recognize the hepta- and nonanucleotide sequences (sequence in text) The cleavage sites within both strands are indicated by arrows. The recognition sequences were established by mapping of the cleavage sites on pBR322 (DraII) and fd109 RF DNA (DraIII). The sequence specifities were confirmed by computer-assisted restriction analyses of the generated fragment patterns of the sequenced DNA's of the bacteriophages lambda, phi X174 RF, M13mp8 RF and fd109 RF, the viruses Adeno2 and SV40, and the plasmids pBR322 and pBR328. The cleavage positions within the recognition sequences were determined by sequencing experiments. Images PMID:2987827

  13. Bidirectional Modulation of Recognition Memory.

    PubMed

    Ho, Jonathan W; Poeta, Devon L; Jacobson, Tara K; Zolnik, Timothy A; Neske, Garrett T; Connors, Barry W; Burwell, Rebecca D

    2015-09-30

    Perirhinal cortex (PER) has a well established role in the familiarity-based recognition of individual items and objects. For example, animals and humans with perirhinal damage are unable to distinguish familiar from novel objects in recognition memory tasks. In the normal brain, perirhinal neurons respond to novelty and familiarity by increasing or decreasing firing rates. Recent work also implicates oscillatory activity in the low-beta and low-gamma frequency bands in sensory detection, perception, and recognition. Using optogenetic methods in a spontaneous object exploration (SOR) task, we altered recognition memory performance in rats. In the SOR task, normal rats preferentially explore novel images over familiar ones. We modulated exploratory behavior in this task by optically stimulating channelrhodopsin-expressing perirhinal neurons at various frequencies while rats looked at novel or familiar 2D images. Stimulation at 30-40 Hz during looking caused rats to treat a familiar image as if it were novel by increasing time looking at the image. Stimulation at 30-40 Hz was not effective in increasing exploration of novel images. Stimulation at 10-15 Hz caused animals to treat a novel image as familiar by decreasing time looking at the image, but did not affect looking times for images that were already familiar. We conclude that optical stimulation of PER at different frequencies can alter visual recognition memory bidirectionally. Significance statement: Recognition of novelty and familiarity are important for learning, memory, and decision making. Perirhinal cortex (PER) has a well established role in the familiarity-based recognition of individual items and objects, but how novelty and familiarity are encoded and transmitted in the brain is not known. Perirhinal neurons respond to novelty and familiarity by changing firing rates, but recent work suggests that brain oscillations may also be important for recognition. In this study, we showed that stimulation of

  14. Bidirectional Modulation of Recognition Memory

    PubMed Central

    Ho, Jonathan W.; Poeta, Devon L.; Jacobson, Tara K.; Zolnik, Timothy A.; Neske, Garrett T.; Connors, Barry W.

    2015-01-01

    Perirhinal cortex (PER) has a well established role in the familiarity-based recognition of individual items and objects. For example, animals and humans with perirhinal damage are unable to distinguish familiar from novel objects in recognition memory tasks. In the normal brain, perirhinal neurons respond to novelty and familiarity by increasing or decreasing firing rates. Recent work also implicates oscillatory activity in the low-beta and low-gamma frequency bands in sensory detection, perception, and recognition. Using optogenetic methods in a spontaneous object exploration (SOR) task, we altered recognition memory performance in rats. In the SOR task, normal rats preferentially explore novel images over familiar ones. We modulated exploratory behavior in this task by optically stimulating channelrhodopsin-expressing perirhinal neurons at various frequencies while rats looked at novel or familiar 2D images. Stimulation at 30–40 Hz during looking caused rats to treat a familiar image as if it were novel by increasing time looking at the image. Stimulation at 30–40 Hz was not effective in increasing exploration of novel images. Stimulation at 10–15 Hz caused animals to treat a novel image as familiar by decreasing time looking at the image, but did not affect looking times for images that were already familiar. We conclude that optical stimulation of PER at different frequencies can alter visual recognition memory bidirectionally. SIGNIFICANCE STATEMENT Recognition of novelty and familiarity are important for learning, memory, and decision making. Perirhinal cortex (PER) has a well established role in the familiarity-based recognition of individual items and objects, but how novelty and familiarity are encoded and transmitted in the brain is not known. Perirhinal neurons respond to novelty and familiarity by changing firing rates, but recent work suggests that brain oscillations may also be important for recognition. In this study, we showed that

  15. Cognitive object recognition system (CORS)

    NASA Astrophysics Data System (ADS)

    Raju, Chaitanya; Varadarajan, Karthik Mahesh; Krishnamurthi, Niyant; Xu, Shuli; Biederman, Irving; Kelley, Troy

    2010-04-01

    We have developed a framework, Cognitive Object Recognition System (CORS), inspired by current neurocomputational models and psychophysical research in which multiple recognition algorithms (shape based geometric primitives, 'geons,' and non-geometric feature-based algorithms) are integrated to provide a comprehensive solution to object recognition and landmarking. Objects are defined as a combination of geons, corresponding to their simple parts, and the relations among the parts. However, those objects that are not easily decomposable into geons, such as bushes and trees, are recognized by CORS using "feature-based" algorithms. The unique interaction between these algorithms is a novel approach that combines the effectiveness of both algorithms and takes us closer to a generalized approach to object recognition. CORS allows recognition of objects through a larger range of poses using geometric primitives and performs well under heavy occlusion - about 35% of object surface is sufficient. Furthermore, geon composition of an object allows image understanding and reasoning even with novel objects. With reliable landmarking capability, the system improves vision-based robot navigation in GPS-denied environments. Feasibility of the CORS system was demonstrated with real stereo images captured from a Pioneer robot. The system can currently identify doors, door handles, staircases, trashcans and other relevant landmarks in the indoor environment.

  16. An introduction to object recognition.

    PubMed

    Liter, J C; Bülthoff, H H

    1998-01-01

    In this report we present a general introduction to object recognition. We begin with brief discussions of the terminology used in the object recognition literature and the psychophysical tasks that are used to investigate object recognition. We then discuss models of shape representation. We dispense with the idea that shape representations are like the 3-D models used in computer aided design and explore instead models of shape representation that are based on future descriptions. As these descriptions encode only the features that are visible from a particular viewpoint, they are generally viewpoint-specific. We discuss various means of achieving viewpoint-invariant recognition using such descriptions, including reliance on diagnostic features visible from a wide range of viewpoints, storage of multiple descriptions for each object, and the use of transformation mechanisms. Finally, we discuss how differences in viewpoint dependence that are often observed for within-category and between-category recognition tasks could be due to differences in the types of features that are naturally available to distinguish among different objects in these tasks.

  17. Kazakh Traditional Dance Gesture Recognition

    NASA Astrophysics Data System (ADS)

    Nussipbekov, A. K.; Amirgaliyev, E. N.; Hahn, Minsoo

    2014-04-01

    Full body gesture recognition is an important and interdisciplinary research field which is widely used in many application spheres including dance gesture recognition. The rapid growth of technology in recent years brought a lot of contribution in this domain. However it is still challenging task. In this paper we implement Kazakh traditional dance gesture recognition. We use Microsoft Kinect camera to obtain human skeleton and depth information. Then we apply tree-structured Bayesian network and Expectation Maximization algorithm with K-means clustering to calculate conditional linear Gaussians for classifying poses. And finally we use Hidden Markov Model to detect dance gestures. Our main contribution is that we extend Kinect skeleton by adding headwear as a new skeleton joint which is calculated from depth image. This novelty allows us to significantly improve the accuracy of head gesture recognition of a dancer which in turn plays considerable role in whole body gesture recognition. Experimental results show the efficiency of the proposed method and that its performance is comparable to the state-of-the-art system performances.

  18. An audiovisual emotion recognition system

    NASA Astrophysics Data System (ADS)

    Han, Yi; Wang, Guoyin; Yang, Yong; He, Kun

    2007-12-01

    Human emotions could be expressed by many bio-symbols. Speech and facial expression are two of them. They are both regarded as emotional information which is playing an important role in human-computer interaction. Based on our previous studies on emotion recognition, an audiovisual emotion recognition system is developed and represented in this paper. The system is designed for real-time practice, and is guaranteed by some integrated modules. These modules include speech enhancement for eliminating noises, rapid face detection for locating face from background image, example based shape learning for facial feature alignment, and optical flow based tracking algorithm for facial feature tracking. It is known that irrelevant features and high dimensionality of the data can hurt the performance of classifier. Rough set-based feature selection is a good method for dimension reduction. So 13 speech features out of 37 ones and 10 facial features out of 33 ones are selected to represent emotional information, and 52 audiovisual features are selected due to the synchronization when speech and video fused together. The experiment results have demonstrated that this system performs well in real-time practice and has high recognition rate. Our results also show that the work in multimodules fused recognition will become the trend of emotion recognition in the future.

  19. Activation of Supraoptic Oxytocin Neurons by Secretin Facilitates Social Recognition.

    PubMed

    Takayanagi, Yuki; Yoshida, Masahide; Takashima, Akihide; Takanami, Keiko; Yoshida, Shoma; Nishimori, Katsuhiko; Nishijima, Ichiko; Sakamoto, Hirotaka; Ya