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Sample records for 111in-dtpa-trastuzumab fab fragments

  1. Single chain Fab (scFab) fragment

    PubMed Central

    Hust, Michael; Jostock, Thomas; Menzel, Christian; Voedisch, Bernd; Mohr, Anja; Brenneis, Mariam; Kirsch, Martina I; Meier, Doris; Dübel, Stefan

    2007-01-01

    Background The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera

  2. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  3. Iodination of Fab fragments: Effect of I/Fab molar ratio

    SciTech Connect

    Kishore, R.; Eary, J.F.; Beaumier, P.L.; Hellstrom, K.E.; Hellstrom, I.; Nelp, W.B.

    1985-05-01

    Radioisotopes of iodine covalently coupled to antibodies have formed the standard against which other radiolabeled antibody tracers are compared. Since the use of monoclonal antibodies (MoAb) for the diagnosis and therapy of malignant diseases is increasing rapidly the authors wished to investigate radiolabeling variables which affect the immunointegrity of radioiodinated antibodies. The authors studied the electrophilic (chloramine-T) iodination of the Fab fragment of a murine MoAb against the high molecular weight proteoglycan antigen of human melanoma. The immunoreactivity of the iodianted Fab was assessed using a cell binding assay with formaldehyde-fixed cells of a selected cell line (number 2669). The in vitro stability of the labeled fragment was studied electrophoretically. The results indicate that the reaction time and concentrations of chloramine-T were not critical within broad limits. On the other hand immunoreactivity and deiodination over time (shelf-life) were inversely related and very sensitive to I/Fab molar ratio even at concentrations below -0.1 atom of I per Fab molecule. This has important implications for the radiotherapy of malignant tumors using I-131 labeled immunoglobulins which often have higher I/Fab molar ratios (100 mCi I-131/10 mg Fab approx. = 0.5 I atoms/Fab) versus diagnostic preparations (10 mCi I-131/5 mg Fab approx. = 0.1 I atoms/Fab). Thus the authors conclude that to maintain high immunointegrity the I/Fab molar ratio should be kept low, especially for therapeutic preparations, by using correspondingly higher amounts of Fab.

  4. Production of single chain Fab (scFab) fragments in Bacillus megaterium

    PubMed Central

    Jordan, Eva; Al-Halabi, Laila; Schirrmann, Thomas; Hust, Michael; Dübel, Stefan

    2007-01-01

    Background The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli. Results The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli. Conclusion B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli. PMID:18042285

  5. GTP-specific fab fragment-based GTPase activity assay.

    PubMed

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri

    2015-03-17

    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  6. Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

    SciTech Connect

    Wilbur, D.S.; Hadley, S.W.; Grant, L.M.; Hylarides, M.D. )

    1991-03-01

    A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2.

  7. Assessment of Digoxin-Specific Fab Fragment Dosages in Digoxin Poisoning.

    PubMed

    Nordt, Sean Patrick; Clark, Richard F; Machado, Carol; Cantrell, F Lee

    2016-01-01

    Digoxin poisoning still remains a common cause of morbidity and mortality. Fortunately, digoxin-specific Fab fragments are commercially available as an antidote. However, these Fab fragments are several thousand dollars per vial. There is a standardized formula to calculate appropriate Fab fragment dosage based on the serum digoxin concentration. This can greatly reduce the amount of Fab fragment administered. There is also an empiric dosing guideline recommending 6-10 vials be given; however, this may result in higher amounts of Fab fragments being administered than required. We performed this study to assess the amounts of digoxin-specific Fab fragments administered in the treatment of digoxin poisonings recorded in a poison control system database from January 1, 2000, to December 31, 2009, in which digoxin serum concentrations were available. This was a retrospective study of 278 patients, 107 with acute poisonings (group A) and 171 following chronic poisoning (group B). In group A, the calculated Fab dose was higher than the calculated dose based on available concentrations in 39 (36%) of group A and 15 (9%) of group B patients. The average wholesale price cost of the excessive dosages ranged from $4818 to as high as $50,589 per patient. Our data suggests that clinician education on digoxin poisoning and the use of the standardized formula to calculate the Fab dose may decrease over utilization and decrease costs associated with the administration of digoxin-specific Fab fragments in the treatment of digoxin poisonings.

  8. Specific recognition of a tetrahedral phosphonamidate transition state analogue group by a recombinant antibody Fab fragment.

    PubMed

    Hua, T D; Lamaty, F; Souriau, C; Rolland-Fulcrand, V; Lazaro, R; Viallefont, P; Lefranc, M P; Weill, M

    1996-06-01

    In order to obtain antibodies able to catalyse a peptide synthesis, a naive combinatorial library of human Fab antibody fragments was screened with the phosphonamidate transition state analogue of the reaction. Several Fab fragments were able to bind the analogue. Competitive binding studies performed with molecules containing representative parts of the hapten showed that two Fabs were able to recognize specifically the tetrahedral phosphorus present in the hapten.

  9. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

    PubMed Central

    Barbas, C F; Björling, E; Chiodi, F; Dunlop, N; Cababa, D; Jones, T M; Zebedee, S L; Persson, M A; Nara, P L; Norrby, E

    1992-01-01

    A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies. PMID:1384050

  10. Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications.

    PubMed

    Crivianu-Gaita, Victor; Thompson, Michael

    2015-08-15

    Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab' fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab' fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab' fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab' fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab' immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab' fragments in different applications and future directions of the field will be addressed, especially regarding biosensors.

  11. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    PubMed

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  12. Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain.

    PubMed

    Nesbeth, Darren N; Perez-Pardo, Miguel-Angel; Ali, Shaukat; Ward, John; Keshavarz-Moore, Eli

    2012-02-01

    Host cell engineering is becoming a realistic option in whole bioprocess strategies to maximize product manufacturability. High molecular weight (MW) genomic DNA currently hinders bioprocessing of Escherichia coli by causing viscosity in homogenate feedstocks. We previously showed that co-expressing Staphylococcal nuclease and human Fab' fragment in the periplasm of E. coli enables auto-hydrolysis of genomic DNA upon cell disruption, with a consequent reduction in feedstock viscosity and improvement in clarification performance. Here we report the impact of periplasmic nuclease expression on stability of DNA and Fab' fragment in homogenates, host-strain growth kinetics, cell integrity at harvest and Fab' fragment productivity. Nuclease and Fab' plasmids were shown to exert comparable levels of growth burden on the host W3110 E. coli strain. Nuclease co-expression did not compromise either the growth performance or volumetric yield of the production strain. 0.5 g/L Fab' fragment (75 L scale) and 0.7 g/L (20 L scale) was achieved for both unmodified and cell-engineered production strains. Unexpectedly, nuclease-modified cells achieved maximum Fab' levels 8-10 h earlier than the original, unmodified production strain. Scale-down studies of homogenates showed that nuclease-mediated hydrolysis of high MW DNA progressed to completion within minutes of homogenization, even when homogenates were chilled on ice, with no loss of Fab' product and no need for additional co-factors or buffering.

  13. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

    PubMed

    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

    2013-12-31

    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

  14. Nebulized anti-IL-13 monoclonal antibody Fab' fragment reduces allergen-induced asthma.

    PubMed

    Hacha, Jonathan; Tomlinson, Kate; Maertens, Ludovic; Paulissen, Geneviève; Rocks, Natacha; Foidart, Jean-Michel; Noel, Agnès; Palframan, Roger; Gueders, Maud; Cataldo, Didier D

    2012-11-01

    IL-13 is a prototypic T helper type 2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis, and eosinophil infiltration. We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness, and remodeling in an experimental model of allergic asthma. Anti-IL-13 Fab' was administered to mice as a liquid aerosol generated by inExpose inhalation system in a tower allowing a nose-only exposure. BALB/c mice were treated by PBS, anti-IL-13 Fab', or A33 Fab' fragment and subjected to ovalbumin exposure for 1 and 5 weeks (short-term and long-term protocols). Our data demonstrate a significant antiasthma effect after nebulization of anti-IL-13 Fab' in a model of asthma driven by allergen exposure as compared with saline and nonimmune Fab fragments. In short- and long-term protocols, administration of the anti-IL-13 Fab' by inhalation significantly decreased bronchial responsiveness to methacholine, bronchoalveolar lavage fluid eosinophilia, inflammatory cell infiltration in lung tissue, and many features of airway remodeling. Levels of proinflammatory mediators and matrix metalloprotease were significantly lower in lung parenchyma of mice treated with anti-IL-13 Fab'. These data demonstrate that an inhaled anti-IL-13 Fab' significantly reduces airway inflammation, hyperresponsiveness, and remodeling. Specific neutralization of IL-13 in the lungs using an inhaled anti-IL-13 Fab' could represent a novel and effective therapy for the treatment of asthma.

  15. Promoter engineering to optimize recombinant periplasmic Fab' fragment production in Escherichia coli.

    PubMed

    Schofield, Desmond M; Templar, Alex; Newton, Joseph; Nesbeth, Darren N

    2016-07-08

    Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840-847, 2016.

  16. Anti-fouling properties of Fab' fragments immobilized on silane-based adlayers

    NASA Astrophysics Data System (ADS)

    Crivianu-Gaita, Victor; Romaschin, Alexander; Thompson, Michael

    2015-12-01

    Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab') in biosensors. One Fab' linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab' fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab' fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab'-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection.

  17. IgG Fab fragments forming bivalent complexes by a conformational mechanism that is reversible by osmolytes.

    PubMed

    Nelson, Alfreda D; Hoffmann, Michele M; Parks, Christopher A; Dasari, Surendra; Schrum, Adam G; Gil, Diana

    2012-12-14

    Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing cross-linking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches, including size exclusion chromatography, SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte l-proline might impact bivalent Fab complexation. We found that l-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of preformed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of preformed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes.

  18. Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

    PubMed

    Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

    2016-11-25

    Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab')2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab')2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications.

  19. Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

    PubMed

    Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

    2015-11-01

    In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.

  20. Obstruction of dengue virus maturation by Fab fragments of the 2H2 antibody.

    PubMed

    Wang, Zhiqing; Li, Long; Pennington, Janice G; Sheng, Ju; Yap, Moh Lan; Plevka, Pavel; Meng, Geng; Sun, Lei; Jiang, Wen; Rossmann, Michael G

    2013-08-01

    The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.

  1. Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

    PubMed Central

    Tachibana, Hiroshi; Cheng, Xun-Jia; Watanabe, Katsuomi; Takekoshi, Masataka; Maeda, Fumiko; Aotsuka, Satoshi; Kaneda, Yoshimasa; Takeuchi, Tsutomu; Ihara, Seiji

    1999-01-01

    Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis. PMID:10225840

  2. Effects of sheep digoxin-specific antibodies and their Fab fragments on digoxin pharmacokinetics in dogs.

    PubMed Central

    Butler, V P; Schmidt, D H; Smith, T W; Haber, E; Raynor, B D; Demartini, P

    1977-01-01

    Intact sheep antidigoxin antibodies and their Fab fragments have both been found to exert profound effects on digoxin pharmacokinetics in [3H] digoxin-treated dogs. Both classes of molecule remove digoxin from the extravascular space and sequester it in the circulation in protein-bound form, a form in which the digoxin is presumably inactive. These two classes of molecule differ, however, in that the intact antibody molecules interfere with digoxin excretion, thereby promoting the retention of the glycoside; this retained digoxin is eventually released in free, active form when the administered antibody is metabolically degraded. In contrast, urinary excretion of digoxin continues in Fab-treated dogs, with significant quantities of digoxin being excreted promptly in the urine in complex with Fab fragments. These differences in urinary excretion, together with the probable decreased immunogenicity of sheep antidigoxin Fab fragments, suggest that such fragments possess potential advantages over intact antibody molecules for use in the therapy of life-threatening digoxin intoxication in man. PMID:299860

  3. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) immunological test system. 866.5520 Section 866.5520 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5520 Immunoglobulin G (Fab fragment specific) immunological test system....

  4. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) immunological test system. 866.5520 Section 866.5520 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5520 Immunoglobulin G (Fab fragment specific) immunological test system....

  5. Development of fragment-based n-FABS NMR screening applied to the membrane enzyme FAAH.

    PubMed

    Lambruschini, Chiara; Veronesi, Marina; Romeo, Elisa; Garau, Gianpiero; Bandiera, Tiziano; Piomelli, Daniele; Scarpelli, Rita; Dalvit, Claudio

    2013-09-02

    Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹⁹F NMR spectroscopic methods, n-fluorine atoms for biochemical screening (n-FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n-FABS to the discovery of novel fragment hits, targeting the membrane-bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP-FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n-FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC₅₀) in the low mM-μM range. To the best of our knowledge, these results represent the first application of a ¹⁹F NMR fragment-based functional assay to a membrane protein.

  6. Optimization of IGF-1R SPECT/CT imaging using 111In-labeled F(ab')2 and Fab fragments of the monoclonal antibody R1507.

    PubMed

    Heskamp, Sandra; van Laarhoven, Hanneke W M; Molkenboer-Kuenen, Janneke D M; Bouwman, Wilbert H; van der Graaf, Winette T A; Oyen, Wim J G; Boerman, Otto C

    2012-08-06

    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-1R antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal antibody directed against IGF-1R. However, antibodies clear slowly from the circulation, resulting in low tumor-to-background ratios early after injection. Therefore, we aimed to accelerate targeting of IGF-1R using radiolabeled R1507 F(ab')2 and Fab fragments. In vitro, immunoreactivity, binding affinity and internalization of R1507 IgG, F(ab')2 and Fab were determined using the triple negative IGF-1R-expressing breast cancer cell line SUM149. In vivo, pharmacokinetics of (111)In-labeled R1507 IgG, F(ab')2 and Fab were studied in mice bearing subcutaneous SUM149 xenografts. SPECT/CT images were acquired and the biodistribution was measured ex vivo. The in vitro binding characteristics of radiolabeled R1507 IgG and F(ab')2 were comparable, whereas the affinity of Fab fragments was significantly lower (Kd: 0.6 nM, 0.7 nM and 3.0 nM for R1507 IgG, F(ab')2 and Fab, respectively). Biodistribution studies showed that the maximum tumor uptake of (111)In-R1507 IgG, F(ab')2 and Fab was 31.8% ID/g (72 h p.i.), 10.0% ID/g (6 h p.i.), and 1.8% ID/g (1 h p.i.), respectively. However, maximal tumor-to-blood ratios for F(ab')2 (24 h p.i.: 7.5) were more than twice as high as those obtained with R1507 (72 h p.i.: 2.8) and Fab (6 h p.i.: 2.8). Injection of an excess of unlabeled R1507 significantly reduced tumor uptake, suggesting that the uptake of R1507 IgG and F(ab')2 was specific for IGF-1R, while the major fraction of the tumor uptake of Fab was nonspecific. IGF-1R-expressing xenografts were visualized with (111)In-F(ab')2 SPECT/CT as early as 6 h p.i., while with R1507 IgG, the tumor could be visualized after 24 h. No specific targeting was observed with (111)In-Fab. (111)In

  7. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    SciTech Connect

    Page, R.L.; Garg, P.K.; Gard, S. ||

    1994-09-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the {sup 18}F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4{prime}-({sup 18}F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T{sub 1/2{beta}} = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of {sup 18}F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10{sup -3}% injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of {sup 18}F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs.

  8. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    PubMed Central

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region. PMID:25692880

  9. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    PubMed

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  10. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    PubMed

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-02

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  11. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    PubMed

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-08

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  12. Immunogenicity of heterologous Fc and Fab immunoglobulin fragments in rabbits, guinea-pigs and rats

    PubMed Central

    Binaghi, R. A.; Oriol, R.; Boussac-Aron, Yolande

    1967-01-01

    Rabbits, guinea-pigs and rats were immunized with various heterologous 7S and 19S immunoglobulins from each other and man, and the antisera obtained were studied by immunoelectrophoresis. Rabbits produced antibodies against the Fc and the Fab fragments of the immunoglobulin injected, while guinea-pigs and rats only produced anti-Fc antibodies. The fact that guinea-pigs and rats only respond to the specific determinants of each class of immunoglobulin provides a simple method for the preparation of class-specific antisera. ImagesFIG. 1FIG. 2 PMID:6027784

  13. Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

    PubMed

    Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

    2017-01-01

    Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

  14. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

    PubMed Central

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions. PMID:25484039

  15. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

    PubMed

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

  16. Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

    PubMed

    Manjappa, Arehalli S; Goel, Peeyush N; Gude, Rajiv P; Ramachandra Murthy, Rayasa S

    2014-09-01

    Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

  17. Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

    PubMed

    Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

    2017-01-01

    Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.

  18. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    PubMed

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  19. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    PubMed

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  20. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  1. Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine beta-lactoglobulin allergen.

    PubMed

    Niemi, Merja; Jänis, Janne; Jylhä, Sirpa; Kallio, Johanna M; Hakulinen, Nina; Laukkanen, Marja-Leena; Takkinen, Kristiina; Rouvinen, Juha

    2008-01-01

    A D1 Fab fragment containing the allergen-binding variable domains of the IgE antibody was characterized by ESI FT-ICR mass spectrometry and crystallized with bovine beta-lactoglobulin (BLG) using the hanging-drop vapour-diffusion method at 293 K. X-ray data suitable for structure determination were collected to 2.8 A resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.0, b = 100.6, c = 168.1 A. The three-dimensional structure of the D1 Fab fragment-BLG complex will provide the first insight into IgE antibody-allergen interactions at the molecular level.

  2. Structure of an anti-Lewis X Fab fragment in complex with its Lewis X antigen.

    PubMed

    van Roon, Anne-Marie M; Pannu, Navraj S; de Vrind, Johannes P M; van der Marel, Gijs A; van Boom, Jacques H; Hokke, Cornelis H; Deelder, André M; Abrahams, Jan Pieter

    2004-07-01

    The Lewis X trisaccharide is pivotal in mediating specific cell-cell interactions. Monoclonal antibody 291-2G3-A, which was generated from mice infected with schistosomes, has been shown to recognize the Lewis X trisaccharide. Here we describe the structure of the Fab fragment of 291-2G3-A, with Lewis X, to 1.8 A resolution. The crystallographic analysis revealed that the antigen binding site is a rather shallow binding pocket, and residues from all six complementary determining regions of the antibody contact all sugar residues. The high specificity of the binding pocket does not result in high affinity; the K(D) determined by isothermal calorimetry is 11 microM. However, this affinity is in the same range as for other sugar-antibody complexes. The detailed understanding of the antibody-Lewis X interaction revealed by the crystal structure may be helpful in the design of better diagnostic tools for schistosomiasis and for studying Lewis X-mediated cell-cell interactions by antibody interference.

  3. Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin.

    PubMed

    Song, Hyung-Nam; Kim, Dong-Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se-Hwan; Woo, Eui-Jeon

    2015-01-01

    Myoglobin is an early biomarker for acute myocardial infarction. Recently, we isolated the antibody IgG-Myo2-7ds, which exhibits unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid dissociation kinetics are thought to be premature IgG forms that are produced during the early stage of in vivo immunization. In the present study, we identified the epitope region of the IgG-Myo2-7ds antibody to be the C-terminal region of myoglobin, which corresponds to 144-154 aa. The Fab fragment was directly purified by papain cleavage and protein G affinity chromatography and demonstrated kinetics of an association constant of 4.02 × 10(4) M(-1) s(-1) and a dissociation constant of 2.28 × 10(-2) s(-1), which retained the unique reaction kinetics of intact IgG-Myo2-7ds antibodies. Because a rapid dissociation antibody can be utilized for antibody recycling, the results from this study would provide a platform for the development of antibody engineering in potential diagnostic areas such as a continuous monitoring system for heart disease.

  4. Topical ocular treatment with monoclonal antibody Fab fragments targeting Japanese cedar pollen Cry j 1 inhibits Japanese cedar pollen-induced allergic conjunctivitis in mice.

    PubMed

    Mizutani, Nobuaki; Nabe, Takeshi; Yoshino, Shin

    2017-03-05

    Fab fragments (Fabs) of antibodies having the ability only to bind to specific allergens lack effector functions due to the absence of the Fc portion. In the present study, we examined whether IgG1 monoclonal antibody (mAb) Fabs targeting Japanese cedar pollen (JCP) Cry j 1 were able to regulate JCP-induced allergic conjunctivitis in mice. BALB/c mice actively sensitized with JCP were repeatedly challenged by topical administration of JCP eye drops. Fabs prepared by the digestion of anti-JCP IgG1 mAbs (P1-3 and P1-8) with papain were applied to the eye 15min before the JCP challenges followed by measurement of the clinical conjunctivitis score. In the in vitro experiments, P1-3 and P1-8 showed specific binding to JCP Cry j 1. Furthermore, intact P1-3 binding to Cry j 1 was inhibited by P1-3 Fabs, but not P1-8 Fabs; additionally, P1-8 Fabs, but not P1-3 Fabs, suppressed the intact P1-8 binding, suggesting that the epitopes of Cry j 1 recognized by P1-3 and P1-8 were different. Topical ocular treatment with P1-3 Fabs or P1-8 Fabs was followed by marked suppression of JCP-induced conjunctivitis (P<0.01). In histological evaluation, P1-8 Fabs showed a reduction in eosinophil infiltration in the conjunctiva (P<0.01). These results demonstrated that topical ocular treatment with IgG1 mAb Fabs to Cry j 1 was effective in suppressing JCP-induced allergic conjunctivitis in mice. Furthermore, it suggests the possibility that some epitopes recognized by Fabs could be used as a tool to regulate allergic conjunctivitis.

  5. Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

    PubMed

    Delgado, C; Pedley, R B; Herraez, A; Boden, R; Boden, J A; Keep, P A; Chester, K A; Fisher, D; Begent, R H; Francis, G E

    1996-01-01

    Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.

  6. Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

    PubMed Central

    Delgado, C.; Pedley, R. B.; Herraez, A.; Boden, R.; Boden, J. A.; Keep, P. A.; Chester, K. A.; Fisher, D.; Begent, R. H.; Francis, G. E.

    1996-01-01

    Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion. PMID:8546903

  7. Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis.

    PubMed

    Dragosits, Martin; Frascotti, Gianni; Bernard-Granger, Lise; Vázquez, Felícitas; Giuliani, Maria; Baumann, Kristin; Rodríguez-Carmona, Escarlata; Tokkanen, Jaana; Parrilli, Ermenegilda; Wiebe, Marilyn G; Kunert, Renate; Maurer, Michael; Gasser, Brigitte; Sauer, Michael; Branduardi, Paola; Pakula, Tiina; Saloheimo, Markku; Penttilä, Merja; Ferrer, Pau; Luisa Tutino, Maria; Villaverde, Antonio; Porro, Danilo; Mattanovich, Diethard

    2011-01-01

    Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.

  8. Qualification of a free ligand assay in the presence of anti-ligand antibody Fab fragments.

    PubMed

    Hansen, Ryan J; Brown, Robin M; Lu, Jirong; Wroblewski, Victor J

    2013-01-01

    The aim of this work was to develop and characterize an ELISA to measure free ligand concentrations in rat serum in the presence of a Fab to the same ligand. A variety of experiments were conducted to understand optimal assay conditions and to verify that only free ligand was detected. The parameters explored included sample incubation time on plate, the initial concentrations of Fab and ligand, and the pre-incubation time required for the Fab-ligand complex concentrations to reach equilibrium. We found the optimal experimental conditions to include a 10-minute on-plate incubation of ligand-containing samples, with a 24-hour pre-incubation time for test samples of Fab and ligand to reach equilibrium. An alternative approach, involving removal of Fab-ligand complexes from the solution prior to measuring concentrations of the ligand, was also used to verify that the assay only measured free ligand. Rats were dosed subcutaneously with Fab and the assay was used to demonstrate dose-dependent suppression of endogenous free ligand levels in vivo.

  9. Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab' fragments.

    PubMed Central

    Casey, J. L.; King, D. J.; Chaplin, L. C.; Haines, A. M.; Pedley, R. B.; Mountain, A.; Yarranton, G. T.; Begent, R. H.

    1996-01-01

    The monoclonal anti-CEA antibody, A5B7, has previously been administered to patients for radioimmunotherapy (RIT). Long circulation time and the formation of an immune response have limited therapeutic success in the clinic. Antibody fragments can be used to reduce the in vivo circulation time, but the best combination of fragment and radioisotope to use for therapy is far from clear. In this study we have compared the biodistribution of A5B7 IgG and F(ab')2 with chemically cross-linked divalent (DFM) and trivalent (TFM) A5B7 Fab' fragments in nude mice bearing human colorectal tumour xenografts. The cross-linkers were designed to allow site-specific labelling using yttrium 90 (90Y), a high-energy beta-emitter. We have also compared the above antibody forms conjugated to both 131I and 90Y. Both DFM and TFM were fully immunoreactive and remained intact after radiolabelling and incubation in serum at 37 degrees C for 24 h. Biodistribution results showed similar tumour uptake levels and an identical blood clearance pattern for F(ab')2 and DFM with high tumour-blood ratios generated in each case. However, unacceptably high kidney accumulation for both F(ab')2 and DFM and elevated splenic uptake of DFM labelled with 90Y was observed. Kinetic analysis of antigen binding revealed that DFM had the fastest association rate (kass = 1.6 x 10(5) Ms-1) of the antibody forms, perhaps owing to increased flexibility of the cross-linker. This advantage implies that DFM may be more suitable than F(ab')2 radiolabelled with 131I for RIT. TFM cleared from the blood significantly faster than A5B7 IgG when labelled with both 131I and 90Y, producing an improved therapeutic tumour-blood ratio. Kidney accumulation was not observed for [90Y]TFM, but a slightly higher splenic uptake was observed that may indicate reticuloendothelial system (RES) uptake. Overall, tumour uptake was higher for 90Y-labelled antibodies than for 131I-labelled antibodies. Because of the faster clearance, it should

  10. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    PubMed

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

  11. Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

    PubMed

    Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts.

  12. Neutralizing activity and protective immunity to ricin toxin conferred by B subunit (RTB)-specific Fab fragments.

    PubMed

    Yermakova, Anastasiya; Mantis, Nicholas J

    2013-09-01

    SylH3 and 24B11 are murine monoclonal antibodies directed against different epitopes on ricin toxin's binding (RTB) subunit that have been shown to passively protect mice against ricin challenge. Here we report that Fab fragments of SylH3 and 24B11 neutralize ricin in a cell based assay, and in a mouse challenge model as effectively as their respective full length parental IgGs. These data demonstrate that immunity to ricin can occur independent of Fc-mediated clearance.

  13. The discovery, engineering and characterisation of a highly potent anti-human IL-13 fab fragment designed for administration by inhalation.

    PubMed

    Lightwood, Daniel; O'Dowd, Victoria; Carrington, Bruce; Veverka, Vaclav; Carr, Mark D; Tservistas, Markus; Henry, Alistair J; Smith, Bryan; Tyson, Kerry; Lamour, Sabrina; Bracher, Marguerite; Sarkar, Kaushik; Turner, Alison; Lawson, Alastair D; Bourne, Tim; Gozzard, Neil; Palframan, Roger

    2013-02-08

    We describe the discovery, engineering and characterisation of a highly potent anti-human interleukin (IL)-13 Fab fragment designed for administration by inhalation. The lead candidate molecule was generated via a novel antibody discovery process, and the selected IgG variable region genes were successfully humanised and reformatted as a human IgG γ1 Fab fragment. Evaluation of the biophysical properties of a selection of humanised Fab fragments in a number of assays allowed us to select the molecule with the optimal stability profile. The resulting lead candidate, CA652.g2 Fab, was shown to have comparable activity to the parental IgG molecule in a range of in vitro assays and was highly stable. Following nebulisation using a mesh nebuliser, CA652.g2 Fab retained full binding affinity, functional neutralisation potency and structural integrity. Epitope mapping using solution nuclear magnetic resonance confirmed that the antibody bound to the region of human IL-13 implicated in the interaction with IL-13Rα1 and IL-13Rα2. The work described here resulted in the discovery and design of CA652.g2 human γ1 Fab, a highly stable and potent anti-IL-13 molecule suitable for delivery via inhalation.

  14. Immunogenicity and kinetics of distribution and elimination of sheep digoxin-specific IgG and Fab fragments in the rabbit and baboon.

    PubMed Central

    Smith, T W; Lloyd, B L; Spicer, N; Haber, E

    1979-01-01

    To evaluate the relative merits of purified IgG and Fab preparations of defined specificity for potential clinical use, immunogenicity studies were carried out in baboon and rabbit experimental models. Distribution and elimination kinetics of purified sheep digoxin-specific IgG and Fab fragments were also studied following intravenous administration to baboons. Serial plasma and urine Fab concentrations were determined from trichloroacetic acid-precipitable 125I counts from pre-labelled preparations and also by measurement of the antibody's functional 3H-digoxin binding capacity. Results were compared with data obtained from IgG by 3H-digoxin binding. Kinetic data analysed by computer-fitted functions demonstrated that plasma Fab disappearance was best described by a tri-exponential function, whereas a bi-exponential function best described the IgG data. Initial distribution half-life (t 1/2) of Fab (0.28-0.32 hr) was considerably shorter than that of IgG (4.0 hr) and contributed a greater proportion of the total fall in plasma level over 24 hr. Fab elimination t 1/2 (9-13 hr) was also shorter than IgG (61 hr), but appreciably longer than earlier estimates in rabbits, guinea-pigs, rats and mice. The total volume of distribution of Fab was 8.7 times greater than that of IgG measured by the same method. Over the first 24 hr after administration 30-45% of administered Fab was recoverable in active form in urine, while 93% of total administered 125I counts from 125I-Fab preparations (bound and free) could be recovered. Less than 1% of administered IgG binding activity was recovered in urine during the initial 24 hr. The relative immunogenicities of sheep digoxin-specific IgG and Fab fragments were studied in six baboons. Both IgG and Fab elicited prompt immune responses when injected intramuscularly with Freund's complete adjuvant. Intravenous injection of soluble sheep IgG resulted in a prompt immune response in one baboon while repeated injections caused only a late

  15. Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach.

    PubMed

    Schlapschy, Martin; Gruber, Helga; Gresch, Oliver; Schäfer, Claudia; Renner, Christoph; Pfreundschuh, Michael; Skerra, Arne

    2004-12-01

    CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an alphaCD30 x alphaCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.

  16. PEGylation prolongs the pulmonary retention of an anti-IL-17A Fab' antibody fragment after pulmonary delivery in three different species.

    PubMed

    Freches, Danielle; Patil, Harshad P; Machado Franco, Maria; Uyttenhove, Catherine; Heywood, Sam; Vanbever, Rita

    2017-04-15

    The PEGylation of antibody fragments has been shown to greatly prolong their residence time in the lungs in mice. The purpose of this research was to confirm the effect of PEGylation in higher animal species, that is, the rat and the rabbit. An anti-IL-17A Fab' antibody fragment was conjugated to a two-armed 40kDa polyethylene glycol (PEG) via site-selective thiol PEGylation. PEGylation did not significantly alter the binding activity of the Fab' fragment but it largely enhanced its inhibitory potency. PEGylation increased the residence time of the Fab' in the lungs of mice, rats and rabbits. Following intratracheal administration, the unconjugated Fab' was cleared from the lungs within 24h while large quantities of the PEGylated Fab' remained present up to 48h. No significant differences in clearance were noted between the three animal species although there was a tendency of longer residence time in higher species. PEGylation represents a promising approach to sustain the presence of antibody fragments in the lungs and to enhance their therapeutic efficacy in respiratory diseases.

  17. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    PubMed

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

  18. Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.

    PubMed

    Schlapschy, Martin; Fogarasi, Marton; Gruber, Helga; Gresch, Oliver; Schäfer, Claudia; Aguib, Yasmine; Skerra, Arne

    2009-03-01

    An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

  19. Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 Bound at the Peripheral Site

    PubMed Central

    Bourne, Yves; Renault, Ludovic; Marchot, Pascale

    2015-01-01

    The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. PMID:25411244

  20. Selectivity evaluation and separation of human immunoglobulin G, Fab and Fc fragments with mixed-mode resins.

    PubMed

    Luo, Ying-Di; Zhang, Qi-Lei; Yuan, Xiao-Ming; Shi, Wei; Yao, Shan-Jing; Lin, Dong-Qiang

    2017-01-01

    Adsorption selectivity is critical important for mixed-mode chromatography with specially-designed ligands. Human immunoglobulin G (hIgG), Fc and Fab fragments were used in the present work to evaluate adsorption behavior and binding selectivity of four mixed-mode resins with the ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. The resins showed an obvious pH-dependent adsorption behavior. High adsorption capacities were found at neutral pH for hIgG, Fc and Fab, and almost no adsorption happened under acidic conditions. An adsorption selectivity index was proposed to evaluate separation efficiency. High specificity of hIgG/Fc was found at pH 8.9 for MEP resin, and for W-ABI resin at pH 8.0 and 8.9. In addition, isothermal titration calorimetry was used to evaluate ligand-protein interactions. Finally, the separation of hIgG and Fc (1:1) was optimized with mixed-mode resins, and the best separation performance was obtained with W-ABI-based resin. Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4% and recovery of 98.8%, while elution at pH 3.6 provided hIgG with purity of 99.7% and recovery of 86.5%.

  1. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

    PubMed Central

    Li, Tong; Tracka, Malgorzata B.; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J.; Livesay, Dennis R.

    2015-01-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation. PMID:26132144

  2. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    PubMed

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2015-07-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  3. Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding.

    PubMed

    He, Kuo; Du, Xinjun; Sheng, Wei; Zhou, Xiaonan; Wang, Junping; Wang, Shuo

    2016-03-30

    The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods.

  4. The Fab Fragment of a Human Anti-Siglec-9 Monoclonal Antibody Suppresses LPS-Induced Inflammatory Responses in Human Macrophages

    PubMed Central

    Chu, Sasa; Zhu, Xuhui; You, Na; Zhang, Wei; Zheng, Feng; Cai, Binggang; Zhou, Tingting; Wang, Yiwen; Sun, Qiannan; Yang, Zhiguo; Zhang, Xin; Wang, Changjun; Nie, Shinan; Zhu, Jin; Wang, Maorong

    2016-01-01

    Sepsis is a major cause of death for hospitalized patients and is characterized by massive overreaction of immune responses to invading pathogens which is mediated by cytokines. For decades, there has been no effective treatment for sepsis. Sialic acid-binding, Ig-like lectin-9 (Siglec-9), is an immunomodulatory receptor expressed primarily on hematopoietic cells which is involved in various aspects of inflammatory responses and is a potential target for treatment of sepsis. The aim of the present study was to develop a human anti-Siglec-9 Fab fragment, which was named hS9-Fab03 and investigate its immune activity in human macrophages. We began by constructing the hS9-Fab03 prokaryotic expression vector from human antibody library and phage display. Then, we utilized a multitude of assays, including SDS-PAGE, Western blotting, ELISA, affinity, and kinetics assay to evaluate the binding affinity and specificity of hS9-Fab03. Results demonstrated that hS9-Fab03 specifically bind to Siglec-9 antigen with high affinity, and pretreatment with hS9-Fab03 could attenuate lipopolysaccharide (LPS)-induced TNF-α, IL-6, IL-1β, IL-8, and IFN-β production in human PBMC-derived macrophages, but slightly increased IL-10 production in an early time point. We also observed similar results in human THP-1-differentiated macrophages. Collectively, we prepared the hS9-Fab03 with efficient activity for blocking LPS-induced pro-inflammatory cytokines production in human macrophages. These results indicated that ligation of Siglec-9 with hS9-Fab03 might be a novel anti-inflammatory therapeutic strategy for sepsis. PMID:28082984

  5. Allergen-specific regulation of allergic rhinitis in mice by intranasal exposure to IgG1 monoclonal antibody Fab fragments against pathogenic allergen.

    PubMed

    Matsuoka, Daiko; Mizutani, Nobuaki; Sae-Wong, Chutha; Yoshino, Shin

    2014-09-01

    Fab fragments (Fabs) have the ability to bind to specific antigens but lack the Fc portion for binding to receptors on immune and inflammatory cells that play a critical role in allergic diseases. In the present study, we investigated whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) inhibited allergic rhinitis in mice. BALB/c mice sensitized by intraperitoneal injections of ovalbumin (OVA) plus alum on days 0 and 14 were intranasally challenged with OVA on days 28-30, and 35. Fabs prepared by the digestion of an anti-OVA IgG1 mAb (O1-10) with papain were also intranasally administered 15min before each OVA challenge. The results showed that treatment with O1-10 Fabs significantly suppressed the sneezing frequency, associated with decrease of OVA-specific IgE in the serum and infiltration by mast cells in the nasal mucosa seen following the fourth antigenic challenge; additionally, the level of mouse mast cell protease-1, a marker of mast cell activation, in serum was decreased. Furthermore, infiltration of eosinophils and goblet cell hyperplasia in the nasal mucosa at the fourth challenge were inhibited by treatment with O1-10 Fabs. In conclusion, these results suggest that intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb may be effective in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen.

  6. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    NASA Technical Reports Server (NTRS)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  7. Detection of pulmonary embolism with 99mTc-labeled F(ab)2 fragment of anti-P-selectin monoclonal antibody in dogs.

    PubMed

    Ji, Shundong; Fang, Wei; Zhu, Mingqing; Bai, Xia; Wang, Chen; Ruan, Changgeng

    2011-01-01

    Pulmonary embolism is a common and potentially life-threatening condition, and its correct diagnosis is highly desirable before anticoagulant therapy is initiated. However, the safe and accurate diagnosis of acute pulmonary embolism remains a challenge. Single photon emission computed tomography (SPECT) is a highly sensitive scintigraphic imaging technique. Pulmonary embolism can be detected by SPECT with (99m)Tc-labeled imaging agents that bind to components present predominantly on thromboemboli. P-selectin is an adhesion glycoprotein that is expressed in platelets and endothelial cells. P-selectin on activated platelets is a suitable biomarker of the active thrombus process. The objective of this study was to evaluate (99m)Tc-labeled F(ab)(2) fragment of anti-P-selectin monoclonal antibody SZ51, (99m)Tc-SZ51-F(ab)(2), for imaging pulmonary embolism in beagle canines. SZ51 was digested to F(ab)(2) fragment, named SZ51-F(ab)(2), and its specific binding to P-selectin on either human or canine platelets was verified by flow cytometry assay. In each dog, an 18-gauge catheter was inserted into left or right pulmonary artery, and a two-stranded spiral stainless-steel coil (20 mm) was inserted through catheter. At 30 min after coil placement, X-ray angiography was performed to document the pulmonary embolism and the locations of the coil. After intravenous injection of (99m)Tc-SZ51-F(ab)(2), experimental thrombi in dogs could be consistently visualized for 2-3 hours by SPECT. Pulmonary embolism showed higher uptake of (99m)Tc-SZ51-F(ab)(2). The present study suggests that (99m)Tc-SZ51-F(ab)(2) may be a promising agent for detecting pulmonary embolism.

  8. Single-step purification of F(ab')2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW.

    PubMed

    Morimoto, K; Inouye, K

    1992-03-01

    Hydrophobic interaction high performance liquid chromatography (HPLC) using TSKgel Phenyl-5PW was applicable to single-step purification of F(ab')2 fragments from pepsin digests of mouse monoclonal antibodies of IgG1 class. The digests were applied to the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate. F(ab')2 fragments were adsorbed onto the gel using the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 fragments was homogeneous (purity: higher than 98%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration HPLC. The recovery of the antigen binding site was 42-58%. The cycle time of the Phenyl-5PW HPLC was 45 min, and F(ab')2 of up to 2200 mg was purified in a cycle. This method could be useful especially for large scale purification of F(ab')2 fragments.

  9. Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

    PubMed

    Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

    2016-12-01

    Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

  10. Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization

    PubMed Central

    Johnson, Jennifer L.; Entzminger, Kevin C.; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P.; Morales, Ivan A.; Sheppard, Aly; Gumbart, James C.; Maynard, Jennifer A.; Lieberman, Raquel L.

    2015-01-01

    Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach. PMID:25849400

  11. Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

    PubMed

    Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

    2015-04-01

    Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

  12. Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.

    PubMed

    Banner, David W; Gsell, Bernard; Benz, Jörg; Bertschinger, Julian; Burger, Dominique; Brack, Simon; Cuppuleri, Simon; Debulpaep, Maja; Gast, Alain; Grabulovski, Dragan; Hennig, Michael; Hilpert, Hans; Huber, Walter; Kuglstatter, Andreas; Kusznir, Eric; Laeremans, Toon; Matile, Hugues; Miscenic, Christian; Rufer, Arne C; Schlatter, Daniel; Steyaert, Jan; Stihle, Martine; Thoma, Ralf; Weber, Martin; Ruf, Armin

    2013-06-01

    The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic β-cells, which leads to inactivation of the β-cell proliferating activity of Tmem27. This role of BACE2 in the control of β-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme β-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

  13. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples.

    PubMed

    Stubenrauch, Kay; Wessels, Uwe; Essig, Ulrich; Kowalewsky, Frank; Vogel, Rudolf; Heinrich, Julia

    2013-01-01

    Generic immunoassay formats in animal serum have been described for pharmacokinetic (PK) analysis of human full-length antibodies, but not of human antigen binding fragment (Fab) proteins. Here we characterize two murine monoclonal antibodies (mAb) raised against human immunoglobulin G (IgG) which bind to unique epitopes in the Fab region of human IgG. mAb M-1.7.10 is directed against the constant domain of the kappa light chain and mAb M-1.19.31 binds to the constant domain 1 (CH1) of the heavy chain. Surface plasmon resonance analysis showed that mAb M-1.7.10 does not cross-react with sera from mouse, rat, rabbit, dog, marmoset, rhesus macaque, baboon and cynomolgus monkey, but binds to human and chimpanzee serum (dissociation constant K(D) of 6.8 × 10(-12) and 3.1 × 10(-11)M, respectively). mAb M-1.19.31 shows a higher K(D) for human and chimpanzee IgG (2.0 × 10(-9)M and 5.8 × 10(-10)M, respectively), but also does not bind to serum of the other species. Therefore, mAb M-1.7.10 was used as capture and mAb M-1.19.31 as detection reagent in a generic enzyme linked immunosorbent assay (ELISA) to quantify the human anti-IGF-1R Fab in mouse serum. The generic human Fab assay showed a limit of detection of 31.5 ng/mL anti-IGF-1R Fab. Intra- and inter-assay precision was less than 12% and the accuracy range for all controls was within ±20% of the target concentration. The generic human Fab ELISA was applied to determine serum levels of human anti-IGF-1R Fab after intravenous (iv) administration of 10mg/kg to mice. The resulting concentration-time profile was nearly identical to that obtained by analysis with a validated specific ELISA for anti-IGF-1R Fab. The mean relative concentration of anti-IGF-1R Fab analyzed by the generic assay was 82-118% of that of the specific assay. This equivalence was confirmed in a cynomolgus monkey study with the full length human mAb anti-TROP-2 IgG. Both specific ELISAs used mAb M-1.7.10 as detection reagent and their targets for

  14. Site specific discrete PEGylation of (124)I-labeled mCC49 Fab' fragments improves tumor MicroPET/CT imaging in mice.

    PubMed

    Ding, Haiming; Carlton, Michelle M; Povoski, Stephen P; Milum, Keisha; Kumar, Krishan; Kothandaraman, Shankaran; Hinkle, George H; Colcher, David; Brody, Rich; Davis, Paul D; Pokora, Alex; Phelps, Mitchell; Martin, Edward W; Tweedle, Michael F

    2013-11-20

    The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab'-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.

  15. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A. N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  16. Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity.

    PubMed Central

    Zalutsky, M R; Garg, P K; Friedman, H S; Bigner, D D

    1989-01-01

    alpha-Particles such as those emitted by 211At may be advantageous for radioimmunotherapy since they are radiation of high linear energy transfer, depositing high energy over a short distance. Here we describe a strategy for labeling monoclonal antibodies and F(ab')2 fragments with 211At by means of the bifunctional reagent N-succinimidyl 3-(trimethylstannyl)benzoate. An intact antibody, 81C6, and the F(ab')2 fragment of Me1-14 (both reactive with human gliomas) were labeled with 211At in high yield and with a specific activity of up to 4 mCi/mg in a time frame compatible with the 7.2-hr half-life of 211At. Quantitative in vivo binding assays demonstrated that radioastatination was accomplished with maintenance of high specific binding and affinity. Comparison of the biodistribution of 211At-labeled Me1-14 F(ab')2 to that of a nonspecific antibody fragment labeled with 211At and 131I in athymic mice bearing D-54 MG human glioma xenografts demonstrated selective and specific targeting of 211At-labeled antibody in this human tumor model. PMID:2476813

  17. First successful curative use of digoxin-specific Fab antibody fragments in a life-threatening coconut crab (Birgus latro L.) poisoning.

    PubMed

    Maillaud, C; Barguil, Y; Mikulski, M; Cheze, M; Pivert, C; Deveaux, M; Lapostolle, F

    2012-11-01

    We wish to report the first curative use of digoxin-specific Fab antibody fragments in a coconut crab Birgus latro L. poisoning in New Caledonia. The female patient, aged sixty-three with a previous history of cardiovascular and metabolic dysfunctions, showed marked first-degree atrio-ventricular block and several atrial pauses, and was given 760 mg of digoxin-specific Fab antibody fragments. Shortly after the perfusion her electrocardiogram returned to close to normal with only slight first-degree atrio-ventricular block and no more atrial pauses. Neriifolin LC-MS/MS tests performed on the patient's serum and urine samples confirmed cardenolide poisoning. Another, younger patient, with high neriifolin levels in her serum and urine samples only experienced gastro-intestinal symptoms and was discharged without specific treatment. The consumption of coconut crab in New Caledonia should be avoided even though the first of the two cases reported suggests that digoxin-specific Fab antibody fragments can be effective in the treatment of life-threatening poisoning caused by the ingestion of this crustacean.

  18. Antitumor effects of ricin A chain immunotoxins prepared from intact antibodies and Fab' fragments on solid human Hodgkin's disease tumors in mice.

    PubMed

    Engert, A; Martin, G; Pfreundschuh, M; Amlot, P; Hsu, S M; Diehl, V; Thorpe, P

    1990-05-15

    Three monoclonal antibodies which strongly bind to Hodgkin and Reed-Sternberg cells and two corresponding Fab' fragments were linked to deglycosylated ricin A chain (dg A) to evaluate their potential as immunotoxins for the treatment of Hodgkin's disease. Two of the antibodies, Ber-H2 and HRS-3, were shown to bind to the same epitope on the CD30 antigen, whereas the third antibody, IRac, bound to a different antigen. None of the antibodies significantly cross-reacted with normal human tissues as judged by indirect immunofluorescence and immunoperoxidase analyses on frozen sections from 28 normal tissues. All three antibodies formed potent and specific immunotoxins. They inhibited protein synthesis of the L540 Hodgkin's disease cell line in vitro by 50% at concentrations of 1 x 10(-11) M for IRac.dgA, 9 x 10(-11) M for HRS-3.dgA, and 2 x 10(-10) M for Ber-H2.dgA. HRS-3 Fab' and IRac Fab' immunotoxins were 7.8- and 60-fold less cytotoxic, respectively, than their intact counterparts in vitro. In vivo, a single i.v. injection of a dose of Ber-H2.dgA, HRS-3.dgA, or IRac.dgA corresponding to 40% of the LD50 induced lasting complete remissions in 38, 44, and 50%, respectively, of mice with solid s.c. L540 tumors of 60 to 80 mm3 size (0.5-cm diameter). At equivalent dosage (40% of the LD50), the HRS-3 Fab'.dgA and the IRac Fab'.dgA both induced lasting complete remissions in 25% of the mice, although the HRS-3 Fab'.dgA was significantly superior to IRac Fab'.dgA at retarding tumor growth in the remaining animals. The effectiveness of the immunotoxins depended on the size of the tumor at the time of injection, since IRac.dgA treatment induced complete remissions in 100% of mice with small tumors (10 to 20 mm3, approximately 0.3 cm in diameter) but only 13% of mice with larger tumors of 400 to 600 mm3 (approximately 1 cm in diameter). Tumors which regrew after IRac.dgA treatment mainly consisted of antigen-deficient mutants having reduced sensitivity to IRac.dgA but normal

  19. Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2017-03-01

    We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

  20. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    PubMed

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability).

  1. Single-step purification of F(ab')2 mu fragments of mouse monoclonal antibodies (immunoglobulins M) by hydrophobic interaction high-performance liquid chromatography using TSKgel ether-5PW.

    PubMed

    Inouye, K; Morimoto, K

    1993-02-01

    A procedure is described for preparation and single-step purification of F(ab')2 fragments, herein designated as F(ab')2 mu' from mouse monoclonal antibodies of the IgM class. Hydrophobic interaction high-performance liquid chromatography (HPLC) using TSKgel Ether-5PW was well applicable to the purification. The IgM was digested with pepsin at the pepsin-to-IgM ratio of 1:200 (w/w) in 100 mM citrate buffer (pH 4.2) at 37 degrees C for 2 h. The digests were applied to the gel equilibrated with the buffer containing 1 M ammonium sulfate. F(ab')2 mu fragments were adsorbed onto the gel with the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 mu fragments was homogeneous (purity higher than 97%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration HPLC. The recovery of the antigen-binding site was 55-72%. The cycle time of the Ether-5PW HPLC was 40 min, and up to 98 mg F(ab')2 mu fragments. The molecular mass of F(ab')2 mu was estimated to be 144-146 kDa. In comparison with IgM, F(ab')2 mu lost entirely the complement C1q binding activity, and the sugar content was greatly reduced. The binding of IgM with non-specific proteins turned to be negligible, when IgM was converted to F(ab')2 mu, suggesting that the fragments are useful for immunological application.

  2. A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG-binding domain of Protein G.

    PubMed

    El Khoury, Graziella; Lowe, Christopher R

    2013-04-01

    This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab-binding domain of Streptococcal Protein G (SpG-domain III). The ligand (A2C7I1) was synthesized by the four-component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer-aided analysis suggests a putative binding site on the CH 1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization-mass spectrometry and (1) H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml(-1) and a dissociation constant of 5.34 × 10(-5)  M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively.

  3. Matching the decay half-life with the biological half-life: ImmunoPET imaging with 44 Sc-labeled Cetuximab Fab fragment

    DOE PAGES

    Chakravarty, Rubel; Goel, Shreya; Valdovinos, Hector F.; ...

    2014-11-11

    Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N'',N''-pentaacetic acid (CHX-A"-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice wasmore » investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ~12% ID/g at 4 h postinjection) of 44Sc–CHX-A"-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. As a result, this successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-''-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.« less

  4. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the A[beta] peptides associated with Alzheimer's disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A.N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-28

    The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid {beta} peptide (A{beta}) associated with Alzheimer's disease. This region of A{beta} has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Az{beta} peptides A{beta}{sub 1-16} and A{beta}{sub 1-28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 {angstrom} resolution. The complexes of WO2 Fab with either A{beta}{sub 1-16} or A{beta}{sub 1-28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 {angstrom} resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble A{beta}{sub 1-42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 {angstrom} resolution.

  5. Matching the decay half-life with the biological half-life: ImmunoPET imaging with 44 Sc-labeled Cetuximab Fab fragment

    SciTech Connect

    Chakravarty, Rubel; Goel, Shreya; Valdovinos, Hector F.; Hernandez, Reinier; Hong, Hao; Nickles, Robert J.; Cai, Weibo

    2014-11-11

    Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N'',N''-pentaacetic acid (CHX-A"-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ~12% ID/g at 4 h postinjection) of 44Sc–CHX-A"-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. As a result, this successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-''-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.

  6. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    SciTech Connect

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  7. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

    PubMed Central

    Roben, P; Moore, J P; Thali, M; Sodroski, J; Barbas, C F; Burton, D R

    1994-01-01

    Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently. Images PMID:7518527

  8. Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

    PubMed

    Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

    2016-10-01

    We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

  9. Structural insights into steroid hormone binding: the crystal structure of a recombinant anti-testosterone Fab fragment in free and testosterone-bound forms.

    PubMed

    Valjakka, Jarkko; Takkinenz, Kristiina; Teerinen, Tuija; Söderlund, Hans; Rouvinen, Juha

    2002-02-08

    The monoclonal anti-testosterone antibody (3-C(4)F(5)) has a relatively high affinity (3 x 10(8) m(-1)) with an overall good specificity profile. However, the earlier characterized binding properties have shown that both the affinity and specificity of this antibody must be improved if it is intended for use in clinical immunoassays. In this paper, the crystal structures of the recombinant anti-testosterone (3-C(4)F(5)) Fab fragment have been determined in the testosterone-bound and free form at resolutions of 2.60 and 2.72 A, respectively. The high affinity binding of the (3-C(4)F(5)) Fab is mainly determined by shape complementarity between the protein and testosterone. Only one direct hydrogen bond is formed between the hydroxyl group of the testosterone D-ring and the main-chain oxygen of Gly100(J)H. The testosterone is deeply bound in a hydrophobic pocket, and the close shape complementarity is mainly formed by the third complementarity-determining regions (CDR) of the heavy and light chain. Comparison of the bound structure with the free structure indicates conformational changes in the protein upon testosterone binding. The conformational changes of the side chains of two residues Glu95H and Tyr99H in the CDR-H3 are particularly essential for the binding. Interesting similarities in the binding of different steroids were also observed upon comparison of the available structures of anti-steroid antibodies.

  10. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  11. Astatine-211 labeling of an anti-melanoma antibody and its Fab fragment using N-succinimidyl para[{sup 211} At]astatobenzoate : comparisons In Vivo with the para-[{sup 125}1]iodobenzoyl conjugate.

    SciTech Connect

    Hadley, S. W.; Wilbur, D. S.; Gray, M. A.; Atcher, R. W.; Chemistry; NeoRx Corp.; Univ. of Washington Medical Center

    1991-01-01

    Astatine-211 labeling of an anti-melanoma antibody, NR-ML-05, and its Fab fragment using N-succinimidyl para[{sup 211} At]astatobenzoate has been described. Preparation of the astatinated intermediate 2a was accomplished by distilling astatine-211 from an irradiated bismuth target directly into a reaction mixture containing an organometallic compound, N-succinimidyl p-(tri-n-butylstannyl)benzoate (1), and an oxidant, N-chlorosuccinimide, in 5% HOAc/MeOH. Trapping of distilled astatine as 2a was found to be efficient, resulting in 70-90% yields based on the amount of astatine-211 which ranged from 20% to 75%. Conjugation of 2a to NR-ML-05 and its Fab fragment was accomplished in 40-60% yields. The [{sup 211}At]astatobenzoyl-conjugated antibodies were found to be stable in vitro when challenged by strong denaturants and nucleophilic reagents. Coinjected dual-labeled studies of the 2a astatinated antibodies and the same antibodies labeled with N-succinimidyl p-[{sup 125}I]iodobenzoate (2b) in athymic mice bearing the human tumor xenograft A375 Met/Mix demonstrated that both radiolabeled antibodies had equivalent tumor localization. Data from the dual-labeled biodistribution of the intact antibody suggests that the astatine is stably attached. Data from the dual-labeled Fab fragment suggests that a portion of the astatine label is released as astatide, either from the astatinated Fab or from a catabolite.

  12. F(ab')2 fragment of a gp41 NHR-trimer-induced IgM monoclonal antibody neutralizes HIV-1 infection and blocks viral fusion by targeting the conserved gp41 pocket.

    PubMed

    Lu, Lu; Wei, Meili; Chen, Yanxia; Xiong, Weiliang; Yu, Fei; Qi, Zhi; Jiang, Shibo; Pan, Chungen

    2013-11-01

    Using a recombinant protein N46FdFc that mimics the HIV-1 gp41 N-helix trimer to immunize mice, we identified the first IgM monoclonal antibody 18D3 that specifically bound to the conserved gp41 pocket. Its F(ab')2 fragment potently inhibited HIV-1 Env-mediated cell-cell fusion and neutralized infection by laboratory-adapted and primary HIV-1 isolates with different subtypes and tropism, including the T20-resistant variants. This F(ab')2 fragment can be used to develop a bispecific broad neutralizing monoclonal antibody or HIV-1 inactivator as a novel immunotherapeutic for treatment and prevention of HIV-1 infection.

  13. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

    SciTech Connect

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  14. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment.

    PubMed

    McKinstry, William J; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T; Martin, Thomas J; Parker, Michael W

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  15. Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

    NASA Astrophysics Data System (ADS)

    Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

    2001-09-01

    To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

  16. Choice of labeling and cell line influences interactions between the Fab fragment AbD15179 and its target antigen CD44v6.

    PubMed

    Stenberg, Jonas; Spiegelberg, Diana; Karlsson, Hampus; Nestor, Marika

    2014-02-01

    Medical imaging by use of immunotargeting generally relies on a labeled molecule binding to a specific target on the cell surface. It is important to utilize both cell-based and time-resolved binding assays in order to understand the properties of such molecular interactions in a relevant setting. In this report we describe the detailed characterization of the interaction properties for AbD15179, a promising CD44v6-targeting antibody fragment for radio-immunotargeting. Influence of labeling and cell-line model on the protein interaction kinetics was assessed using three different labeling approaches ((111)In, (125)I and FITC) on three different squamous carcinoma cell lines. Interactions were measured using time-resolved assays on living cells, and further analyzed with Interaction Map®. Results demonstrated a general biphasic appearance of a high- and a low-affinity binding event in all cases. The relative contribution from these two interactions differed between conjugates. For (125)I-Fab, the population of low-affinity binders could be significantly increased by extending the chloramine T exposure during labeling, whereas the (111)In-labeling predominantly resulted in a high-affinity interaction. Interactions were also shown to be cell line dependent, with e.g. SCC-25 cells generally mediating a faster dissociation of conjugates compared to the other cell lines. In conclusion, we report both cell line dependent and labeling associated variations in interaction kinetics for AbD15179 binding to CD44v6. This has implications for cell-based kinetic assays and applications based on labeled conjugates in general, as well as in a clinical setting, where each individual tumor may create different kinetic profiles for the same conjugate.

  17. In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study

    PubMed Central

    2014-01-01

    Background Patients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours. Methods The fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup. Results Both species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys. Conclusions We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies. PMID:24598405

  18. Iodine-131-labeled MAb F(ab')2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice

    SciTech Connect

    Buchegger, F.; Pelegrin, A.; Delaloye, B.; Bischof-Delaloye, A.; Mach, J.P. )

    1990-06-01

    During one week, beginning 18 days after transplantation, nude mice bearing human colon carcinoma ranging from 115 to 943 mm3 (mean 335 mm3) were treated by repeated intravenous injections of either iodine-131-({sup 131}I) labeled intact antibodies or {sup 131}I-labeled corresponding F(ab')2 fragments of a pool of four monoclonal antibodies (MAbs) directed against distinct epitopes of carcinoembryonic antigen (CEA). Complete tumor remission was observed in 8 of 10 mice after therapy with F(ab')2 and 6 of the animals survived 10 mo in good health. In contrast, after treatment with intact MAbs, tumors relapsed in 7 of 8 mice after remission periods of 1 to 3.5 mo despite the fact that body weight loss and depression of peripheral white blood cells, symptoms of radiation toxicity, and the calculated radiation doses for liver, spleen, bone, and blood were increased or equal in these animals as compared to mice treated with F(ab')2.

  19. Demonstration by pulsed neutron scattering that the arrangement of the Fab and Fc fragments in the overall structures of bovine IgG1 and IgG2 in solution is similar.

    PubMed Central

    Mayans, M O; Coadwell, W J; Beale, D; Symons, D B; Perkins, S J

    1995-01-01

    The bovine IgG1 and IgG2 isotypes exhibit large differences in effector functions. To examine the structural basis for this, the 12-domain structures of IgG1 and IgG2 were investigated by pulsed neutron scattering using a recently developed camera LOQ. This method reports on the average relative disposition in solution of the Fab and Fc fragments in IgG. The radii of gyration (RG) were found to be similar at 5.64 and 5.71 nm for IgG1 and IgG2 respectively in 100% 2H2O buffers. The two cross-sectional radii of gyration (RXS) were also similar at 2.38-2.41 and 0.98-1.02 nm. Similar values were obtained for porcine IgG. Both bovine IgG1 and IgG2 possess similar overall solution structures, despite sequence differences at the hinge region at the centre of their structures. An automated computer survey of possible IgG structures was developed, in which coordinates for the two Fab fragments were displaced in a two-dimensional plane relative to those of the Fc fragment in 0.25 nm steps. The scattering curves calculated from these structures were found to be sensitive to relative displacements of the three fragments, but not on their rotational orientation about their longest axes. Good agreement with the solution scattering data was obtained with a planar IgG model in which the C-terminus of the CH1 domain of Fab was 3.6 nm from the N-terminus of Fc in both IgG1 and IgG2, with a precision of 0.7 nm. Energy refinement showed that this spatial separation is compatible with the hinge sequences of bovine IgG1 and IgG2. The results show that multidomain protein structures can be modelled using LOQ data, and that a long hinge sequence does not necessarily reflect a large distance between Fab and Fc. The steric accessibility of Fc sites for interactions with cell-surface Fc receptors and C1q of complement is shown to be generally similar for IgG1 and IgG2, and the difference in effector function between IgG1 and IgG2 is probably based on deletions in the IgG2 hinge sequence

  20. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity.

    PubMed

    Graille, M; Stura, E A; Corper, A L; Sutton, B J; Taussig, M J; Charbonnier, J B; Silverman, G J

    2000-05-09

    Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-A resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (V(H)) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human V(H)3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig V(H) regions and the T-cell receptor V(beta) regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor V(beta) backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

  1. Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

    PubMed

    Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

    2015-05-15

    The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.

  2. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    PubMed

    Louis, John M; Aniana, Annie; Lohith, Katheryn; Sayer, Jane M; Roche, Julien; Bewley, Carole A; Clore, G Marius

    2014-01-01

    We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  3. Preparation and evaluation of an immunoaffinity sorbent with Fab' antibody fragments for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry.

    PubMed

    Medina-Casanellas, Silvia; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

    2013-07-30

    An immunoaffinity (IA) sorbent with antibody fragments was prepared for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry (IA-SPE-CE-MS). The antibody fragmentation was evaluated by MALDI-TOF-MS. Fab' fragments obtained from a polyclonal IgG antibody against Endomorphins 1 and 2 (End1 and End2) were covalently attached to succinimidyl silica particles to prepare the IA sorbent. An IA-SPE-CE-MS methodology was established analyzing standard solutions of End1 and End2 and acceptable repeatability, linearity ranges and LODs (0.5 and 5 ng mL(-1), respectively) were obtained. The LOD of End1 was slightly better than that previously obtained using an IA sorbent with intact antibodies (1 ng mL(-1)). In human plasma samples, End1 and End2 could be detected at 1 and 50 ng mL(-1), respectively, which meant an improvement of 100 and 2-fold with regard to the LODs using an IA sorbent with intact antibodies (100 ng mL(-1)).

  4. Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site.

    PubMed

    Talavera, Ariel; Mackenzie, Jenny; Garrido, Greta; Friemann, Rosmarie; López-Requena, Alejandro; Moreno, Ernesto; Krengel, Ute

    2011-07-01

    The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806.

  5. Anchoring a cationic ligand: the structure of the Fab fragment of the anti-morphine antibody 9B1 and its complex with morphine.

    PubMed

    Pozharski, Edwin; Wilson, Mark A; Hewagama, Anura; Shanafelt, Armen B; Petsko, Gregory; Ringe, Dagmar

    2004-03-26

    The crystal structures of an anti-morphine antibody 9B1 (to 1.6A resolution) and its complex with morphine (to 2.0 A resolution) are reported. The morphine-binding site is described as a shallow depression on the protein surface, an unusual topology for a high-affinity ( Ka approximately 10(9) M(-1)) antibody against a small antigen. The polar part of the ligand is exposed to solvent, and the cationic nitrogen atom of the morphine molecule is anchored at the bottom of the binding site by a salt-bridge to a glutamate side-chain. Additional affinity is provided by a double cation-pi interaction with two tryptophan residues. Comparison of the morphine complex with the structure of the free Fab shows that a domain closure occurs upon binding of the ligand.

  6. Site-specific conjugation of chain-terminal chelating polymers to Fab' fragments of anti-CEA mAb: effect of linkage type and polymer size on conjugate biodistribution in nude mice bearing human colorectal carcinoma.

    PubMed

    Slinkin, M A; Curtet, C; Sai-Maurel, C; Gestin, J F; Torchilin, V P; Chatal, J F

    1992-01-01

    Polylysine-based chelating polymers were used for site-specific modification of anti-CEA mAb Fab' fragments via their SH group distal to the antigen-binding site of the antibody molecule. Conjugation was performed using chain-terminal (pyridyldithio)propionate or 4-(p-maleimidophenyl)butyrate moieties to form reducible (S-S) or stable (S-C) bonds between a polymer and Fab' molecule, respectively. One S-S conjugate (S-S9) and two different S-C conjugates (S-C3 and S-C9) were prepared using 3- and 9-kDa molecular weight polymers. No significant loss of immunoreactivity was observed in solid-phase immunoassay, 90-95% of 111In-labeled conjugates being bound to CEA-coated Sepharose beads. After labeling with 111In, the conjugates had a specific radioactivity of 90-120 microCi/micrograms. Injected in nude mice bearing LS 174T carcinoma, the conjugates produced different biodistribution patterns. S-S9 was practically unable to accumulate in tumor and produced very rapid blood clearance of radioactivity and high uptake of radioactivity in liver, spleen, and especially kidneys (225% ID/g 24 h postinjection). S-C3 and S-C9 produced practically the same blood clearances (much slower than that of S-S9) and significant tumor uptake (9-10% ID/g at 24 h). S-C3 gave significantly lower radioactivity in spleen, skin, and bones, and cleared more rapidly from liver and kidneys. Renal uptake for S-C3 and S-C9 was rather high (45% ID/g at 24 h), but much lower than for S-S9.

  7. Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

    SciTech Connect

    Obmolova, Galina Malia, Thomas J.; Teplyakov, Alexey; Sweet, Raymond W.; Gilliland, Gary L.

    2014-07-23

    The power of microseed matrix screening is demonstrated in the crystallization of a panel of antibody Fab fragments. The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.

  8. Production of anti-horse antibodies induced by IgG, F(ab')2 and Fab applied repeatedly to rabbits. Effect on antivenom pharmacokinetics.

    PubMed

    Vázquez, Hilda; Olvera, Felipe; Alagón, Alejandro; Sevcik, Carlos

    2013-12-15

    We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.

  9. Conversion of a Mouse Fab into a Whole Humanized IgG Antibody for Detecting Botulinum Toxin

    DTIC Science & Technology

    2006-04-01

    pentavalent toxoid; Fab, antibody fragment; HRP, horseradish peroxidase; LCκ, kappa light chain ; scFv, single- chain antibody fragments; VL, variable light ...The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified...from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated

  10. Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

    PubMed

    Ohkuri, Takatoshi; Murase, Eri; Sun, Shu-Lan; Sugitani, Jun; Ueda, Tadashi

    2013-10-01

    A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

  11. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

    PubMed

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

    2015-07-01

    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

  12. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    PubMed

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  13. Pharmacokinetics of heterologous and homologous immunoglobulin G, F(ab')2 and Fab after intravenous administration in the rat.

    PubMed

    Bazin-Redureau, M I; Renard, C B; Scherrmann, J M

    1997-03-01

    Because few pharmacokinetic studies of antibodies and their fragments have compared the influence of species origin and antibody size, the plasma pharmacokinetics of a single intravenous dose (0.7 mg kg-1) of 125I-labelled mouse, rat and human immunoglobulin G (IgG), and mouse F(ab')2 and Fab were investigated in the rat. IgG reached equilibrium after six distribution half-lives, i.e. only 36-50 h post-dosing, and the distribution volume was about four times the rat plasma volume. IgG elimination half-lives ranged from 5.33 to 8.10 days. Fragmentation of IgG into smaller fragments, F(ab')2 and Fab, resulted in pharmacokinetics that were molecular-weight-dependent with volume of distribution and systemic clearance values inversely related to antibody size. We conclude that antibody variability in terms of species origin and size influences antibody pharmacokinetics and should be carefully studied before selection of the best antibody for a clinical application.

  14. Fab-PEG-Fab as a potential antibody mimetic.

    PubMed

    Khalili, Hanieh; Godwin, Antony; Choi, Ji-Won; Lever, Rebecca; Khaw, Peng T; Brocchini, Steve

    2013-11-20

    IgG antibodies have evolved to be flexible so that they can bind to epitopes located over a wide spatial range. The two Fabs in an IgG antibody are linked together as if each Fab is at the end of a linear, flexible molecule. PEG was used as a scaffold molecule to link two Fabs together to give Fab-PEG-Fab molecules, or FpFs. Preparation of FpFs was achieved with reagents that undergo site-specific conjugation at each PEG terminus by bis-alkylation with the two cysteine thiols from a disulfide bond. This allowed each Fab to be conjugated to the PEG scaffold in essentially the same region that each Fab is linked in an IgG. Fabs were sourced directly (e.g., ranibizumab) or monoclonal IgG antibodies were proteolytically digested to obtain the Fabs. This allowed the resulting FpFs to be directly compared to parent IgGs. PEG scaffolds of 6, 10, and 20 kDa were used to make the corresponding FpFs. Dynamic light scatting data suggested the resulting FpFs were similar in size to an IgG antibody and about half the size of a 20 kDa PEGylated-Fab. The solution size of PEG-conjugated proteins is known to be dominated by the extended solution structure of PEG, so it is thought that the smaller size of the FpFs is due to interactions between the two Fabs. Anti-VEGF and anti-Her2 FpFs were prepared and evaluated. The FpFs displayed similar apparent affinities to their parent IgGs. Slower dissociation rates were observed for the anti-VEGF FpFs compared to bevacizumab. The anti-VEGF FpFs also displayed in vitro anti-angiogenic properties comparable to or better than bevacizumab. These first studies indicate that FpFs warrant further examination in a therapeutic indication where the presence of the Fc may not be required.

  15. 'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

    PubMed

    Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

    2016-04-01

    A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli.

  16. Construction and selection of human Fab antibody phage display library of liver cancer.

    PubMed

    Shui, Xuan; Huang, Jian; Li, Yue-Hui; Xie, Ping-Li; Li, Guan-Cheng

    2009-10-01

    The aim of this study was to construct the fully humanized anti-hepatoma Fab fragment phage libraries and select antibodies against hepatoma specifically. PBMCs of liver cancer patients were immunized in vitro with HpeG(2) cells and were then transformed by Epstein-Barr virus (EBV). After total RNA was extracted, the heavy chain Fd and kappa/lambda light chain were amplified by RT-PCR and cloned into the vector pComb3 to construct the libraries of Fab fragments. The libraries were then panned by HpeG(2) cells. By means of ELISA and immunochemistry, the Fab phage antibodies binding with hepatoma were selected and identified. The Fd and light chain PCR products were subsequently inserted into pComb3, and the volume of Fab libraries reached 1.7 x 10(7). The libraries were enriched about 138-fold by three cycles of panning. 540 phage clones were picked randomly. Using cell ELISA and immunohistochemistry with cultured cells, one clone Fab phage antibody, which had binding activity with hepatoma, was picked out. Fully humanized anti-hepatoma Fab antibody phage display libraries were constructed. One phage clone was selected and confirmed to specifically bind to hepatoma cells. The selected Fab antibody may be further developed and applied to clinical diagnosis and therapy.

  17. Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

    PubMed

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

    2015-01-01

    A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

  18. Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.

    PubMed

    Hayashi-Takanaka, Yoko; Yamagata, Kazuo; Wakayama, Teruhiko; Stasevich, Timothy J; Kainuma, Takashi; Tsurimoto, Toshiki; Tachibana, Makoto; Shinkai, Yoichi; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2011-08-01

    Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

  19. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    PubMed

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

  20. Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

    PubMed

    Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

    2015-11-01

    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.

  1. Fab MOR03268 triggers absorption shift of a diagnostic dye via packaging in a solvent-shielded Fab dimer interface.

    PubMed

    Hillig, Roman C; Urlinger, Stefanie; Fanghänel, Jörg; Brocks, Bodo; Haenel, Cornelia; Stark, Yvonne; Sülzle, Detlev; Svergun, Dmitri I; Baesler, Siegfried; Malawski, Guido; Moosmayer, Dieter; Menrad, Andreas; Schirner, Michael; Licha, Kai

    2008-03-14

    Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm. This is the largest shift reported for a biological system so far. Crystal structure determination and absorption spectroscopy in the crystal in combination with microcalorimetry and small-angle X-ray scattering in solution revealed that the redshift is triggered by formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed protein cavity within the dimer interface. The derived principle of shifting the absorption peak of a symmetric dye via packaging within a Fab dimer interface may be transferred to other diagnostic fluorophores, opening the way towards smart imaging probes that change their wavelength upon interaction with an antibody.

  2. Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

    PubMed

    Organtini, Lindsey J; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Parrish, Colin R; Hafenstein, Susan

    2016-11-01

    Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM.

  3. "Fab 13": The Learning Factory.

    ERIC Educational Resources Information Center

    Crooks, Steven M.; Eucker, Tom R.

    2001-01-01

    Describes how situated learning theory was employed in the design of Fab 13, a four-day simulation-based learning experience for manufacturing professionals at Intel Corporation. Presents a conceptual framework for understanding situated learning and discusses context, content, anchored instruction, facilitation, scaffolding, collaborating,…

  4. Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid.

    PubMed

    Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

    2014-01-15

    Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate.

  5. FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

    PubMed Central

    Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.

    2014-01-01

    Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus. PMID:25403676

  6. Analysis of an anti-progesterone antibody: variable crystal morphology of the Fab' and steroid-Fab' complexes.

    PubMed Central

    Stura, E A; Arevalo, J H; Feinstein, A; Heap, R B; Taussig, M J; Wilson, I A

    1987-01-01

    Anti-progesterone monoclonal antibodies are being used for structural studies of antibody-antigen interaction, for their ability to block pregnancy shortly after fertilization, and for hormone immunoassay. A mouse anti-progesterone monoclonal Fab' fragment has been crystallized in its native form and co-crystallized with seven different, but structurally related, steroids. The crystals show interesting preferences in their crystal morphology, depending on the bound steroid ligand. The X-ray crystallographic analysis of this Fab', complexed with a series of related steroid ligands, should reveal details of the chemistry of antibody-antigen union and provide insights into how steroids interact with proteins. Images Figure 3 Figure 4 PMID:3123368

  7. Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

    PubMed

    Caballero, A; Foletti, D; Bierdeman, M; Tang, A; Arana, A; Hasa-Moreno, A; Sangalang, E; O'Callaghan, R J

    2014-06-09

    Abstract Purpose: To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. Methods: A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. Results: LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). Conclusions: The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

  8. The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies.

    PubMed

    Quintero-Hernández, Veronica; Juárez-González, Victor R; Ortíz-León, Mauricio; Sánchez, Rosalba; Possani, Lourival D; Becerril, Baltazar

    2007-02-01

    The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.

  9. Characterization of human colorectal cancer MDR1/P-gp Fab antibody.

    PubMed

    Zhang, Xuemei; Xiao, Gary Guishan; Gao, Ying

    2013-01-01

    In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29). Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG). After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT) comprising residues 883-898 within the transmembrane (TM) domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.

  10. Characterization of a Pseudomonas aeruginosa Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD)

    PubMed Central

    Kutchma, Alecksandr J.; Hoang, Tung T.; Schweizer, Herbert P.

    1999-01-01

    A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding β-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding β-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding β-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption–ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4′-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H6-FabD exhibited malonyl-CoA:ACP transacylase activity. PMID:10464226

  11. Anti-Fab antibodies in humans. Predominance of minor immunoglobulin G subclasses in rheumatoid arthritis.

    PubMed Central

    Persselin, J E; Stevens, R H

    1985-01-01

    Isoelectric focusing analyses of sera from patients with rheumatoid arthritis (RA) demonstrate two populations of antibodies directed against the Fab portion of pooled human IgG. One population is composed of polyclonal alkaline anti-Fab antibodies (alpha FABA) and the other, acidic alpha FABA which are more clonally restricted. In this study we have identified the immunoglobulin classes and subclasses of these antibodies in RA sera. Enzyme-linked immunosorbent assays (ELISA) demonstrated alpha FABA in RA sera to be predominantly IgG. A large portion of IgG alpha FABA existed as immune complexes, inasmuch as dialysis of RA sera against 6 M urea before ELISA analysis was necessary for maximal detection of alpha FABA activity. Chromatofocusing of RA sera isolated alpha FABA of different charges and revealed the acidic clonally restricted alpha FABA to be IgG4 and IgG3, whereas the polyclonal alkaline group contained IgG1, IgG2, and IgG3. Overall, acidic IgG3 and IgG4 comprised 70% of IgG alpha FABA, and high levels of IgG4 were seen in most RA sera. When alpha FABA were elevated in normal sera, they were primarily of the IgG4 subclass, and also existed as immune complexes. Serum anti-Fab activity was removed by adsorption of sera with Fab fragments. Anti-Fab antibodies of both kappa and lambda light-chain types were present in RA sera, and F(ab')2 fragments of RA serum immunoglobulin were found to possess anti-Fab activity. These studies indicate that alpha FABA in RA sera are limited to the IgG class, and that most of these antibodies exist as immune complexes and display clonal and minor IgG subclass restriction. Images PMID:3928684

  12. Effect of acetylation on monoclonal antibody ZCE-025 Fab': Distribution in normal and tumor-bearing mice

    SciTech Connect

    Tarburton, J.P.; Halpern, S.E.; Hagan, P.L.; Sudora, E.; Chen, A.; Fridman, D.M.; Pfaff, A.E. )

    1990-04-01

    Studies were performed to determine in vitro and in vivo effects of acetylation on Fab' fragments of ZCE-025, a monoclonal anti-CEA antibody. Isoelectric focusing revealed a drop in isoelectric point of 1.7 pI units following acetylation. Biodistribution studies of acetylated and nonacetylated (111In)Fab' were performed in normal BALB/c mice and in nude mice bearing the T-380 CEA-producing human colon tumor. The acetylated fragments remained in the vascular compartment longer and had significantly diminished renal uptake of 111In compared to controls. While acetylation itself effected a 50% drop in immunoreactivity, tumor uptake of the acetylated and nonacetylated 111In-labeled Fab' fragments was comparable, with the exception of one data point, through 72 h.

  13. In vitro Fab display: a cell-free system for IgG discovery.

    PubMed

    Stafford, Ryan L; Matsumoto, Marissa L; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R; Baliga, Ramesh; Murray, Christopher J; Thanos, Christopher D; Hallam, Trevor J; Sato, Aaron K

    2014-04-01

    Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

  14. Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

    SciTech Connect

    Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.; Greenberg, Harry B.; Harrison, Stephen C.; Dormitzer, Philip R.

    2009-06-17

    Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.

  15. A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain.

    PubMed

    Clausen, Rasmus P; Mohr, Andreas Ø; Riise, Erik; Jensen, Anders A; Gill, Avinash; Madden, Dean R; Kastrup, Jette S; Skottrup, Peter D

    2016-11-01

    A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor.

  16. QSAR models for prediction of chromatographic behavior of homologous Fab variants.

    PubMed

    Robinson, Julie R; Karkov, Hanne S; Woo, James A; Krogh, Berit O; Cramer, Steven M

    2016-12-12

    While quantitative structure activity relationship (QSAR) models have been employed successfully for the prediction of small model protein chromatographic behavior, there have been few reports to date on the use of this methodology for larger, more complex proteins. Recently our group generated focused libraries of antibody Fab fragment variants with different combinations of surface hydrophobicities and electrostatic potentials, and demonstrated that the unique selectivities of multimodal resins can be exploited to separate these Fab variants. In this work, results from linear salt gradient experiments with these Fabs were employed to develop QSAR models for six chromatographic systems, including multimodal (Capto MMC, Nuvia cPrime, and two novel ligand prototypes), hydrophobic interaction chromatography (HIC; Capto Phenyl), and cation exchange (CEX; CM Sepharose FF) resins. The models utilized newly developed "local descriptors" to quantify changes around point mutations in the Fab libraries as well as novel cluster descriptors recently introduced by our group. Subsequent rounds of feature selection and linearized machine learning algorithms were used to generate robust, well-validated models with high training set correlations (R(2)  > 0.70) that were well suited for predicting elution salt concentrations in the various systems. The developed models then were used to predict the retention of a deamidated Fab and isotype variants, with varying success. The results represent the first successful utilization of QSAR for the prediction of chromatographic behavior of complex proteins such as Fab fragments in multimodal chromatographic systems. The framework presented here can be employed to facilitate process development for the purification of biological products from product-related impurities by in silico screening of resin alternatives. Biotechnol. Bioeng. 2016;9999: 1-10. © 2016 Wiley Periodicals, Inc.

  17. Structure of the omalizumab Fab.

    PubMed

    Jensen, Rasmus K; Plum, Melanie; Tjerrild, Luna; Jakob, Thilo; Spillner, Edzard; Andersen, Gregers Rom

    2015-04-01

    Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor FcℇRI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc Cℇ2-4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.

  18. Crystallization of antibody fragments and their complexes with antigen

    NASA Astrophysics Data System (ADS)

    Boulot, G.; Guillon, V.; Mariuzza, R. A.; Poljak, R. J.; Riottot, M.-M.; Souchon, H.; Spinelli, S.; Tello, D.

    1988-07-01

    Immunoglobulins, myeloma light chains and their fragments, and Fab fragments from monoclonal antibodies of predefined specificity have been crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab and Fc fragments separately. Intrasegmental mobility in Fabs has not been an obstacle to their crystallization, although this has been a low frequency event, occuring in about 1 in 25 to 1 in 50 trials with different Fabs. However, the immune system provides a large functional and structural diversity of antibody molecules so that an active search may eventually reveal antibodies of the desired specificity suitable for crystallization and X-ray diffraction studies.

  19. Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer.

    PubMed

    Duska, L R; Hamblin, M R; Bamberg, M P; Hasan, T

    1997-01-01

    The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer.

  20. Noninvasive detection of human cardiac transplant rejection with indium-111 antimyosin (Fab) imaging

    SciTech Connect

    Frist, W.; Yasuda, T.; Segall, G.; Khaw, B.A.; Strauss, H.W.; Gold, H.; Stinson, E.; Oyer, P.; Baldwin, J.; Billingham, M.

    1987-11-01

    Diagnosis of rejection after cardiac transplantation is currently made by right ventricular endomyocardial biopsy. To evaluate antimyosin imaging as a noninvasive means of detecting human cardiac rejection, the Fab fragment of murine monoclonal antimyosin antibodies was labeled with indium-111 and given intravenously to 18 patients (age 45 +/- 12 years) in 20 studies 7 days to 9 years after transplantation. Endomyocardial biopsy specimens were obtained at the time of each imaging study. Eight patients had positive scans confirmed by biopsy as rejection, and eight patients had negative scans and no evidence of rejection on biopsy. Discordance was observed in four studies, two with positive scans and no rejection on biopsy and two with negative scans and positive biopsy. The sensitivity, specificity, and overall accuracy of the technique were each 80%. Imaging with radiolabeled antimyosin antibody Fab fragments may be of value in the noninvasive identification of rejection in the cardiac transplant recipient.

  1. A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties

    PubMed Central

    Unverdorben, Felix; Hutt, Meike; Seifert, Oliver; Kontermann, Roland E.

    2015-01-01

    Background Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions. Methodology/Principal Findings Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics. Conclusions/Significance The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity. PMID:26430884

  2. FAB (Functionally Alert Behavior Strategies) to Improve Self-Control

    ERIC Educational Resources Information Center

    Pagano, John

    2015-01-01

    This paper describes the FAB (Functionally Alert Behavior) Strategies approach to improve behavior in children and adolescents with complex behavioral challenges. FAB Strategies include evidence-based environmental adaptations, sensory modulation, positive behavioral support, and physical self-regulation strategies. FAB Strategies can be used by…

  3. Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup.

    PubMed

    Sasso, E H; Silverman, G J; Mannik, M

    1991-09-15

    Staphylococcal protein A (SPA) is a bacterial membrane protein that possesses, in addition to its Fc gamma-binding activity, a distinct specificity for the Fab region of some IgM, IgA, IgG, and IgE. The Fab site that binds to SPA has been localized to the V region of the Ig H chain. In a previous study of human monoclonal and polyclonal IgM, we demonstrated that binding to SPA was highly restricted to molecules of the VHIII subgroup, and that nearly all VHIII IgM were able to bind SPA. The present study examines the VH composition of SPA-binding and SPA-nonbinding fractions of purified human polyclonal IgA, and IgG F(ab')2 fragments. We found that 22% of the IgA and 15% of the IgG F(ab')2 bound to SPA-agarose. Analysis with VH subgroup-specific antisera indicated that the SPA-binding fraction of IgA was dominated by the VHIII subgroup, and the SPA-binding fraction of IgG F(ab')2 contained only VHIII molecules. Furthermore, substantial portions of the total VHIII protein in IgA and in IgG F(ab')2 bound to SPA. We conclude that Fab binding to SPA is both restricted to and highly prevalent among human VHIII molecules, regardless of Ig class. These results suggest that protein A is an Ig superantigen.

  4. Comparison of F(ab')2 versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial

    PubMed Central

    Ruha, Anne-Michelle; Seifert, Steven A.; Morgan, David L.; Lewis, Brandon J.; Arnold, Thomas C.; Clark, Richard F.; Meggs, William J.; Toschlog, Eric A.; Borron, Stephen W.; Figge, Gary R.; Sollee, Dawn R.; Shirazi, Farshad M.; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D.; Boyer, Leslie V.

    2015-01-01

    Background. Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. Methods. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count < 150 K/mm3, fibrinogen level < 150 mg/dL) between end of maintenance dosing and day 8. Results. 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. Conclusions. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation

  5. Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

    PubMed

    Kyrchanova, Olga; Mogila, Vladic; Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

    2016-07-01

    Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

  6. Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains.

    PubMed

    Valjakka, Jarkko; Hemminki, Ari; Niemi, Seija; Söderlund, Hans; Takkinen, Kristiina; Rouvinen, Juha

    2002-11-15

    A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C(4)F(5)). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K(d) = 3 x 10(-10) m) when compared with the wild-type (3-C(4)F(5)) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 A) and without testosterone (2.10 A) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.

  7. Preparation and characterization of an immuno-electron microscope tracer consisting of a heme-octapeptide coupled to fab.

    PubMed

    Kraehenbuhl, J P; Galardy, R E; Jamieson, J D

    1974-01-01

    A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent K(m) of 0.2 M, an apparent v(max) of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of approximately 2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 A, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent K(m) of 0.4 M, an apparent v(max) of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits.

  8. Fabs enable single particle cryoEM studies of small proteins.

    PubMed

    Wu, Shenping; Avila-Sakar, Agustin; Kim, JungMin; Booth, David S; Greenberg, Charles H; Rossi, Andrea; Liao, Maofu; Li, Xueming; Alian, Akram; Griner, Sarah L; Juge, Narinobu; Yu, Yadong; Mergel, Claudia M; Chaparro-Riggers, Javier; Strop, Pavel; Tampé, Robert; Edwards, Robert H; Stroud, Robert M; Craik, Charles S; Cheng, Yifan

    2012-04-04

    In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of an ∼65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.

  9. Fractionation of Fab glycosylated immunoglobulin G with concanavalin A chromatography unveils new structural properties of the molecule

    PubMed Central

    Gu, Huan; Zhao, Conghui; Liu, Xingmu; Yan, Meiling; Deng, Xiaodong; Zhang, Zaiping; Gu, Jiang

    2016-01-01

    Concanavalin A (ConA) chromatography has been extensively used to separate asymmetric Immunoglobulin G (IgG), which possesses oligosaccharide attached to one of the two F(ab')2 arms, from symmetric IgG with no glycan attached to Fab fragments. In this study, applying affinity chromatography, silver stain, Western blot and lectin stain techniques, N- linked oligosaccharide attached to Fab fragment was demonstrated to be exposed on the surface of the protein and be accessible by ConA. In contrast, N- linked oligosaccharide attached to asparagine (Asn) 297 of IgG Fc was located in the inside of the natural protein and was inaccessible by ConA. In addition to asymmetric IgG, there are also detectable level of IgG with both F(ab')2 arms glycosylated that has not been reported previously. The discoveries of new basic molecular structure of IgG would have implications in understanding the function and properties of this important immune molecule with clinical applications. PMID:27145274

  10. Site-directed immobilisation of antibody fragments for detection of C-reactive protein.

    PubMed

    Vikholm-Lundin, Inger; Albers, Willem M

    2006-01-15

    C-reactive protein, CRP antibody Fab'-fragments have been attached on pre-cleaned gold slides and protein repellent polymers have been used to block the remaining free space in between the antibody fragments. At optimal conditions the antibody fragments are site-directly immobilised on the surface and non-specific binding is reduced. The amount of Fab'-fragments in the polymer host monolayer has been optimised for various buffers. Binding of CRP to Fab'-fragment/polymer layers produced in phosphate buffered saline decreased with NaCl salt concentration. In a 1M NaCl phosphate buffer, the antibodies seem to be randomly oriented on the surface with a similar response to CRP as that of an antibody F(ab)(2)-fragment layer. In a 150 mM NaCl phosphate buffer, on the other hand, the fragments seem to be site-directly oriented and the response to CRP was fivefold. The highest response to CRP was obtained to a layer with a Fab'-fragment concentration of 60 microg/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 microg/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 microg/ml from serum/PBS. CRP was, moreover, successfully detected in patient samples with good reproducibility. The layer would thus be sensitive enough to analyse the CRP concentration in human serum for predicting cardiovascular disease.

  11. Approach toward minimizing chemical interference in FAB mass spectra: the development and application of thermally - assisted FAB

    SciTech Connect

    Ackermann, B.L.

    1987-01-01

    Interferences with fast atom bombardment (FAB) mass spectrometry can be classified into two major categories. The first includes impurities which remain after analyte isolation/purification, and is especially problematic in samples of biological origin. The second type of chemical interference originates from the matrix used for FAB. An example of the first type, also known as sample-related interference, is presented in the context of the analysis of the urinary metabolites of the analgesic acetaminophen by means of the off-line combination of reverse phase HPLC and FAB. Recommendations are made for efficient use of these two methods with specific regard to minimizing chemical interferences. In addition, a method for calculating analyte signal to background (S/B) values is introduced as a means of evaluating the quality of the FAB mass spectrum. A method known as thermally-assisted FAB (TA-FAB) is introduced as a means of minimizing matrix-related background. Success to date has been achieved using aqueous saccharide solutions as TA-FAB matrices. Several important improvements to FAB result from thermal control of the matrix including a selection against matrix background, and the possibility of valid background subtraction. The development of TA-FAB is described in the context of applications of the technique to the analysis of several representative nonvolatile biomolecules including a series of cyclic tetrapeptide mycotoxins. In the final section, the hypothesis of ternary perculation (TP) is submitted to account for behavior observed during TA-FAB.

  12. Monitoring strategy to match the advanced fabs

    NASA Astrophysics Data System (ADS)

    Ackmann, Paul W.

    2004-06-01

    The reduction in feature size below the exposure wavelength, the requirement for high yields, the expectation for consistent cycletime and shipment to mix, all mean that the reticle industry must be like advanced wafer fabrication centers. Due to the lower output of write tools versus steppers, and the fact that a reticle is a lot of one instead of 25 or 50 wafers as well as the need to match ship data to Fab ramp, the reticle line monitoring strategy must be optimized for small sample size. The use of tool time and alternative inspection strategies can lead to the early detection of problems. Because every reticle is a customer specific design, the monitoring strategy takes on a new look compared to the Fab. We have organized the AMTC to resemble a wafer fab. We have a dedicated Integration group that works with the customers and technologists, to monitor the needs of the customers and then drive the development programs that improve reticle capability. We have dedicated yield team to identify and classify the yield loss mechanisms and define probable causes. The teams then work with the Process owners to fix the source of yield loss and track the corrective actions. All sources of variations must be modeled and then sources of errors reduced to levels below the tool specification. The manufacturing organization has all the process and tool experts to focus on Pilot Line and Development tasks to meet the advance needs of our customers. With the organization in place we can then develop the methods based on Reticle and Fab manufacturing to best control the line and provide development with manufacturing cycle times.

  13. Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

    PubMed

    Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

    2014-11-01

    The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.

  14. A Polycomb and Gaga Dependent Silencer Adjoins the Fab-7 Boundary in the Drosophila Bithorax Complex

    PubMed Central

    Hagstrom, K.; Muller, M.; Schedl, P.

    1997-01-01

    The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-7(1)) dominantly transforms parasegment 11 into parasegment 12. Previous studies suggested that removal of a domain boundary element on the proximal side of Fab-7(1) is responsible for this gain-of-function phenotype. In this article we demonstrate that the Fab-7(1) deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of a BXD, Ubx/lacZ reporter transgene. Both silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste(1) allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans a nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor. PMID:9258680

  15. Reagents for astatination of biomolecules. 5. Evaluation of hydrazone linkers in (211)At- and (125)I-labeled closo-decaborate(2-) conjugates of Fab' as a means of decreasing kidney retention.

    PubMed

    Wilbur, D Scott; Chyan, Ming-Kuan; Hamlin, Donald K; Nguyen, Holly; Vessella, Robert L

    2011-06-15

    Evaluation of monoclonal antibody (mAb) fragments (e.g., Fab', Fab, or engineered fragments) as cancer-targeting reagents for therapy with the α-particle emitting radionuclide astatine-211 ((211)At) has been hampered by low in vivo stability of the label and a propensity of these proteins localize to kidneys. Fortunately, our group has shown that the low stability of the (211)At label, generally a meta- or para-[(211)At]astatobenzoyl conjugate, on mAb Fab' fragments can be dramatically improved by the use of closo-decaborate(2-) conjugates. However, the higher stability of radiolabeled mAb Fab' conjugates appears to result in retention of radioactivity in the kidneys. This investigation was conducted to evaluate whether the retention of radioactivity in kidney might be decreased by the use of an acid-cleavable hydrazone between the Fab' and the radiolabeled closo-decaborate(2-) moiety. Five conjugation reagents containing sulfhydryl-reactive maleimide groups, a hydrazone functionality, and a closo-decaborate(2-) moiety were prepared. In four of the five conjugation reagents, a discrete poly(ethylene glycol) (PEG) linker was used, and one substituent adjacent to the hydrazone was varied (phenyl, benzoate, anisole, or methyl) to provide varying acid sensitivity. In the initial studies, the five maleimido-closo-decaborate(2-) conjugation reagents were radioiodinated ((125)I or (131)I), then conjugated with an anti-PSMA Fab' (107-1A4 Fab'). Biodistributions of the five radioiodinated Fab' conjugates were obtained in nude mice at 1, 4, and 24 h post injection (pi). In contrast to closo-decaborate(2-) conjugated to 107-1A4 Fab' through a noncleavable linker, two conjugates containing either a benzoate or a methyl substituent on the hydrazone functionality displayed clearance rates from kidney, liver, and spleen that were similar to those obtained with directly radioiodinated Fab' (i.e., no conjugate). The maleimido-closo-decaborate(2-) conjugation reagent containing a

  16. Dimerization kinetics of the IgE-class antibodies by divalent haptens. I. The Fab-hapten interactions.

    PubMed Central

    Schweitzer-Stenner, R; Licht, A; Pecht, I

    1992-01-01

    The binding of divalent haptens to IgE-class antibodies leads predominantly to their oligomerization into open and closed dimers. Kinetics of the open dimer formation was investigated by fluorescence titrations of Fab fragments of monoclonal DNP-specific IgE antibodies with divalent haptens having different spacer length (gamma = 14-130 A). Binding was monitored by quenching of intrinsic tryptophan emission of the Fab. Addition of divalent haptens with short spacers (gamma = 14-21 A) to the Fabs at rates larger than a distinct threshold value caused a significant decrease of Fab-binding site occupation in the initial phase of the titration. This finding was interpreted to reflect a nonequilibrium state of hapten-Fab-binding. Such nonequilibrium titrations were analyzed by inserting a kinetic model into a theory of antibody aggregation as presented by Dembo and Golstein (Histamine release due to bivalent penicilloyl haptens. 1978. J. Immunol. 121, 345). Fitting of this model to the fluorescence titrations yielded dissociation rate constants of 7.8 x 10(-3) s-1 and 6 x 10(-3) s-1 for the Fab dimers formed by the flexible divalent haptens N alpha, N epsilon-di(dinitrophenyl)-L-lysine (gamma = 16 A) and bis(N beta-2,4-dinitrophenyl-alanyl)-meso-diamino-succinate (gamma = 21 A). Making the simplifying assumption that a single step binding equilibrium prevails, the corresponding dimer formation rate constants were calculated to be 1.9 x 10(5) M-1 s-1 and 1.1 x 10(4) M-1 s-1, respectively. In contrast, all haptens with spacers longer than 40 A (i.e., bis(N alpha-2,4-dinitrophenyl-tri-D-alanyl)-1,7-diamino-heptane, and di(N epsilon-2,4-dinitrophenyl)-6-aminohexanoate-aspartyl-(prolyl)n-L-l ysyl (n = 24, 27, 33) exhibit a relative fast dimerization rate of the Fab fragments (greater than 7 x 10(6) M-1 s-1). These observations were interpreted as being caused by orientational constraints set by the limited solid angle of the reaction between the macromolecular reactants

  17. Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

    PubMed

    Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D; Heywood, Sam; Humphreys, David P

    2016-10-01

    An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

  18. Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

    PubMed Central

    Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E.; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D.; Heywood, Sam; Humphreys, David P.

    2016-01-01

    ABSTRACT An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG. PMID:27532598

  19. Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis

    PubMed Central

    2009-01-01

    Background The original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in vivo labeling studies in Clostridium butyricum ATCC 6015 (now C. beijerinckii) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. C. acetobutylicium synthesizes the same species of unsaturated fatty acids as E. coli, but lacks all of the known unsaturated fatty acid synthetic genes identified in E. coli and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of C. acetobutylicium, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci). Results We report that the FabF homologue located within the fatty acid biosynthetic gene cluster of C. acetobutylicium functions in synthesis of both unsaturated fatty acids and saturated fatty acids. Expression of this protein in E. coli functionally replaced both the FabB and FabF proteins of the host in vivo and replaced E. coli FabB in a defined in vitro fatty acid synthesis system. In contrast the single C. acetobutylicium FabZ homologue, although able to functionally replace E. coli FabZ in vivo and in vitro, was unable to replace FabA, the key dehydratase-isomerase of E. coli unsaturated fatty acid biosynthesis in vivo and lacked isomerase activity in vitro. Conclusion Thus, C. acetobutylicium introduces the double of unsaturated fatty acids by use of a novel and unknown enzyme. PMID:19493359

  20. Targeting Mast Cells and Basophils with Anti-FcεRIα Fab-Conjugated Celastrol-Loaded Micelles Suppresses Allergic Inflammation.

    PubMed

    Peng, Xia; Wang, Juan; Li, Xianyang; Lin, Lihui; Xie, Guogang; Cui, Zelin; Li, Jia; Wang, Yuping; Li, Li

    2015-12-01

    Mast cells and basophils are effector cells in the pathophysiology of allergic diseases. Targeted elimination of these cells may be a promising strategy for the treatment of allergic disorders. Our present study aims at targeted delivery of anti-FcεRIα Fab-conjugated celastrol-loaded micelles toward FcεRIα receptors expressed on mast cells and basophils to have enhanced anti-allergic effect. To achieve this aim, we prepared celastrol-loaded (PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles using thin-film hydration method. The anti-FcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The anti-FcεRIα Fab-conjugated celastrol-loaded micelles revealed uniform particle size (93.43 ± 12.93 nm) with high loading percentage (21.2 ± 1.5% w/w). The image of micelles showed oval and rod like. The anti-FcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake and cytotoxity toward target KU812 cells than non-conjugated micelles in vitro. Furthermore, diffusion of the drug into the cells allowed an efficient induction of cell apoptosis. In mouse model of allergic asthma, treatment with anti-FcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-sIgE, histamine and Th2 cytokines (IL-4, IL-5, TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, anti-FcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evan's in the ear. These results indicate that anti-FcεRIα Fab-conjugated celastrol-loaded micelles can target and selectively kill mast cells and basophils which express FcεRIα, and may be efficient reagents for the treatment of allergic disorders and mast cell related diseases.

  1. Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

    PubMed

    Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

    2016-04-04

    The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

  2. Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.

    PubMed

    Mourão, Cecília Alves; Carmignotto, Gabriela Pannunzio; Bueno, Sonia Maria Alves

    2016-04-01

    This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution.

  3. The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors.

    PubMed

    Houimel, Mehdi

    2014-08-01

    A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74-92% and 90-96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3-IGKV1, IGHV3-IGVL1, and IGHV3-IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.

  4. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    PubMed

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.

  5. A Case Study of a High School Fab Lab

    NASA Astrophysics Data System (ADS)

    Lacy, Jennifer E.

    This dissertation examines making and design-based STEM education in a formal makerspace. It focuses on how the design and implementation of a Fab Lab learning environment and curriculum affect how instructors and students see themselves engaging in science, and how the Fab Lab relates to the social sorting practices that already take place at North High School. While there is research examining design-based STEM education in informal and formal learning environments, we know little about how K-12 teachers define STEM in making activities when no university or museum partnership exists. This study sought to help fill this gap in the research literature. This case study of a formal makerspace followed instructors and students in one introductory Fab Lab course for one semester. Additional observations of an introductory woodworking course helped build the case and set it into the school context, and provided supplementary material to better understand the similarities and differences between the Fab Lab course and a more traditional design-based learning course. Using evidence from observational field notes, participant interviews, course materials, and student work, I found that the North Fab Lab relies on artifacts and rhetoric symbolic of science and STEM to set itself apart from other design-based courses at North High School. Secondly, the North Fab Lab instructors and students were unable to explain how what they were doing in the Fab Lab was science, and instead relied on vague and unsupported claims related to interdisciplinary STEM practices and dated descriptions of science. Lastly, the design and implementation of the Fab Lab learning environment and curriculum and its separation from North High School's low tech, design-based courses effectively reinforced social sorting practices and cultural assumptions about student work and intelligence.

  6. High-level iodination of monoclonal antibody fragments for radiotherapy

    SciTech Connect

    Ferens, J.M.; Krohn, K.A.; Beaumier, P.L.; Brown, J.P.; Hellstroem, I.; Hellstroem, K.E.; Carrasquillo, J.A.; Larson, S.M.

    1984-03-01

    Two different murine monoclonal antibody Fab fragments specific for p97, a melanoma-associated antigen, were labeled with I-131 at high activity levels without excessive chemical damage. Up to 20 mg of Fab were labeled with up to 300 mCi of I-131 using the chloramine-T method and large working volumes at room temperature. As much as 90% of the initial activity was recovered as labeled product. The labeled Fabs varied in their sensitivity to radioiodination damage, as measured by an in vitro cell-binding assay. Radioiodination was performed safely using a remote iodination apparatus. The final product was of radiopharmaceutical quality suitable for clinical diagnosis and experimental radiotherapy in humans.

  7. Increased Fab thermoresistance via VH-targeted directed evolution

    PubMed Central

    Entzminger, Kevin C.; Johnson, Jennifer L.; Hyun, Jeongmin; Lieberman, Raquel L.; Maynard, Jennifer A.

    2015-01-01

    Antibody aggregation is frequently mediated by the complementarity determining regions within the variable domains and can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. Aggregation-resistant antibodies could offset these risks; accordingly, we have developed a directed evolution strategy to improve Fab stability. A Fab-phage display vector was constructed and the VH domain targeted for mutagenesis by error-prone PCR. To enrich for thermoresistant clones, the resulting phage library was transiently heated, followed by selection for binding to an anti-light chain constant domain antibody. Five unique variants were identified, each possessing one to three amino acid substitutions. Each engineered Fab possessed higher, Escherichia coli expression yield, a 2–3°C increase in apparent melting temperature and improved aggregation resistance upon heating at high concentration. Select mutations were combined and shown to confer additive improvements to these biophysical characteristics. Finally, the wild-type and most stable triple variant Fab variant were converted into a human IgG1 and expressed in mammalian cells. Both expression level and aggregation resistance were similarly improved in the engineered IgG1. Analysis of the wild-type Fab crystal structure provided a structural rationale for the selected residues changes. This approach can help guide future Fab stabilization efforts. PMID:26283664

  8. Increased Fab thermoresistance via VH-targeted directed evolution.

    PubMed

    Entzminger, Kevin C; Johnson, Jennifer L; Hyun, Jeongmin; Lieberman, Raquel L; Maynard, Jennifer A

    2015-10-01

    Antibody aggregation is frequently mediated by the complementarity determining regions within the variable domains and can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. Aggregation-resistant antibodies could offset these risks; accordingly, we have developed a directed evolution strategy to improve Fab stability. A Fab-phage display vector was constructed and the VH domain targeted for mutagenesis by error-prone PCR. To enrich for thermoresistant clones, the resulting phage library was transiently heated, followed by selection for binding to an anti-light chain constant domain antibody. Five unique variants were identified, each possessing one to three amino acid substitutions. Each engineered Fab possessed higher, Escherichia coli expression yield, a 2-3°C increase in apparent melting temperature and improved aggregation resistance upon heating at high concentration. Select mutations were combined and shown to confer additive improvements to these biophysical characteristics. Finally, the wild-type and most stable triple variant Fab variant were converted into a human IgG1 and expressed in mammalian cells. Both expression level and aggregation resistance were similarly improved in the engineered IgG1. Analysis of the wild-type Fab crystal structure provided a structural rationale for the selected residues changes. This approach can help guide future Fab stabilization efforts.

  9. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    SciTech Connect

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

    2006-09-01

    The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K{sup +} channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments.

  10. Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

    PubMed Central

    Hoang, T T; Schweizer, H P

    1997-01-01

    The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemophilus influenzae. Chromosomal fabA and fabB mutants were isolated; the mutants were auxotrophic for unsaturated fatty acids. A temperature-sensitive fabA mutant was obtained by site-directed mutagenesis of a single base that induced a G101D change; this mutant grew normally at 30 degrees C but not at 42 degrees C, unless the growth medium was supplemented with oleate. By physical and genetic mapping, the fabAB genes were localized between 3.45 and 3.6 Mbp on the 5.9-Mbp chromosome, which corresponds to the 58- to 59.5-min region of the genetic map. PMID:9286984

  11. Escherichia coli unsaturated fatty acid synthesis: complex transcription of the fabA gene and in vivo identification of the essential reaction catalyzed by FabB.

    PubMed

    Feng, Youjun; Cronan, John E

    2009-10-23

    Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the years. The key players are the fabA and fabB genes. Earlier studies of fabA transcription showed that the gene was transcribed from two promoters, with one being positively regulated by the FadR protein. The other weaker promoter (which could not be mapped with the technology then available) was considered constitutive because its function was independent of FadR. However, the FabR negative regulator was recently shown to represses fabA transcription. We report that the weak promoter overlaps the FadR-dependent promoter and is regulated by FabR. This promoter is strictly conserved in all E. coli and Salmonella enterica genomes sequenced to date and is thought to provide insurance against inappropriate regulation of fabA transcription by exogenous saturated fatty acids. Also, the fabAup promoter, a mutant promoter previously isolated by selection for increased FabA activity, was shown to be a promoter created de novo by a four-base deletion within the gene located immediately upstream of fabA. Demonstration of the key UFA synthetic reaction catalyzed by FabB has been elusive, although it was known to catalyze an elongation reaction. Strains lacking FabB are UFA auxotrophs indicating that the enzyme catalyzes an essential step in UFA synthesis. Using thioesterases specific for hydrolysis of short chain acyl-ACPs, the intermediates of the UFA synthetic pathway have been followed in vivo for the first time. These experiments showed that a fabB mutant strain accumulated less cis-5-dodecenoic acid than the parental wild-type strain. These data indicate that the key reaction in UFA synthesis catalyzed by FabB is elongation of the cis-3-decenoyl-ACP produced by FabA.

  12. Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

    PubMed Central

    Kornberger, Petra; Skerra, Arne

    2014-01-01

    We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins. PMID:24492291

  13. Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin.

    PubMed

    Kornberger, Petra; Skerra, Arne

    2014-01-01

    We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH 6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly 2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography--utilizing the Strep-tag II appended to gelonin--and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC 50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.

  14. Oriented immobilisation of anti-pneumolysin Fab through a histidine tag for electrochemical immunosensors.

    PubMed

    Vallina-García, Romina; del Mar García-Suárez, María; Fernández-Abedul, M Teresa; Méndez, Francisco Javier; Costa-García, Agustín

    2007-09-30

    Orientation of reagents is a key step in the construction of immunosensors. When the immunoreagent is a recombinant protein, this can be achieved by employing hexahistidine tags. The orientation of recombinant histidine-tagged Fab fragments of monoclonal anti-pneumolysin antibodies on gold films is evaluated. Using histidine as a chelator of Ni or employing an anti-polyhistidine antibody for capturing the His6 residue is considered. Measurements are based in the signal of indigo, which comes from the hydrolysis of 3-indoxylphosphate by alkaline phosphatase (AP). The attachment of the enzyme occurs through the interaction of biotin with AP-labelled streptavidin or employing AP-conjugated immunoreagents. In the case of the interaction Ni-histidine, for the study of the self-assembling process a His-tagged and biotinylated protein (His6-GST-B) was employed. General conditions were studied and non-specific adsorption was avoided with the use of 1-hexanethiol. Improvements of the signal compared with the direct adsorption were only achieved by the use of histidine capturing antibodies. With an optimised ratio anti-polyhis:His6-Fab the signal increases approximately a 100%. Precision is adequate and the response is linear with the concentration of pneumolysin between 0.1 and 10 ng/mL.

  15. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.

    PubMed

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-10-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse

  16. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    PubMed

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  17. Role of Fc fragments in antibody-mediated recovery from ocular and subcutaneous herpes simplex virus infections.

    PubMed Central

    Oakes, J E; Lausch, R N

    1981-01-01

    The contributions of the Fc fragment of virus-specific antibody in the resistance of mice to peripheral herpes simplex virus infection were investigated. Rabbit anti-herpes simplex virus-specific F(ab')2 fragments prepared by pepsin digestion of immune immunoglobulin G (IgG) were found to be inactive in complement-mediated cytolysis while retaining their capacity to neutralize virus infectivity in vitro. When F(ab')2 fragments were passively transferred either before or simultaneously with virus inoculation, they were as efficient as intact IgG was in protecting animals from virus challenge. However, if passive transfer was delayed until 8 h after herpes simplex virus infection, only IgG antibody was protective. The loss of protective activity could not be attributed to a rapid disappearance of F(ab')2 fragments, because comparable levels of F(ab')2 fragments and IgG antibody were maintained in the blood of recipients during the time that antibody mediated its protective effects. The inability of F(ab')2 subunits to activate complement was also not a factor, because complement-deficient A/J mice and complement-sufficient SJL/J mice recovered from herpes simplex virus infection after the passive transfer of IgG. We concluded that the Fc component of the antibody molecule is needed to resolve intracellular infection and that the mechanism by which antibody mediates recovery remains undefined but does not appear to involve virus neutralization or complement activation. PMID:6266961

  18. Construction of a large synthetic human Fab antibody library on yeast cell surface by optimized yeast mating.

    PubMed

    Baek, Du-San; Kim, Yong-Sung

    2014-03-28

    Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here, we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and Vkappa1-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than 10(9) by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ~10(7). The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.

  19. Protection from infection with influenza A H7N9 virus in a mouse model by equine neutralizing F(ab')2.

    PubMed

    Zhao, Zhongpeng; Fan, Chuanbo; Duan, Yueqiang; Zhang, Liangyan; Li, Min; Yang, Xiaolan; Li, Ruisheng; Yang, Penghui; Wang, Xiliang

    2014-11-01

    Influenza A H7N9 virus has demonstrated considerable pandemic potential in China ever since early spring 2013. Until now, there have been no specific medicines to treat influenza A H7N9 virus infected patients. Development of a safe and effective H7N9 therapeutic preparation is urgently needed. To this end, we prepared and evaluated the pepsin-digested F(ab')2 fragments of serum IgGs from the horses inoculated with a inactivated influenza A H7N9 whole virus antigens. The protective effects of the F(ab')2 fragments against H7N9 virus infection were determined in cultured MDCK cells by cytopathic effect (CPE) and evaluated in a BALB/c mouse model by observing death, weight loss and viral load. The in vitro results showed that the F(ab')2 fragments had an HI titer of 1:2048 and a neutralization titer of 1: 31,623. The in vivo assays suggested that 600 U of the preparations could efficiently protect BALB/c mice from a lethal dose of A/Anhui/01/2013 (H7N9) infection even when administered two days post infection. Thus, this highly purified preparation should be a potential candidate for treating severe patients suffering from influenza A H7N9.

  20. Comparative analysis of anti-polyglutamine Fab crystals grown on Earth and in microgravity

    PubMed Central

    Owens, Gwen E.; New, Danielle M.; Olvera, Alejandra I.; Manzella, Julia Ashlyn; Macon, Brittney L.; Dunn, Joshua C.; Cooper, David A.; Rouleau, Robyn L.; Connor, Daniel S.; Bjorkman, Pamela J.

    2016-01-01

    Huntington’s disease is one of nine neurodegenerative diseases caused by a polyglutamine (polyQ)-repeat expansion. An anti-polyQ antigen-binding fragment, MW1 Fab, was crystallized both on Earth and on the International Space Station, a microgravity environment where convection is limited. Once the crystals returned to Earth, the number, size and morphology of all crystals were recorded, and X-ray data were collected from representative crystals. The results generally agreed with previous microgravity crystallization studies. On average, microgravity-grown crystals were 20% larger than control crystals grown on Earth, and microgravity-grown crystals had a slightly improved mosaicity (decreased by 0.03°) and diffraction resolution (decreased by 0.2 Å) compared with control crystals grown on Earth. However, the highest resolution and lowest mosaicity crystals were formed on Earth, and the highest-quality crystal overall was formed on Earth after return from microgravity. PMID:27710941

  1. Comparative analysis of anti-polyglutamine Fab crystals grown on Earth and in microgravity.

    PubMed

    Owens, Gwen E; New, Danielle M; Olvera, Alejandra I; Manzella, Julia Ashlyn; Macon, Brittney L; Dunn, Joshua C; Cooper, David A; Rouleau, Robyn L; Connor, Daniel S; Bjorkman, Pamela J

    2016-10-01

    Huntington's disease is one of nine neurodegenerative diseases caused by a polyglutamine (polyQ)-repeat expansion. An anti-polyQ antigen-binding fragment, MW1 Fab, was crystallized both on Earth and on the International Space Station, a microgravity environment where convection is limited. Once the crystals returned to Earth, the number, size and morphology of all crystals were recorded, and X-ray data were collected from representative crystals. The results generally agreed with previous microgravity crystallization studies. On average, microgravity-grown crystals were 20% larger than control crystals grown on Earth, and microgravity-grown crystals had a slightly improved mosaicity (decreased by 0.03°) and diffraction resolution (decreased by 0.2 Å) compared with control crystals grown on Earth. However, the highest resolution and lowest mosaicity crystals were formed on Earth, and the highest-quality crystal overall was formed on Earth after return from microgravity.

  2. Single-Chain Fragment Variable Antibody Piezoimmunosensors

    PubMed Central

    Shen, Zhihong; Stryker, Gabrielle A.; Mernaugh, Ray L.; Yu, Lei; Yan, Heping; Zeng, Xiangqun

    2008-01-01

    In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled mono-layer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW ≈ 27000) and the ease with which they can be modified using genetic engineering, scFv’s have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples. PMID:15679346

  3. Generation, characterization, and docking studies of DNA-hydrolyzing recombinant F(ab) antibodies.

    PubMed

    Zein, Haggag S; El-Sehemy, Ahmed A; Fares, Mohamed O; ElHefnawi, Mahmoud; da Silva, Jaime A Teixeira; Miyatake, Kazutaka

    2011-01-01

    Previously we established a series of catalytic antibodies (catAbs) capable of hydrolyzing DNA prepared by hybridoma technology. A group of these catAbs exhibited high reactivity and substrate specificity. To determine the molecular basis for these catAbs, we cloned, sequenced, and expressed the variable regions of this group of antibodies as functional F(ab) fragments. The nucleotide and deduced amino acid sequences of the expressed light chain (Vκ) germline gene assignments confidently belonged to germline family Vκ1A, gene bb1.1 and GenBank accession number EF672207 while heavy chain variable region V(H) genes belonged to V(H) 1/V(H) J558, gene V130.3 and GenBank accession number EF672221. A well-established expression system based on the pARA7 vector was examined for its ability to produce catalytically active antibodies. Recombinant F(ab) (rF(ab) ) fragments were purified and their hydrolyzing activity was analyzed against supercoiled pUC19 plasmid DNA (scDNA). The study of rF(ab) provides important information about the potential catalytic activities of antibodies whose structure allows us to understand their basis of catalysis. Molecular surface analysis and docking studies were performed on the molecular interactions between the antibodies and poly(dA9), poly(dG9), poly(dT9), and poly(dC9) oligomers. Surface analysis identified the important sequence motifs at the binding sites, and different effects exerted by arginine and tyrosine residues at different positions in the light and heavy chains. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies. These studies may define important features of DNA catAbs.

  4. Daunting Challenge of Fab Access for U. S. Universities

    DTIC Science & Technology

    2009-03-01

    CMP  33,250 Euros ($42,106) (STM) 5 mm x 5 mm 0.13m CMOS MOSIS has a 5 mm x 5 mm minimum. Both CMP and Europractice allow smaller dice to be...trips to Taiwan. Some U. S. faculty with ties to China are getting free fab through SMIC (Shanghai), which offers technologies down to 65-nm CMOS 7...number. 1. REPORT DATE MAR 2009 2. REPORT TYPE 3. DATES COVERED 00-00-2009 to 00-00-2009 4. TITLE AND SUBTITLE Daunting challenge of fab

  5. 75 FR 9438 - Samsung Austin Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung Electronics Corporation...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-02

    ... Employment and Training Administration Samsung Austin Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung..., DRAM Fab 1, including on-site leased workers from Manpower, Austin, Texas. The notice will be published... subsidiary of Samsung Electronics Corporation, DRAM Fab 1. The Department has determined that these...

  6. 20 CFR 30.316 - How does the FAB issue a final decision on a claim?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How does the FAB issue a final decision on a... Adjudicatory Process Hearings and Final Decisions on Claims § 30.316 How does the FAB issue a final decision on... waives any objections to all or part of the recommended decision, the FAB may issue a final...

  7. Fragmentation, labeling and biodistribution studies of KS1/4, a monoclonal antibody

    SciTech Connect

    Mohd, S.B.

    1987-01-01

    In this study, an IgG2a (KS1/4), a monoclonal antibody (MoAb) specific against a human lung adenocarcinoma (UCLA P-3) was successfully fragmented enzymatically to yield F(ab')/sub 2/ and Fab by using pepsin and papain, respectively. The kinetic of fragmentation of the MoAb was compared to that of human immunoglobulin G (IgG). A similar pattern of fragmentation was observed with both antibodies with a higher percentage yield of the F(ab')/sub 2/ and Fab obtained upon the fragmentation of the IgG by the enzymes. The KS1/4 and the two fragments were labeled with three different radionuclides, namely iodine-131, indium-111 and selenium-75. The radioiodination of the MoAb and the fragments was carried out by using a modified chloramine-T method. Radiometal labeling of the MoAb and the fragments with indium-111 was performed by using DTPA as a bifunctional chelating agent, while intrinsic labeling of the MoAb was done by culturing the hybridoma in the presence of /sup 75/Se-methionine. The biodistribution of the radiolabeled MoAb, F(ab')/sub 2/ and Fab fragments were performed by injecting the preparations intravenously into nude mice bearing human lung adenocarcinoma.

  8. Pollution prevention opportunity assessment for MicroFab and SiFab facilities at Sandia National Laboratories.

    SciTech Connect

    Gerard, Morgan Evan

    2011-12-01

    This Pollution Prevention Opportunity Assessment (PPOA) was conducted for the MicroFab and SiFab facilities at Sandia National Laboratories/New Mexico in Fiscal Year 2011. The primary purpose of this PPOA is to provide recommendations to assist organizations in reducing the generation of waste and improving the efficiency of their processes and procedures. This report contains a summary of the information collected, the analyses performed, and recommended options for implementation. The Sandia National Laboratories Environmental Management System (EMS) and Pollution Prevention (P2) staff will continue to work with the organizations to implement the recommendations.

  9. Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis.

    PubMed

    Li, Meng; Meng, Qiu; Fu, Huihui; Luo, Qixia; Gao, Haichun

    2016-11-15

    As type II fatty acid synthesis is essential for the growth of Escherichia coli, its many components are regarded as potential targets for novel antibacterial drugs. Among them, β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) FabB is the exclusive factor for elongation of the cis-3-decenoyl-ACP (cis-3-C10-ACP). In our previous study, we presented evidence to suggest that this may not be the case in Shewanella oneidensis, an emerging model gammaproteobacterium renowned for its respiratory versatility. Here, we identified FabF1, another KAS, as a functional replacement for FabB in S. oneidensis In fabB(+) or desA(+) (encoding a desaturase) cells, which are capable of making unsaturated fatty acids (UFA), FabF1 is barely produced. However, UFA auxotroph mutants devoid of both fabB and desA genes can be spontaneously converted to suppressor strains, which no longer require exogenous UFAs for growth. Suppression is caused by a TGTTTT deletion in the region upstream of the fabF1 gene, resulting in enhanced FabF1 production. We further demonstrated that the deletion leads to transcription read-through of the terminator for acpP, an acyl carrier protein gene immediately upstream of fabF1 There are multiple tandem repeats in the region covering the terminator, and the TGTTTT deletion, as well as others, compromises the terminator efficacy. In addition, FabF2 also shows an ability to complement the FabB loss, albeit substantially less effectively than FabF1.

  10. Scatterometry on pelliclized masks: an option for wafer fabs

    NASA Astrophysics Data System (ADS)

    Gallagher, Emily; Benson, Craig; Higuchi, Masaru; Okumoto, Yasuhiro; Kwon, Michael; Yedur, Sanjay; Li, Shifang; Lee, Sangbong; Tabet, Milad

    2007-03-01

    Optical scatterometry-based metrology is now widely used in wafer fabs for lithography, etch, and CMP applications. This acceptance of a new metrology method occurred despite the abundance of wellestablished CD-SEM and AFM methods. It was driven by the desire to make measurements faster and with a lower cost of ownership. Over the last year, scatterometry has also been introduced in advanced mask shops for mask measurements. Binary and phase shift masks have been successfully measured at all desired points during photomask production before the pellicle is mounted. There is a significant benefit to measuring masks with the pellicle in place. From the wafer fab's perspective, through-pellicle metrology would verify mask effects on the same features that are characterized on wafer. On-site mask verification would enable quality control and trouble-shooting without returning the mask to a mask house. Another potential application is monitoring changes to mask films once the mask has been delivered to the fab (haze, oxide growth, etc.). Similar opportunities apply to the mask metrologist receiving line returns from a wafer fab. The ability to make line-return measurements without risking defect introduction is clearly attractive. This paper will evaluate the feasibility of collecting scatterometry data on pelliclized masks. We explore the effects of several different pellicle types on scatterometry measurements made with broadband light in the range of 320-780 nm. The complexity introduced by the pellicles' optical behavior will be studied.

  11. 20 CFR 30.319 - May a claimant request reconsideration of a final decision of the FAB?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... final decision of the FAB? 30.319 Section 30.319 Employees' Benefits OFFICE OF WORKERS' COMPENSATION... reconsideration of a final decision of the FAB? (a) A claimant may request reconsideration of a final decision of the FAB by filing a written request with the FAB within 30 days from the date of issuance of...

  12. Characterization of FAB1 phosphatidylinositol kinases in Arabidopsis pollen tube growth and fertilization.

    PubMed

    Serrazina, Susana; Dias, Fernando Vaz; Malhó, Rui

    2014-08-01

    In yeast and animal cells, phosphatidylinositol-3-monophosphate 5-kinases produce phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) and have been implicated in endomembrane trafficking and pH control in the vacuole. In plants, PtdIns(3,5)P2 is synthesized by the Fab1 family, four orthologs of which exist in Arabidopsis: FAB1A and FAB1B, both from the PIKfyve/Fab1 family; FAB1C and FAB1D, both without a PIKfyve domain and of unclear role. Using a reverse genetics and cell biology approach, we investigated the function of the Arabidopsis genes encoding FAB1B and FAB1D, both highly expressed in pollen. Pollen viability, germination and tube morphology were not significantly affected in homozygous mutant plants. In vivo, mutant pollen fertilized ovules leading to normal seeds and siliques. The same result was obtained when mutant ovules were fertilized with wild-type pollen. Double mutant pollen for the two genes was able to fertilize and develop plants no different from the wild-type. At the cellular level, fab1b and fab1d pollen tubes were found to exhibit perturbations in membrane recycling, vacuolar acidification and decreased production of reactive oxygen species (ROS). Subcellular imaging of FAB1B-GFP revealed that the protein localized to the endomembrane compartment, whereas FAB1D-GFP localized mostly to the cytosol and sperm cells. These results were discussed considering possible complementary roles of FAB1B and FAB1D.

  13. A strain-specific epitope of enterovirus 71 identified by cryo-electron microscopy of the complex with fab from neutralizing antibody.

    PubMed

    Lee, Hyunwook; Cifuente, Javier O; Ashley, Robert E; Conway, James F; Makhov, Alexander M; Tano, Yoshio; Shimizu, Hiroyuki; Nishimura, Yorihiro; Hafenstein, Susan

    2013-11-01

    Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody.

  14. A Strain-Specific Epitope of Enterovirus 71 Identified by Cryo-Electron Microscopy of the Complex with Fab from Neutralizing Antibody

    PubMed Central

    Lee, Hyunwook; Cifuente, Javier O.; Ashley, Robert E.; Conway, James F.; Makhov, Alexander M.; Tano, Yoshio; Shimizu, Hiroyuki; Nishimura, Yorihiro

    2013-01-01

    Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody. PMID:23946455

  15. Chameleon fragmentation

    SciTech Connect

    Brax, Philippe

    2014-02-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ{sup 4} and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  16. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    PubMed

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

  17. Spherical cows in the sky with fab four

    SciTech Connect

    Kaloper, Nemanja; Sandora, McCullen E-mail: mesandora@ucdavis.edu

    2014-05-01

    We explore spherically symmetric static solutions in a subclass of unitary scalar-tensor theories of gravity, called the 'Fab Four' models. The weak field large distance solutions may be phenomenologically viable, but only if the Gauss-Bonnet term is negligible. Only in this limit will the Vainshtein mechanism work consistently. Further, classical constraints and unitarity bounds constrain the models quite tightly. Nevertheless, in the limits where the range of individual terms at large scales is respectively Kinetic Braiding, Horndeski, and Gauss-Bonnet, the horizon scale effects may occur while the theory satisfies Solar system constraints and, marginally, unitarity bounds. On the other hand, to bring the cutoff down to below a millimeter constrains all the couplings scales such that 'Fab Fours' can't be heard outside of the Solar system.

  18. FAB overlapping: a strategy for sequencing homologous proteins

    NASA Astrophysics Data System (ADS)

    Ferranti, P.; Malorni, A.; Marino, G.; Pucci, P.; di Luccia, A.; Ferrara, L.

    1991-12-01

    Extensive similarity has been shown to exist between the primary structures of closely related proteins from different species, the only differences being restricted to a few amino acid variations. A new mass spectrometric procedure, which has been called FAB-overlapping, has been developed for sequencing highly homologous proteins based on the detection of these small differences as compared with a known protein used as a reference. Several complementary peptide maps are constructed using fast atom bombardment mass spectrometry (FAB-MS) analysis of different proteolytic digests of the unknown protein and the mass values are related to those expected on the basis of the sequence of the reference protein. The mass signals exhibiting unusual mass values identify those regions where variations have taken place; fine location of the mutations can be obtained by coupling simple protein chemistry methodologies with FAB-MS. Using the FAB-overlapping procedure, it was possible to determine the sequence of [alpha]1, [alpha]3 and [beta] globins from water buffalo (Bubalus bubalis hemoglobins (phenotype AA). Two amino acid substitutions were detected in the buffalo [beta] chain (Lys16 --> His and Asn118 --> His) whereas the [alpha]1 chains were found the [alpha]1 and [alpha]3 chains were found to contain four amino acid replacements, three of which were identical (Glu23 --> Asp, Glu71 --> Gly, Phe117 --> Cys), and the insertion of an alanine residue in position 124. The only differences between [alpha]1 and [alpha]3 globins were identified in the C -terminal region; [alpha]1 contains a Phe residue at position 130 whereas [alpha]3 shows serine at position 132.

  19. Strategy optimization for mask rule check in wafer fab

    NASA Astrophysics Data System (ADS)

    Yang, Chuen Huei; Lin, Shaina; Lin, Roger; Wang, Alice; Lee, Rachel; Deng, Erwin

    2015-07-01

    Photolithography process is getting more and more sophisticated for wafer production following Moore's law. Therefore, for wafer fab, consolidated and close cooperation with mask house is a key to achieve silicon wafer success. However, generally speaking, it is not easy to preserve such partnership because many engineering efforts and frequent communication are indispensable. The inattentive connection is obvious in mask rule check (MRC). Mask houses will do their own MRC at job deck stage, but the checking is only for identification of mask process limitation including writing, etching, inspection, metrology, etc. No further checking in terms of wafer process concerned mask data errors will be implemented after data files of whole mask are composed in mask house. There are still many potential data errors even post-OPC verification has been done for main circuits. What mentioned here are the kinds of errors which will only occur as main circuits combined with frame and dummy patterns to form whole reticle. Therefore, strategy optimization is on-going in UMC to evaluate MRC especially for wafer fab concerned errors. The prerequisite is that no impact on mask delivery cycle time even adding this extra checking. A full-mask checking based on job deck in gds or oasis format is necessary in order to secure acceptable run time. Form of the summarized error report generated by this checking is also crucial because user friendly interface will shorten engineers' judgment time to release mask for writing. This paper will survey the key factors of MRC in wafer fab.

  20. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... multiple myeloma (tumor of bone marrow cells), Waldenstrom's macroglobulinemia (increased immunoglobulin production by the spleen and bone marrow cells), and lymphoma (tumor of the lymphoid tissues)....

  1. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... multiple myeloma (tumor of bone marrow cells), Waldenstrom's macroglobulinemia (increased immunoglobulin production by the spleen and bone marrow cells), and lymphoma (tumor of the lymphoid tissues)....

  2. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... multiple myeloma (tumor of bone marrow cells), Waldenstrom's macroglobulinemia (increased immunoglobulin production by the spleen and bone marrow cells), and lymphoma (tumor of the lymphoid tissues)....

  3. Inefficient translation renders the Enterococcus faecalis fabK enoyl-acyl carrier protein reductase phenotypically cryptic.

    PubMed

    Bi, Hongkai; Zhu, Lei; Wang, Haihong; Cronan, John E

    2014-01-01

    Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the bacterial fatty acid elongation cycle. Enterococcus faecalis is unusual in that it encodes two unrelated enoyl-ACP reductases, FabI and FabK. We recently reported that deletion of the gene encoding FabI results in an unsaturated fatty acid (UFA) auxotroph despite the presence of fabK, a gene encoding a second fully functional enoyl-ACP reductase. By process of elimination, our prior report argued that poor expression was the reason that fabK failed to functionally replace FabI. We now report that FabK is indeed poorly expressed and that the expression defect is at the level of translation rather than transcription. We isolated four spontaneous mutants that allowed growth of the E. faecalis ΔfabI strain on fatty acid-free medium. Each mutational lesion (single base substitution or deletion) extended the fabK ribosome binding site. Inactivation of fabK blocked growth, indicating that the mutations acted only on fabK rather than a downstream gene. The mutations activated fabK translation to levels that supported fatty acid synthesis and hence cell growth. Furthermore, site-directed and random mutagenesis experiments showed that point mutations that resulted in increased complementarity to the 3' end of the 16S rRNA increased FabK translation to levels sufficient to support growth, whereas mutations that decreased complementarity blocked fabK translation.

  4. A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

    PubMed

    Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

    2015-01-01

    Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

  5. Stable supply of large amounts of human Fab from the inclusion bodies in E. coli.

    PubMed

    Fujii, Testuro; Ohkuri, Takatoshi; Onodera, Reiko; Ueda, Tadashi

    2007-05-01

    Recombinant human Fab-H chain and L chain were separately expressed as inclusion body using Escherichia coli. After solubilization of Fab-H chain and L chain by the reduction and S-alkyldisulphidation in 8 M urea, about 100 mg of purified Fab-H chain and about 160 mg of L chain could be obtained from 1 l of each culture by ion-exchange chromatogram in the presence of 8 M urea. Combination of the lyophilized Fab-H chain and L chain could be efficiently folded to native human Fab by using the stepwise dialysis method and the human Fab was purified with cation-exchange chromatogram. In the folding procedure, it was found that cysteamine and cystamine with positive charge were effective to improve the folding yield of human Fab. Moreover, from comparison of folding yield in the presence of ten kinds of additives, it was suggested that taurine was effective to improve the folding of human Fab. Consequently, we could obtain about 60 mg of folded human Fab from 1 l of each culture under the optimum conditions.

  6. Fragmentation Processes

    NASA Astrophysics Data System (ADS)

    Whelan, Colm T.

    2012-12-01

    Preface; 1. Direct and resonant double-photoionization: from atoms to solids L. Avaldi and G. Stefani; 2. The application of propagation exterior complex scaling to atomic collisions P. L. Bartlett and A. T. Stelbovics; 3. Fragmentation of molecular-ion beams in intense ultra-short laser pulses I. Ben-Itzhak; 4. Atoms with one and two active electrons in strong laser fields I. A. Ivanov and A. S. Kheifets; 5. Experimental aspects of ionization studies by positron and positronium impact G. Laricchia, D. A. Cooke, Á. Kövér and S. J. Brawley; 6. (e,2e) spectroscopy using fragmentation processes J. Lower, M. Yamazaki and M. Takahashi; 7. A coupled pseudostate approach to the calculation of ion-atom fragmentation processes M. McGovern, H. R. J. Walters and C. T. Whelan; 8. Electron Impact Ionization using (e,2e) coincidence techniques from threshold to intermediate energies A. J. Murray; 9. (e,2e) processes on atomic inner shells C. T. Whelan; 10. Spin resolved atomic (e,2e) processes J. Lower and C. T. Whelan; Index.

  7. The role of indium-111 antimyosin (Fab) imaging as a noninvasive surveillance method of human heart transplant rejection

    SciTech Connect

    De Nardo, D.; Scibilia, G.; Macchiarelli, A.G.; Cassisi, A.; Tonelli, E.; Papalia, U.; Gallo, P.; Antolini, M.; Pitucco, G.; Reale, A. )

    1989-09-01

    The identification of rejection after heart transplantation in patients receiving cyclosporine immunosuppressive therapy requires the endomyocardial biopsy, an invasive method associated with a finite morbidity. To evaluate the role of indium-111 antimyosin (Fab) scintigraphy as a noninvasive surveillance method of heart transplant rejection, the Fab fragment of murine monoclonal antimyosin antibodies labeled with indium-111 was administered intravenously in 30 scintigraphic studies to 10 consecutive heart transplant recipients. Endomyocardial biopsy specimens were obtained 72 hours after each scintigraphic study. Nineteen scintigraphic studies had negative findings; no false negative finding was obtained. Eleven antimyosin scintigraphic studies had positive findings, and in these studies endomyocardial biopsy revealed mild rejection in two cases, moderate acute rejection with myocyte necrosis in two cases, myocyte necrosis as a consequence of ischemic injury in six cases, and possibly cytotoxic damage in one case. Antimyosin scintigraphy may represent a reliable screening method for the surveillance of heart transplant patients. In the presence of a negative finding from antimyosin scintigraphy, it may be possible to avoid endomyocardial biopsy. Conversely, in patients who have a positive finding from antimyosin scintigraphy, the endomyocardial biopsy is mandatory to establish the definitive diagnosis by histologic examination of the myocardium.

  8. Mercaptan-induced fragmentation of a subunit-like proteolytic fragment of immunoglobulin M

    PubMed Central

    Butchko, G. M.; Inman, F. P.

    1972-01-01

    Limited papain hydrolysis of immunoglobulin M (IgM) produces a subunit-like proteolytic fragment designated IgMp (Inman & Hazen, 1968). In the presence of mercaptans, IgMp partially dissociated into Fcμ-like and Fabμ fragments. Treatment of residual IgM (that remaining after a papain digestion) with 2mm-mercaptoethylamine resulted in fragmentation of the same type that occurs in a routine limited digestion of IgM with papain, although exogenous enzyme was not added to the mixture. When IgM was hydrolysed with 14C-labelled papain, a small quantity of the enzyme was found to be associated with the residual IgM and IgMp fractions. IgM and IgM 7S subunit (IgMs) that had been exposed to papain in the absence of activating mercaptan and separated from the enzyme by gel filtration also fragmented when subsequently treated with 2mm-mercaptoethylamine. The fragments resembled those produced during a typical limited papain digestion of IgM. It was concluded that mercaptoethylamine induced fragmentation of IgMp by activating adsorbed papain. ImagesFig. 1.PLATE 1 PMID:5076228

  9. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

    PubMed

    Florinas, Stelios; Liu, Marc; Fleming, Ryan; Van Vlerken-Ysla, Lilian; Ayriss, Joanne; Gilbreth, Ryan; Dimasi, Nazzareno; Gao, Changshou; Wu, Herren; Xu, Ze-Qi; Chen, Shaoyi; Dirisala, Anjaneyulu; Kataoka, Kazunori; Cabral, Horacio; Christie, R James

    2016-05-09

    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.

  10. Extending green technology innovations to enable greener fabs

    NASA Astrophysics Data System (ADS)

    Takahisa, Kenji; Yoo, Young Sun; Fukuda, Hitomi; Minegishi, Yuji; Enami, Tatsuo

    2015-03-01

    Semiconductor manufacturing industry has growing concerns over future environmental impacts as fabs expand and new generations of equipment become more powerful. Especially rare gases supply and price are one of prime concerns for operation of high volume manufacturing (HVM) fabs. Over the past year it has come to our attention that Helium and Neon gas supplies could be unstable and become a threat to HVM fabs. To address these concerns, Gigaphoton has implemented various green technologies under its EcoPhoton program. One of the initiatives is GigaTwin deep ultraviolet (DUV) lithography laser design which enables highly efficient and stable operation. Under this design laser systems run with 50% less electric energy and gas consumption compared to conventional laser designs. In 2014 we have developed two technologies to further reduce electric energy and gas efficiency. The electric energy reduction technology is called eGRYCOS (enhanced Gigaphoton Recycled Chamber Operation System), and it reduces electric energy by 15% without compromising any of laser performances. eGRYCOS system has a sophisticated gas flow design so that we can reduce cross-flow-fan rotation speed. The gas reduction technology is called eTGM (enhanced Total gas Manager) and it improves gas management system optimizing the gas injection and exhaust amount based on laser performances, resulting in 50% gas savings. The next steps in our roadmap technologies are indicated and we call for potential partners to work with us based on OPEN INNOVATION concept to successfully develop faster and better solutions in all possible areas where green innovation may exist.

  11. Composition and method for detecting cancer with technetium labeled antibody fragments

    SciTech Connect

    Burchiel, S. W.; Crockford, D. R.; Rhodes, B. A.

    1984-10-23

    F(ab')/sub 2/ or Fab fragments of antibodies to: (a) human chorionic gonadotropin (hCG), hCG alpha subunit, hCG beta subunit, or an hCG-like material; or (b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, are radiolabeled with technetium-99m (Tc-99m). When the F(ab')/sub 2/ or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body. In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab')/sub 2/ or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab')/sub 2/ or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab')/sub 2/ or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody. This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased.

  12. Functionally Approached Body (FAB) Strategies for Young Children Who Have Behavioral and Sensory Processing Challenges

    ERIC Educational Resources Information Center

    Pagano, John

    2005-01-01

    Functionally Approached Body (FAB) Strategies offer a clinical approach to help parents of young children with behavioral and sensory processing strategies. This article introduces the FAB Strategies, clinical strategies developed by the author for understanding and addressing young children's behavioral and sensory processing challenges. The FAB…

  13. Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

    PubMed

    Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

    2017-04-01

    Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (VH+CH1) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli.

  14. The Emerging Importance of IgG Fab Glycosylation in Immunity.

    PubMed

    van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

    2016-02-15

    Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity.

  15. Conformational diversity of bacterial FabH: Implications for molecular recognition specificity

    PubMed Central

    Mittal, Anuradha; Johnson, Michael E.

    2015-01-01

    The molecular basis of variable substrate and inhibitor specificity of the highly conserved bacterial fatty acid synthase enzyme, FabH, across different bacterial species remains poorly understood. In the current work, we explored the conformational diversity of FabH enzymes to understand the determinants of diverse interaction specificity across Gram-positive and Gram-negative bacteria. Atomistic molecular dynamics simulations reveal that FabH from E. coli and E. faecalis exhibit distinct native state conformational ensembles and dynamic behaviors. Despite strikingly similar substrate binding pockets, hot spot assessment using computational solvent mapping identified quite different favorable binding interactions between the two homologs. Our data suggest that FabH utilizes protein dynamics and seemingly minor sequence and structural differences to modulate its molecular recognition and substrate specificity across bacterial species. These insights will potentially facilitate the rational design and development of antibacterial inhibitors against FabH enzymes. PMID:25437098

  16. 20 CFR 30.320 - Can a claim be reopened after the FAB has issued a final decision?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can a claim be reopened after the FAB has... AMENDED Adjudicatory Process Reopening Claims § 30.320 Can a claim be reopened after the FAB has issued a final decision? (a) At any time after the FAB has issued a final decision pursuant to § 30.316,...

  17. Atomic Basis for the Species-specific Inhibition of αV Integrins by Monoclonal Antibody 17E6 Is Revealed by the Crystal Structure of αVβ3 Ectodomain-17E6 Fab Complex*

    PubMed Central

    Mahalingam, Bhuvaneshwari; Van Agthoven, Johannes F.; Xiong, Jian-Ping; Alonso, José Luis; Adair, Brian D.; Rui, Xianliang; Anand, Saurabh; Mehrbod, Mehrdad; Mofrad, Mohammad R. K.; Burger, Christa; Goodman, Simon L.; Arnaout, M. Amin

    2014-01-01

    The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn2+. The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins. PMID:24692540

  18. Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVβ3 ectodomain-17E6 Fab complex.

    PubMed

    Mahalingam, Bhuvaneshwari; Van Agthoven, Johannes F; Xiong, Jian-Ping; Alonso, José Luis; Adair, Brian D; Rui, Xianliang; Anand, Saurabh; Mehrbod, Mehrdad; Mofrad, Mohammad R K; Burger, Christa; Goodman, Simon L; Arnaout, M Amin

    2014-05-16

    The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.

  19. Comparing domain interactions within antibody Fabs with kappa and lambda light chains

    PubMed Central

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W.; Dickey, Mark; Froning, Karen; Conner, Elaine M.; Cujec, Thomas P.; Demarest, Stephen J.

    2016-01-01

    ABSTRACT IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates. PMID:27454112

  20. Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

    PubMed

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

    2016-10-01

    IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

  1. Structural and dynamical aspects of Streptococcus gordonii FabH through molecular docking and MD simulations.

    PubMed

    Shamim, Amen; Abbasi, Sumra Wajid; Azam, Syed Sikander

    2015-07-01

    β-Ketoacyl-ACP-synthase III (FabH or KAS III) has become an attractive target for the development of new antibacterial agents which can overcome the multidrug resistance. Unraveling the fatty acid biosynthesis (FAB) metabolic pathway and understanding structural coordinates of FabH will provide valuable insights to target Streptococcus gordonii for curing oral infection. In this study, we designed inhibitors against therapeutic target FabH, in order to block the FAB pathway. As compared to other targets, FabH has more interactions with other proteins, located on the leading strand with higher codon adaptation index value and associated with lipid metabolism category of COG. Current study aims to gain in silico insights into the structural and dynamical aspect of S. gordonii FabH via molecular docking and molecular dynamics (MD) simulations. The FabH protein is catalytically active in dimerization while it can lock in monomeric state. Current study highlights two residues Pro88 and Leu315 that are close to each other by dimerization. The active site of FabH is composed of the catalytic triad formed by residues Cys112, His249, and Asn279 in which Cys112 is involved in acetyl transfer, while His249 and Asn279 play an active role in decarboxylation. Docking analysis revealed that among the studied compounds, methyl-CoA disulfide has highest GOLD score (82.75), binding affinity (-11 kcal/mol) and exhibited consistently better interactions. During MD simulations, the FabH structure remained stable with the average RMSD value of 1.7 Å and 1.6 Å for undocked protein and docked complex, respectively. Further, crucial hydrogen bonding of the conserved catalytic triad for exhibiting high affinity between the FabH protein and ligand is observed by RDF analysis. The MD simulation results clearly demonstrated that binding of the inhibitor with S. gordonii FabH enhanced the structure and stabilized the dimeric FabH protein. Therefore, the inhibitor has the potential to become

  2. The community FabLab platform: applications and implications in biomedical engineering.

    PubMed

    Stephenson, Makeda K; Dow, Douglas E

    2014-01-01

    Skill development in science, technology, engineering and math (STEM) education present one of the most formidable challenges of modern society. The Community FabLab platform presents a viable solution. Each FabLab contains a suite of modern computer numerical control (CNC) equipment, electronics and computing hardware and design, programming, computer aided design (CAD) and computer aided machining (CAM) software. FabLabs are community and educational resources and open to the public. Development of STEM based workforce skills such as digital fabrication and advanced manufacturing can be enhanced using this platform. Particularly notable is the potential of the FabLab platform in STEM education. The active learning environment engages and supports a diversity of learners, while the iterative learning that is supported by the FabLab rapid prototyping platform facilitates depth of understanding, creativity, innovation and mastery. The product and project based learning that occurs in FabLabs develops in the student a personal sense of accomplishment, self-awareness, command of the material and technology. This helps build the interest and confidence necessary to excel in STEM and throughout life. Finally the introduction and use of relevant technologies at every stage of the education process ensures technical familiarity and a broad knowledge base needed for work in STEM based fields. Biomedical engineering education strives to cultivate broad technical adeptness, creativity, interdisciplinary thought, and an ability to form deep conceptual understanding of complex systems. The FabLab platform is well designed to enhance biomedical engineering education.

  3. Antibody Fragments as Probe in Biosensor Development

    PubMed Central

    Saerens, Dirk; Huang, Lieven; Bonroy, Kristien; Muyldermans, Serge

    2008-01-01

    Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed. PMID:27873779

  4. Detection and quantification of affinity ligand leaching and specific antibody fragment concentration within chromatographic fractions using surface plasmon resonance.

    PubMed

    Thillaivinayagalingam, Pranavan; Newcombe, Anthony R; O'Donovan, Kieran; Francis, Richard; Keshavarz-Moore, Eli

    2007-12-01

    Rapid analyses of chromatographic steps within a biopharmaceutical manufacturing process are often desirable to evaluate column performance, provide mass balance data and to permit accurate calculations of yields and recoveries. Using SPR (surface plasmon resonance) biosensor (Biacore) technology, we have developed a sandwich immunoassay to quantify polyclonal anti-digoxin Fab fragments used for the production of the FDA (Food and Drug Administration)-approved biotherapeutic DigiFab. The results show that specific Fab may be quantified in all affinity process streams and accurate yield and mass balance data calculated. Control experiments using sheep Fab and Fc indicate that the assay is specific to DigiFab. The quantification of potential leached ligand within chromatographic fractions may also be technically challenging, particularly when low-molecular-mass ligands are covalently coupled with an affinity absorbent. Typical methods to assess ligand leakage such as DDMA (digoxin-dicarboxymethoxylamine; digoxin analogue) often involve the use of labelled ligands and relatively complex and labour-intensive analytical techniques. Using the same analytical methodologies, an assay to detect leached or eluted ligand off the column was developed. The results indicate minimal levels of leached ligand in all chromatographic fractions, with total levels of leached DDMA calculated to be 1.52 microg. This is less than 0.01% of the total amount of DDMA coupled with the laboratory-scale affinity column. The SPR methods described in the present study may be applicable for the rapid in-process analysis of specific polyclonal Fab fragments (within a polyclonal mixture) and to rapidly assess leakage of small molecule ligands covalently attached to chromatographic supports.

  5. A Review on Platensimycin: A Selective FabF Inhibitor

    PubMed Central

    Sakha Ghosh, Partha; Manna, Kuntal

    2016-01-01

    Emerging resistance to existing antibiotics is an inevitable matter of concern in the treatment of bacterial infection. Naturally occurring unique class of natural antibiotic, platensimycin, a secondary metabolite from Streptomyces platensis, is an excellent breakthrough in recent antibiotic research with unique structural pattern and significant antibacterial activity. β-Ketoacyl-(acyl-carrier-protein (ACP)) synthase (FabF) whose Gram-positive bacteria need to biosynthesize cell membranes is the target of inhibition of platensimycin. So, isolation, retrosynthetic analysis, synthesis of platensimycin, and analogues of platensimycin synthesized till today are the objectives of this review which may be helpful to further investigate and to reveal untouched area on this molecule and to obtain a potential antibacterial lead with enhanced significant antibacterial activity. PMID:26942008

  6. Automated reticle inspection data analysis for wafer fabs

    NASA Astrophysics Data System (ADS)

    Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

    2009-03-01

    To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity DefectTM data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

  7. Automated reticle inspection data analysis for wafer fabs

    NASA Astrophysics Data System (ADS)

    Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

    2009-04-01

    To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity Defect(R) data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

  8. Automated reticle inspection data analysis for wafer fabs

    NASA Astrophysics Data System (ADS)

    Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

    2008-10-01

    To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity DefecTM data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

  9. Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody.

    PubMed

    Ramsland, Paul A; Terzyan, Simon S; Cloud, Gwendolyn; Bourne, Christina R; Farrugia, William; Tribbick, Gordon; Geysen, H Mario; Moomaw, Carolyn R; Slaughter, Clive A; Edmundson, Allen B

    2006-05-01

    The 2.6 A (1 A=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611-39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024-14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.

  10. Antibody fragment-conjugated polymeric micelles incorporating platinum drugs for targeted therapy of pancreatic cancer.

    PubMed

    Ahn, Jooyeon; Miura, Yutaka; Yamada, Naoki; Chida, Tsukasa; Liu, Xueying; Kim, Ahram; Sato, Ryuta; Tsumura, Ryo; Koga, Yoshikatsu; Yasunaga, Masahiro; Nishiyama, Nobuhiro; Matsumura, Yasuhiro; Cabral, Horacio; Kataoka, Kazunori

    2015-01-01

    Antibody-mediated therapies including antibody-drug conjugates (ADCs) have shown much potential in cancer treatment by tumor-targeted delivery of cytotoxic drugs. However, there is a limitation of payloads that can be delivered by ADCs. Integration of antibodies to drug-loaded nanocarriers broadens the applicability of antibodies to a wide range of therapeutics. Herein, we developed antibody fragment-installed polymeric micelles via maleimide-thiol conjugation for selectively delivering platinum drugs to pancreatic tumors. By tailoring the surface density of maleimide on the micelles, one tissue factor (TF)-targeting Fab' was conjugated to each carrier. Fab'-installed platinum-loaded micelles exhibited more than 15-fold increased cellular binding within 1 h and rapid cellular internalization compared to non-targeted micelles, leading to superior in vitro cytotoxicity. In vivo, Fab'-installed micelles significantly suppressed the growth of pancreatic tumor xenografts for more than 40 days, outperforming non-targeted micelles and free drugs. These results indicate the potential of Fab'-installed polymeric micelles for efficient drug delivery to solid tumors.

  11. The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis.

    PubMed

    Cavalli, G; Paro, R

    1998-05-15

    Polycomb group (PcG) and trithorax group (trxG) gene products are responsible for the maintenance of repressed and active expression patterns of many developmentally important regulatory genes including the homeotic genes. In Drosophila embryos, Polycomb protein and the trxG protein GAGA factor colocalize at the Fab-7 DNA element of the bithorax complex. In transgenic lines, the Fab-7 element induces extensive silencing on a flanking GAL4-driven lacZ reporter and mini-white genes. However, a short single pulse of GAL4 during embryogenesis is sufficient to release PcG-dependent silencing from the transgene. Such an activated state of Fab-7 is mitotically inheritable through development and can be transmitted in a GAL4-independent manner to the subsequent generations through female meiosis. Thus, Fab-7 is a switchable chromosomal element, which can convey memory of epigenetically determined active and repressed chromatin states.

  12. Novel PI(4)P 5-kinase homologue, Fab1p, essential for normal vacuole function and morphology in yeast.

    PubMed

    Yamamoto, A; DeWald, D B; Boronenkov, I V; Anderson, R A; Emr, S D; Koshland, D

    1995-05-01

    The FAB1 gene of budding yeast is predicted to encode a protein of 257 kDa that exhibits significant sequence homology to a human type II PI(4)P 5-kinase (PIP5K-II). The recently cloned human PIP5K-II specifically converts PI(4)P to PI(4,5)P2 (Boronenkov and Anderson, 1995). The region of highest similarity between Fab1p and PIP5K-II includes a predicted nucleotide binding motif, which is likely to correspond to the catalytic domain of the protein. Interestingly, neither PIP5K-II nor Fab1p exhibit significant homology with cloned PI 3-kinases or PI 4-kinases. fab1 mutations result in the formation of aploid and binucleate cells (hence the name FAB). In addition, loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth. Experiments with a temperature conditional fab1 mutant revealed that their vacuoles rapidly (within 30 min) enlarge to more than double the size upon shifting cells to the nonpermissive temperature. Additional experiments with the fab1 ts mutant together with results obtained with fab1 vps (vacuolar protein sorting defective) double mutants indicate that the nuclear division and cell surface integrity defects observed in fab1 mutants are secondary to the vacuole morphology defects. Based on these data, we propose that Fab1p is a PI(4)P 5-kinase and that the product of the Fab1p reaction, PIP2, functions as an important regulator of vacuole homeostasis perhaps by controlling membrane flux to and/or from the vacuole. Furthermore, a comparison of the phenotypes of fab1 mutants and other yeast mutants affecting PI metabolism suggests that phosphoinositides may serve as general regulators of several different membrane trafficking pathways.

  13. Functional Characterization of Triclosan-Resistant Enoyl-acyl-carrier Protein Reductase (FabV) in Pseudomonas aeruginosa

    PubMed Central

    Huang, Yong-Heng; Lin, Jin-Shui; Ma, Jin-Cheng; Wang, Hai-Hong

    2016-01-01

    Pseudomonas aeruginosa is extremely resistant to triclosan. Previous studies have shown that P. aeruginosa encodes a triclosan-resistant enoyl-acyl-carrier protein reductase (ENR), FabV, and that deletion of fabV causes P. aeruginosa to be extremely sensitive to triclosan. In this report, we complemented a P. aeruginosa fabV deletion strain with several triclosan-resistant ENR encoding genes, including Vibrio cholerae fabV, Bacillus subtilis fabL and Enterococcus faecalis fabK. All complemented strains restored triclosan resistance to the level of the wild-type strain, which confirmed that triclosan-resistant ENR allows P. aeruginosa to be extremely resistant to triclosan. Moreover, fabV exhibits pleiotropic effects. Deletion of fabV led P. aeruginosa to show attenuated swarming motility, decreased rhamnolipid, pyoverdine and acyl-homoserine lactones (AHLs) production. Complementation of the fabV mutant with any one ENR encoding gene could restore these features to some extent, in comparison with the wild-type strain. Furthermore, we found that addition of exogenous AHLs could restore the fabV mutant strain to swarm on semisolid plates and to produce more virulence factors than the fabV mutant strain. These findings indicate that deletion of fabV reduced the activity of ENR in P. aeruginosa, decreased fatty acid synthesis, and subsequently depressed the production of AHLs and other virulence factors, which finally may led to a reduction in the pathogenicity of P. aeruginosa. Therefore, fabV should be an ideal target for the control of P. aeruginosa infectivity. PMID:27965638

  14. Structural characterisation of the fatty acid biosynthesis enzyme FabF from the pathogen Listeria monocytogenes

    PubMed Central

    Soares da Costa, Tatiana P.; Nanson, Jeffrey D.; Forwood, Jade K.

    2017-01-01

    Development of new antimicrobial agents is required against the causative agent for listeriosis, Listeria monocytogenes, as the number of drug resistant strains continues to increase. A promising target is the β-ketoacyl-acyl carrier protein synthase FabF, which participates in the catalysis of fatty acid synthesis and elongation, and is required for the production of phospholipid membranes, lipoproteins, and lipopolysaccharides. In this study, we report the 1.35 Å crystal structure of FabF from L. monocytogenes, providing an excellent platform for the rational design of novel inhibitors. By comparing the structure of L. monocytogenes FabF with other published bacterial FabF structures in complex with known inhibitors and substrates, we highlight conformational changes within the active site, which will need to be accounted for during drug design and virtual screening studies. This high-resolution structure of FabF represents an important step in the development of new classes of antimicrobial agents targeting FabF for the treatment of listeriosis. PMID:28045020

  15. Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

    PubMed

    Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

    2015-11-01

    Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

  16. Conformation changes and dissociation of Fc fragments of rabbit immunoglobulin G as a function of pH

    PubMed Central

    Charlwood, P. A.; Utsumi, S.

    1969-01-01

    1. The sedimentation coefficients of rabbit immunoglobulin G, four types of Fc fragments, univalent Fab and bivalent F(ab)2 fragments were measured as a function of pH. 2. In conjunction with molecular-weight determinations by sedimentation equilibrium, and with the behaviour on gel filtration, this enabled the state of association of the Fc fragments to be followed. 3. The type possessing an interchain disulphide bond, 1Fc fragment, changed extensively in structure, but not in molecular weight. 4. There was good correlation between the readiness to dissociate and the chain length of the shorter Fc fragments that do not contain the interchain covalent bond. 5. The increasing resistance to dissociation as the fragments became shorter ran parallel with the ability to resist enzymic attack. 6. The site of the strong association between component chains of Fc fragment is located in the C-terminal half. 7. The gel-filtration behaviour of the Fc fragments clearly confirms that the process is governed by the Stokes radius rather than molecular weight. 8. The ultracentrifugal results were used to estimate the separations of the hydrodynamic subunits in intact immunoglobulin G, and as a basis for a schematic structure. ImagesFig. 3. PMID:5801306

  17. Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm.

    PubMed

    Levy, Raphael; Ahluwalia, Kiran; Bohmann, David J; Giang, Hoa M; Schwimmer, Lauren J; Issafras, Hassan; Reddy, Nithin B; Chan, Chung; Horwitz, Arnold H; Takeuchi, Toshihiko

    2013-08-30

    Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.

  18. Triclosan Resistance in a Bacterial Fish Pathogen, Aeromonas salmonicida subsp. salmonicida, is Mediated by an Enoyl Reductase, FabV.

    PubMed

    Khan, Raees; Lee, Myung Hwan; Joo, Hae-Jin; Jung, Yong-Hoon; Ahmad, Shabir; Choi, Jin-Hee; Lee, Seon-Woo

    2015-04-01

    Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX(8)K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria.

  19. Shewanella oneidensis FabB: A β-ketoacyl-ACP Synthase That Works with C16:1-ACP.

    PubMed

    Luo, Qixia; Li, Meng; Fu, Huihui; Meng, Qiu; Gao, Haichun

    2016-01-01

    It is established that Escherichia coli β-ketoacyl-ACP synthase (KAS) I (encoded by EcfabB) is the primary, if not exclusive, factor for elongation of the cis-3-decenoyl-ACP (C10:1-ACP) but not effective with C16:1- or longer-chain-ACPs. To test the extent to which these features apply to KAS I proteins in other species, in this study, we examined the physiological role of FabB in Shewanella oneidensis, an excellent model for researching type II fatty acid synthetic (FAS) system and its regulation. We showed that the loss of either FabA (the enzyme that introduces double bond) or FabB, in the absence of DesA which desaturizes C16 and C18 to generate respective C16:1 and C18:1, leads to a UFA auxotroph. However, fatty acid profiles of membrane phospholipid of the fabA and fabB mutants are significantly different, suggesting that FabB participates in steps beyond elongation of C10:1-ACP. Further analyses demonstrated that S. oneidensis FabB differs from EcFabB in that (i) it is not the only enzyme capable of catalyzing elongation of the cis-3-decenoyl-ACP produced by FabA, (ii) it plays a critical role in elongation of C16:1- and longer-chain-ACPs, and (iii) its overproduction is detrimental.

  20. Management of Tissue Loss After Agkistrodon Snakebite: Appropriate Use of Crotalidae-Fab Antivenin.

    PubMed

    Larson, Kenneth W; Schaefer, Keith R; Austin, Cindy; Norton, Rhy; Finley, Phillip J

    2016-01-01

    Although initially created for the treatment of rattlesnake (genus: Crotalus) bites, Crotalidae-Fab antivenin is used to treat many different pit viper envenomations. However, the efficacy of Crotalidae-Fab in preventing tissue loss from copperhead (Agkistrodon contortrix) or cottonmouth (Agkistrodon piscivorus) snakebites remains unclear. Recent reports show that Agkistrodon-related bites rarely require treatment beyond simple observation and pain control. The purpose of this study was to examine the amount of tissue loss in patients who received Crotalidae-Fab compared with those who did not after an Agkistrodon bite. After institutional review board approval, a retrospective study was completed at a Level 1 trauma center. Between 2009 and 2013, a total of 57 snakebites were identified. Of the 57 bites, the snake species was documented in 36 cases including 31 copperheads, 1 cottonmouth, and 4 rattlesnakes. The other 21 bites were from unknown or nonvenomous species. Of the 32 Agkistrodon-related bites, 15 patients received Crotalidae-Fab (average of 3 vials administered) and 17 did not receive Crotalidae-Fab. None of the 32 patients, regardless of treatment option, had tissue loss or required surgical interventions. Only 1 patient received Crotalidae-Fab and debridement of a vesicle associated with the bite. No clinically significant differences were observed between the groups. These findings support previous literature that failed to show added benefit of Crotalidae-Fab treatment for Agkistrodon bites beyond patient comfort and pain control. Evaluation of current protocols for Agkistrodon envenomations is warranted. Snakebite wound education in trauma physicians and nurses may decrease unnecessary use of antivenom medication.

  1. Pharmacokinetics of /sup 99m/Tc(Sn)- and /sup 131/I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments in nude mice

    SciTech Connect

    Zimmer, A.M.; Kazikiewicz, J.M.; Rosen, S.T.; Spies, S.M.

    1987-03-15

    The biodistribution, radioimmunoimaging, and high pressure liquid chromatography activity profiles of /sup 99m/Tc(Sn) and /sup 131/I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments were compared. Nude mice, bearing specific (colon carcinoma, LS174T) and nonspecific (pancreatic carcinoma, MIA) xenografts were given injections of the respective radiolabeled antibody fragments and also of irrelevant /sup 125/I-labeled antibody fragments (MOPC-21). The animals were imaged at 24 h after being given injections, they were sacrificed, and biodistribution studies were performed. Results of the study showed high kidney uptake (48.6% injected dose (ID)/g +/- 8.1% (SD)) and low tumor uptake (1.5% ID/g +/- 0.6%) for /sup 99m/Tc(Sn)-labeled fragments and higher uptake (4.4% ID/g +/- 0.6%) for /sup 131/I-labeled fragments, resulting in a higher localization index for the radioiodinated monoclonal antibody fragments. Imaging results showed good tumor visualization at 24 h after injection for the /sup 131/I-labeled fragments and poor tumor visualization with predominant kidney uptake for /sup 99m/Tc(Sn)-labeled fragments. After radiolabeling, high pressure liquid chromatography analysis indicated that 131I was primarily associated with F(ab')2 fragments, whereas 99mTc was mostly associated with Fab' fragments.

  2. A system for repertoire cloning and phage display of murine and leporid antibody fragments.

    PubMed

    Schüller, Carolin; Wiebe, Julia C; Pegel, Antje; Kramer, Karl; Skerra, Arne; Hock, Bertold

    2010-01-01

    Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tet(p/o)-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.

  3. Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

    PubMed

    Rose, D R; Przybylska, M; To, R J; Kayden, C S; Oomen, R P; Vorberg, E; Young, N M; Bundle, D R

    1993-07-01

    The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.

  4. A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA

    PubMed Central

    Moynié, Lucile; Hope, Anthony G.; Finzel, Kara; Schmidberger, Jason; Leckie, Stuart M.; Schneider, Gunter; Burkart, Michael D.; Smith, Andrew D.; Gray, David W.; Naismith, James H.

    2016-01-01

    Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 = 5.7 ± 0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials. PMID:26562505

  5. Crystallization and preliminary X-ray diffraction analysis of FabG from Yersinia pestis.

    PubMed

    Nanson, Jeffrey David; Forwood, Jade Kenneth

    2014-01-01

    The type II fatty-acid biosynthesis pathway of bacteria provides enormous potential for antibacterial drug development owing to the structural differences between this and the type I fatty-acid biosynthesis system found in mammals. β-Ketoacyl-ACP reductase (FabG) is responsible for the reduction of the β-ketoacyl group linked to acyl carrier protein (ACP), and is essential for the formation of fatty acids and bacterial survival. Here, the cloning, expression, purification, crystallization and diffraction of FabG from Yersinia pestis (ypFabG), the highly virulent causative agent of plague, are reported. Recombinant FabG was expressed, purified to homogeneity and crystallized via the hanging-drop vapour-diffusion technique. Diffraction data were collected at the Australian Synchrotron to 2.30 Å resolution. The crystal displayed P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 68.22, b = 98.68, c = 169.84 Å, and four ypFabG molecules in the asymmetric unit.

  6. Development of tools to study personal weight control strategies: OxFAB taxonomy

    PubMed Central

    Aveyard, Paul; Koshiaris, Constantinos; Jebb, Susan A.

    2016-01-01

    Objective To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts. Methods The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think‐aloud interviews were conducted to test the face/concept validity of the questionnaire, and test–retest reliability was assessed in a sample of 138 participants. Results The OxFAB taxonomy consists of 117 strategies grouped into 23 domains. Compared to taxonomies used to describe interventions, around half of the domains and strategies identified are unique to the OxFAB taxonomy. The OxFAB questionnaire consists of 117 questions, one for each strategy from the taxonomy. Test–retest resulted in a mean PABAK score of 0.61 (SD 0.15). Questions were revised where appropriate. Conclusions The OxFAB taxonomy and questionnaire provide a conceptual framework to identify the cognitive and behavioral strategies used by individuals during attempts at weight control. PMID:26748902

  7. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    PubMed

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S; Greenblatt, Jack F; Marcon, Edyta; Arrowsmith, Cheryl H; Edwards, Aled M; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.

  8. A human/murine chimeric fab antibody neutralizes anthrax lethal toxin in vitro.

    PubMed

    Ding, Guipeng; Chen, Ximin; Zhu, Jin; Duesbery, Nicholas S; Cheng, Xunjia; Cao, Brian

    2013-01-01

    Human anthrax infection caused by exposure to Bacillus anthracis cannot always be treated by antibiotics. This is mostly because of the effect of the remaining anthrax toxin in the body. Lethal factor (LF) is a component of lethal toxin (LeTx), which is the major virulence of anthrax toxin. A murine IgG monoclonal antibody (mAb) against LF with blocking activity (coded LF8) was produced in a previous study. In this report, a human/murine chimeric Fab mAb (coded LF8-Fab) was developed from LF8 by inserting murine variable regions into human constant regions using antibody engineering to reduce the incompatibility of the murine antibody for human use. The LF8-Fab expressed in Escherichia coli could specifically identify LF with an affinity of 3.46 × 10(7) L/mol and could neutralize LeTx with an EC50 of 85  μ g/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cells in vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection.

  9. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  10. Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

    SciTech Connect

    Bzymek, Krzysztof P.; Ma, Yuelong; Avery, Kendra A.; Horne, David A.; Williams, John C.

    2016-05-23

    An overview of cyclization strategies of a Fab-binding peptide to maximize affinity. Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic ‘pocket’ and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex.

  11. Mapping of Fab-1:VEGF Interface Using Carboxyl Group Footprinting Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Wecksler, Aaron T.; Kalo, Matt S.; Deperalta, Galahad

    2015-12-01

    A proof-of-concept study was performed to demonstrate that carboxyl group footprinting, a relatively simple, bench-top method, has utility for first-pass analysis to determine epitope regions of therapeutic mAb:antigen complexes. The binding interface of vascular endothelial growth factor (VEGF) and the Fab portion of a neutralizing antibody (Fab-1) was analyzed using carboxyl group footprinting with glycine ethyl ester (GEE) labeling. Tryptic peptides involved in the binding interface between VEGF and Fab-1 were identified by determining the specific GEE-labeled residues that exhibited a reduction in the rate of labeling after complex formation. A significant reduction in the rate of GEE labeling was observed for E93 in the VEGF tryptic peptide V5, and D28 and E57 in the Fab-1 tryptic peptides HC2 and HC4, respectively. Results from the carboxyl group footprinting were compared with the binding interface identified from a previously characterized crystal structure (PDB: 1BJ1). All of these residues are located at the Fab-1:VEGF interface according to the crystal structure, demonstrating the potential utility of carboxyl group footprinting with GEE labeling for mapping epitopes.

  12. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein.

    PubMed

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V; Xia, Di

    2016-08-01

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space group P1), with unit-cell parameters a = 40.67, b = 44.91, c = 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  13. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

    SciTech Connect

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

    2016-07-27

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  14. Parton fragmentation functions

    NASA Astrophysics Data System (ADS)

    Metz, A.; Vossen, A.

    2016-11-01

    The field of fragmentation functions of light quarks and gluons is reviewed. In addition to integrated fragmentation functions, attention is paid to the dependence of fragmentation functions on transverse momenta and on polarization degrees of freedom. Higher-twist and di-hadron fragmentation functions are considered as well. Moreover, the review covers both theoretical and experimental developments in hadron production in electron-positron annihilation, deep-inelastic lepton-nucleon scattering, and proton-proton collisions.

  15. Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen

    PubMed Central

    Selis, Fabio; Focà, Giuseppina; Sandomenico, Annamaria; Marra, Carla; Di Mauro, Concetta; Saccani Jotti, Gloria; Scaramuzza, Silvia; Politano, Annalisa; Sanna, Riccardo; Ruvo, Menotti; Tonon, Giancarlo

    2016-01-01

    PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments. PMID:27043557

  16. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange.

    PubMed

    van der Neut Kolfschoten, Marijn; Schuurman, Janine; Losen, Mario; Bleeker, Wim K; Martínez-Martínez, Pilar; Vermeulen, Ellen; den Bleker, Tamara H; Wiegman, Luus; Vink, Tom; Aarden, Lucien A; De Baets, Marc H; van de Winkel, Jan G J; Aalberse, Rob C; Parren, Paul W H I

    2007-09-14

    Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

  17. Fragmentation Analysis - Fundamental Processes

    DTIC Science & Technology

    Wausau quartzite and anorthosite of 3.0 to 3.5 inch size were fragmented in this device. An analysis of the fragment distribution results of the drop...disc-shaped specimens of Wausau quartzite, anorthosite , and Felch marble were then fragmented with the impact pendulum device. Computer programs were

  18. 20 CFR 30.908 - How will the FAB evaluate new medical evidence submitted to challenge the impairment...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How will the FAB evaluate new medical... Medical Evidence of Impairment § 30.908 How will the FAB evaluate new medical evidence submitted to... impairment evaluation that differs from the impairment evaluation relied upon by the district office, the...

  19. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

    PubMed

    Yao, Jiangwei; Ericson, Megan E; Frank, Matthew W; Rock, Charles O

    2016-12-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes.

  20. Reference metrology in a research fab: the NIST clean calibrations thrust

    NASA Astrophysics Data System (ADS)

    Dixson, Ronald; Fu, Joe; Orji, Ndubuisi; Renegar, Thomas; Zheng, Alan; Vorburger, Theodore; Hilton, Al; Cangemi, Marc; Chen, Lei; Hernandez, Mike; Hajdaj, Russell; Bishop, Michael; Cordes, Aaron

    2009-03-01

    In 2004, the National Institute of Standards and Technology (NIST) commissioned the Advanced Measurement Laboratory (AML) - a state-of-the-art, five-wing laboratory complex for leading edge NIST research. The NIST NanoFab - a 1765 m2 (19,000 ft2) clean room with 743 m2 (8000 ft2) of class 100 space - is the anchor of this facility and an integral component of the new Center for Nanoscale Science and Technology (CNST) at NIST. Although the CNST/NanoFab is a nanotechnology research facility with a different strategic focus than a current high volume semiconductor fab, metrology tools still play an important role in the nanofabrication research conducted here. Some of the metrology tools available to users of the NanoFab include stylus profiling, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Since 2001, NIST has collaborated with SEMATECH to implement a reference measurement system (RMS) using critical dimension atomic force microscopy (CD-AFM). NIST brought metrology expertise to the table and SEMATECH provided access to leading edge metrology tools in their clean room facility in Austin. Now, in the newly launched "clean calibrations" thrust at NIST, we are implementing the reference metrology paradigm on several tools in the CNST/NanoFab. Initially, we have focused on calibration, monitoring, and uncertainty analysis for a three-tool set consisting of a stylus profiler, an SEM, and an AFM. Our larger goal is the development of new and supplemental calibrations and standards that will benefit from the Class 100 environment available in the NanoFab and offering our customers calibration options that do not require exposing their samples to less clean environments. Toward this end, we have completed a preliminary evaluation of the performance of these instruments. The results of these evaluations suggest that the achievable uncertainties are generally consistent with our measurement goals.

  1. Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

    SciTech Connect

    Donaldson, Joshua M.; Zer, Cindy; Avery, Kendra N.; Bzymek, Krzysztof P.; Horne, David A.; Williams, John C.

    2013-10-07

    Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximab (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Lastly, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.

  2. International Foot and Ankle Biomechanics Community (i-FAB): past, present and beyond

    PubMed Central

    Nester, Christopher J; Leardini, Alberto; Cavanagh, Peter R; Rosenbaum, Dieter; Burns, Joshua

    2009-01-01

    The International Foot and Ankle Biomechanics Community (i-FAB) is an international collaborative activity which will have an important impact on the foot and ankle biomechanics community. It was launched on July 2nd 2007 at the foot and ankle session of the International Society of Biomechanics (ISB) meeting in Taipei, Taiwan. i-FAB is driven by the desire to improve our understanding of foot and ankle biomechanics as it applies to health, disease, and the design, development and evaluation of foot and ankle surgery, and interventions such as footwear, insoles and surfaces. PMID:19531239

  3. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

    DOE PAGES

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; ...

    2016-01-28

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. In addition, we havemore » discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. Our results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

  4. 20 CFR 30.317 - Can the FAB request a further response from the claimant or return a claim to the district office?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can the FAB request a further response from....317 Can the FAB request a further response from the claimant or return a claim to the district office? At any time before the issuance of its final decision, the FAB may request that the claimant...

  5. 20 CFR 30.312 - What will the FAB do if the claimant objects to the recommended decision but does not request a...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false What will the FAB do if the claimant objects....312 What will the FAB do if the claimant objects to the recommended decision but does not request a... period of time allotted in § 30.310 but does not request a hearing, the FAB will consider any...

  6. Selectable fragmentation warhead

    DOEpatents

    Bryan, Courtney S.; Paisley, Dennis L.; Montoya, Nelson I.; Stahl, David B.

    1993-01-01

    A selectable fragmentation warhead capable of producing a predetermined number of fragments from a metal plate, and accelerating the fragments toward a target. A first explosive located adjacent to the plate is detonated at selected number of points by laser-driven slapper detonators. In one embodiment, a smoother-disk and a second explosive, located adjacent to the first explosive, serve to increase acceleration of the fragments toward a target. The ability to produce a selected number of fragments allows for effective destruction of a chosen target.

  7. Highly specific PET imaging of prostate tumors in mice with an iodine-124-labeled antibody fragment that targets phosphatidylserine.

    PubMed

    Stafford, Jason H; Hao, Guiyang; Best, Anne M; Sun, Xiankai; Thorpe, Philip E

    2013-01-01

    Phosphatidylserine (PS) is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab')2, that binds to complexes of PS and β2-glycoprotein I. PGN635 F(ab')2 was labeled with the positron-emitting isotope iodine-124 ((124)I) and the resulting probe was injected into nude mice bearing subcutaneous or orthotopic human PC3 prostate tumors. Biodistribution studies showed that (124)I-PGN635 F(ab')2 localized with remarkable specificity to the tumors with little uptake in other organs, including the liver and kidneys. Clear delineation of the tumors was achieved by PET 48 hours after injection. Radiation of the tumors with 15 Gy or systemic treatment of the mice with 10 mg/kg docetaxel increased localization in the tumors. Tumor-to-normal (T/N) ratios were inversely correlated with tumor growth measured over 28 days. These data indicate that (124)I-PGN635 F(ab')2 is a promising new imaging agent for predicting tumor response to therapy.

  8. AGILE integration into APC for high mix logic fab

    NASA Astrophysics Data System (ADS)

    Gatefait, M.; Lam, A.; Le Gratiet, B.; Mikolajczak, M.; Morin, V.; Chojnowski, N.; Kocsis, Z.; Smith, I.; Decaunes, J.; Ostrovsky, A.; Monget, C.

    2015-09-01

    mix logic Fab) in term of product and technology portfolio AGILE corrects for up to 120nm of product topography error on process layer with less than 50nm depth of focus Based on tool functionalities delivered by ASML and on high volume manufacturing requirement, AGILE integration is a real challenge. Regarding ST requirements "Automatic AGILE" functionality developed by ASML was not a turnkey solution and a dedicated functionality was needed. A "ST homemade AGILE integration" has been fully developed and implemented within ASML and ST constraints. This paper describes this integration in our Advanced Process Control platform (APC).

  9. High quality mask storage in an advanced Logic-Fab

    NASA Astrophysics Data System (ADS)

    Jähnert, Carmen; Fritsche, Silvio

    2012-02-01

    High efficient mask logistics as well as safe and high quality mask storage are essential requirements within an advanced lithography area of a modern logic waferfab. Fast operational availability of the required masks at the exposure tool with excellent mask condition requires a safe mask handling, safeguarding of high mask quality over the whole mask usage time without any quality degradation and an intelligent mask logistics. One big challenge is the prevention of haze on high advanced phase shift masks used in a high volume production line for some thousands of 248nm or 193nm exposures. In 2008 Infineon Dresden qualified a customer specific developed semi-bare mask storage system from DMSDynamic Micro Systems in combination with a high advanced mask handling and an interconnected complex logistic system. This high-capacity mask storage system DMS M1900.22 for more than 3000 masks with fully automated mask and box handling as well as full-blown XCDA purge has been developed and adapted to the Infineon Lithotoollandscape using Nikon and SMIF reticle cases. Advanced features for ESD safety and mask security, mask tracking via RFID and interactions with the exposure tools were developed and implemented. The stocker is remote controlled by the iCADA-RSM system, ordering of the requested mask directly from the affected exposure tool allows fast access. This paper discusses the advantages and challenges for this approach as well as the practical experience gained during the implementation of the new system which improves the fab performance with respect to mask quality, security and throughput. Especially the realization of an extremely low and stable humidity level in addition with a well controlled air flow at each mask surface, preventing masks from haze degradation and particle contamination, turns out to be a notable technical achievement. The longterm stability of haze critical masks has been improved significantly. Relevant environmental parameters like

  10. Universality of fragment shapes

    PubMed Central

    Domokos, Gábor; Kun, Ferenc; Sipos, András Árpád; Szabó, Tímea

    2015-01-01

    The shape of fragments generated by the breakup of solids is central to a wide variety of problems ranging from the geomorphic evolution of boulders to the accumulation of space debris orbiting Earth. Although the statistics of the mass of fragments has been found to show a universal scaling behavior, the comprehensive characterization of fragment shapes still remained a fundamental challenge. We performed a thorough experimental study of the problem fragmenting various types of materials by slowly proceeding weathering and by rapid breakup due to explosion and hammering. We demonstrate that the shape of fragments obeys an astonishing universality having the same generic evolution with the fragment size irrespective of materials details and loading conditions. There exists a cutoff size below which fragments have an isotropic shape, however, as the size increases an exponential convergence is obtained to a unique elongated form. We show that a discrete stochastic model of fragmentation reproduces both the size and shape of fragments tuning only a single parameter which strengthens the general validity of the scaling laws. The dependence of the probability of the crack plan orientation on the linear extension of fragments proved to be essential for the shape selection mechanism. PMID:25772300

  11. Universality of fragment shapes

    NASA Astrophysics Data System (ADS)

    Domokos, Gábor; Kun, Ferenc; Sipos, András Árpád; Szabó, Tímea

    2015-03-01

    The shape of fragments generated by the breakup of solids is central to a wide variety of problems ranging from the geomorphic evolution of boulders to the accumulation of space debris orbiting Earth. Although the statistics of the mass of fragments has been found to show a universal scaling behavior, the comprehensive characterization of fragment shapes still remained a fundamental challenge. We performed a thorough experimental study of the problem fragmenting various types of materials by slowly proceeding weathering and by rapid breakup due to explosion and hammering. We demonstrate that the shape of fragments obeys an astonishing universality having the same generic evolution with the fragment size irrespective of materials details and loading conditions. There exists a cutoff size below which fragments have an isotropic shape, however, as the size increases an exponential convergence is obtained to a unique elongated form. We show that a discrete stochastic model of fragmentation reproduces both the size and shape of fragments tuning only a single parameter which strengthens the general validity of the scaling laws. The dependence of the probability of the crack plan orientation on the linear extension of fragments proved to be essential for the shape selection mechanism.

  12. Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments

    PubMed Central

    Schrum, Adam G.

    2015-01-01

    Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacokinetic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding; 2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig

  13. Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex.

    PubMed

    Lala, Puloma; Higashiyama, Tadayoshi; Erman, Mary; Griswold, Jennifer; Wagner, Traci; Osawa, Yoshio; Ghosh, Debashis

    2004-03-01

    Human cytochrome P450 aromatase (P450arom) is responsible for biosynthesis of estrogens from androgens. Monoclonal antibody MAb3-2C2 to P450arom specifically binds to a conformational epitope and suppresses the enzyme activity in a dose-dependent manner. The crystal structure of the Fab fragment of MAb3-2C2 has been used to engineer a recombinant single chain antibody fragment (scFv) and a homodimeric variable domain of the light chain (VL(2)). These recombinant antibody fragments have been expressed in Escherichia coli and purified. Here, we show that the recombinant scFv suppresses P450arom activity with an IC(50) value similar to that of natural MAb3-2C2 F(ab')(2). The recombinant VL(2) also exhibits dose-dependent suppression of the P450arom activity, but at a reduced level, demonstrating that the homodimer is unable to fully mimic the complementarity determining region (CDR) of a variable heavy chain (VH)-VL heterodimer. We prepare and purify a stable complex of P450arom with MAb3-2C2 F(ab')(2) and show that the complex migrates and precipitates as a single molecular assembly. Efforts to crystallize P450arom for structure-function studies have yielded small single crystals. Our results suggest that formation of stable complexes with fragments of the monoclonal antibody could provide an alternative method for crystallization of P450arom.

  14. Preexisting Antibodies to an F(ab')2 Antibody Therapeutic and Novel Method for Immunogenicity Assessment.

    PubMed

    Ruppel, Jane; Brady, Ann; Elliott, Rebecca; Leddy, Cecilia; Palencia, Marco; Coleman, Daniel; Couch, Jessica A; Wakshull, Eric

    2016-01-01

    Anti-therapeutic antibodies (ATAs) may impact drug exposure and activity and induce immune complex mediated toxicity; therefore the accurate measurement of ATA is important for the analysis of drug safety and efficacy. Preexisting ATAs to the hinge region of anti-Delta like ligand 4 (anti-DLL4) F(ab')2, a potential antitumor therapeutic, were detected in cynomolgus monkey serum, which presented a challenge in developing assays for detecting treatment induced ATA. A total ATA assay was developed using a bridging ELISA that detected both anti-CDR and anti-framework ATA including anti-hinge reactivity. A competition assay that could detect 500 ng/mL of anti-CDR ATA in the presence of preexisting ATA was also developed to determine ATA specific to the anti-DLL4 F(ab')2 CDR using anti-DLL4 F(ab')2 and a control F(ab')2. We used these assay methods in a cynomolgus monkey in vivo study to successfully evaluate total and anti-CDR ATA. The preexisting anti-hinge reactivity was also observed to a lesser extent in human serum, and a similar approach could be applied for specific immunogenicity assessment in clinical trials.

  15. Quench-condensing superconducting thin films using the Fab on a Chip approach

    NASA Astrophysics Data System (ADS)

    Han, Han; Imboden, Matthias; Del Corro, Pablo; Stark, Thomas; Lally, Richard; Pardo, Flavio; Bolle, Cristian; Bishop, David

    Micro-electromechanical systems (MEMS) being manufactured in a macroscopic fab inspires the idea of getting the process further down to fabricate even smaller structures, namely nano-structures, using MEMS. The Fab on a Chip concept was proposed based on such ideas. By implementing the final-step, additive fabrication approach, manufacturing, characterization and experiments of nano-structures are integrated in-situ. Due to the miniature size of MEMS, the thickness precision is significantly improved while the power consumption is significantly depressed, making the quench-condensation of very thin films well controlled and easily achievable. Among various types of nano-structures, quench-condensed superconducting thin films are of great interest for physicists. Here we present such experiments done on superconducting thin films quench-condensed using the Fab on a Chip. We show that we are able to fabricate very thin films with its thickness precisely controlled, and the base temperature kept under ~3K during the process. The resistivity data demonstrates the high purity and uniformity of the film, as well as the annealing effect when cycling to higher temperatures. Based on the tremendous results obtained from the superconducting thin films, more complex nano-circuits can be fabricated and investigated using the Fab on a Chip, enabling a new approach for novel condensed matter physics experiments. This research is funded by the NSF through their CMMI division. This research is funded by the NSF through their CMMI division.

  16. Nurturing Creativity and Innovation through FabKids: A Case Study

    ERIC Educational Resources Information Center

    Beyers, Ronald Noel

    2010-01-01

    This paper will report on a case study that was conducted involving Grade 10 learners who were exposed to a high-tech rapid-prototyping environment of a Fabrication Laboratory as part of a FabKids experience. This project must be viewed in the context of a global shortage of key skills placing a higher priority on the initiation and development of…

  17. Part I, FAB evaluation & application trials AFUE measurements: Part II, Integrated heating system (IHS) development

    SciTech Connect

    Leigh, R.W.; Fisher, L.

    1996-07-01

    An oil burner/boiler efficiency test stand has been set up in the BNL oil heat laboratory which can measure the Annual Fuel Utilization Efficiency (AFUE) of burner/boiler combinations in accordance with ASHRAE and DOE standards. Measurements include both steady state efficiencies and heat-up and cool-down characteristics so that cycling effects can be included in an estimate of seasonal average performance. In addition to AFUE measurements, the direct conversion of fuel energy content to enthalpy increase in the boiler water is monitored. The system is largely automated, with most control functions under computer control and data taken electronically and permanently recorded on disks for future reference. To date, a retention-head burner and a fan atomized burner (FAB) have been tested in a steel boiler, the latter operating at two different fuel flow rates. The results are presented below, and verify that the very tight construction of the FAB`s fan results in a significant decrease in off-cycle sensible heat losses. Tests were also performed on a center-flue water heater fired with a conventional retention-head burner and with an FAB. The tests conformed to DOE standard procedures for hot water heaters, and the results are discussed below.

  18. Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

    PubMed Central

    Bzymek, Krzysztof P.; Ma, Yuelong; Avery, Kendra A.; Horne, David A.; Williams, John C.

    2016-01-01

    Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic ‘pocket’ and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex. PMID:27303895

  19. Impact of Fab Lab Tulsa on Student Self-Efficacy toward STEM Education

    ERIC Educational Resources Information Center

    Dubriwny, Nicholas; Pritchett, Nathan; Hardesty, Michelle; Hellman, Chan M.

    2016-01-01

    Student self-confidence is important to any attempt to increase interest and achievement in Science, Technology, Engineering, and Math (STEM) education. This study presents a longitudinal examination of Fab Lab Tulsa's impact on attitude and self-efficacy toward STEM education among middle-school aged students. Paired samples t-test showed a…

  20. Fragmentation properties of metals

    SciTech Connect

    Grady, D.E.; Kipp, M.E.

    1996-06-01

    In the present study we are developing an experimental fracture material property test method specific to dynamic fragmentation. Spherical test samples of the metals of interest are subjected to controlled impulsive stress loads by acceleration to high velocities with a light-gas launcher facility and subsequent normal impact on thin plates. Motion, deformation and fragmentation of the test samples are diagnosed with multiple flash radiography methods. The impact plate materials are selected to be transparent to the x-ray method so that only test metal material is imaged. Through a systematic series of such tests, both strain-to-failure and fragmentation resistance properties are determined through this experimental method. Fragmentation property data for several steels, copper, aluminum, tantalum and titanium have been obtained to date. Aspects of the dynamic data have been analyzed with computational methods to achieve a better understanding of the processes leading to failure and fragmentation, and to test an existing computational fragmentation model.

  1. Loss-of-function and gain-of-function mutations in FAB1A/B impair endomembrane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis.

    PubMed

    Hirano, Tomoko; Matsuzawa, Tomohiko; Takegawa, Kaoru; Sato, Masa H

    2011-02-01

    In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P(2) from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B in fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-green fluorescent proteins localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis.

  2. FabQ, a dual-function dehydratase/isomerase, circumvents the last step of the classical fatty acid synthesis cycle.

    PubMed

    Bi, Hongkai; Wang, Haihong; Cronan, John E

    2013-09-19

    In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynthesis. Most strikingly, FabQ in combination with E. coli FabB imparts the surprising ability to bypass reduction of the trans-2-acyl-ACP intermediates of classical fatty acid synthesis. FabQ allows elongation by progressive isomerization reactions to form the polyunsaturated fatty acid, 3-hydroxy-cis-5, 7-hexadecadienoic acid, both in vitro and in vivo. FabQ therefore provides a potential pathway for bacterial synthesis of polyunsaturated fatty acids.

  3. FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

    PubMed Central

    Bi, Hongkai; Wang, Haihong; Cronan, John E.

    2015-01-01

    SUMMARY In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynthesis. Most strikingly, FabQ in combination with E. coli FabB imparts the surprising ability to bypass reduction of the trans-2-acyl-ACP intermediates of classical fatty acid synthesis. FabQ allows elongation by progressive isomerization reactions to form the polyunsaturated fatty acid, 3-hydroxy-cis-5, 7-hexadecadienoic acid, both in vitro and in vivo. FabQ therefore provides a potential pathway for bacterial synthesis of polyunsaturated fatty acids. PMID:23972938

  4. Crystallization and preliminary X-ray crystallographic analysis of enoyl-ACP reductase III (FabL) from Bacillus subtilis

    SciTech Connect

    Kim, Kook-Han; Park, Joon Kyu; Ha, Byung Hak; Moon, Jin Ho; Kim, Eunice EunKyeong

    2007-03-01

    Enoyl-ACP reductase III (FabL) from B. subtilis has been overexpressed, purified and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120°, and data were collected to 2.5 Å resolution using synchrotron radiation. Enoyl-[acyl-carrier protein] reductase (enoyl-ACP reductase; ENR) is a key enzyme in type II fatty-acid synthase that catalyzes the last step in each elongation cycle. It has been considered as an antibiotic target since it is an essential enzyme in bacteria. However, recent studies indicate that some pathogens have more than one ENR. Bacillus subtilis is reported to have two ENRs, namely BsFabI and BsFabL. While BsFabI is similar to other FabIs, BsFabL shows very little sequence similarity and is NADPH-dependent instead of NADH-dependent as in the case of FabI. In order to understand these differences on a structural basis, BsFabL has been cloned, expressed and and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120° and one molecule of FabL in the asymmetric unit. Data were collected using synchrotron radiation (beamline 4A at the Pohang Light Source, Korea). The crystal diffracted to 2.5 Å resolution.

  5. FabSim: Facilitating computational research through automation on large-scale and distributed e-infrastructures

    NASA Astrophysics Data System (ADS)

    Groen, Derek; Bhati, Agastya P.; Suter, James; Hetherington, James; Zasada, Stefan J.; Coveney, Peter V.

    2016-10-01

    We present FabSim, a toolkit developed to simplify a range of computational tasks for researchers in diverse disciplines. FabSim is flexible, adaptable, and allows users to perform a wide range of tasks with ease. It also provides a systematic way to automate the use of resources, including HPC and distributed machines, and to make tasks easier to repeat by recording contextual information. To demonstrate this, we present three use cases where FabSim has enhanced our research productivity. These include simulating cerebrovascular bloodflow, modelling clay-polymer nanocomposites across multiple scales, and calculating ligand-protein binding affinities.

  6. Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction

    NASA Astrophysics Data System (ADS)

    Zhang, Ying; Cui, Weidong; Wecksler, Aaron T.; Zhang, Hao; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2016-07-01

    Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis.

  7. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

    PubMed Central

    Yin, Gang; Garces, Eudean D; Yang, Junhao; Zhang, Juan; Tran, Cuong; Steiner, Alexander R; Roos, Christine; Bajad, Sunil; Hudak, Susan; Penta, Kalyani; Zawada, James; Pollitt, Sonia

    2012-01-01

    We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics. PMID:22377750

  8. Fragments and Coherence

    ERIC Educational Resources Information Center

    Watson, Anne

    2008-01-01

    Can teachers contact the inner coherence of mathematics while working in a context fragmented by always-new objectives, criteria, and initiatives? How, more importantly, can learners experience the inner coherence of mathematics while working in a context fragmented by testing, modular curricular, short-term learning objectives, and lessons that…

  9. Fragment Hazard Investigation Program

    DTIC Science & Technology

    1978-10-01

    53 Ballistic Density (k) ............................................. 53 Ejection A ngle (a...54 Ejection Velocity (V) ................................................. 54 DEVELOPMENT OF EMPIRICAL RELATION...5S 54 Fragment Weight Versus Gamma for Test QD-155-08 ......................... 56 55 Fragment Range Versus Ejection Angle as a Function of

  10. Fragmentation of fullerenes

    NASA Astrophysics Data System (ADS)

    Chancey, Ryan T.; Oddershede, Lene; Harris, Frank E.; Sabin, John R.

    2003-04-01

    We have performed classical molecular-dynamics simulations of the fragmentation collisions of neutral fullerenes (C24, C60, C100, and C240) with a hard wall. The interactions between the carbon atoms are modeled by a Tersoff potential and the position of each carbon atom at each time step is calculated using a sixth-order predictor-corrector method. The statistical distribution of the fragments depends on impact energy. At low energies, the fragment distribution appears symmetric, with both the large and small fragment distributions well fitted by an exponential function of the same exponent, the value of which decreases with impact energy. At intermediate energies, the distribution of the smallest fragments can be fitted equally well by a power law or an exponential function. At high impact energies, the entire fragmentation pattern is well described by a single exponential function, the exponent increasing with energy. The observed tendencies in fragment distributions as well as the obtained exponents are in agreement with experimental observations. The fragmentation behavior of the four investigated fullerenes is very similar, and it is noted that C60 appears to be the most stable.

  11. Effect of size of radiolabeled antibody and fragments on tumor uptake and distribution in nephrectomized mice

    SciTech Connect

    Halpern, S.E.; Buchegger, F.; Schreyer, M.; Mach, J.P.

    1984-01-01

    The importance of molecular size in tumor (T) uptake of intact monoclonal antibody (MAb) of MAb fragments (frag.) is difficult to assess because frag. are excreted by the kidney. To obviate this problem nephrectomized nude mice (M) bearing carcinoembryonic (CEA) secreting human colon (T) were used in the following experiments. Following nephrectomy 3 groups of M were injected intravenously with intact In-111 anti-CEA MAb and simultaneously with I-125 intact, F(ab')2 or Fab anti-CEA-MAb. Iodination was by chloramine T and In-111 labeling by bifunctional chelation. All M were killed 8 h after injection, T and normal tissues (NT) dissected, weighed, and counted against a standard of the injectate. The distribution of intact I-125 and In-111-MAb were nearly identical allowing In-111-MAb to be used for comparison with the I-125 frag. T concentration (conc.) of I-125-F(ab')2 were 25% greater and L conc. were 63% lower than for intact In-111-MAb. T conc. of I-125-Fab were 86% greater and L conc. 64% lower than for intact In-111-MAb. Large T, which produce higher serum CEA levels, increased the L conc. of intact MAb but not I-125 frag. The authors conclude that when renal excretion is prevented there is (a) an inverse relationship between size of a MAb moiety and its T conc. indicating that the improved T/NT ratios observed with frag. are not due only to renal excretion, and (b) the Fc portion of the MAb appears to be critical for L uptake of MAb or immune complexes.

  12. Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab)

    PubMed Central

    Wanczyk, Heather; Barker, Tolga; Rood, Debra; Zapata, Daniel I.; Howell, Amy R.; Richardson, Stewart K.; Zinckgraf, John; Marusov, Gregory P.; Lynes, Michael A.; Silbart, Lawrence K.

    2013-01-01

    Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers. PMID:23518474

  13. Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense.

    PubMed

    Crottet, P; Corthésy, B

    1998-11-15

    Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

  14. Structure of an anti-DNA fab complexed with a non-DNA ligand provides insights into cross-reactivity and molecular mimicry.

    PubMed

    Schuermann, Jonathan P; Henzl, Michael T; Deutscher, Susan L; Tanner, John J

    2004-11-01

    Antibodies that recognize DNA (anti-DNA) are part of the autoimmune response underlying systemic lupus erythematosus. To better understand molecular recognition by anti-DNA antibodies, crystallographic studies have been performed using an anti-ssDNA antigen-binding fragment (Fab) known as DNA-1. The previously determined structure of a DNA-1/dT5 complex revealed that thymine bases insert into a narrow groove, and that ligand recognition primarily involves the bases of DNA. We now report the 1.75-A resolution structure of DNA-1 complexed with the biological buffer HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid). All three light chain complementarity-determining regions (CDRs) and HCDR3 contribute to binding. The HEPES sulfonate hydrogen bonds to His L91, Asn L50, and to the backbone of Tyr H100 and Tyr H100A. The Tyr side-chains of L32, L92, H100, and H100A form nonpolar contacts with the HEPES ethylene and piperazine groups. Comparison to the DNA-1/dT5 structure reveals that the dual recognition of dT5 and HEPES requires a 13-A movement of HCDR3. This dramatic structural change converts the combining site from a narrow groove, appropriate for the edge-on insertion of thymine bases, to one sufficiently wide to accommodate the HEPES sulfonate and piperazine. Isothermal titration calorimetry verified the association of HEPES with DNA-1 under conditions similar those used for crystallization (2 M ammonium sulfate). Interestingly, the presence of 2 M ammonium sulfate increases the affinities of DNA-1 for both HEPES and dT5, suggesting that non-polar Fab-ligand interactions are important for molecular recognition in highly ionic solvent conditions. The structural and thermodynamic data suggest a molecular mimicry mechanism based on structural plasticity and hydrophobic interactions.

  15. Opaque rock fragments

    SciTech Connect

    Abhijit, B.; Molinaroli, E.; Olsen, J.

    1987-05-01

    The authors describe a new, rare, but petrogenetically significant variety of rock fragments from Holocene detrital sediments. Approximately 50% of the opaque heavy mineral concentrates from Holocene siliciclastic sands are polymineralic-Fe-Ti oxide particles, i.e., they are opaque rock fragments. About 40% to 70% of these rock fragments show intergrowth of hm + il, mt + il, and mt + hm +/- il. Modal analysis of 23,282 opaque particles in 117 polished thin sections of granitic and metamorphic parent rocks and their daughter sands from semi-arid and humid climates show the following relative abundances. The data show that opaque rock fragments are more common in sands from igneous source rocks and that hm + il fragments are more durable. They assume that equilibrium conditions existed in parent rocks during the growth of these paired minerals, and that the Ti/Fe ratio did not change during oxidation of mt to hm. Geothermometric determinations using electron probe microanalysis of opaque rock fragments in sand samples from Lake Erie and the Adriatic Sea suggest that these rock fragments may have equilibrated at approximately 900/sup 0/ and 525/sup 0/C, respectively.

  16. Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of [beta]-hydroxyacyl-ACP dehydratase (FabZ)

    SciTech Connect

    Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2009-08-14

    Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence

  17. Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

    NASA Technical Reports Server (NTRS)

    Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

    1990-01-01

    A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

  18. Fragmentation of monoclonal antibodies

    PubMed Central

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  19. Universality in Fragmentation

    NASA Astrophysics Data System (ADS)

    Åström, J. A.; Holian, B. L.; Timonen, J.

    2000-04-01

    Fragmentation of a two-dimensional brittle solid by impact and ``explosion,'' and a fluid by ``explosion'' are all shown to become critical. The critical points appear at a nonzero impact velocity, and at infinite explosion duration, respectively. Within the critical regimes, the fragment-size distributions satisfy a scaling form qualitatively similar to that of the cluster-size distribution of percolation, but they belong to another universality class. Energy balance arguments give a correlation length exponent that is exactly one-half of its percolation value. A single crack dominates fragmentation in the slow-fracture limit, as expected.

  20. From triclosan toward the clinic: discovery of nonbiocidal, potent FabI inhibitors for the treatment of resistant bacteria.

    PubMed

    Gerusz, Vincent; Denis, Alexis; Faivre, Fabien; Bonvin, Yannick; Oxoby, Mayalen; Briet, Sophia; LeFralliec, Géraldine; Oliveira, Chrystelle; Desroy, Nicolas; Raymond, Cédric; Peltier, Laëtitia; Moreau, François; Escaich, Sonia; Vongsouthi, Vanida; Floquet, Stéphanie; Drocourt, Elodie; Walton, Armelle; Prouvensier, Laure; Saccomani, Marc; Durant, Lionel; Genevard, Jean-Marie; Sam-Sambo, Vanessa; Soulama-Mouze, Coralie

    2012-11-26

    In this paper, we present some elements of our optimization program to decouple triclosan's specific FabI effect from its nonspecific cytotoxic component. The implementation of this strategy delivered highly specific, potent, and nonbiocidal new FabI inhibitors. We also disclose some preclinical data of one of their representatives, 83, a novel antibacterial compound active against resistant staphylococci and some clinically relevant Gram negative bacteria that is currently undergoing clinical trials.

  1. Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

    DOE PAGES

    Donaldson, Joshua M.; Zer, Cindy; Avery, Kendra N.; ...

    2013-10-07

    Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximabmore » (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Lastly, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.« less

  2. A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo.

    PubMed

    Lin, Hong; Zhang, Huiling; Wang, Jun; Lu, Meiping; Zheng, Feng; Wang, Changjun; Tang, Xiaojun; Xu, Ning; Chen, Renjie; Zhang, Dawei; Zhao, Ping; Zhu, Jin; Mao, Yuan; Feng, Zhenqing

    2014-03-01

    Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer.

  3. Fragmentation in Biaxial Tension

    SciTech Connect

    Campbell, G H; Archbold, G C; Hurricane, O A; Miller, P L

    2006-06-13

    We have carried out an experiment that places a ductile stainless steel in a state of biaxial tension at a high rate of strain. The loading of the ductile metal spherical cap is performed by the detonation of a high explosive layer with a conforming geometry to expand the metal radially outwards. Simulations of the loading and expansion of the metal predict strain rates that compare well with experimental observations. A high percentage of the HE loaded material was recovered through a soft capture process and characterization of the recovered fragments provided high quality data, including uniform strain prior to failure and fragment size. These data were used with a modified fragmentation model to determine a fragmentation energy.

  4. Family of Advanced Beyond Line-of-Sight Terminals (FAB-T)

    DTIC Science & Technology

    2015-12-01

    Selected Acquisition Report (SAR) RCS: DD-A&T(Q&A)823-199 Family of Advanced Beyond Line-of-Sight Terminals (FAB-T) As of FY 2017 President’s...Program Office Estimate RDT&E - Research, Development, Test, and Evaluation SAR - Selected Acquisition Report SCP - Service Cost Position TBD - To Be...the B-2, B-52, and select RC-135 aircraft and will not provide satellite control or PNVC functionality. The initial installation and integration is a

  5. Fabricating quench condensed lead thin film circuits using MEMS Fab on a Chip technology

    NASA Astrophysics Data System (ADS)

    Imboden, Matthias; Han, Han; Del Corro, Pablo; Pardo, Flavio; Bolle, Cristian; Bishop, David

    2015-03-01

    We have developed a MEMS Fab on a Chip consisting of micro-sources, mass sensors, heaters/thermometers, shutters and a dynamic stencil. The fab only occupies a volume of a few cubic millimeters and consumes milliwatts of power, and hence can be operated in a cryostat. Thin film patterns of arbitrary shapes using multiple materials can be manufactured, while strongly suppressing thermal annealing effects. We demonstrate deposition of quench condensed lead films with fractions of a monolayer thickness control. Furthermore, using low deposition rates it is estimated that the surface temperature of the target heats by only 1.7 K. We study the effects of growing quench condensed films with different evaporation rates to demonstrate thermal annealing effects which occur during deposition. We measure the minimum conduction thickness (insulator to metal transition) as well as the superconducting transition temperature as a function of film thickness in order to shed light on growth of amorphous films and the transition to nanocluster formations. The Fab on a Chip will allow us to build nanocircuits made of ultra-thin materials. Annealing and doping is controlled and measurements occur in situ, without exposing the fabricated circuits to thermal fluctuations or foreign contaminants. This enables new types of experiments based on quantum circuits which cannot be fabricated using standard lithography techniques.

  6. Acute myocardial infarct imaging with indium-111-labeled monoclonal antimyosin Fab

    SciTech Connect

    Khaw, B.A.; Yasuda, T.; Gold, H.K.; Leinbach, R.C.; Johns, J.A.; Kanke, M.; Barlai-Kovach, M.; Strauss, H.W.; Haber, E.

    1987-11-01

    Indium-111 monoclonal antimyosin Fab scintigraphy was used to detect myocardial necrosis in 52 of 54 patients (96.3%) with acute myocardial infarction. Infarcts were visualized when coronary arteries were persistently occluded (n = 10), became patent after thrombolysis (n = 33), or became patent after spontaneous reperfusion (n = 7). Posteroinferolateral visualizations were obtained in two patients with clinical and enzymatic evidence of infarction but normal electrocardiograms. Of the two patients in whom no infarcts were visualized, one had an anterior myocardial infarct. This patient underwent successful thrombolytic therapy, with attendant minimization of creatine kinase release. The other patient had a small, nonreperfused inferior myocardial infarct. Five patients with a history of remote infarction and acute necrosis showed antimyosin uptake only in regions concordant with the acute episodes of infarction, and radiolabeled antimyosin Fab localized in neither old infarcts nor normal, noninfarcted myocardium. Antimyosin Fab scintigraphy, thus, appears to be a highly specific means of delineating necrotic myocardium, at least in this limited and selected group of patients.

  7. An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.

    PubMed

    Demissie, Robel D; Kabre, Pauline; Tuntland, Micheal L; Fung, Leslie W-M

    2016-04-01

    Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

  8. Production of a single-chain fragment of the murine anti-idiotypic antibody ACA125 as phage-displayed and soluble antibody by recombinant phage antibody technique.

    PubMed

    Schlebusch, H; Reinartz, S; Kaiser, R; Grünn, U; Wagner, U

    1997-02-01

    The F(ab')2 fragment of the murine monoclonal anti-idiotypic antibody ACA125 mimicking the tumor-associated antigen CA125 is used as a vaccine for the induction of an anti-tumoral immunity in patients with ovarian carcinoma. We tried to generate a single-chain fragment (ScFv) composed of ACA125 heavy- and light-chain variable domains connected by a polypeptide linker as an alternative to the corresponding F(ab')2 fragment. Heavy- and light-chain genes of antibody-producing mouse hybridoma cell line were amplified separately and assembled into a ScFv gene with linker DNA by the polymerase chain reaction (PCR). The ScFv gene was ligated into the phagemid vector pCANTAB5E, which allows the production of both phage-displayed and soluble ScFv. Transformed Escherichia coli TG1 cells were infected with M13K07 helper phage to yield recombinant phage, which display ScFv fragments as a g3p fusion protein on the surface of the filamentous phage M13. Recombinant phages could be selected by binding to the idiotypic antibody OC125 after one round of panning and directly used to reinfect E. coli TG1 cells. The E. coli nonsuppressor strain HB2151 was infected with an antigen-positive phage clone, previously screened by enzyme-linked immunosorbent assay (ELISA), to express soluble ScFv fragments. Functional soluble ScFv binding to the idiotypic antibody OC125 F(ab')2 could be detected in the bacterial periplasm by Western blot and ELISA. The variable heavy- and light-chain genes of the ACA125 ScFv fragment were further sequenced and compared with known antibody sequences.

  9. Yeast vacuoles fragment in an asymmetrical two-phase process with distinct protein requirements.

    PubMed

    Zieger, Martin; Mayer, Andreas

    2012-09-01

    Yeast vacuoles fragment and fuse in response to environmental conditions, such as changes in osmotic conditions or nutrient availability. Here we analyze osmotically induced vacuole fragmentation by time-lapse microscopy. Small fragmentation products originate directly from the large central vacuole. This happens by asymmetrical scission rather than by consecutive equal divisions. Fragmentation occurs in two distinct phases. Initially, vacuoles shrink and generate deep invaginations that leave behind tubular structures in their vicinity. Already this invagination requires the dynamin-like GTPase Vps1p and the vacuolar proton gradient. Invaginations are stabilized by phosphatidylinositol 3-phosphate (PI(3)P) produced by the phosphoinositide 3-kinase complex II. Subsequently, vesicles pinch off from the tips of the tubular structures in a polarized manner, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol-3,5-bisphosphate and the Fab1 complex. It is accelerated by the PI(3)P- and phosphatidylinositol 3,5-bisphosphate-binding protein Atg18p. Thus vacuoles fragment in two steps with distinct protein and lipid requirements.

  10. Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    SciTech Connect

    Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.; Grooms, Michael; Daines, Robert A.; Lonsdale, John T.; Khandekar, Sanjay S.

    2010-07-20

    {beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

  11. [One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity].

    PubMed

    Liu, Yin-Xing; Xiong, Dong-Sheng; Fan, Dong-Mei; Shao, Xiao-Feng; Xu, Yuan-Fu; Zhu, Zhen-Ping; Yang, Chun-Zheng

    2003-05-01

    Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.

  12. IMPACT fragmentation model developments

    NASA Astrophysics Data System (ADS)

    Sorge, Marlon E.; Mains, Deanna L.

    2016-09-01

    The IMPACT fragmentation model has been used by The Aerospace Corporation for more than 25 years to analyze orbital altitude explosions and hypervelocity collisions. The model is semi-empirical, combining mass, energy and momentum conservation laws with empirically derived relationships for fragment characteristics such as number, mass, area-to-mass ratio, and spreading velocity as well as event energy distribution. Model results are used for several types of analysis including assessment of short-term risks to satellites from orbital altitude fragmentations, prediction of the long-term evolution of the orbital debris environment and forensic assessments of breakup events. A new version of IMPACT, version 6, has been completed and incorporates a number of advancements enabled by a multi-year long effort to characterize more than 11,000 debris fragments from more than three dozen historical on-orbit breakup events. These events involved a wide range of causes, energies, and fragmenting objects. Special focus was placed on the explosion model, as the majority of events examined were explosions. Revisions were made to the mass distribution used for explosion events, increasing the number of smaller fragments generated. The algorithm for modeling upper stage large fragment generation was updated. A momentum conserving asymmetric spreading velocity distribution algorithm was implemented to better represent sub-catastrophic events. An approach was developed for modeling sub-catastrophic explosions, those where the majority of the parent object remains intact, based on estimated event energy. Finally, significant modifications were made to the area-to-mass ratio distribution to incorporate the tendencies of different materials to fragment into different shapes. This ability enabled better matches between the observed area-to-mass ratios and those generated by the model. It also opened up additional possibilities for post-event analysis of breakups. The paper will discuss

  13. Target fragmentation in radiobiology

    NASA Technical Reports Server (NTRS)

    Wilson, John W.; Cucinotta, Francis A.; Shinn, Judy L.; Townsend, Lawrence W.

    1993-01-01

    Nuclear reactions in biological systems produce low-energy fragments of the target nuclei seen as local high events of linear energy transfer (LET). A nuclear-reaction formalism is used to evaluate the nuclear-induced fields within biosystems and their effects within several biological models. On the basis of direct ionization interaction, one anticipates high-energy protons to have a quality factor and relative biological effectiveness (RBE) of unity. Target fragmentation contributions raise the effective quality factor of 10 GeV protons to 3.3 in reasonable agreement with RBE values for induced micronuclei in bean sprouts. Application of the Katz model indicates that the relative increase in RBE with decreasing exposure observed in cell survival experiments with 160 MeV protons is related solely to target fragmentation events. Target fragment contributions to lens opacity given an RBE of 1.4 for 2 GeV protons in agreement with the work of Lett and Cox. Predictions are made for the effective RBE for Harderian gland tumors induced by high-energy protons. An exposure model for lifetime cancer risk is derived from NCRP 98 risk tables, and protraction effects are examined for proton and helium ion exposures. The implications of dose rate enhancement effects on space radiation protection are considered.

  14. The Fragmentation of Learning.

    ERIC Educational Resources Information Center

    Downes, Stephen

    2001-01-01

    Information and communication technologies, especially the Internet, have vastly increased access to information and educational opportunities. Steadily increasing consumer demand is driving the development of online educational materials. The end result may be a "fragmentation" of learning involving multiple learning providers and delivery modes,…

  15. Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA

    PubMed Central

    Finzel, Kara; Nguyen, Chi; Jackson, David R.; Gupta, Aarushi; Tsai, Shiou-Chuan; Burkart, Michael D.

    2015-01-01

    Summary Microbial fatty acid biosynthetic enzymes are important targets for areas as diverse as antibiotic development to biofuel production. Elucidating the molecular basis of chain length control during fatty acid biosynthesis is crucial for the understanding of regulatory processes of this fundamental metabolic pathway. In Escherichia coli, the acyl carrier protein (AcpP) plays a central role by sequestering and shuttling the growing acyl chain between fatty acid biosynthetic enzymes. FabA, a β-hydroxylacyl-AcpP dehydratase, is an important enzyme in controlling fatty acid chain length and saturation levels. FabA-AcpP interactions are transient in nature and thus difficult to visualize. In this study, four mechanistic crosslinking probes mimicking varying acyl chain lengths were synthesized to systematically probe for modified chain length specificity of fourteen FabA mutants. These studies provide evidence for the AcpP interacting “positive patch,” FabA mutations that altered substrate specificity, and the roles that the FabA “gating residues” play in chain-length selection. PMID:26526101

  16. Deletion of fabN in Enterococcus faecalis results in unsaturated fatty acid auxotrophy and decreased release of inflammatory cytokines.

    PubMed

    Diederich, Ann-Kristin; Duda, Katarzyna A; Romero-Saavedra, Felipe; Engel, Regina; Holst, Otto; Huebner, Johannes

    2016-05-01

    The Gram-positive bacterium Enterococcus faecalis can cause life-threatening infections and is resistant to several commonly used antibiotics. The type II fatty acid pathway in bacteria is discussed as a potential target for antimicrobial therapy. However, it was shown that inhibition or deletion of its enzymes can be rescued in Gram-positive bacteria by supplementation with fatty acids. Here we show that by deletion of the fabN gene, which is essential for unsaturated fatty acid (UFA) synthesis in E. faecalis, growth is impaired but can be rescued by supplementation with oleic acid or human serum. Nonetheless, we demonstrate alterations of the UFA profile after supplementation with oleic acid in the ΔfabN mutant using a specific glycolipid. In addition, we demonstrate that cytokine release in vitro is almost abolished after stimulation of mouse macrophages by the mutant in comparison to the wild type. The results indicate that fabN is not a suitable target for antimicrobials as UFA auxotrophy can be overcome. However, deletion of fabN resulted in a decreased inflammatory response indicating that fabN and resulting UFA synthesis are relevant for virulence.

  17. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    SciTech Connect

    Baum, Bernhard; Lecker, Laura S. M.; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N.; Brenk, Ruth

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  18. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    SciTech Connect

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  19. The FermiFab toolbox for fermionic many-particle quantum systems

    NASA Astrophysics Data System (ADS)

    Mendl, Christian B.

    2011-06-01

    This paper introduces the FermiFab toolbox for many-particle quantum systems. It is mainly concerned with the representation of (symbolic) fermionic wavefunctions and the calculation of corresponding reduced density matrices (RDMs). The toolbox transparently handles the inherent antisymmetrization of wavefunctions and incorporates the creation/annihilation formalism. Thus, it aims at providing a solid base for a broad audience to use fermionic wavefunctions with the same ease as matrices in Matlab, say. Leveraging symbolic computation, the toolbox can greatly simply tedious pen-and-paper calculations for concrete quantum mechanical systems, and serves as "sandbox" for theoretical hypothesis testing. FermiFab (including full source code) is freely available as a plugin for both Matlab and Mathematica. Program summaryProgram title:FermiFab Catalogue identifier: AEIN_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEIN_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Special license provided by the author No. of lines in distributed program, including test data, etc.: 1 165 461 No. of bytes in distributed program, including test data, etc.: 15 557 308 Distribution format: tar.gz Programming language: MATLAB 7.9, Mathematica 7.0, C Computer: PCs, Sun Solaris workstation Operating system: Any platform supporting MATLAB or Mathematica; tested with Windows (32 and 64 bit) and Sun Solaris. RAM: Case dependent Classification: 4.15 Nature of problem: Representation of fermionic wavefunctions, computation of RDMs (reduced density matrices) and handing of the creation/annihilation operator formalism. Solution method: Mapping of Slater determinants to bitfields, implementation of the creation/annihilation and RDM formalism by bit operations. Running time: Depends on the problem size; several seconds for the provided demonstration files.

  20. Statistical models of brittle fragmentation

    NASA Astrophysics Data System (ADS)

    Åström, J. A.

    2006-06-01

    Recent developments in statistical models for fragmentation of brittle material are reviewed. The generic objective of these models is understanding the origin of the fragment size distributions (FSDs) that result from fracturing brittle material. Brittle fragmentation can be divided into two categories: (1) Instantaneous fragmentation for which breakup generations are not distinguishable and (2) continuous fragmentation for which generations of chronological fragment breakups can be identified. This categorization becomes obvious in mining industry applications where instantaneous fragmentation refers to blasting of rock and continuous fragmentation to the consequent crushing and grinding of the blasted rock fragments. A model of unstable cracks and crack-branch merging contains both of the FSDs usually related to instantaneous fragmentation: the scale invariant FSD with the power exponent (2-1/D) and the double exponential FSD which relates to Poisson process fragmentation. The FSDs commonly related to continuous fragmentation are: the lognormal FSD originating from uncorrelated breakup and the power-law FSD which can be modeled as a cascade of breakups. Various solutions to the generic rate equation of continuous fragmentation are briefly listed. Simulations of crushing experiments reveal that both cascade and uncorrelated fragmentations are possible, but that also a mechanism of maximizing packing density related to Apollonian packing may be relevant for slow compressive crushing.

  1. Xanthomonas campestris FabH is required for branched-chain fatty acid and DSF-family quorum sensing signal biosynthesis

    PubMed Central

    Yu, Yong-Hong; Hu, Zhe; Dong, Hui-Juan; Ma, Jin-Cheng; Wang, Hai-Hong

    2016-01-01

    Xanthomonas campestris pv. campestris (Xcc), a Gram-negative phytopathogenic bacterium, causes black rot disease of cruciferous vegetables. Although Xcc has a complex fatty acid profile comprised of straight-chain fatty acids and branched-chain fatty acids (BCFAs), and encodes a complete set of genes required for fatty acid synthesis, there is still little known about the mechanism of BCFA synthesis. We reported that expression of Xcc fabH restores the growth of Ralstonia solanacearum fabH mutant, and this allows the R. solanacearum fabH mutant to produce BCFAs. Using in vitro assays, we demonstrated that Xcc FabH is able to condense branched-chain acyl-CoAs with malonyl-ACP to initiate BCFA synthesis. Moreover, although the fabH gene is essential for growth of Xcc, it can be replaced with Escherichia coli fabH, and Xcc mutants failed to produce BCFAs. These results suggest that Xcc does not have an obligatory requirement for BCFAs. Furthermore, Xcc mutants lost the ability to produce cis-11-methyl-2-dodecenoic acid, a diffusible signal factor (DSF) required for quorum sensing of Xcc, which confirms that the fatty acid synthetic pathway supplies the intermediates for DSF signal biosynthesis. Our study also showed that replacing Xcc fabH with E. coli fabH affected Xcc pathogenesis in host plants. PMID:27595587

  2. Multivariate PLS Modeling of Apicomplexan FabD-Ligand Interaction Space for Mapping Target-Specific Chemical Space and Pharmacophore Fingerprints

    PubMed Central

    Surolia, Avadhesha

    2015-01-01

    Biomolecular recognition underlying drug-target interactions is determined by both binding affinity and specificity. Whilst, quantification of binding efficacy is possible, determining specificity remains a challenge, as it requires affinity data for multiple targets with the same ligand dataset. Thus, understanding the interaction space by mapping the target space to model its complementary chemical space through computational techniques are desirable. In this study, active site architecture of FabD drug target in two apicomplexan parasites viz. Plasmodium falciparum (PfFabD) and Toxoplasma gondii (TgFabD) is explored, followed by consensus docking calculations and identification of fifteen best hit compounds, most of which are found to be derivatives of natural products. Subsequently, machine learning techniques were applied on molecular descriptors of six FabD homologs and sixty ligands to induce distinct multivariate partial-least square models. The biological space of FabD mapped by the various chemical entities explain their interaction space in general. It also highlights the selective variations in FabD of apicomplexan parasites with that of the host. Furthermore, chemometric models revealed the principal chemical scaffolds in PfFabD and TgFabD as pyrrolidines and imidazoles, respectively, which render target specificity and improve binding affinity in combination with other functional descriptors conducive for the design and optimization of the leads. PMID:26535573

  3. The frontal assessment battery (FAB) reveals neurocognitive dysfunction in substance-dependent individuals in distinct executive domains: Abstract reasoning, motor programming, and cognitive flexibility.

    PubMed

    Cunha, Paulo Jannuzzi; Nicastri, Sergio; de Andrade, Arthur Guerra; Bolla, Karen I

    2010-10-01

    Substance-dependence is highly associated with executive cognitive function (ECF) impairments. However, considering that it is difficult to assess ECF clinically, the aim of the present study was to examine the feasibility of a brief neuropsychological tool (the Frontal Assessment Battery - FAB) to detect specific ECF impairments in a sample of substance-dependent individuals (SDI). Sixty-two subjects participated in this study. Thirty DSM-IV-diagnosed SDI, after 2weeks of abstinence, and 32 healthy individuals (control group) were evaluated with FAB and other ECF-related tasks: digits forward (DF), digits backward (DB), Stroop Color Word Test (SCWT), and Wisconsin Card Sorting Test (WCST). SDI did not differ from the control group on sociodemographic variables or IQ. However, SDI performed below the controls in DF, DB, and FAB. The SDI were cognitively impaired in 3 of the 6 cognitive domains assessed by the FAB: abstract reasoning, motor programming, and cognitive flexibility. The FAB correlated with DF, SCWT, and WCST. In addition, some neuropsychological measures were correlated with the amount of alcohol, cannabis, and cocaine use. In conclusion, SDI performed more poorly than the comparison group on the FAB and the FAB's results were associated with other ECF-related tasks. The results suggested a negative impact of alcohol, cannabis, and cocaine use on the ECF. The FAB may be useful in assisting professionals as an instrument to screen for ECF-related deficits in SDI.

  4. Fragmentation of Fractal Random Structures

    NASA Astrophysics Data System (ADS)

    Elçi, Eren Metin; Weigel, Martin; Fytas, Nikolaos G.

    2015-03-01

    We analyze the fragmentation behavior of random clusters on the lattice under a process where bonds between neighboring sites are successively broken. Modeling such structures by configurations of a generalized Potts or random-cluster model allows us to discuss a wide range of systems with fractal properties including trees as well as dense clusters. We present exact results for the densities of fragmenting edges and the distribution of fragment sizes for critical clusters in two dimensions. Dynamical fragmentation with a size cutoff leads to broad distributions of fragment sizes. The resulting power laws are shown to encode characteristic fingerprints of the fragmented objects.

  5. Structure of FabH and factors affecting the distribution of branched fatty acids in Micrococcus luteus

    SciTech Connect

    Pereira, Jose H.; Goh, Ee-Been; Keasling, Jay D.; Beller, Harry R.; Adams, Paul D.

    2012-10-01

    In an effort to better understand the control of the formation of branched fatty acids in Micrococcus luteus, the structure of β-ketoacyl-ACP synthase III, which catalyzes the initial step of fatty-acid biosynthesis, has been determined. Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or β-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition.

  6. Phage display and bacterial expression of a recombinant Fab specific for Pseudomonas aeruginosa serotype O6 lipopolysaccharide.

    PubMed Central

    Tout, N L; Lam, J S

    1997-01-01

    Immunotherapy with antibodies (Abs) against the lipopolysaccharide (LPS) of Pseudomonas aeruginosa remains an alternative to serotype-specific LPS-based vaccines due to their limited use and to antibiotics due to the intrinsic resistance to antimicrobials observed in P. aeruginosa. We have chosen a monoclonal Ab (MAb), MF23-1, that binds to the O antigen of the most clinically relevant serotype, IATS O6, for producing a recombinant antibody. Heavy (H) and light (L) chain genes were isolated from MF23-1 to form a functional Fab molecule in the periplasm of Escherichia coli and on the surface of phage by using phagemid vector pComb3. The entire kappa L chain gene was used, but the H chain gene was amplified to 2 amino acids past cysteine 128 which is involved in interchain disulfide bond formation with the L chain. The truncated H chain associated with the L chain in the periplasm of E. coli to form a functional Fab molecule that bound in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay to O6 LPS. Therefore, the remainder of the CH1 past cysteine 128 is not essential for stable formation of the Fab portion of MF23-1. This recombinant Fab (r-Fab) was shown to be specific for the LPS of the most predominant clinical isolate, serotype O6, while no cross-reactivity was detected to the LPS of the other 19 remaining serotypes. This r-Fab was also expressed on the surface of filamentous phage upon addition of helper phage to recombinant E. coli containing phagemid. Recombinant phage from clones MT13 and MT24 bound specifically to O6 LPS in ELISA. These results represent an important step toward the design of therapeutic Abs to be used against P. aeruginosa infections. PMID:9067648

  7. Pharmacokinetics of the toxic fraction of Centruroides limpidus limpidus venom in experimentally envenomed rabbits and effects of immunotherapy with specific F(ab')2.

    PubMed

    Calderón-Aranda, E S; Rivière, G; Choumet, V; Possani, L D; Bon, C

    1999-05-01

    Envenomations after scorpion stings are a major health problem throughout the world. Their specific treatment is immunotherapy which consists of the injection of specific antibody. In this article, we studied the pharmacokinetics of the toxic fraction of Centruroides limpidus limpidus venom (fraction II) in experimentally envenomed rabbits. After an intravenous injection, fraction II (FII) was rapidly distributed and eliminated from the body (terminal half-life of 1.9 h). When injected subcutaneously, high concentrations of FII were measured in the vascular space rapidly after the injection (Tmax = 1 h) and FII was eliminated with a terminal half-life of 1.8 h, close to that determined after intravenous injection. These observations go along with the rapid onset of clinical symptoms observed after accidental envenomations. To investigate the mechanism of action of antivenom, we examined the effects of the intravenous administration of antivenom (horse F(ab')2 directed against Centruroides venoms) on the pharmacokinetics of FII. Immunotherapy performed 2 h after the experimental envenomation largely increased the area under the concentration time curve of FII compared to that calculated in absence of immunotherapy (13,000 versus 170 ng h ml(-1), respectively). These observations agree with previous findings which showed that specific antibody fragments are able to remove drugs from their site of action and sequester them in the vascular space. These studies provide a powerful tool to determine an excellent procedure for further improvement of immunotherapy.

  8. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

    PubMed Central

    Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

    2016-01-01

    ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. PMID:27853644

  9. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

    SciTech Connect

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; Swaminathan, Subramanyam

    2016-01-28

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. In addition, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. Our results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.

  10. Recombinant Human Respiratory Syncytial Virus (RSV) Monoclonal Antibody Fab is Effective Therapeutically when Introduced Directly into the Lungs of RSV-Infected Mice

    NASA Astrophysics Data System (ADS)

    Crowe, James E., Jr.; Murphy, Brian R.; Chanock, Robert M.; Williamson, R. Anthony; Barbas, Carlos F., III; Burton, Dennis R.

    1994-02-01

    Previously, recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fabs were generated by antigen selection from random combinatorial libraries displayed at the tip of filamentous phage. Two such Fabs, which exhibited high binding affinity for RSV F glycoprotein (a major protective antigen), were evaluated for therapeutic efficacy in infected mice just before or at the time of peak virus replication in the lungs. Fab 19, which neutralized RSV infectivity with high efficiency in tissue culture, was effective therapeutically when delivered directly into the lungs by intranasal instillation under anesthesia. In contrast, RSV Fab 126, which failed to neutralize virus in cell culture, did not exhibit a therapeutic effect under these conditions. The amount of Fab 19 required to effect a 5000- to 12,000-fold reduction in titer of RSV in the lungs within 24 hr was rather small. In four separate experiments, a single instillation of 12.9-50 μg of RSV Fab 19 was sufficient to achieve such a reduction in pulmonary virus in a 25g mouse. The use of Fabs instead of the whole immunoglobulin molecules from which they are derived reduced the protein content of a therapeutic dose. This is important because the protein load that can be delivered effectively into the lungs is limited. The therapeutic effect of a single treatment with Fab 19 was not sustained, so that a rebound in pulmonary virus titer occurred on the 2nd day after treatment. This rebound in pulmonary RSV titer could be prevented by treating infected mice with a single dose of Fab 19 daily for 3 days. These observations suggest that human monoclonal Fabs grown in Escherichia coli may prove useful in the treatment of serious RSV disease as well as diseases caused by other viruses where replication in vivo is limited primarily to the lumenal lining of the respiratory tract.

  11. Production of stable bispecific IgG1 by controlled Fab-arm exchange

    PubMed Central

    Gramer, Michael J; van den Bremer, Ewald TJ; van Kampen, Muriel D; Kundu, Amitava; Kopfmann, Peter; Etter, Eric; Stinehelfer, David; Long, Justin; Lannom, Tom; Noordergraaf, Esther H; Gerritsen, Jolanda; Labrijn, Aran F; Schuurman, Janine; van Berkel, Patrick HC; Parren, Paul WHI

    2013-01-01

    The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of >95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques. PMID:23995617

  12. Comparison between therapeutic antitoxin F(ab)2 fractionated with ammonium sulfate and caprylic acid.

    PubMed

    Redwan, El-Rashdy M

    2006-01-01

    To date, animal derived therapeutic antibodies represent the best and only choice source of antitoxins, especially in developing countries. Furthermore, this industry needs to develop a production protocol to achieve safer products. Recently, several laboratories changed their production protocol from ammonium sulfate (AS) protocol to caprylic acid (CA) fractionation. Our results showed that using the CA protocol leads to improvement in the product quality, as assessed by the albumin and protein content decrease (from 4.75 to 3.54 g/dL and 0.64 to 0.18 g/dL, respectively), which yielded a purer antitoxin product. The F(ab)2 protein aggregate formation and turbidity have been significantly reduced, 4.60 versus 2.55 and 0.046 versus 0.021 (p < 0.01), respectively. However, the anti-complementary activity was also reduced, from 42 to 33. The total IgG content was higher in CA fractionated products than AS materials. The endotoxin content was worrisome in some F(ab)2 products.

  13. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    PubMed

    Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo; Schuck, Peter; Louis, John M; Pierson, Jason; Rao, Prashant; Subramaniam, Sriram; Gustchina, Alla; Clore, G Marius; Wlodawer, Alexander

    2013-01-01

    A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.

  14. New Scalings in Nuclear Fragmentation

    SciTech Connect

    Bonnet, E.; Bougault, R.; Galichet, E.; Gagnon-Moisan, F.; Guinet, D.; Lautesse, P.; Marini, P.; Parlog, M.

    2010-10-01

    Fragment partitions of fragmenting hot nuclei produced in central and semiperipheral collisions have been compared in the excitation energy region 4-10 MeV per nucleon where radial collective expansion takes place. It is shown that, for a given total excitation energy per nucleon, the amount of radial collective energy fixes the mean fragment multiplicity. It is also shown that, at a given total excitation energy per nucleon, the different properties of fragment partitions are completely determined by the reduced fragment multiplicity (i.e., normalized to the source size). Freeze-out volumes seem to play a role in the scalings observed.

  15. Fragmentation of random trees

    NASA Astrophysics Data System (ADS)

    Kalay, Z.; Ben-Naim, E.

    2015-01-01

    We study fragmentation of a random recursive tree into a forest by repeated removal of nodes. The initial tree consists of N nodes and it is generated by sequential addition of nodes with each new node attaching to a randomly-selected existing node. As nodes are removed from the tree, one at a time, the tree dissolves into an ensemble of separate trees, namely, a forest. We study statistical properties of trees and nodes in this heterogeneous forest, and find that the fraction of remaining nodes m characterizes the system in the limit N\\to ∞ . We obtain analytically the size density {{φ }s} of trees of size s. The size density has power-law tail {{φ }s}˜ {{s}-α } with exponent α =1+\\frac{1}{m}. Therefore, the tail becomes steeper as further nodes are removed, and the fragmentation process is unusual in that exponent α increases continuously with time. We also extend our analysis to the case where nodes are added as well as removed, and obtain the asymptotic size density for growing trees.

  16. Fragmentation of random trees

    NASA Astrophysics Data System (ADS)

    Kalay, Ziya; Ben-Naim, Eli

    2015-03-01

    We investigate the fragmentation of a random recursive tree by repeated removal of nodes, resulting in a forest of disjoint trees. The initial tree is generated by sequentially attaching new nodes to randomly chosen existing nodes until the tree contains N nodes. As nodes are removed, one at a time, the tree dissolves into an ensemble of separate trees, namely a forest. We study the statistical properties of trees and nodes in this heterogeneous forest. In the limit N --> ∞ , we find that the system is characterized by a single parameter: the fraction of remaining nodes m. We obtain analytically the size density ϕs of trees of size s, which has a power-law tail ϕs ~s-α , with exponent α = 1 + 1 / m . Therefore, the tail becomes steeper as further nodes are removed, producing an unusual scaling exponent that increases continuously with time. Furthermore, we investigate the fragment size distribution in a growing tree, where nodes are added as well as removed, and find that the distribution for this case is much narrower.

  17. Environmental DNA-encoded antibiotics fasamycins A and B inhibit FabF in type II fatty acid biosynthesis.

    PubMed

    Feng, Zhiyang; Chakraborty, Debjani; Dewell, Scott B; Reddy, Boojala Vijay B; Brady, Sean F

    2012-02-15

    In a recent study of polyketide biosynthetic gene clusters cloned directly from soil, we isolated two antibiotics, fasamycins A and B, which showed activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. To identify the target of the fasamycins, mutants with elevated fasamycin A minimum inhibitory concentrations were selected from a wild-type culture of E. faecalis OG1RF. Next-generation sequencing of these mutants, in conjunction with in vitro biochemical assays, showed that the fasamycins inhibit FabF of type II fatty acid biosynthesis (FASII). Candidate gene overexpression studies also showed that fasamycin resistance is conferred by fabF overexpression. On the basis of comparisons with known FASII inhibitors and in silico docking studies, the chloro-gem-dimethyl-anthracenone substructure seen in the fasamycins is predicted to represent a naturally occurring FabF-specific antibiotic pharmacophore. Optimization of this pharmacophore should yield FabF-specific antibiotics with increased potencies and differing spectra of activity. This study demonstrates that culture-independent antibiotic discovery methods have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use.

  18. 20 CFR 30.319 - May a claimant request reconsideration of a final decision of the FAB?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... PROGRAMS, DEPARTMENT OF LABOR ENERGY EMPLOYEES OCCUPATIONAL ILLNESS COMPENSATION PROGRAM ACT OF 2000 CLAIMS FOR COMPENSATION UNDER THE ENERGY EMPLOYEES OCCUPATIONAL ILLNESS COMPENSATION PROGRAM ACT OF 2000, AS... decision after granting a request for reconsideration, the FAB may return the claim to the district...

  19. 75 FR 21353 - Intel Corporation, Fab 20 Division, Including On-Site Leased Workers From Volt Technical...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-23

    ... From Volt Technical Resources, Staff Finders Technical, Kelly Services, Retronix International... Services, Hillsboro, Oregon. The notice will be published soon in the Federal Register. At the request of... the Hillsboro, Oregon location of Intel Corporation, Fab 20 Division. The Department has...

  20. IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients.

    PubMed

    Koneczny, Inga; Stevens, Jo A A; De Rosa, Anna; Huda, Saif; Huijbers, Maartje G; Saxena, Abhishek; Maestri, Michelangelo; Lazaridis, Konstantinos; Zisimopoulou, Paraskevi; Tzartos, Socrates; Verschuuren, Jan; van der Maarel, Silvère M; van Damme, Philip; De Baets, Marc H; Molenaar, Peter C; Vincent, Angela; Ricciardi, Roberta; Martinez-Martinez, Pilar; Losen, Mario

    2017-02-01

    Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies

  1. A human recombinant Fab identifies a human immunodeficiency virus type 1-induced conformational change in cell surface-expressed CD4.

    PubMed Central

    Bachelder, R E; Bilancieri, J; Lin, W; Letvin, N L

    1995-01-01

    To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection. PMID:7637018

  2. Fab'-bearing siRNA TNFα-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis.

    PubMed

    Laroui, Hamed; Viennois, Emilie; Xiao, Bo; Canup, Brandon S B; Geem, Duke; Denning, Timothy L; Merlin, Didier

    2014-07-28

    Patients suffering from inflammatory bowel disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNFα siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNFα siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab' portion of the F4/80 Ab (Fab'-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab'-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab'-bearing TNFα-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNFα-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab'-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and Iκbα accumulation) were more attenuated Fab'-bearing NPs loaded with TNFα siRNA than without the Fab'-bearing. Grafting the Fab'-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab'-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages.

  3. The Largest Fragment of a Homogeneous Fragmentation Process

    NASA Astrophysics Data System (ADS)

    Kyprianou, Andreas; Lane, Francis; Mörters, Peter

    2017-03-01

    We show that in homogeneous fragmentation processes the largest fragment at time t has size e^{-t Φ '(overline{p})}t^{-3/2 (log Φ )'(overline{p})+o(1)}, where Φ is the Lévy exponent of the fragmentation process, and overline{p} is the unique solution of the equation (log Φ )'(bar{p})=1/1+bar{p}. We argue that this result is in line with predictions arising from the classification of homogeneous fragmentation processes as logarithmically correlated random fields.

  4. Solid state of CG-400549, a novel FabI inhibitor: characterization, dissolution, transformation.

    PubMed

    Kim, Bo-Yeon; Sohn, Young-Taek

    2011-05-01

    The polymorphic and pseudopolymorphic forms of CG-400549, a novel FabI inhibitor with potent in vivo activity were prepared and characterized by differential scanning calorimetry (DSC), powder X-ray diffractometry (PXRD) and thermogravimetric analysis (TG). Seven crystal forms of CG-400549, one anhydrate and six solvates, have been isolated by recrystallization and the DSC and PXRD patterns of the seven crystal forms of CG-400549 were different respectively. The dissolution patterns of these seven crystal forms of CG-400549 were studied and they showed significant differences in the dissolution rate. After storage of 1 month at 0% RH (silica gel, 20°C), 52% RH (saturated solution of Na(2)Cr(2)O(7)2H(2)O/20°C) and 95% RH (saturated solution of Na(2)HPO(4)/20°C), all crystal forms were not transformed.

  5. Photomask film degradation effects in the wafer fab: how to detect and monitor over time

    NASA Astrophysics Data System (ADS)

    Whittey, John; Hess, Carl; Garcia, Edgardo; Wagner, Mark; Duffy, Brian

    2012-11-01

    As a result of repeated cleanings and exposure effects such as chrome migration or MoSi oxidation some photomasks in the semiconductor fabs exhibit changes in critical dimension uniformity (CDU) over time. Detecting these effects in a timely manner allows for better risk management and process control in manufacturing. By monitoring changes in film reflectance intensity due to the various degradation mechanisms it is possible to predict when they may begin to influence across chip line width variations (ACLV). By accurately predicting the magnitude of these changes it is possible for semiconductor manufacturers to replace the photomasks before they have an impact on yields. This paper looks at possible causes of CDU variations on reticles during use and how this information might be used to improve or monitor reticle CDU changes over time.

  6. Building blocks X-FAB SOI 0.18 μm

    NASA Astrophysics Data System (ADS)

    Cizel, J.-B.; Ahmad, S.; Callier, S.; Cornat, R.; Dulucq, F.; Fleury, J.; Martin-Chassard, G.; Raux, L.; de La Taille, C.; Thienpont, D.

    2015-02-01

    This work has been done in order to study a new technology provided by X-FAB named xt018. It is an SOI (Silicon On Insulator) technology with a minimal gate length of 180 nm. Building blocks have been done to test the advantages and drawbacks of this technology compared to the one currently used (AMS SiGe 0.35 μm). These building blocks have been designed to fit in an existing experience housed by the CALICE collaboration: the read-out chip for the Electromagnetic CALorimeter (ECAL) of the foreseen International Linear Collider (ILC). Performances will be compared to those of the SKIROC2 chip designed by the OMEGA laboratory, trying to fit the same requirements. The chip is being manufactured and will be back for measurements in December, the displayed results are only simulation results and thus the conclusions concerning the performances of these building blocks are subject to change.

  7. A novel Fab-based antivenom for the treatment of mass bee attacks.

    PubMed

    Jones, R G; Corteling, R L; Bhogal, G; Landon, J

    1999-09-01

    The frequency of mass bee attacks has dramatically increased in the Americas following the introduction and spread of the aggressive Africanized 'killer' bee (Apis mellifera scutellata). As yet no specific therapy is available, which led us to develop an ovine Fab-based antivenom as a potential new treatment. Sera from sheep immunized against the venom contained high levels of specific antibodies, as demonstrated by ELISA and by small-scale affinity chromatography, against both whole (A. m. mellifera) venom and purified melittin. A nerve muscle preparation was used to show the myotoxic effects of the venom and neutralization by the antivenom. Antivenom neutralizing ability was also demonstrated using assays for venom phospholipase A2 and in vivo activities. Venom from both European and Africanized bees appeared identical when analyzed by acid-urea gel electrophoresis. This antivenom may therefore provide the first specific therapy for the treatment of mass envenomation by either European or Africanized 'killer' bees.

  8. Binary stars - Formation by fragmentation

    NASA Technical Reports Server (NTRS)

    Boss, Alan P.

    1988-01-01

    Theories of binary star formation by capture, separate nuclei, fission and fragmentation are compared, assessing the success of theoretical attempts to explain the observed properties of main-sequence binary stars. The theory of formation by fragmentation is examined, discussing the prospects for checking the theory against observations of binary premain-sequence stars. It is concluded that formation by fragmentation is successful at explaining many of the key properties of main-sequence binary stars.

  9. Species–fragmented area relationship

    PubMed Central

    Hanski, Ilkka; Zurita, Gustavo A.; Bellocq, M. Isabel; Rybicki, Joel

    2013-01-01

    The species–area relationship (SAR) gives a quantitative description of the increasing number of species in a community with increasing area of habitat. In conservation, SARs have been used to predict the number of extinctions when the area of habitat is reduced. Such predictions are most needed for landscapes rather than for individual habitat fragments, but SAR-based predictions of extinctions for landscapes with highly fragmented habitat are likely to be biased because SAR assumes contiguous habitat. In reality, habitat loss is typically accompanied by habitat fragmentation. To quantify the effect of fragmentation in addition to the effect of habitat loss on the number of species, we extend the power-law SAR to the species–fragmented area relationship. This model unites the single-species metapopulation theory with the multispecies SAR for communities. We demonstrate with a realistic simulation model and with empirical data for forest-inhabiting subtropical birds that the species–fragmented area relationship gives a far superior prediction than SAR of the number of species in fragmented landscapes. The results demonstrate that for communities of species that are not well adapted to live in fragmented landscapes, the conventional SAR underestimates the number of extinctions for landscapes in which little habitat remains and it is highly fragmented. PMID:23858440

  10. Effect of chloramine-T labeling conditions on the stability of monoclonal antibodies and their fragments

    SciTech Connect

    DeNardo, G.L.; DeNardo, S.J.; Miyao, N.P.; Peng, J.S.; Epstein, A.L.; Cardiff, R.D.

    1985-05-01

    Rapid in vivo degradation of radioiodinated monoclonal antibodies (MAb) has been reported. Conditions for radioiodination have varied. The purposes of this study were to compare the stability of MAb and their fragments when iodinated with chloramine-T (CT) under different conditions, and to compare methods for quality assessment of the radioiodinated molecules. A B-cell lymphoma MAb (Lym-1, IgG2a) and its FAb fragment, and a mammary cancer MAb(B6.01, IgG1) and its F(Ab')/sub 2/ fragment were iodinated with I-125 at CT:AB and I:Ab ratios of 1:1 and 1:10. Molecular sieving (TSK-3000) high performance liquid chromatography (HPLC), cellulose acetate electrophoresis (CAE) at 11 and 45 minutes and solid phase immunoreactivity (IRA) were used to observe stability of the molecules when stored at 4/sup 0/C. Radiochemical yield was greater than 95% in all instances. Iodination at CT:Ab and I:Ab ratios of 1:1 induced progressive degradation in all species which was most marked for the fragments. Iodination at CT:Ab and I:Ab ratios of 1:10 resulted in no observable degradation over 21 days. There was no significant difference in degradation between the IgG2a and IgG1 antibody when iodinated under identical circumstances. HPLC, CAE for 11 minutes and IRA, but not CAE for 45 minutes, revealed comparable changes. The authors conclude that lesser amounts of chloramine-T can be used to iodinate MAb and their fragments without loss of radiochemical efficiency and with improved stability of the species. MAb fragments are more vulnerable to chloramine-T. These observations may explain, at least in part, rapid in vivo degradation of radioiodinated MAb.

  11. Thermodynamical string fragmentation

    NASA Astrophysics Data System (ADS)

    Fischer, Nadine; Sjöstrand, Torbjörn

    2017-01-01

    The observation of heavy-ion-like behaviour in pp collisions at the LHC suggests that more physics mechanisms are at play than traditionally assumed. The introduction e.g. of quark-gluon plasma or colour rope formation can describe several of the observations, but as of yet there is no established paradigm. In this article we study a few possible modifications to the Pythia event generator, which describes a wealth of data but fails for a number of recent observations. Firstly, we present a new model for generating the transverse momentum of hadrons during the string fragmentation process, inspired by thermodynamics, where heavier hadrons naturally are suppressed in rate but obtain a higher average transverse momentum. Secondly, close-packing of strings is taken into account by making the temperature or string tension environment-dependent. Thirdly, a simple model for hadron rescattering is added. The effect of these modifications is studied, individually and taken together, and compared with data mainly from the LHC. While some improvements can be noted, it turns out to be nontrivial to obtain effects as big as required, and further work is called for.

  12. Validated LC-MS/MS analysis of immune checkpoint inhibitor Nivolumab in human plasma using a Fab peptide-selective quantitation method: nano-surface and molecular-orientation limited (nSMOL) proteolysis.

    PubMed

    Iwamoto, Noriko; Shimada, Takashi; Terakado, Hiroyuki; Hamada, Akinobu

    2016-06-15

    We previously reported the nano-surface and molecular-orientation limited (nSMOL) proteolysis, which is a novel method for selective quantitation of monoclonal antibody Fab. The nSMOL strategy is a Fab-selective limited proteolysis which utilizes the size difference between the protease nanoparticle (200nm) and the antibody resin pore (100nm). Here, we applied this method to a fully validated LCMS analysis of Nivolumab in human plasma. The immunoglobulin fraction was collected using Protein A resin, which was then followed by nSMOL reaction using the FG nanoparticle surface-immobilized trypsin under a nondenaturing physiological condition at 50°C for 7h. After removal of resin and nanoparticles by filter centrifugation, signature peptides were separated using the ODS column liquid chromatography. The signature peptide ASGITFSNSGMHWVR from Nivolumab complementarity-determining region (CDR) and the P14R internal standard were simultaneously quantified by multiple-reaction monitoring (MRM) LCMS, with parent m/z 550.8>fragment m/z 661.5 (y11 2+). The lower limit of quantification (LLOQ) of Nivolumab using the nSMOL method was 0.977μg/ml, with a linear dynamic range of from 0.977 to 250μg/ml. The intra- and inter-assay precision of LLOQ, low quality control (LQC), middle quality control (MQC), and high quality control (HQC) were 7.56-17.9% and 15.6%, 6.99-9.25% and 7.51%, 2.51-8.85% and 8.01%, and 4.78-7.33% and 6.75%, respectively. Our study demonstrates that the nSMOL bioanalysis can be utilized as a reliable method for clinical pharmacokinetic studies of Nivolumab and other antibody drugs.

  13. Structure-directed construction of a high-performance version of the enzyme FabG from the photosynthetic microorganism Synechocystis sp. PCC 6803.

    PubMed

    Liu, Yinghui; Feng, Yanbin; Cao, Xupeng; Li, Xia; Xue, Song

    2015-10-07

    PhaB (acetoacetyl-CoA reductase) catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA in polyhydroxybutyrate (PHB) synthesis and FabG (3-ketoacyl-acyl-carrier-protein reductase) catalyzes the β-ketoacyl-ACP to yield (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis. Both of them have been classified into the same group EC 1.1.1. PhaB is limited with substrate specificities, while FabG was considered as a potential PhaB due to broad substrate selectivity despite of low activity. Here, X-ray crystal structures of FabG and PhaB from the photosynthetic microorganism Synechocystis sp. PCC 6803 were resolved. Based on them, a high-performance FabG on acyl-CoA directed by structural evolution was constructed that may serve as a critical enzyme to partition carbon flow from fatty acid synthesis to PHA.

  14. Fragment screening and HIV therapeutics.

    PubMed

    Bauman, Joseph D; Patel, Disha; Arnold, Eddy

    2012-01-01

    Fragment screening has proven to be a powerful alternative to traditional methods for drug discovery. Biophysical methods, such as X-ray crystallography, NMR spectroscopy, and surface plasmon resonance, are used to screen a diverse library of small molecule compounds. Although compounds identified via this approach have relatively weak affinity, they provide a good platform for lead development and are highly efficient binders with respect to their size. Fragment screening has been utilized for a wide range of targets, including HIV-1 proteins. Here, we review the fragment screening studies targeting HIV-1 proteins using X-ray crystallography or surface plasmon resonance. These studies have successfully detected binding of novel fragments to either previously established or new sites on HIV-1 protease and reverse transcriptase. In addition, fragment screening against HIV-1 reverse transcriptase has been used as a tool to better understand the complex nature of ligand binding to a flexible target.

  15. Fragmentation functions in nuclear media

    NASA Astrophysics Data System (ADS)

    Sassot, Rodolfo; Stratmann, Marco; Zurita, Pia

    2010-03-01

    We perform a detailed phenomenological analysis of how well hadronization in nuclear environments can be described in terms of effective fragmentation functions. The medium modified fragmentation functions are assumed to factorize from the partonic scattering cross sections and evolve in the hard scale in the same way as the standard or vacuum fragmentation functions. Based on precise data on semi-inclusive deep-inelastic scattering off nuclei and hadron production in deuteron-gold collisions, we extract sets of effective fragmentation functions for pions and kaons at next-to-leading order accuracy. The obtained sets provide a rather accurate description of the kinematical dependence of the analyzed cross sections and are found to differ significantly from standard fragmentation functions both in shape and magnitude. Our results support the notion of factorization and universality in the studied nuclear environments, at least in an effective way and within the precision of the available data.

  16. Acute myeloid leukemias M2 potentially misdiagnosed as M3 variant French-American-Britain (FAB) subtype: a transitional form?

    PubMed

    Fenu, S; Carmini, D; Mancini, F; Guglielmi, C; Alimena, G; Riccioni, R; Barsotti, P; Mancini, M; Avvisati, G; Mandelli, F

    1995-01-01

    From 1990 to 1994, 3 patients with de novo acute myeloid leukemia (AML) in whom light microscopy and cytochemistry suggested a FAB subtype M3 variant were observed at our Institute. Immunophenotype showed HLA-DR-, CD13+, CD33+, CD2+, CD9+; promyelocytic features were also detected by electron microscopy. However, leukemic cells lacked both translocation t(15;17) and RAR alpha/PML genes rearrangement. These cases were considered to be 'M2 atypical' subtypes and they contribute to point out how cytogenetics and molecular biology are mandatory for a correct diagnosis of acute promyelocytic leukemia (APL) particularly because therapy with all trans retinoic acid (ATRA) is now the best treatment for APL. Nevertheless these 3 cases indicate that the atypical M2 subtype may be confused with the M3v if only cytochemistry, immunophenotype and electron microscopy are used in the defining the FAB subtypes.

  17. Resistance Mechanisms and the Future of Bacterial Enoyl-Acyl Carrier Protein Reductase (FabI) Antibiotics

    PubMed Central

    Yao, Jiangwei; Rock, Charles O.

    2016-01-01

    Missense mutations leading to clinical antibiotic resistance are a liability of single-target inhibitors. The enoyl-acyl carrier protein reductase (FabI) inhibitors have one intracellular protein target and drug resistance is increased by the acquisition of single base pair mutations that alter drug binding. The spectrum of resistance mechanisms to FabI inhibitors suggests criteria that should be considered during the development of single-target antibiotics that would minimize the impact of missense mutations on their clinical usefulness. These criteria include high-affinity, fast on/off kinetics, few drug contacts with residue side chains, and no toxicity. These stringent criteria are achievable by structure-guided design, but this approach will only yield pathogen-specific drugs. Single-step acquisition of resistance may limit the clinical application of broad-spectrum, single-target antibiotics, but appropriately designed, pathogen-specific antibiotics have the potential to overcome this liability. PMID:26931811

  18. Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

    PubMed

    Ghadbane, Hemza; Brown, Alistair K; Kremer, Laurent; Besra, Gurdyal S; Fütterer, Klaus

    2007-10-01

    Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

  19. 3-Substituted Indole Inhibitors Against Francisella tularensis FabI Identified by Structure-Based Virtual Screening

    DTIC Science & Technology

    2013-07-01

    Seto, W. H.; Ng, T. K.; Yam, W. C.; Ng, W. W. Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong...Brenwald, N. P.; Gill, M. J.; Walker, R. A.; Livermore, D. M.; George, R. C. Emergence of a fluoroquinolone-resistant strain of Streptococcus pneumoniae ...Payne, D. J.; Rock, C. O.; Wallis, N. G. Characterization of Streptococcus pneumoniae enoyl-(acyl-carrier protein) reductase (FabK). Biochem. J. 2003

  20. Rationalizing the Binding Kinetics for the Inhibition of the Burkholderia pseudomallei FabI1 Enoyl-ACP Reductase.

    PubMed

    Neckles, Carla; Eltschkner, Sandra; Cummings, Jason E; Hirschbeck, Maria; Daryaee, Fereidoon; Bommineni, Gopal R; Zhang, Zhuo; Spagnuolo, Lauren; Yu, Weixuan; Davoodi, Shabnam; Slayden, Richard A; Kisker, Caroline; Tonge, Peter J

    2017-04-04

    There is growing awareness of the link between drug-target residence time and in vivo drug activity, and there are increasing efforts to determine the molecular factors that control the lifetime of a drug-target complex. Rational alterations in the drug-target residence time require knowledge of both the ground and transition states on the inhibition reaction coordinate, and we have determined the structure-kinetic relationship for 22 ethyl- or hexyl-substituted diphenyl ethers that are slow-binding inhibitors of bpFabI1, the enoyl-ACP reductase FabI1 from Burkholderia pseudomallei. Analysis of enzyme inhibition using a two-dimensional kinetic map demonstrates that the ethyl and hexyl diphenyl ethers fall into two distinct clusters. Modifications to the ethyl diphenyl ether B ring result in changes to both on and off rates, where residence times of up to ∼700 min (∼11 h) are achieved by either ground state stabilization (PT444) or transition state destabilization (slower on rate) (PT404). By contrast, modifications to the hexyl diphenyl ether B ring result in residence times of 300 min (∼5 h) through changes in only ground state stabilization (PT119). Structural analysis of nine enzyme:inhibitor complexes reveals that the variation in structure-kinetic relationships can be rationalized by structural rearrangements of bpFabI1 and subtle changes to the orientation of the inhibitor in the binding pocket. Finally, we demonstrate that three compounds with residence times on bpFabI1 from 118 min (∼2 h) to 670 min (∼11 h) have in vivo efficacy in an acute B. pseudomallei murine infection model using the virulent B. pseudomallei strain Bp400.

  1. Three-dimensional structures of a humanized anti-IFN-gamma Fab (HuZAF) in two crystal forms.

    PubMed

    Bourne, Philip C; Terzyan, Simon S; Cloud, Gwendolyn; Landolfi, Nicholas F; Vásquez, Maximiliano; Edmundson, Allen B

    2004-10-01

    Three-dimensional structures were determined for two crystal forms (orthorhombic P2(1)2(1)2(1) and monoclinic C2) of the Fab from the humanized version of a murine monoclonal antibody (AF2) that possesses binding and potent neutralizing activity against human interferon gamma (IFN-gamma). This humanized antibody (HuZAF; USAN name fontolizumab) is currently in phase II clinical trials for the treatment of Crohn's disease. HuZAF exhibits binding and IFN-gamma neutralizing capacities that closely approximate those of the original antibody. It is shown that HuZAF, whose VH domain was designed using a best-sequence-fit approach, is closer structurally to its mouse precursor than is a version whose VH was constructed using a human sequence with lower homology to the original mouse sequence. This work thus offers direct structural evidence in support of the best-sequence-fit approach and adds to previous results of biological and biochemical evaluations of distinctly engineered antibodies that also favored the use of a best-sequence-fit strategy. A second crystal type appeared during attempts to crystallize the Fab-IFN-gamma complex. The antibody-antigen complex that existed in solution dissociated in the crystallization mixture. A conformationally altered but unliganded HuZAF protein crystallized in a different space group (C2), with two Fab molecules in the asymmetric unit. In this crystal lattice, no space was available for accommodating the IFN-gamma antigen. Thus, there are currently three slightly different structures of the HuZAF Fab.

  2. Comparative study of cadmium and lead accumulations in Cambarus bartoni (Fab. ) (Decapoda, Crustacea) from an acidic and a neutral lake

    SciTech Connect

    Keenan, S.; Alikhan, M.A. )

    1991-07-01

    The purpose of the study reported in this paper was to compare concentrations of lead and cadmium in the sediment and water, as well as in the crayfish, Cambarus Bartoni (Fab.) (Decapoda - Crustacea) trapped from an acidic and a neutral lake in the Sudbury district of Northeastern Ontario. Hepatopancreatic, alimentary canal, tail muscles and exoskeletal concentrations in the crayfish are also examined to determine specific tissue sites for these accumulations.

  3. Driven fragmentation of granular gases.

    PubMed

    Cruz Hidalgo, Raúl; Pagonabarraga, Ignacio

    2008-06-01

    The dynamics of homogeneously heated granular gases which fragment due to particle collisions is analyzed. We introduce a kinetic model which accounts for correlations induced at the grain collisions and analyze both the kinetics and relevant distribution functions these systems develop. The work combines analytical and numerical studies based on direct simulation Monte Carlo calculations. A broad family of fragmentation probabilities is considered, and its implications for the system kinetics are discussed. We show that generically these driven materials evolve asymptotically into a dynamical scaling regime. If the fragmentation probability tends to a constant, the grain number diverges at a finite time, leading to a shattering singularity. If the fragmentation probability vanishes, then the number of grains grows monotonously as a power law. We consider different homogeneous thermostats and show that the kinetics of these systems depends weakly on both the grain inelasticity and driving. We observe that fragmentation plays a relevant role in the shape of the velocity distribution of the particles. When the fragmentation is driven by local stochastic events, the long velocity tail is essentially exponential independently of the heating frequency and the breaking rule. However, for a Lowe-Andersen thermostat, numerical evidence strongly supports the conjecture that the scaled velocity distribution follows a generalized exponential behavior f(c) approximately exp(-cn) , with n approximately 1.2 , regarding less the fragmentation mechanisms.

  4. Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

    PubMed Central

    Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

    2016-01-01

    Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

  5. Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase III (mtFabH) assay: principles and method.

    PubMed

    Sachdeva, Sarbjot; Reynolds, Kevin A

    2008-01-01

    Fatty acid biosynthesis is one of the relatively newer targets in antibacterial drug discovery. The presence of distinct fatty acid synthases (FAS) in mammals and bacteria and the fact that most bacterial FAS enzymes are essential for viability make this a very attractive antimicrobial drug target. The enzyme beta-ketoacyl ACP synthase (KASIII or FabH) is the key enzyme that initiates fatty acid biosynthesis in a type II dissociated FAS. This enzyme catalyzes the condensation of acyl CoA and malonyl ACP (acyl carrier protein) to form a beta-ketoacyl ACP product, which is further processed to form mature fatty acids that are involved in various essential cellular processes and structures like phospholipid biosynthesis, cell wall formation, etc. Herein we describe a new assay for the Mycobacterium tuberculosis FabH (mtFabH) enzyme involved in a key initiation step in the synthesis of mycolic acids, which are an integral component of the cell wall. The assay eliminates the need for the cumbersome washing steps or specialty scintillation proximity assay beads and the preparation of acyl carrier proteins required in other assay formats. This discontinuous assay involves the reduction of radiolabled long-chain beta-ketoacyl CoA product to its dihydroxy derivative, which partitions into a nonpolar phase for quantitation, while the reduced radiolabeled substrate derivative remains in the aqueous phase.

  6. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

    PubMed Central

    Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

  7. An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels.

    PubMed

    Lang, Michael J; Strunk, Bethany S; Azad, Nadia; Petersen, Jason L; Weisman, Lois S

    2017-04-01

    Phosphorylated phosphoinositide lipids (PPIs) are low-abundance signaling molecules that control signal transduction pathways and are necessary for cellular homeostasis. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is essential in multiple organ systems. PI(3,5)P2 is generated from PI3P by the conserved lipid kinase Fab1/PIKfyve. Defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here we report an intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We identify mutations in the kinase domain that lead to elevated levels of PI(3,5)P2 and impair the interaction between the kinase and CCR domain. We also identify mutations in the CCR domain that lead to elevated levels of PI(3,5)P2 Together these findings reveal a regulatory mechanism that involves the CCR domain of Fab1 and contributes to dynamic control of cellular PI(3,5)P2 synthesis.

  8. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae.

    PubMed

    Mohedano, Maria L; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  9. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    SciTech Connect

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  10. Crystallization and preliminary X-ray analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae

    SciTech Connect

    Saito, Jun Yamada, Mototsugu; Watanabe, Takashi; Kitagawa, Hideo; Takeuchi, Yasuo

    2006-06-01

    Enoyl-acyl carrier protein (ACP) reductases are responsible for bacterial type II fatty-acid biosynthesis and are attractive targets for developing novel antibiotics. The S. pneumoniae enoyl-ACP reductase (FabK) was crystallized and selenomethionine MAD data were collected to 2 Å resolution. The enoyl-acyl carrier protein (ACP) reductase from Streptococcus pneumoniae (FabK; EC 1.3.1.9) is responsible for catalyzing the final step in each elongation cycle of fatty-acid biosynthesis. Selenomethionine-substituted FabK was purified and crystallized by the hanging-drop vapour-diffusion method at 277 K. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 50.26, b = 126.70, c = 53.63 Å, β = 112.46°. Diffraction data were collected to 2.00 Å resolution using synchrotron beamline BL32B2 at SPring-8. Two molecules were estimated to be present in the asymmetric unit, with a solvent content of 45.1%.

  11. Antigen binding of human IgG Fabs mediate ERK-associated proliferation of human breast cancer cells.

    PubMed

    Wen, Yue-Jin; Mancino, Anne; Pashov, Anastas; Whitehead, Tracy; Stanley, Joseph; Kieber-Emmons, Thomas

    2005-02-01

    Serum-circulating antibody can be linked to poor outcomes in some cancer patients. To investigate the role of human antibodies in regulating tumor cell growth, we constructed a recombinant cDNA expression library of human IgG Fab from a patient with breast cancer. Clones were screened from the library with breast tumor cell lysate. Sequence analysis of the clones showed somatic hypermutations when compared to their closest VH/VL germ-line genes. Initial characterizations focused on five clones. All tested clones displayed stronger binding to antigen derived from primary breast cancers and established breast cancer cell lines than to normal breast tissues. In vitro functional studies showed that four out of five tested clones could stimulate the growth of MDA-MB-231 breast cancer cell lines, and one out of five was able to promote MCF-7 cell growth as well. Involvement of ERK2 pathway was observed. By 1H-NMR spectra and Western blot analysis, it was evident that two tested antibody Fabs are capable of interacting with sialic acid. Our study suggests a possible role for human antibody in promoting tumor cell growth by direct binding of IgG Fab to breast tumor antigen. Such studies prompt speculation regarding the role of serum antibodies in mediating tumor growth as well as their contribution to disease progression.

  12. Computational and Experimental Determination of Fragmentation for Naturally Fragmenting Warheads

    DTIC Science & Technology

    1981-05-01

    Table Page I Chemical analysis of Armco iron and HF-I steel ....................... 3 2 Summary of tensile-pull measurements for transverse-direction...ntered) REPORT DOCUMENTATION PAGE - E-EFORE COMTLETING FORM I REPORT NUMBER 2 GOVT ACCESSION NO. 3 RECIPIENT’S CATALOG NUMBERNSWC TR 80-238 4 TITLE (and...Sulbtitle) S TYPE OF REPORT & PERIOD COVERED COMPUTATIONAL AND EXPERIMENTAL 1 Final DETERMINATION OF FRAGMENTATION FOR NATURALLY FRAGMENTING WARHEADS

  13. Depleted Uranium Test Range Fragment Reclamation

    DTIC Science & Technology

    1982-07-01

    fragment drying was necessary in order to obtain adequate vacuum levels in the VIR furnaces . e. Vacuujm Induction Remelting Fragments and Casting...Acid Pickle and Water Rinse .... ........ 2 d. Drying the Fragments .... ............... 2 e. Vacuum Induction Remelting Fragments and Casting...feasibility of reclaiming test range fragments by vacuum induction remelting (VIR). The technical direction of Phase 11 was highly dependent upon the

  14. Identification of the sea urchin egg receptor for sperm using an antiserum raised against a fragment of its extracellular domain

    PubMed Central

    1992-01-01

    Sea urchin egg fertilization requires the species-specific interaction of molecules on the sperm and egg surfaces. Previously, we isolated an extracellular, 70-kD glycosylated fragment of the S. purpuratus egg receptor for sperm by treating the eggs with lysylendoproteinase C (Foltz, K. R., and W. J. Lennarz. 1990. J. Cell Biol. 111:2951-2959). To characterize the receptor further, we have generated a polyclonal antiserum (anti-70KL) against the purified 70-kD fragment. Anti-70KL was found to react with a single polypeptide of approximately 350 kD on Western blots, presumed to be the intact receptor, in an egg cell surface preparation. This polypeptide appeared to be tightly associated with the plasma membrane/vitelline layer complex, as it was released from these preparations only by detergent treatment. Immunofluorescence microscopy revealed that the receptor was distributed evenly over the egg surface. The anti-70KL was species specific both in its ability to recognize the egg surface protein and to inhibit sperm binding. Fab fragments generated from affinity-purified anti-70KL also bound to the egg surface and inhibited sperm binding in a concentration-dependent manner. Interestingly, treatment with Fabs caused a small percentage of eggs to undergo cortical granule exocytosis, even in the absence of external Ca2+. These results confirm earlier findings indicating that the receptor is a cell surface glycoprotein of high molecular weight that species specifically binds sperm. This antiserum provides a powerful tool for further investigation of gamete interactions and the structure of the sperm receptor. PMID:1309817

  15. Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism

    PubMed Central

    Main, Sarah; Newton, Philip; Chodorge, Matthieu; Cadwallader, Karen; Humphreys, Robin; Albert, Vivian; Vaughan, Tristan J; Minter, Ralph R; Edwards, Bryan M

    2009-01-01

    Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer. PMID:20068388

  16. Fragmentation of drying paint layers

    NASA Astrophysics Data System (ADS)

    Bakos, Katinka; Dombi, András; Járai-Szabó, Ferenc; Néda, Zoltán

    2013-11-01

    Fragmentation of thin layers of drying granular materials on a frictional surface are studied both by experiments and computer simulations. Besides a qualitative description of the fragmentation phenomenon, the dependence of the average fragment size as a function of the layer thickness is thoroughly investigated. Experiments are done using a special nail polish, which forms characteristic crack structures during drying. In order to control the layer thickness, we diluted the nail polish in acetone and evaporated in a controlled manner different volumes of this solution on glass surfaces. During the evaporation process we managed to get an instable paint layer, which formed cracks as it dried out. In order to understand the obtained structures a previously developed spring-block model was implemented in a three-dimensional version. The experimental and simulation results proved to be in excellent qualitative and quantitative agreement. An earlier suggested scaling relation between the average fragment size and the layer thickness is reconfirmed.

  17. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants.

    PubMed

    Paul, Suparna; Connor, Judy; Nesspor, Tom; Haytko, Peter; Boakye, Ken; Chiu, Mark L; Jiang, Haiyan

    2016-05-01

    Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.

  18. Characterization of silicon photomultipliers at National Nano-Fab Center for PET-MR

    SciTech Connect

    Kim, Hyoungtaek; Cho, Gyuseong; Sul, Woo Suk

    2014-10-15

    The silicon photomultipliers (SiPMs) were fabricated for magnetic resonance compatible positron emission tomography (PET) applications using customized CMOS processes at National NanoFab Center. Each micro-cell consists of a shallow n+/p well junction on a p-type epitaxial wafer and passive quenching circuit was applied. The size of the SiPM is 3 × 3 mm{sup 2} and the pitch of each micro-cell is 65 μm. In this work, several thousands of SiPMs were packaged and tested to build a PET ring detector which has a 60 mm axial and 390 mm radial field of view. I-V characteristics of the SiPMs are shown good uniformity and breakdown voltage is around 20 V. The photon detection efficiency was measured via photon counting method and the maximum value was recorded as 16% at 470 nm. The gamma ray spectrum of a Ge-68 isotope showed nearly 10% energy resolution at 511 keV with a 3 × 3 × 20 mm{sup 3} LYSO crystal.

  19. A semiempirical nuclear fragmentation model

    NASA Technical Reports Server (NTRS)

    Wilson, John W.; Townsend, Lawrence W.; Badavi, F. F.

    1987-01-01

    An abrasion/ablation model of heavy ion fragmentation is derived which includes a second order correction for the surface energy term and provides a reasonable representation of the present elemental fragmentation cross sections. The full development of the model must await the resolution of disagreement among different experiments and an expansion of the experimental data base to a broader set of projectile-target combinations.

  20. High Fragmentation Steel Production Process

    DTIC Science & Technology

    1984-01-01

    phase of the project entailed the purchase and metallurgical characterization of two heats of HF-1 steel from different vendors. Performed by...At>-A 13^ nzt AD AD-E401 117 CONTRACTOR REPORT ARLCD-CR-83049 HIGH FRAGMENTATION STEEL PRODUCTION PROCESS ^"fP-PTTMirj A 1 James F. Kane...Report 6. PERFORMING ORG. REPORT NUMBER High Fragmentation Steel Production Process 7. AUTHORfs; James F. Kane, Ronald L. Kivak, Colin C. MacCrindle

  1. QGP and Modified Jet Fragmentation

    SciTech Connect

    Wang, Xin-Nian

    2005-04-18

    Recent progresses in the study of jet modification in hotmedium and their consequences in high-energy heavy-ion collisions are reviewed. In particular, I will discuss energy loss for propagating heavy quarks and the resulting modified fragmentation function. Medium modification of the parton fragmentation function due to quark recombination are formulated within finite temperature field theory and their implication on the search for deconfined quark-gluon plasma is also discussed.

  2. Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

    PubMed Central

    Vick, Jacob E.; Clomburg, James M.; Blankschien, Matthew D.; Chou, Alexander; Kim, Seohyoung

    2014-01-01

    We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ΔfabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli. PMID:25527535

  3. Cross-reactive binding of cyclic peptides to an anti-TGFalpha antibody Fab fragment: an X-ray structural and thermodynamic analysis.

    PubMed

    Hahn, M; Winkler, D; Welfle, K; Misselwitz, R; Welfle, H; Wessner, H; Zahn, G; Scholz, C; Seifert, M; Harkins, R; Schneider-Mergener, J; Höhne, W

    2001-11-23

    The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis. Two cyclic peptides which are cross-reactive binders for tAb2 were selected earlier using combinatorial peptide libraries. One is cyclized by an amide bond between the N-alpha group and the side-chain of the last residue (cyclo-SHFNEYE), and the other by a disulfide bridge (cyclo-CSHFNDYC). The complex structures of tAb2 with the linear epitope peptide VVSHFND and with cyclo-SHFNEYE were determined by X-ray diffraction. Both peptides show a similar conformation and binding pattern in the complex. The linear peptide SHFNEYE does not bind tAb2, but cyclo-SHFNEYE is stabilized in a loop conformation suitable for binding. Hence the cyclization counteracts the exchange of aspartate in the epitope sequence to glutamate. Isothermal titration calorimetry was used to characterize the binding energetics of tAb2 with the two cyclic peptides and the epitope peptide. The binding reactions are enthalpically driven with an unfavorable entropic contribution under all measured conditions. The association reactions are characterized by negative DeltaC(p) changes and by the uptake of one proton per binding site. A putative candidate for proton uptake during binding is the histidine residue in each of the peptides. Hydrogen bonds and the putative formation of an electrostatic pair between the protonated histidine and a carboxy group may contribute markedly to the favorable enthalpy of complex formation. Implications to cyclization of peptides for stabilization are discussed.

  4. Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

    PubMed

    Broughton, Sophie E; Hercus, Timothy R; Nero, Tracy L; Dhagat, Urmi; Owczarek, Catherine M; Hardy, Matthew P; Fabri, Louis J; Scotney, Pierre D; Nash, Andrew D; Wilson, Nicholas J; Lopez, Angel F; Parker, Michael W

    2014-03-01

    Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined.

  5. Rabies Post-Exposure Prophylaxis in the Philippines: Health Status of Patients Having Received Purified Equine F(ab')2 Fragment Rabies Immunoglobulin (Favirab)

    PubMed Central

    Quiambao, Beatriz P.; DyTioco, Hazel Z.; Dizon, Ruby M.; Crisostomo, Marilyn E.; Laot, Thelma M.; Teuwen, Dirk E.

    2008-01-01

    Background Recommended treatment for severe rabies exposure in unvaccinated individuals includes wound cleaning, administration of rabies immunoglobulins (RIG), and rabies vaccination. We conducted a survey of rabies treatment outcomes in the Philippines. Methods This was a case series involving 7,660 patients (4 months to 98 years of age) given purified equine RIG (pERIG) at the Research Institute for Tropical Medicine (Muntinlupa, Philippines) from July 2003 to August 2004 following Category II or III exposures. Data on local and systemic adverse reactions (AR) within 28 days and biting animal status were recorded; outcome data were obtained by telephone or home visit 6–29 months post-exposure. Results Follow-up data were collected for 6,464 patients. Of 151 patients with laboratory-confirmed rabies exposure, 143 were in good health 6–48 months later, seven could not be contacted, and one 4-year-old girl died. Of 16 deaths in total, 14 were unrelated to rabies exposure or treatment. Two deaths were considered PEP failures: the 4-year old girl, who had multiple deep lacerated wounds from a rabid dog of the nape, neck, and shoulders requiring suturing on the day of exposure, and an 8-year-old boy who only received rabies PEP on the day of exposure. Conclusions This extensive review of outcomes in persons with Category III exposure shows the recommended treatment schedule at RITM using pERIG is well tolerated, while survival of 143 laboratory-confirmed rabies exposures confirms the intervention efficacy. Two PEP intervention failures demonstrate that sustained education and training is essential in rabies management. PMID:18509475

  6. Ultra scale-down approach for the prediction of full-scale recovery of ovine polycolonal immunoglobulins used in the manufacture of snake venom-specific Fab fragment.

    PubMed

    Neal, G; Christie, J; Keshavarz-Moore, E; Shamlou, P Ayazi

    2003-01-20

    In this article, we describe a new approach that allows the prediction of the performance of a large-scale integrated process for the primary recovery of a therapeutic antibody from an analysis of the individual unit operations and their interactions in an ultra scale-down mimic of the process. The recovery process consisted of four distinct unit operations. Using the new approach we defined the important engineering parameters in each operation that impacted the overall recovery process and in each case verified its effect by a combination of modelling and experimentation. Immunoglobulins were precipitated from large volumes of dilute blood plasma and the precipitated flocs were recovered by centrifugal separation from the liquor containing contaminating proteins, including albumin. The fluid mechanical forces acting on the precipitate and the time of exposure to these forces were used to define a time-integrated fluid stress. This was used as a scaling factor to predict the properties of the precipitated flocs at large scale. In the case of centrifugation, the performance of a full-scale disc stack centrifuge was predicted. This was achieved from a computational fluid dynamics (CFD) analysis of the flow field in the centrifuge coupled with experimental data obtained from the precipitated immunoglobulin flocs using the scale-down precipitation tank, a rotating shear device, and a standard swing-out rotor centrifuge operating under defined conditions. In this way, the performance of the individual unit operations, and their linkage, was successfully analysed from a combination of modelling and experiments. These experiments required only millilitre quantities of the process material. The overall performance of the large-scale process was predicted by tracking the changes in physical and biological properties of the key components in the system, including the size distribution of the antibody precipitates and antibody activity through the individual unit operations in the ultra scale-down process flowsheet.

  7. Fragmentation Pathways in the Uracil Radical Cation

    SciTech Connect

    Zhou, Congyi; Matsika, Spiridoula; Kotur, Marija; Weinacht, Thomas C.

    2012-08-24

    We investigate pathways for fragmentation in the uracil radical cation using ab initio electronic structure calculations. We focus on the main fragments produced in pump–probe dissociative ionization experiments. These are fragments with mass to charge ratios (m/z) of 69, 28, 41, and 42. Barriers to dissociation along the ground ionic surface are reported, which provide an estimate of the energetic requirements for the production of the main fragments. Finally, direct and sequential fragmentation mechanisms have been analyzed, and it is concluded that sequential fragmentation after production of fragment with m/z 69 is the dominant mechanism for the production of the smaller fragments.

  8. Particle size statistics in dynamic fragmentation

    SciTech Connect

    Grady, D.E. )

    1990-12-15

    Condensed matter, when subjected to intense disrupting forces through impact or radiation deposition, will break up into a randomly distributed array of fragments. An earlier analysis of random fragmentation is extended to account for fragmentation in bodies which are finite in extent and for bodies within which the minimum fragment size is bounded. The statistical fragment size relations are compared with molecular dynamic simulations of dynamic fragmentation, with fragmentation caused by the high-energy collision of nuclear particles, and with the distribution of galaxies in the universe which are assumed to be fragment debris from the primordial Big Bang.

  9. The combined fragmentation and systematic molecular fragmentation methods.

    PubMed

    Collins, Michael A; Cvitkovic, Milan W; Bettens, Ryan P A

    2014-09-16

    Conspectus Chemistry, particularly organic chemistry, is mostly concerned with functional groups: amines, amides, alcohols, ketones, and so forth. This is because the reactivity of molecules can be categorized in terms of the reactions of these functional groups, and by the influence of other adjacent groups in the molecule. These simple truths ought to be reflected in the electronic structure and electronic energy of molecules, as reactivity is determined by electronic structure. However, sophisticated ab initio quantum calculations of the molecular electronic energy usually do not make these truths apparent. In recent years, several computational chemistry groups have discovered methods for estimating the electronic energy as a sum of the energies of small molecular fragments, or small sets of groups. By decomposing molecules into such fragments of adjacent functional groups, researchers can estimate the electronic energy to chemical accuracy; not just qualitative trends, but accurate enough to understand reactivity. In addition, this has the benefit of cutting down on both computational time and cost, as the necessary calculation time increases rapidly with an increasing number of electrons. Even with steady advances in computer technology, progress in the study of large molecules is slow. In this Account, we describe two related "fragmentation" methods for treating molecules, the combined fragmentation method (CFM) and systematic molecular fragmentation (SMF). In addition, we show how we can use the SMF approach to estimate the energy and properties of nonconducting crystals, by fragmenting the periodic crystal structure into relatively small pieces. A large part of this Account is devoted to simple overviews of how the methods work. We also discuss the application of these approaches to calculating reactivity and other useful properties, such as the NMR and vibrational spectra of molecules and crystals. These applications rely on the ability of these

  10. Fragmentation and ablation during entry

    SciTech Connect

    Canavan, G.H.

    1997-09-01

    This note discusses objects that both fragment and ablate during entry, using the results of previous reports to describe the velocity, pressure, and fragmentation of entering objects. It shows that the mechanisms used there to describe the breakup of non-ablating objects during deceleration remain valid for most ablating objects. It treats coupled fragmentation and ablation during entry, building on earlier models that separately discuss the entry of objects that are hard, whose high heat of ablation permits little erosion, and those who are strong whose strength prevents fragmentation, which are discussed in ``Radiation from Hard Objects,`` ``Deceleration and Radiation of Strong, Hard, Asteroids During Atmospheric Impact,`` and ``Meteor Signature Interpretation.`` This note provides a more detailed treatment of the further breakup and separation of fragments during descent. It replaces the constraint on mass per unit area used earlier to determine the altitude and magnitude of peak power radiation with a detailed analytic solution of deceleration. Model predictions are shown to be in agreement with the key features of numerical calculations of deceleration. The model equations are solved for the altitudes of maximum radiation, which agree with numerical integrations. The model is inverted analytically to infer object size and speed from measurements of peak power and altitude to provide a complete model for the approximate inversion of meteor data.

  11. An Immunosensor Based on Antibody Binding Fragments Attached to Gold Nanoparticles for the Detection of Peptides Derived from Avian Influenza Hemagglutinin H5

    PubMed Central

    Jarocka, Urszula; Sawicka, Róża; Góra-Sochacka, Anna; Sirko, Agnieszka; Zagórski-Ostoja, Włodzimierz; Radecki, Jerzy; Radecka, Hanna

    2014-01-01

    This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i) modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii) immobilization of antibody-binding fragments (Fab') of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii) covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab' fragments and hemagglutinin (HA) variants have been explored with electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6]3−/4− as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues) of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues) A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues) of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL. PMID:25157550

  12. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD[superscript +] and triclosan

    SciTech Connect

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2010-11-19

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD{sup +} has been solved to a resolution of 2.1 {angstrom}. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD{sup +} reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD{sup +} cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors.

  13. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

    PubMed

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  14. Three-dimensional structure of the Fab from a human IgM cold agglutinin.

    PubMed

    Cauerhff, A; Braden, B C; Carvalho, J G; Aparicio, R; Polikarpov, I; Leoni, J; Goldbaum, F A

    2000-12-01

    Cold agglutinins (CAs) are IgM autoantibodies characterized by their ability to agglutinate in vitro RBC at low temperatures. These autoantibodies cause hemolytic anemia in patients with CA disease. Many diverse Ags are recognized by CAs, most frequently those belonging to the I/i system. These are oligosaccharides composed of repeated units of N:-acetyllactosamine, expressed on RBC. The three-dimensional structure of the Fab of KAU, a human monoclonal IgM CA with anti-I activity, was determined. The KAU combining site shows an extended cavity and a neighboring pocket. Residues from the hypervariable loops V(H)CDR3, V(L)CDR1, and V(L)CDR3 form the cavity, whereas the small pocket is defined essentially by residues from the hypervariable loops V(H)CDR1 and V(H)CDR2. This fact could explain the V(H)4-34 germline gene restriction among CA. The KAU combining site topography is consistent with one that binds a polysaccharide. The combining site overall dimensions are 15 A wide and 24 A long. Conservation of key binding site residues among anti-I/i CAs indicates that this is a common feature of this family of autoantibodies. We also describe the first high resolution structure of the human IgM C(H)1:C(L) domain. The structural analysis shows that the C(H)1-C(L) interface is mainly conserved during the isotype switch process from IgM to IgG1.

  15. Best-practice evaluation-methods for wafer-fab photomask-requalification inspection tools

    NASA Astrophysics Data System (ADS)

    Cho, Chan Seob; Mungmode, Ashish; Taylor, Ron; Cho, David; Koh, Hui Peng

    2014-10-01

    Requalifying semiconductor photomasks remains critically important and is increasingly challenging for 20nm and 14nm node logic reticles. Patterns are becoming more complex on the photomask, and defect sensitivity requirements are more stringent than ever before. Reticle inspection tools are equally important for effective process development and the successful ramp and sustained yield for high volume manufacturing. The inspection stages considered were: incoming inspection to match with Mask Shop Outgoing result and to detect defects generated during transport; requalification by routine cycle inspection to detect Haze and any other defects; and inspection by in-house or Mask shop at the post cleaning. There are many critical capability and capacity factors for the decision for best inspection tool and strategy for high volume manufacturing, especially objective Lens NA, wavelength, power, pixel size, throughput, full-automation inspection linked with Overhead Transport, algorithm application, engineering application function, and inspection of PSM and OMOG . These tools are expensive but deliver differentiated value in terms of performance and throughput as well as extendibility. Performing a thorough evaluation and making a technically sound choice which explores these many factors is critical for success of a fab. This paper examines the methodology for evaluating two different photomask inspection tools. The focus is on ensuring production worthiness on real and advanced product photomasks requiring accurate evaluation of sensitivity, throughput, data analysis function and engineering work function on those product photomasks. Photomasks used for data collection are production reticles, PDM(Program defect Mask), SiN spray defect Reticle which is described that evaluates how the tools would perform on a contaminated plate.

  16. Fragmentation of DNA by sonication.

    PubMed

    Sambrook, Joseph; Russell, David W

    2006-09-01

    INTRODUCTIONDNA fragmentation is often necessary prior to library construction or subcloning for DNA sequencing. This protocol describes a method for DNA fragmentation by sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonication. DNA that has been sonicated for excessive periods of time is extremely difficult to clone. Most sonicators will not shear DNA to a size of less than 300-500 bp, and it is tempting to continue sonication until the entire DNA population has been reduced in size. However, the yield of subclones is usually greater if sonication is stopped when the fragments of the target DNA first reach a size of ~700 bp.

  17. Understanding the molecular recognition between antibody fragments and protein A biomimetic ligand.

    PubMed

    Branco, Ricardo J F; Dias, Ana M G C; Roque, Ana C A

    2012-06-29

    Affinity chromatography with protein A from Staphylococcus aureus (SpA) is the most widespread and accepted methodology for antibody capture during the downstream process of antibody manufacturing. A triazine based ligand (ligand 22/8) was previously developed as an inexpensive and robust alternative to SpA chromatography (Li et al. and Teng et al.). Despite the experimental success, there is no structural information on the binding modes of ligand 22/8 to antibodies, namely to Immunoglobulin G (IgG) molecules and fragments. In this work, we addressed this issue by a molecular docking approach allied to molecular dynamics simulations. Theoretical results confirmed the preference of the synthetic ligand to bind IgG through the binding site found in the crystallographic structure of the natural complex between SpA and the Fc fragment of IgG. Our studies also suggested other unknown "hot-spots" for specific binding of the affinity ligand at the hinge between V(H) and C(H)1 domains of Fab fragment. The best docking poses were further analysed by molecular dynamics studies at three different protonation states (pH 3, 7 and 11). The main interactions between ligand 22/8 and the IgG fragments found at pH 7 were weaker at pH 3 and pH 11 and in these conditions the ligand start losing tight contact with the binding site, corroborating the experimental evidence for protein elution from the chromatographic adsorbents at these pH conditions.

  18. Manufacture of β-TCP/alginate scaffolds through a Fab@home model for application in bone tissue engineering.

    PubMed

    Diogo, G S; Gaspar, V M; Serra, I R; Fradique, R; Correia, I J

    2014-06-01

    The growing need to treat bone-related diseases in an elderly population compels the development of novel bone substitutes to improve patient quality of life. In this context, the advent of affordable and effective rapid prototyping equipment, such as the Fab@home plotter, has contributed to the development of novel scaffolds for bone tissue engineering. In this study, we report for the first time the use of a Fab@home plotter for the production of 3D scaffolds composed by beta-tricalcium phosphate (β-TCP)/alginate hybrid materials. β-TCP/alginate mixtures were used in a proportion of 50/50% (w/w), 30/70% (w/w) and 20/80% (w/w). The printing parameters were optimized to a nozzle diameter of 20 Gauge for the production of rigid scaffolds with pre-defined architectures. We observed that, despite using similar printing parameters, both the precision and resolution of the scaffolds were significantly affected by the blend's viscosity. In particular, we demonstrate that the higher viscosity of 50/50 scaffolds (150.0 ± 3.91 mPa s) provides a higher precision in the extrusion process. The physicochemical and biological characterization of the samples demonstrated that the 50/50 scaffolds possessed a resistance to compression comparable to that of native trabecular bone. Moreover, this particular formulation also exhibited a Young's modulus that was higher than that of trabecular bone. Scanning electron microscopy and fluorescence microscopy analysis revealed that osteoblasts were able to adhere, proliferate and also penetrate into the scaffold's architecture. Altogether, our findings suggest that the Fab@home printer can be employed in the manufacture of reproducible scaffolds, using a formulation 50/50 alginate-β-TCP that has suitable properties to be applied as bone substitutes in the future.

  19. Production, purification and biological characterization of mono-PEGylated anti-IL-17A antibody fragments.

    PubMed

    Koussoroplis, Salome-Juliette; Heywood, Sam; Uyttenhove, Catherine; Barilly, Céline; Van Snick, Jacques; Vanbever, Rita

    2013-09-15

    The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥ 20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab')2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies.

  20. A thermodynamic theory of dynamic fragmentation

    SciTech Connect

    Yew, Ching H.; Taylor, P.A.

    1993-08-01

    We present a theory of dynamic fragmentation of brittle materials based on thermodynamic arguments. We recover the expressions for average fragment size and number as originally derived by Grady. We extend the previous work by obtaining descriptions of fragment size distribution and compressibility change due to the fragmentation process. The size distribution is assumed to be proportional to the spectral power of the strain history and a sample distribution is presented for a fragmentation process corresponding to a constant rate strain history. The description of compressibility change should be useful in computational studies of fragmentation. These results should provide insight into the process of fragmentation of brittle materials from hypervelocity impact.

  1. Structural and Enzymatic Analyses Reveal the Binding Mode of a Novel Series of Francisella tularensis Enoyl Reductase (FabI) Inhibitors

    SciTech Connect

    Mehboob, Shahila; Hevener, Kirk E.; Truong, Kent; Boci, Teuta; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-10-10

    Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.

  2. Impact of FAB classification on predicting outcome in acute myeloid leukemia, not otherwise specified, patients undergoing allogeneic stem cell transplantation in CR1: An analysis of 1690 patients from the acute leukemia working party of EBMT.

    PubMed

    Canaani, Jonathan; Beohou, Eric; Labopin, Myriam; Socié, Gerard; Huynh, Anne; Volin, Liisa; Cornelissen, Jan; Milpied, Noel; Gedde-Dahl, Tobias; Deconinck, Eric; Fegueux, Nathalie; Blaise, Didier; Mohty, Mohamad; Nagler, Arnon

    2017-04-01

    The French, American, and British (FAB) classification system for acute myeloid leukemia (AML) is extensively used and is incorporated into the AML, not otherwise specified (NOS) category in the 2016 WHO edition of myeloid neoplasm classification. While recent data proposes that FAB classification does not provide additional prognostic information for patients for whom NPM1 status is available, it is unknown whether FAB still retains a current prognostic role in predicting outcome of AML patients undergoing allogeneic stem cell transplantation. Using the European Society of Blood and Bone Marrow Transplantation registry we analyzed outcome of 1690 patients transplanted in CR1 to determine if FAB classification provides additional prognostic value. Multivariate analysis revealed that M6/M7 patients had decreased leukemia free survival (hazard ratio (HR) of 1.41, 95% confidence interval (CI), 1.01-1.99; P = .046) in addition to increased nonrelapse mortality (NRM) rates (HR, 1.79; 95% CI, 1.06-3.01; P = .028) compared with other FAB types. In the NPM1(wt) AML, NOS cohort, FAB M6/M7 was also associated with increased NRM (HR, 2.17; 95% CI, 1.14-4.16; P = .019). Finally, in FLT3-ITD(+) patients, multivariate analyses revealed that specific FAB types were tightly associated with adverse outcome. In conclusion, FAB classification may predict outcome following transplantation in AML, NOS patients.

  3. The Fragmentation of Literary Theory

    ERIC Educational Resources Information Center

    Howard, Jennifer

    2005-01-01

    Syllabi from some 20 colleges and universities were reviewed with prominent English and literature departments and a discussion was held with a number of professors who teach literary theory. It is suggested that devolution and fragmentation of theory might be a survival strategy, an adaptation to the new realties of academic institutions.

  4. Percolative fragmentation and spontaneous agglomeration

    SciTech Connect

    Hurt, R.; Davis, K.

    1999-03-01

    Captive particle imaging experiments were performed on over 200 coal and char particles in the pulverized size range from four coals of various rank at oxygen concentration from 3--19 mol% and at gas temperatures of about 1250 K. Despite wide variations in single-particle behavior, the data set reveals two clear trends that provide new information on the nature of char combustion. First, the low-rank coal chars are observed to maintain their high reactivity through the late stages of combustion, thus avoiding the near-extinction events and long burnout tails observed for bituminous coal chars. Secondly, percolative fragmentation in the late stages of combustion is a rare event under these conditions. Some particles reach a percolation threshold rate in combustion, but typically undergo spontaneous agglomeration rather than liberation of the incipient fragments. It is concluded that percolative fragmentation behavior in the pulverized size range is determined not only by solid-phase connectivity, but also by a real competition between disruptive and cohesive forces present at the time of formation of the colloidal-sized incipient fragments.

  5. Controlled Dynamic Fragmentation of Ceramics

    DTIC Science & Technology

    2009-12-14

    mechanism determining ballistic impact performance of ceramic armors as well as reliability of gun barrels. This program, which was sponsored by the...Research Office Fragmentation is a key damage mechanism determining ballistic impact performance of ceramic armors as well as reliability of gun ...the defect distributions that were considered (Weibull, Gauss and Uniform) fall on a single universal curve. Sdf

  6. Nuclear energy release from fragmentation

    NASA Astrophysics Data System (ADS)

    Li, Cheng; Souza, S. R.; Tsang, M. B.; Zhang, Feng-Shou

    2016-08-01

    It is well known that binary fission occurs with positive energy gain. In this article we examine the energetics of splitting uranium and thorium isotopes into various numbers of fragments (from two to eight) with nearly equal size. We find that the energy released by splitting 230,232Th and 235,238U into three equal size fragments is largest. The statistical multifragmentation model (SMM) is applied to calculate the probability of different breakup channels for excited nuclei. By weighing the probability distributions of fragment multiplicity at different excitation energies, we find the peaks of energy release for 230,232Th and 235,238U are around 0.7-0.75 MeV/u at excitation energy between 1.2 and 2 MeV/u in the primary breakup process. Taking into account the secondary de-excitation processes of primary fragments with the GEMINI code, these energy peaks fall to about 0.45 MeV/u.

  7. Fission fragment driven neutron source

    DOEpatents

    Miller, Lowell G.; Young, Robert C.; Brugger, Robert M.

    1976-01-01

    Fissionable uranium formed into a foil is bombarded with thermal neutrons in the presence of deuterium-tritium gas. The resulting fission fragments impart energy to accelerate deuterium and tritium particles which in turn provide approximately 14 MeV neutrons by the reactions t(d,n).sup.4 He and d(t,n).sup.4 He.

  8. Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

    PubMed Central

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A.

    2012-01-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes. PMID:22753059

  9. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

    PubMed

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

    2014-02-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy.

  10. Metabolic flux between unsaturated and saturated fatty acids is controlled by the FabA:FabB ratio in the fully reconstituted fatty acid biosynthetic pathway of Escherichia coli.

    PubMed

    Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

    2013-11-19

    The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s(-1) was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.

  11. Experimental Volcanology: Fragmentation and Permeability

    NASA Astrophysics Data System (ADS)

    Spieler, O.

    2005-12-01

    An increasing number of scientists design new experiments to analyse processes that control the dynamics of explosive eruptions. There research is mostly coupled to numerical models and aims toward its controlling parameters. The fragmentation process, its threshold and the speed of the fragmentation wave as well as the energy consumed by the fragmentation are some hot spots of the experimental volcanology. Analysing the fragmentation behaviour of volcaniclastics as close to the natural system as possible, we found a number of physical constrains. Identifying the porosity as determining factor of the threshold, we realised that neither threshold nor the speed of the fragmentation process are solely controlled by the rock density. The later results of the shock tube type apparatus lead to the analysis of the specific surface area and permeability as direct links to textural features. Permeability analysis performed in a modified shock tube type apparatus, show two clear, distinct trends for dome rock and pyroclastic samples. The specific surface determined by Argon sorbtion (BET) as well as textural features of pumices from Campi Flegrei, Montserrat and Krakatoa (1883) give in contrary evidence of a more complex story. Large spherical, or ellipsoidal bubbles around fractured crystals prove that the high permeability of the pumice has partially developed after the fixing of the bubble size distribution. This puts up the question, if permeability measurements on pyroclastic samples reveal relevant numbers! The surface tension controlled 'self sealing' behaviour of surfaces from foaming obsidian hinders in situ measurements. Close textural investigations will have to clarify how the 'post process' samples deviate from the syneruptive conduit filling.

  12. Fragments

    ERIC Educational Resources Information Center

    Moreira, Claudio

    2008-01-01

    This performance autoethnography shows the author's struggle in finding his place, scholarship, voice, and body, into the academic setting. Mixing together memories of his lived experience with sugar cane workers, notes, and leftovers of different fieldworks, plus 6 years of life as grad student at the University of Illinois, the author looks for…

  13. A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.

    PubMed

    Ni, Yan G; Condra, Jon H; Orsatti, Laura; Shen, Xun; Di Marco, Stefania; Pandit, Shilpa; Bottomley, Matthew J; Ruggeri, Lionello; Cummings, Richard T; Cubbon, Rose M; Santoro, Joseph C; Ehrhardt, Anka; Lewis, Dale; Fisher, Timothy S; Ha, Sookhee; Njimoluh, Leila; Wood, Dana D; Hammond, Holly A; Wisniewski, Douglas; Volpari, Cinzia; Noto, Alessia; Lo Surdo, Paola; Hubbard, Brian; Carfí, Andrea; Sitlani, Ayesha

    2010-04-23

    PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

  14. Metabolism-directed structure optimization of benzimidazole-based Francisella tularensis enoyl-reductase (FabI) inhibitors.

    PubMed

    Zhang, Yan-Yan; Liu, Yong; Mehboob, Shahila; Song, Jin-Hua; Boci, Teuta; Johnson, Michael E; Ghosh, Arun K; Jeong, Hyunyoung

    2014-05-01

    1. FabI is a potential antibiotic target against Francisella tularensis, which has been classified as a Category A biowarfare agent of high risk to public health. Our previous work demonstrated that N-benzyl benzimidazole compounds possess promising FabI inhibitory activity, but their druggability properties, including metabolic stability, are unknown. 2. The objective of this study was to characterize structure-metabolism relationships of a series of N-benzyl benzimidazole compounds to guide chemical optimization for better metabolic stability. To this end, metabolic stability data were obtained for 22 initial lead compounds using mouse hepatic microsomes. 3. Metabolic hotspots on the benzimidazole core structure as well as the benzyl ring were identified and verified by metabolite identification studies of four model compounds. Interestingly, the proposed structure-metabolism relationships did not apply to nine newly synthesized cyclopentane or oxacyclopentane derivatives of N-benzyl benzimidazole. 4. Subsequently, in silico quantitative structure-property relationship models were developed. Four molecular descriptors representing molecular polarity/polarisability, symmetry and size were identified to best explain variability in metabolic stability of different compounds. Multi-linear and non-linear regression models based on the selected molecular descriptors were developed and validated. 5. The structure-metabolism relationships for N-benzyl benzimidazole compounds should help optimization of N-benzyl benzimidazole compounds for better pharmacokinetic behavior.

  15. Mode of Action, In Vitro Activity, and In Vivo Efficacy of AFN-1252, a Selective Antistaphylococcal FabI Inhibitor

    PubMed Central

    Albert, Monique; Awrey, Donald; Bardouniotis, Elias; Berman, Judd; Clarke, Teresa; Dorsey, Mandy; Hafkin, Barry; Ramnauth, Jaillal; Romanov, Vladimir; Schmid, Molly B.; Thalakada, Rosanne; Yethon, Jeremy; Pauls, Henry W.

    2012-01-01

    The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252–FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log10 reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC90, 0.015 μg/ml) and coagulase-negative staphylococci (MIC90, 0.12 μg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections. PMID:22948878

  16. Metabolism-Directed Structure Optimization of Benzimidazole-Based Francisella tularensis Enoyl-Reductase (FabI) Inhibitors

    PubMed Central

    Zhang, Yan-Yan; Liu, Yong; Mehboob, Shahila; Song, Jin-Hua; Boci, Teuta; Johnson, Michael E.; Ghosh, Arun K.; Jeong, Hyunyoung

    2015-01-01

    FabI is a potential antibiotic target against Francisella tularensis, which has been classified as a Category A biowarfare agent of high risk to public health. Our previous work demonstrated that N-benzyl benzimidazole compounds possess promising FabI inhibitory activity, but their druggability properties including metabolic stability are unknown. The objective of this study was to characterize structure-metabolism relationships of a series of N-benzyl benzimidazole compounds to guide chemical optimization for better metabolic stability. To this end, metabolic stability data were obtained for 22 initial lead compounds using mouse hepatic microsomes. Metabolic hotspots on the benzimidazole core structure as well as the benzyl ring were identified and verified by metabolite identification studies of 4 model compounds. Interestingly, the proposed structure-metabolism relationships did not apply to 9 newly synthesized cyclopentane or oxacyclopentane derivatives of N-benzyl benzimidazole. Subsequently, in silico quantitative structure-property relationship models were developed. Four molecular descriptors representing molecular polarity/polarisability, symmetry and size were identified to best explain variability in metabolic stability of different compounds. Multi-linear and nonlinear regression models based on the selected molecular descriptors were developed and validated. The structure-metabolism relationships for N-benzyl benzimidazole compounds should help optimization of N-benzyl benzimidazole compounds for better pharmacokinetic behavior. PMID:24171690

  17. Assessment of myocardial damage in dilated-phase hypertrophic cardiomyopathy by using indium-111-antimyosin Fab myocardial scintigraphy

    SciTech Connect

    Nishimura, T.; Nagata, S.; Uehara, T.; Hayashida, K.; Mitani, I.; Kumita, S. )

    1991-07-01

    For the detection of myocardial cell damage, an 111In-antimyosin Fab study was carried out on seven patients (Group A) in the dilated phase of hypertrophic cardiomyopathy, seven patients (Group B) with dilated cardiomyopathy, and eight control patients (Group C). Imaging was done 48 hr after intravenous injection of 74 MBq of 111In-antimyosin Fab. Myocardial antimyosin uptake was visually graded as 0, +1, +2 or +3. A score of +2 or +3 was considered positive. The heart/lung ratio of antimyosin uptake (antimyosin index) also was determined. Antimyosin uptake was positive in seven (100%), nine (90%) and no (0%) patients in Groups A, B, and C, respectively. The antimyosin index in Groups A and B was 2.46 {plus minus} 0.49 and 2.04 {plus minus} 0.24, respectively, findings were significantly higher than that in Group C (1.51 {plus minus} 0.13) (p less than 0.01). Positive biopsy findings were noted in only two patients in Group A. Thus, antimyosin uptake was increased in dilated phase hypertrophic cardiomyopathy and dilated cardiomyopathy, which suggests ongoing necrotic changes in these patients.

  18. Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays.

    PubMed

    Kumada, Yoichi

    2014-11-01

    The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

  19. Energy production using fission fragment rockets

    SciTech Connect

    Chapline, G.; Matsuda, Y.

    1991-08-01

    Fission fragment rockets are nuclear reactors with a core consisting of thin fibers in a vacuum, and which use magnetic fields to extract the fission fragments from the reactor core. As an alternative to ordinary nuclear reactors, fission fragment rockets would have the following advantages: Approximately twice as efficient if one can directly convert the fission fragment energy into electricity; by reducing the buildup of a fission fragment inventory in the reactor one could avoid a Chernobyl type disaster; and collecting the fission fragments outside the reactor could simplify the waste disposal problem. 6 refs., 4 figs., 2 tabs.

  20. Fragmentation of suddenly heated liquids

    SciTech Connect

    Blink, J.A.

    1985-03-01

    Fragmentation of free liquids in Inertial Confinement Fusion reactors could determine the upper bound on reactor pulse rate. The x-ray ablated materials must cool and recondense to allow driver beam propagation. The increased surface area caused by fragmentation will enhance the cooling and condensation rates. Relaxation from the suddenly heated state will move a liquid into the negative pressure region under the liquid-vapor P-V dome. The lithium equation of state was used to demonstrate that neutron-induced vaporization uses only a minor fraction of the added heat, much less than would be required to drive the expansion. A 77% expansion of the lithium is required before the rapid vaporization process of spinodal decomposition could begin, and nucleation and growth are too slow to contribute to the expansion.

  1. Asymmetry effects in fragment production

    NASA Astrophysics Data System (ADS)

    Kaur, Manpreet; Kaur, Varinderjit

    2016-05-01

    The production of different fragments has been studied by taking into account the mass asymmetry of the reaction and employing the momentum dependent interactions. Two different set of asymmetric reactions have been analyzed while keeping Atotal fixed using soft momentum dependent equation of state. Our results indicate that the impact of momentum dependent interactions is different in lighter projectile systems as compared to heavier ones. The comparative analysis of IQMD simulations with the experimental data in case of heavier projectile and lighter target system for the reaction of 197Au+27Al (η = 0.7) at E = 600 MeV/nucleon shows that with the inclusion of MDI we are able, upto some extent, to reproduce the experimental universality of rise and fall of intermediate mass fragments (IMFs).

  2. Fragmentation of the Chelyabinsk Fireball

    NASA Astrophysics Data System (ADS)

    Melosh, J.

    2013-12-01

    The most intense bolide since the 1908 Tunguska event occurred in the early morning hours of 15 February 2013 near the Russian town of Chelyabinsk. The impacting asteroid ranged between 17 and 20 m diameter with a mass of about 10,000 tons. Its estimated pre-atmospheric velocity was about 18.6 km/sec at a low angle of 20° from the horizontal. The resulting airburst occurred at an altitude of about 23 km and released an estimated total energy of about 440 kT (1.7 x 1015 J). The blast wave shattered windows on the ground over a wide area and collapsed the roof of a zinc factory. In spite of the size of the initital asteroid, only small fragments (a few kg, so far) have been recovered. The entry of an asteroid into Earth's atmosphere and its aerodynamic fragmentation and deceleration has been modeled by a number of authors over the past few decades. Full-featured numerical simulations are presently limited in their ability to simultaneously incorporate fragmentation, energy coupling between solid fragments and the atmosphere, and thermal radiation, but an approximate treatment of the fragmentation and dispersion of a large entering meteoroid called the 'pancake model' has achieved good fits to other observed events, including the Tunguska explosion, the 1947 Shikote-Alin fall and strewn fields from larger iron meteorite falls, such as the Henbury craters. Simulations using the pancake model can fit the overall observations of the Chelyabinsk event using an 18 m diameter asteroid of density 3000 kg/m3 following the observed trajectory and possessing an initial strength of about 7 MPa, which is relatively high for a stony meteoroid. This suggests that the asteroid was not a strengthless rubble pile, but in fact possessed considerable strength, compared to other stony meteorites of similar type. Aerodynamic breakup begins at an altitude of 31 km and the final airburst occurs at 22 km, releasing about 250 kT at this time. Subsequent to this airburst, large fragments

  3. Golgi fragmentation in Alzheimer's disease

    PubMed Central

    Joshi, Gunjan; Bekier, Michael E.; Wang, Yanzhuang

    2015-01-01

    The Golgi apparatus is an essential cellular organelle for post-translational modifications, sorting, and trafficking of membrane and secretory proteins. Proper functionality of the Golgi requires the formation of its unique cisternal-stacking morphology. The Golgi structure is disrupted in a variety of neurodegenerative diseases, suggesting a common mechanism and contribution of Golgi defects in neurodegenerative disorders. A recent study on Alzheimer's disease (AD) revealed that phosphorylation of the Golgi stacking protein GRASP65 disrupts its function in Golgi structure formation, resulting in Golgi fragmentation. Inhibiting GRASP65 phosphorylation restores the Golgi morphology from Aβ-induced fragmentation and reduces Aβ production. Perturbing Golgi structure and function in neurons may directly impact trafficking, processing, and sorting of a variety of proteins essential for synaptic and dendritic integrity. Therefore, Golgi defects may ultimately promote the development of AD. In the current review, we focus on the cellular impact of impaired Golgi morphology and its potential relationship to AD disease development. PMID:26441511

  4. Modeling of Fragmentation of Asteroids

    NASA Technical Reports Server (NTRS)

    Agrawal, Parul; Prabhu, Dinesh K.; Carlozzi, Alexander; Hart, Kenneth; Bryson, Katie; Sears, Derek

    2015-01-01

    The objective of this study is to understand fragmentation and fracture of a given asteroid and mechanisms of break-up. The focus of the present work is to develop modeling techniques for stony asteroids in 10m-100m range to answer two questions: 1) What is the role of material makeup of an asteroid in the stress distribution? 2)How is stress distribution altered in the presence of pre-existing defects?

  5. Fragment Penetration Tests of Armor

    DTIC Science & Technology

    1983-03-15

    Identify by block numbhr) Provides techniques for evaluating armor resistance to attack by HE projectile fragments. Includes static detonations of shell...DISTRIBUTION D. REFERENCES * . . . ........ . ........... D-1 1. SCOPE. This TOP describes the available techniques for testing armor for resistance to attack by...Projectiles Against Armor Plates ("Yankee Stadium" Test-). 4.1.1 hCjective. The objective is to determine the resistance to penetration of various armor

  6. Fragmentation: Is the message clear?

    USGS Publications Warehouse

    Bissonette, J.A.; Storch, Ilse

    2002-01-01

    In this paper, we briefly discuss some of the fundamental problems arising from the inherent complexity of larger-scale ecological systems. We examine the tenuous assumption of a direct correspondence between ecological data and theory, we comment on a recent report that evaluated the efficacy of fragmentation experiments, and we briefly assess its implications for ecological research and conservation practice on the landscape scale. Copyright ?? 2002 by the author(s). Published here under licence by The Resilience Alliance.

  7. The fragmentation of Kosmos 2163

    NASA Technical Reports Server (NTRS)

    1992-01-01

    On 6 Dec. 1991 Kosmos 2163, a maneuverable Soviet spacecraft which had been in orbit for 58 days, experienced a major breakup at an altitude of approximately 210 km. Although numerous pieces of debris were created, the fragments decayed rapidly leaving no long-term impact on the near-Earth environment. The assessed cause of the event is the deliberate detonation of an explosive device. Details of this event are presented.

  8. Fragmentation in Science and in Society

    ERIC Educational Resources Information Center

    Bohm, David

    1971-01-01

    Fragmentation" is a general social condition. The author presents a case for wholeness and its dynamic, cyclic character. Science and society need not be fragmented. They should be considered part of a holocyclation." (LS)

  9. Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis.

    PubMed

    Zhang, Lin; Xiao, Jianfeng; Xu, Jianrong; Fu, Tianran; Cao, Zhiwei; Zhu, Liang; Chen, Hong-Zhuan; Shen, Xu; Jiang, Hualiang; Zhang, Liang

    2016-12-01

    Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS.

  10. Myocardial distribution of indium-111-antimyosin Fab in acute inferior and right ventricular infarction: comparison with technetium-99m-pyrophosphate imaging and histologic examination

    SciTech Connect

    Nakata, T.; Sakakibara, T.; Noto, T.; Shoji, T.; Tsuda, T.; Kubota, M.; Hattori, A.; Iimura, O. )

    1991-05-01

    In a postmortem study of a 69-yr-old female patient who had suffered 2 yr previously a non-Q-wave anterior infarction and who had sustained just seven days earlier a left inferior and right ventricular infarction, the distribution of {sup 111}In-antimyosin Fab was compared to the results of {sup 99}mTc-pyrophosphate imaging and histologic examination. Indium-111-antimyosin Fab imaging could not be performed because of cardiogenic shock. However, postmortem gamma scintillation counting revealed increased activities of antimyosin Fab in the inferoapical and right ventricular infarcted regions in which {sup 99}mTc-pyrophosphate positive imagings were observed; in contrast, a histologically confirmed old subendocardial anterior infarction had no definite activity. Thus, the myocardial distribution of {sup 111}In-antimyosin Fab corresponded well to the results of {sup 99}mTc scintigrams and histologic examinations in a human heart, suggesting that this technique could be useful in vivo for detecting several-day-old myocardial infarction of the right ventricle as well as the left ventricle. Tissue from the 2-yr-old infarction was not identified by this technique.

  11. Identification of a conserved protein involved in anaerobic unsaturated fatty acid synthesis in Neiserria gonorrhoeae: implications for facultative and obligate anaerobes that lack FabA.

    PubMed

    Isabella, Vincent M; Clark, Virginia L

    2011-10-01

    Transcriptome analysis of the facultative anaerobe, Neisseria gonorrhoeae, revealed that many genes of unknown function were induced under anaerobic conditions. Mutation of one such gene, NGO1024, encoding a protein belonging to the 2-nitropropane dioxygenase-like superfamily of proteins, was found to result in an inability of gonococci to grow anaerobically. Anaerobic growth of an NG1024 mutant was restored upon supplementation with unsaturated fatty acids (UFA), but not with the saturated fatty acid palmitate. Gonococcal fatty acid profiles confirmed that NGO1024 was involved in UFA synthesis anaerobically, but not aerobically, demonstrating that gonococci contain two distinct pathways for the production of UFAs, with a yet unidentified aerobic mechanism, and an anaerobic mechanism involving NGO1024. Expression of genes involved in classical anaerobic UFA synthesis, fabA, fabM and fabB, was toxic in gonococci and unable to complement a NGO1024 mutation, suggesting that the chemistry involved in gonococcal anaerobic UFA synthesis is distinct from that of the classical pathway. NGO1024 homologues, which we suggest naming UfaA, form a distinct lineage within the 2-nitropropane dioxygenase-like superfamily, and are found in many facultative and obligate anaerobes that produce UFAs but lack fabA, suggesting that UfaA is part of a widespread pathway involved in UFA synthesis.

  12. Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis

    PubMed Central

    Zhang, Lin; Xiao, Jianfeng; Xu, Jianrong; Fu, Tianran; Cao, Zhiwei; Zhu, Liang; Chen, Hong-Zhuan; Shen, Xu; Jiang, Hualiang; Zhang, Liang

    2016-01-01

    Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS. PMID:27874013

  13. Enhanced production of branched-chain fatty acids by replacing β-ketoacyl-(acyl-carrier-protein) synthase III (FabH).

    PubMed

    Jiang, Wen; Jiang, Yanfang; Bentley, Gayle J; Liu, Di; Xiao, Yi; Zhang, Fuzhong

    2015-08-01

    Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and/or the co-production of a large amount of straight-chain fatty acids (SCFAs), making it nearly impossible for further conversion of BCFAs to branched biofuels. Synthesis of both SCFAs and BCFAs requires FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase in Escherichia coli that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. In this study, we demonstrated that replacement of the acetyl-CoA-specific E. coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in a significant enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further demonstrated that the composition of BCFAs can be tuned by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, leading to the production either even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Overall, the top-performing strain from this study produced BCFAs at 126 mg/L, comprising 52% of the total free fatty acids.

  14. Evaluating Fragment Construction Policies for SDT Systems

    DTIC Science & Technology

    2006-01-01

    allocates a fragment and begins translation. Once a termination condition is met, Strata emits any trampolines that are necessary. Trampolines are pieces... trampolines (unless its target previously exists in the fragment cache). Once a CTI’s target instruction becomes available in the fragment cache, the CTI is...linked directly to the destination, avoiding future uses of the trampoline . This mechanism is called Fragment Linking and avoids significant overhead

  15. Comparison of calculations of fragment production

    SciTech Connect

    Canavan, G.H.

    1998-01-01

    This note compares estimates of fragment production rates in debris collisions through calculations performed with consistent debris distribution functions implicit in integrated collision frequencies provided by Attachment A. Differences between estimates of fragment production rates in space debris collisions are shown to be due primarily to different choices of the exponent in the fragment production function and the distinction between catastrophic and all collisions. Sensitivity to the fragment production parameter over the range of values consistent with experimental data is discussed.

  16. Fragmentation of cosmic-string loops

    NASA Technical Reports Server (NTRS)

    York, Thomas

    1989-01-01

    The fragmentation of cosmic string loops is discussed, and the results of a simulation of this process are presented. The simulation can evolve any of a large class of loops essentially exactly, including allowing fragments that collide to join together. Such reconnection enhances the production of small fragments, but not drastically. With or without reconnections, the fragmentation process produces a collection of nonself-intersecting loops whose typical length is on the order of the persistence length of the initial loop.

  17. Changes in membrane fatty acid composition through proton-induced fabF mutation enhancing 1-butanol tolerance in E. coli

    NASA Astrophysics Data System (ADS)

    Jeong, Haeyoung; Kim, Sun Hong; Han, Sang Soo; Kim, Myung Hee; Lee, Keun Chul

    2012-07-01

    While a rational approach based on genomic data has become the preferred method for microbial strain development, radiation-induced random mutagenesis is still a robust method for organisms such as plants whose genome or target gene information is unavailable. We previously reported on a combined approach that consists of proton irradiation and a long-term experimental evolution to enhance 1-butanol tolerance of the E. coli C strain so that it can be used as a basal strain for the production of 1-butanol, a potential biofuel along with ethanol. Genome sequencing of one randomly chosen clone (PKH5000) from the endpoint population revealed eleven mutations occurring in the coding regions, and we found that a mutation (F74C) in fabF gene encoding β-ketoacyl-ACP synthases II is associated with a twofold increase in the major unsaturated fatty acid, cis-vaccenic acid. The increase of cis-vaccenic acid by wild-type FabF, which is more active at low temperatures or in the presence of organic compounds, is considered to be a protective mechanism against cold stress. A structural analysis of the FabF protein suggests that the F74C mutation may affect the enzyme activity through a change in flexibility around the catalytic site. The expression of a plasmid that harbors mutant fabF gene in the fabF knockout strain enhanced growth in a medium containing butanol with a concomitant elevation of the cis-vaccenic acid level. Among the eight available Keio knockout strains for genes that have amino acid substitution in the PKH5000 strain, the fabF mutant showed the slowest growth in the presence of 0.7% butanol. We propose that fabF, as probably the gene most responsible for butanol tolerance in wild-type form, contributes further when converted into a F74C missense mutation, which is beneficial as it increases the level of cis-vaccenic acid.

  18. Robust Object Tracking Using Valid Fragments Selection

    PubMed Central

    Li, Bo; Tian, Peng; Luo, Gang

    2016-01-01

    Local features are widely used in visual tracking to improve robustness in cases of partial occlusion, deformation and rotation. This paper proposes a local fragment-based object tracking algorithm. Unlike many existing fragment-based algorithms that allocate the weights to each fragment, this method firstly defines discrimination and uniqueness for local fragment, and builds an automatic pre-selection of useful fragments for tracking. Then, a Harris-SIFT filter is used to choose the current valid fragments, excluding occluded or highly deformed fragments. Based on those valid fragments, fragment-based color histogram provides a structured and effective description for the object. Finally, the object is tracked using a valid fragment template combining the displacement constraint and similarity of each valid fragment. The object template is updated by fusing feature similarity and valid fragments, which is scale-adaptive and robust to partial occlusion. The experimental results show that the proposed algorithm is accurate and robust in challenging scenarios. PMID:27430036

  19. Evaluating Thermodynamic Integration Performance of the New Amber Molecular Dynamics Package and Assess Potential Halogen Bonds of Enoyl-ACP Reductase (FabI) Benzimidazole Inhibitors

    PubMed Central

    Su, Pin-Chih; Johnson, Michael E.

    2015-01-01

    Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard-Jones and Coulomb Softcore potentials parameters in the one-step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one-step TI in a protein-ligand binding system. The best performing model, the one-step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM-GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure-activity relationship insights, and suggest the para-halogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well-known halogen bond favoring enzyme. PMID:26666582

  20. Evaluating thermodynamic integration performance of the new amber molecular dynamics package and assess potential halogen bonds of enoyl-ACP reductase (FabI) benzimidazole inhibitors.

    PubMed

    Su, Pin-Chih; Johnson, Michael E

    2016-04-05

    Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard-Jones and Coulomb Softcore potentials parameters in the one-step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one-step TI in a protein-ligand binding system. The best performing model, the one-step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM-GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure-activity relationship insights, and suggest the parahalogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well-known halogen bond favoring enzyme.

  1. The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

    PubMed

    Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

    2015-04-10

    Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes.

  2. Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching.

    PubMed

    Hevener, Kirk E; Mehboob, Shahila; Su, Pin-Chih; Truong, Kent; Boci, Teuta; Deng, Jiangping; Ghassemi, Mahmood; Cook, James L; Johnson, Michael E

    2012-01-12

    Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with submicromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented.

  3. Molecular dynamics and docking simulations as a proof of high flexibility in E. coli FabH and its relevance for accurate inhibitor modeling

    NASA Astrophysics Data System (ADS)

    Pérez-Castillo, Yunierkis; Froeyen, Matheus; Cabrera-Pérez, Miguel Ángel; Nowé, Ann

    2011-04-01

    Bacterial β-ketoacyl-acyl carrier protein synthase III (FabH) has become an attractive target for the development of new antibacterial agents which can overcome the increased resistance of these pathogens to antibiotics in clinical use. Despite several efforts have been dedicated to find inhibitors for this enzyme, it is not a straightforward task, mainly due its high flexibility which makes difficult the structure-based design of FabH inhibitors. Here, we present for the first time a Molecular Dynamics (MD) study of the E. colil FabH enzyme to explore its conformational space. We compare the flexibility of this enzyme for the unliganded protein and an enzyme-inhibitor complex and find a correspondence between our modeling results and the experimental evidence previously reported for this enzyme. Furthermore, through a 100 ns MD simulation of the unliganded enzyme we extract useful information related to the concerted motions that take place along the principal components of displacement. We also establish a relation between the presence of water molecules in the oxyanion hole with the conformational stability of structural important loops. Representative conformations of the binding pocket along the whole trajectory of the unliganded protein are selected through cluster analysis and we find that they contain a conformational diversity which is not provided by the X-ray structures of ecFabH. As a proof of this last hypothesis, we use a set of 10 FabH inhibitors and show that they cannot be correctly modeled in any available X-ray structure, while by using our set of conformations extracted from the MD simulations, this task can be accomplish. Finally, we show the ability of short MD simulations for the refinement of the docking binding poses and for MM-PBSA calculations to predict stable protein-inhibitor complexes in this enzyme.

  4. Homology modeling and docking studies of FabH (β-ketoacyl-ACP synthase III) enzyme involved in type II fatty acid biosynthesis of Chlorella variabilis: a potential algal feedstock for biofuel production.

    PubMed

    Misra, Namrata; Patra, Mahesh Chandra; Panda, Prasanna Kumar; Sukla, Lala Bihari; Mishra, Barada Kanta

    2013-03-01

    The concept of using microalgae as an alternative renewable source of biofuel has gained much importance in recent years. However, its commercial feasibility is still an area of concern for researchers. Unraveling the fatty acid metabolic pathway and understanding structural features of various key enzymes regulating the process will provide valuable insights to target microalgae for augmented oil content. FabH (β-ketoacyl-acyl carrier protein synthase; KAS III) is a condensing enzyme catalyzing the initial elongation step of type II fatty acid biosynthetic process and acyl carrier protein (ACP) facilitates the shuttling of the fatty acyl intermediates to the active site of the respective enzymes in the pathway. In the present study, a reliable three-dimensional structure of FabH from Chlorella variabilis, an oleaginous green microalga was modeled and subsequently the key residues involved in substrate binding were determined by employing protein-protein docking and molecular dynamics (MD) simulation protocols. The FabH-ACP complex having the lowest docking energy score showed the binding of ACP to the electropositive FabH surface with strong hydrogen bond interactions. The MD simulation results indicated that the substrate-complexed FabH adopted a more stable conformation than the free enzyme. Further, the FabH structure retained its stability throughout the simulation although noticeable displacements were observed in the loop regions. Molecular simulation studies suggested the importance of crucial hydrogen bonding of the conserved Arg(91) of FabH with Glu(53) and Asp(56) of ACP for exhibiting high affinity between the enzyme and substrate. The molecular modeling results are consistent with available experimental results on the flexibility of FabH and the present study provides first in silico insights into the structural and dynamical aspect of catalytic mechanism of FabH, which could be used for further site-specific mutagenic experiments to develop

  5. Plumes, orogenesis, and supercontinental fragmentation

    NASA Astrophysics Data System (ADS)

    Dalziel, I. W. D.; Lawver, L. A.; Murphy, J. B.

    2000-05-01

    A time-space relationship between large igneous provinces (LIPS), present day hot spots, and the fragmentation of Pangea has been documented over several decades, but the cause of fragmentation has remained elusive. LIPS are regarded either as the result of impingement of a mantle plume on the base of the lithosphere, or as the initial products of adiabatic decompression melting of anomalously hot mantle. Do LIPS therefore constitute evidence of an active role for plumes from the deep mantle in supercontinental fragmentation, or are they merely the first indications of a large-scale but near-surface tectonic process? Two long recognized and enigmatic orogenic events may offer a solution to this geologically important 'chicken or egg' conundrum. The reconstructed early Mesozoic Gondwanide fold belt of South America, southern Africa, and Antarctica, could have resulted from 'plume-modified orogeny', flattening of a downgoing lithospheric slab due to the buoyancy of a plume rising beneath a continental margin subduction zone. If so, the ˜180 Ma Karroo and Ferrar LIPS associated with the opening of the ocean basin between East and West Gondwanaland at ˜165 Ma resulted from impingement of this plume and are unrelated to the thermal insulation of the shallow mantle beneath Gondwanaland. It would then follow that the plume itself played an active, possibly critical, role in the initial breakup of the supercontinent. The Late Paleozoic 'Ancestral Rockies' deformation in the southwestern United States could be yet another example of orogeny driven by a plume that initiated the break-up of Pangea approximately 15 Myr earlier in the Central Atlantic region.

  6. Fission fragment excited laser system

    DOEpatents

    McArthur, David A.; Tollefsrud, Philip B.

    1976-01-01

    A laser system and method for exciting lasing action in a molecular gas lasing medium which includes cooling the lasing medium to a temperature below about 150 K and injecting fission fragments through the lasing medium so as to preferentially excite low lying vibrational levels of the medium and to cause population inversions therein. The cooled gas lasing medium should have a mass areal density of about 5 .times. 10.sup.-.sup.3 grams/square centimeter, relaxation times of greater than 50 microseconds, and a broad range of excitable vibrational levels which are excitable by molecular collisions.

  7. Burried broken extraction instrument fragment

    PubMed Central

    Balaji, S. M.

    2013-01-01

    Despite adequate effort to perform tooth removal carefully, some accidents may happen when defective instruments are unknowingly used. This article reports of a non-symptomatic case of a retained fractured dental elevator tip during an uneventful extraction a decade earlier. Patient was not aware till routine radiographic examination revealed its presence. Use of three dimensional imaging techniques in this case is highlighted. Rarely, instruments breakage may occur during surgical procedures. It is duty of the dentists to check the surgical instrument for signs of breakage and be prepared to solve a possible emergency. Retained fragments should be carefully studied prior to attempt of removal. PMID:23662269

  8. Fragmentation in massive star formation.

    PubMed

    Beuther, Henrik; Schilke, Peter

    2004-02-20

    Studies of evolved massive stars indicate that they form in a clustered mode. During the earliest evolutionary stages, these regions are embedded within their natal cores. Here we present high-spatial-resolution interferometric dust continuum observations disentangling the cluster-like structure of a young massive star-forming region. The derived protocluster mass distribution is consistent with the stellar initial mass function. Thus, fragmentation of the initial massive cores may determine the initial mass function and the masses of the final stars. This implies that stars of all masses can form via accretion processes, and coalescence of intermediate-mass protostars appears not to be necessary.

  9. Ternary fission of nuclei into comparable fragments

    SciTech Connect

    Karpeshin, F. F.

    2015-07-15

    The problem of nuclear fission into three comparable fragments is considered. A mechanism of true ternary fission is proposed. In contrast to sequential fission, where the three fragments arise upon two sequential events of binary fission, the mechanism in question relies on a scenario that originally involves fission into three fragments. This mechanism is driven by a hexadecapole deformation of the fissioning nucleus, in contrast to binary fission associated with quadrupole vibrations of the nuclear surface. The fragment-mass ratios are estimated. The dynamics of formation of collinear fragments and their subsequent motion in opposite directions is traced. The calculated probability of true ternary fission complies with observed values.

  10. Ternary fission of nuclei into comparable fragments

    NASA Astrophysics Data System (ADS)

    Karpeshin, F. F.

    2015-07-01

    The problem of nuclear fission into three comparable fragments is considered. A mechanism of true ternary fission is proposed. In contrast to sequential fission, where the three fragments arise upon two sequential events of binary fission, the mechanism in question relies on a scenario that originally involves fission into three fragments. This mechanism is driven by a hexadecapole deformation of the fissioning nucleus, in contrast to binary fission associated with quadrupole vibrations of the nuclear surface. The fragment-mass ratios are estimated. The dynamics of formation of collinear fragments and their subsequent motion in opposite directions is traced. The calculated probability of true ternary fission complies with observed values.

  11. NuFab{trademark} anti-contamination suit - OST reference No. 1855. Deactivation and decommissioning focus area

    SciTech Connect

    1998-02-01

    Radiation workers at all US Department of Energy (DOE) sites require some form of protective clothing when performing radiological work. A large number of contaminated facilities at DOE site are currently or will eventually undergo some form of decontamination and decommissioning (D&D), requiring some type of protective clothing, often in multiple layers. Protective clothing that does not allow perspiration to escape causes heat stress, which lowers worker comfort and productivity. This report describes the NuFab{trademark} anti-contamination. The suit is a one-piece, disposable, breathable, waterproof coverall with a single front zipper. Constructed of tri-laminated composite material using spun-bonded polypropylene and microporous film layers, the suit is certified as incineratorable.

  12. Detailed Characterization of AR Coatings on Si Solar Cells: A New Application of GT-FabScan 6000; Preprint

    SciTech Connect

    Sopori, B.; Butterfield, B.; Amieva, J.

    2004-08-01

    We have developed a new application of GT-FabScan for rapid mapping of AR coatings on Si solar cells. The system generates an image of the AR thickness and presents it in a color format using false colors. This measurement is made in less than 100 ms. The development of this application enables the system to generate thickness maps of the AR coating to determine the repeatability of the deposition system, as well as to ensure that downstream processing can be controlled. These data can also be used to determine the average thickness of the coating. Downstream processing is an important issue in current solar cell technology. This paper describes its importance to the PV industry and discusses the principles and method of this measurement.

  13. Impact of numerical models on fragmentation processes

    NASA Astrophysics Data System (ADS)

    Renouf, Mathieu; Gezahengn, Belien; Abbas, Micheline; Bourgeois, Florent

    2013-06-01

    Simulated fragmentation process in granular assemblies is a challenging problem which date back the beginning of the 90'. If first approaches have focus on the fragmentation on a single particle, with the development of robust, fast numerical method is is possible today to simulated such process in a large collection of particles. But the question of the fragmentation problem is still open: should the fragmentation be done dynamically (one particle becoming two fragments) and according which criterion or should the fragment paths be defined initially and which is the impact of the discretization and the model of fragments? The present contribution proposes to investigate the second aspect i.e. the impact of fragment modeling on the fragmentation processes. First to perform such an analysis, the geometry of fragments (disks/sphere or polygon/polyhedra), their behavior (rigid/deformable) and the law governing their interactions are investigated. Then such model will be used in a grinding application where the evolution of fragments and impact on the behavior of the whole packing are investigate.

  14. Fragmentation in filamentary molecular clouds

    NASA Astrophysics Data System (ADS)

    Contreras, Yanett; Garay, Guido; Rathborne, Jill M.; Sanhueza, Patricio

    2016-02-01

    Recent surveys of dust continuum emission at sub-mm wavelengths have shown that filamentary molecular clouds are ubiquitous along the Galactic plane. These structures are inhomogeneous, with overdensities that are sometimes associated with infrared emission and active of star formation. To investigate the connection between filaments and star formation, requires an understanding of the processes that lead to the fragmentation of filaments and a determination of the physical properties of the overdensities (clumps). In this paper, we present a multiwavelength study of five filamentary molecular clouds, containing several clumps in different evolutionary stages of star formation. We analyse the fragmentation of the filaments and derive the physical properties of their clumps. We find that the clumps in all filaments have a characteristic spacing consistent with the prediction of the `sausage' instability theory, regardless of the complex morphology of the filaments or their evolutionary stage. We also find that most clumps have sufficient mass and density to form high-mass stars, supporting the idea that high-mass stars and clusters form within filaments.

  15. Cooperative mixtures of bispecific F(ab')2 antibodies for delivering saporin to lymphoma in vitro and in vivo

    SciTech Connect

    French, R.R.; Courtenay, A.E.; Ingamells, S.; Stevenson, G.T.; Glennie, M.J. )

    1991-05-01

    We report that selected combinations of two or more monoclonal bispecific F(ab')2 antibodies (BsAbs) far outperform single derivatives in the delivery of the ribosome-inactivating protein, saporin, to guinea pig L2C leukemic cells. Throughout the work, BsAbs were constructed by thioether-linking the hinges of two Fab'gamma, one from monoclonal anti-L2C-idiotype antibody and the other from anti-saporin antibody. The latter was either affinity-purified rabbit polyclonal or one of a panel of five mouse monoclonal antibodies. In vitro cytotoxicity studies showed that, though all derivatives were effective, the BsAb made with the polyclonal antibody was always 10 to 20 times more potent than those made with a monoclonal antibody in yielding 50% inhibition of (3H)leucine uptake. This superior activity could be matched by selective mixtures of two or more of the monoclonal derivatives. Furthermore, in immunotherapeutic delivery of saporin to tumor, a pair of BsAbs performed significantly better than did either individually. Binding and uptake studies with radiolabeled saporin demonstrated a 20-fold increase in functional affinity when saporin was held at the cell surface by an appropriate BsAb mixture rather than by a single BsAb. In contrast, only small differences were recorded in the rate at which saporin was internalized as a result of the same maneuver. We conclude that the improved performance of combinations of BsAbs arises from their ability to provide multiple linkages between saporin molecules and cell surfaces, increasing the functional affinity with which saporin is tethered to the cell, but, in this system at least, having only a minor effect on the rate at which it is internalized. Cocktails of two or more BsAbs, selected to bind to multiple epitopes on ribosome-inactivating proteins could provide an important new strategy in immunotherapy.

  16. Reframing landscape fragmentation's effects on ecosystem services.

    PubMed

    Mitchell, Matthew G E; Suarez-Castro, Andrés F; Martinez-Harms, Maria; Maron, Martine; McAlpine, Clive; Gaston, Kevin J; Johansen, Kasper; Rhodes, Jonathan R

    2015-04-01

    Landscape structure and fragmentation have important effects on ecosystem services, with a common assumption being that fragmentation reduces service provision. This is based on fragmentation's expected effects on ecosystem service supply, but ignores how fragmentation influences the flow of services to people. Here we develop a new conceptual framework that explicitly considers the links between landscape fragmentation, the supply of services, and the flow of services to people. We argue that fragmentation's effects on ecosystem service flow can be positive or negative, and use our framework to construct testable hypotheses about the effects of fragmentation on final ecosystem service provision. Empirical efforts to apply and test this framework are critical to improving landscape management for multiple ecosystem services.

  17. Heavy Ion Fragmentation Experiments at the Bevatron

    NASA Technical Reports Server (NTRS)

    Heckman, H. H.

    1975-01-01

    Fragmentation processes of heavy nuclei in matter using the heavy-ion capability of the Bevatron were studied. The purpose was to obtain the single particle inclusive spectra of secondary nuclei produced at 0 deg by the fragmentation of heavy ion beam projectiles. The process being examined is B+T yields F + anything, where B is the beam nucleus, T is the target nucleus, and F is the detected fragment. The fragments F are isotopically identified by experimental procedures involving magnetic analysis, energy loss and time-of-flight measurements. Attempts were also made to: (1) measure the total and partial production cross section for all isotopes, (2) test the applicability of high-energy multi-particle interaction theory to nuclear fragmentation, (3) apply the cross-section data and fragmentation probabilities to cosmic ray transport theory, and (4) search for systematic behavior of fragment production as a means to improve existing semi-empirical theories of cross sections.

  18. Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-alpha toxin antibody fragment and alpha toxin.

    PubMed

    Oganesyan, Vaheh; Barnes, Arnita; Tkaczyk, Christine; Ferguson, Andrew; Wu, Herren; Dall'Acqua, William F

    2013-03-01

    Staphylococcus aureus alpha toxin (AT) has been crystallized in complex with the Fab fragment of a human antibody (MEDI4893). This constitutes the first reported crystals of AT bound to an antibody. The monoclinic crystals belonged to space group P2₁, with unit-cell parameters a=85.52, b=148.50, c=93.82 Å, β=99.82°. The diffraction of the crystals extended to 2.56 Å resolution. The asymmetric unit contained two MEDI4893 Fab-AT complexes. This corresponds to a crystal volume per protein weight (VM) of 2.3 Å3 Da(-1) and a solvent content of 47%. The three-dimensional structure of this complex will contribute to an understanding of the molecular basis of the interaction of MEDI4893 with AT. It will also shed light on the mechanism of action of this antibody, the current evaluation of which in the field of S. aureus-mediated diseases makes it a particularly interesting case study. Finally, this study will provide the three-dimensional structure of AT in a monomeric state for the first time.

  19. Exploring fragment spaces under multiple physicochemical constraints

    NASA Astrophysics Data System (ADS)

    Pärn, Juri; Degen, Jörg; Rarey, Matthias

    2007-06-01

    We present a new algorithm for the enumeration of chemical fragment spaces under constraints. Fragment spaces consist of a set of molecular fragments and a set of rules that specifies how fragments can be combined. Although fragment spaces typically cover an infinite number of molecules, they can be enumerated in case that a physicochemical profile of the requested compounds is given. By using min-max ranges for a number of corresponding properties, our algorithm is able to enumerate all molecules which obey these properties. To speed up the calculation, the given ranges are used directly during the build-up process to guide the selection of fragments. Furthermore, a topology based fragment filter is used to skip most of the redundant fragment combinations. We applied the algorithm to 40 different target classes. For each of these, we generated tailored fragment spaces from sets of known inhibitors and additionally derived ranges for several physicochemical properties. We characterized the target-specific fragment spaces and were able to enumerate the complete chemical subspaces for most of the targets.

  20. Permeable Gas Flow Influences Magma Fragmentation Speed.

    NASA Astrophysics Data System (ADS)

    Richard, D.; Scheu, B.; Spieler, O.; Dingwell, D.

    2008-12-01

    Highly viscous magmas undergo fragmentation in order to produce the pyroclastic deposits that we observe, but the mechanisms involved remain unclear. The overpressure required to initiate fragmentation depends on a number of physical parameters, such as the magma's vesicularity, permeability, tensile strength and textural properties. It is clear that these same parameters control also the speed at which a fragmentation front travels through magma when fragmentation occurs. Recent mathematical models of fragmentation processes consider most of these factors, but permeable gas flow has not yet been included in these models. However, it has been shown that permeable gas flow through a porous rock during a sudden decompression event increases the fragmentation threshold. Fragmentation experiments on natural samples from Bezymianny (Russia), Colima (Mexico), Krakatau (Indonesia) and Augustine (USA) volcanoes confirm these results and suggest in addition that high permeable flow rates may increase the speed of fragmentation. Permeability from the investigated samples ranges from as low as 5 x 10-14 to higher than 9 x 10- 12 m2 and open porosity ranges from 16 % to 48 %. Experiments were performed for each sample series at applied pressures up to 35 MPa. Our results indicate that the rate of increase of fragmentation speed is higher when the permeability is above 10-12 m2. We confirm that it is necessary to include the influence of permeable flow on fragmentation dynamics.

  1. FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium-Copy Number Plasmids in Escherichia coli.

    PubMed

    Ali, Syed A; Chew, Yik Wei

    2015-01-01

    Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium-copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium-copy number plasmid vectors in E. coli.

  2. Fragmentation of interstellar clouds and star formation

    NASA Technical Reports Server (NTRS)

    Silk, J.

    1982-01-01

    The principal issues are addressed: the fragmentation of molecular clouds into units of stellar mass and the impact of star formation on molecular clouds. The observational evidence for fragmentation is summarized, and the gravitational instability described of a uniform spherical cloud collapsing from rest. The implications are considered of a finite pressure for the minimum fragment mass that is attainable in opacity-limited fragmentation. The role of magnetic fields is discussed in resolving the angular momentum problem and in making the collapse anisotropic, with notable consequences for fragmentation theory. Interactions between fragments are described, with emphasis on the effect of protostellar winds on the ambient cloud matter and on inhibiting further star formation. Such interactions are likely to have profound consequences for regulating the rate of star formation and on the energetics and dynamics of molecular clouds.

  3. Fragmentation processes in two-phase materials

    NASA Astrophysics Data System (ADS)

    Carmona, H. A.; Guimarães, A. V.; Andrade, J. S.; Nikolakopoulos, I.; Wittel, F. K.; Herrmann, H. J.

    2015-01-01

    We investigate the fragmentation process of solid materials with crystalline and amorphous phases using the the discrete element method. Damage initiates inside spherical samples above the contact zone in a region where the circumferential stress field is tensile. Cracks initiated in this region grow to form meridional planes. If the collision energy exceeds a critical value which depends on the material's internal structure, cracks reach the sample surface resulting in fragmentation. We show that this primary fragmentation mechanism is very robust with respect to the internal structure of the material. For all configurations, a sharp transition from the damage to the fragmentation regime is observed, with smaller critical collision energies for crystalline samples. The mass distribution of the fragments follows a power law for small fragments with an exponent that is characteristic for the branching merging process of unstable cracks. Moreover this exponent depends only on the dimensionally of the system and not on the microstructure.

  4. Coal char fragmentation during pulverized coal combustion

    SciTech Connect

    Baxter, L.L.

    1995-07-01

    A series of investigations of coal and char fragmentation during pulverized coal combustion is reported for a suite of coals ranging in rank from lignite to low-volatile (lv) bituminous coal under combustion conditions similar to those found in commercial-scale boilers. Experimental measurements are described that utilize identical particle sizing characteristics to determine initial and final size distributions. Mechanistic interpretation of the data suggest that coal fragmentation is an insignificant event and that char fragmentation is controlled by char structure. Chars forming cenospheres fragment more extensively than solid chars. Among the chars that fragment, large particles produce more fine material than small particles. In all cases, coal and char fragmentation are seen to be sufficiently minor as to be relatively insignificant factors influencing fly ash size distribution, particle loading, and char burnout.

  5. Impact failure and fragmentation properties of metals

    SciTech Connect

    Grady, D.E.; Kipp, M.E.

    1998-03-01

    In the present study we describe the development of an experimental fracture material property test method specific to dynamic fragmentation. Spherical test samples of the metals of interest are subjected to controlled impulsive stress loads by acceleration to high velocities with a light-gas launcher facility and subsequent normal impact on thin plates. Motion, deformation and fragmentation of the test samples are diagnosed with multiple flash radiography methods. The impact plate materials are selected to be transparent to the x-ray method so that only test metal material is imaged. Through a systematic series of such tests both strain-to-failure and fragmentation resistance properties are determined through this experimental method. Fragmentation property data for several steels, copper, aluminum, tantalum and titanium have been obtained to date. Aspects of the dynamic data have been analyzed with computational methods to achieve a better understanding of the processes leading to failure and fragmentation, and to test an existing computational fragmentation model.

  6. Production of a PEGylated Fab' of the anti-LINGO-1 Li33 antibody and assessment of its biochemical and functional properties in vitro and in a rat model of remyelination.

    PubMed

    Pepinsky, R Blake; Walus, Lee; Shao, Zhaohui; Ji, Benxiu; Gu, Sheng; Sun, Yaping; Wen, Dingyi; Lee, Xinhua; Wang, Qin; Garber, Ellen; Mi, Sha

    2011-02-16

    The use of LINGO-1 antagonists to promote repair of damaged myelin is an emerging therapeutic opportunity for treatment of CNS diseases caused by demyelination such as multiple sclerosis. The Li33 anti-LINGO-1 antibody is a potent inducer of myelination in vitro and in vivo, but aggregation issues prevented the engineering of an optimal development candidate. PEGylated Li33 Fab' is one of several versions of the Li33 antibody that is being investigated in an attempt to identify the most favorable anti-LINGO-1 antibody design. For targeted PEGylation, a Li33 Fab' construct was engineered with a single unpaired cysteine in the heavy-chain hinge sequence. The Fab' was expressed in CHO cells, purified, and PEGylated with 20 kDa methoxy-poly(ethylene glycol) maleimide using a reaction strategy optimized to improve the yield of the PEG-Fab'. Biochemical analysis of the Li33 PEG-Fab' verified the selectivity of the PEGylation reaction. The in vitro and in vivo attributes of the PEG-Fab' were benchmarked against a Li33 full antibody. Both the Li33 PEG-Fab' and intact antibody bound LINGO-1 with nanomolar affinity, promoted myelination in an in vitro signaling assay, and promoted the repair of damaged myelin in the rat lysolecithin model. These studies extend our understanding of the biological activity of the Li33 mAb and validate the use of an anti-LINGO-1 PEG-Fab' for treatment of CNS diseases caused by demyelination.

  7. Tooth fragment reattachment: An esthetic, biological restoration

    PubMed Central

    Choudhary, Ajay; Garg, Rakesh; Bhalla, Anindya; Khatri, Rohit Kumar

    2015-01-01

    Coronal fractures of the anterior teeth are a common form of dental trauma. If the original tooth fragment is retained following fracture, reattachment of the fractured fragment to the remaining tooth can provide better and long lasting esthetics, improved function, a positive psychological response, and is a faster and less complicated procedure. This paper reports on coronal tooth fracture case that was successfully treated using adhesive reattachment of fractured fragment and post placement. PMID:25810662

  8. Complex fragment emission from hot compound nuclei

    SciTech Connect

    Moretto, L.G.

    1986-03-01

    The experimental evidence for compound nucleus emission of complex fragments at low energies is used to interpret the emission of the same fragments at higher energies. The resulting experimental picture is that of highly excited compound nuclei formed in incomplete fusion processes which decay statistically. In particular, complex fragments appear to be produced mostly through compound nucleus decay. In the appendix a geometric-kinematic theory for incomplete fusion and the associated momentum transfer is outlined. 10 refs., 19 figs.

  9. Filter for interpretation of fragmentation during entry

    SciTech Connect

    Canavan, G.H.

    1997-10-01

    Objects that fragment cascade and decelerate abruptly, producing short, bright, signatures which can be used to estimate object diameter and speed. Other objects can be incorporated into a generalized fragmentation filter. This note summarizes the results of previous reports on the prediction and inversion of signatures from objects that radiate, ablate, and fragment during entry and uses them to produce models for the parameters of entering objects.

  10. Interaction of three fission fragments and yields of various ternary fragments

    NASA Astrophysics Data System (ADS)

    Denisov, V. Yu.; Pilipenko, N. A.; Sedykh, I. Yu.

    2017-01-01

    The interaction potential energy of the three deformed fragments formed in fission of 252Cf is studied for various combinations of three-fragment fission. The lowest height of the potential energy ridge between three touching and separated deformed fragments is sought. The excitation energies of various three-deformed-fragment configurations, at the lowest barrier heights related to the yield of the corresponding configuration, are considered in detail. The most probable three-fragment fission configurations are discussed. The yields of various ternary fragments in fission of 250Cf agree well with available experimental data.

  11. Expression, purification and characterization of B72.3 Fv fragments.

    PubMed Central

    King, D J; Byron, O D; Mountain, A; Weir, N; Harvey, A; Lawson, A D; Proudfoot, K A; Baldock, D; Harding, S E; Yarranton, G T

    1993-01-01

    The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv. Images Figure 1 Figure 2 PMID:8457200

  12. The dihadron fragmentation function and its evolution

    SciTech Connect

    Majumder, Abhijit; Wang, Xin-Nian

    2004-02-24

    Dihadron fragmentation functions and their evolution arestudied in the process of e+e- annihilation. Under the collinearfactorization approximation and facilitated by the cut-vertex technique,the two hadron inclusive cross section at leading order (LO) is shown tofactorize into a short distance parton cross section and a long distancedihadron fragmentation function. We provide the definition of such adihadron fragmentation function in terms of parton matrix elements andderive its DGLAP evolution equation at leading log. The evolutionequation for the non-singlet quark fragmentation function is solvednumerically with a simple ansatz for the initial condition and resultsare presented for cases of physical interest.

  13. Ligation-based assembly for constructing mouse synthetic scFv libraries by chain shuffling with in vivo-amplified VH and VL fragments.

    PubMed

    Nishi, Michiru; Jian, Nan; Yamamoto, Keiko; Seto, Haruyo; Nishida, Yuichi; Tonoyama, Yasuhiro; Shimizu, Nobuyoshi; Nishi, Yoshisuke

    2014-10-01

    In vitro assembly of two or three PCR fragments using primers is a common method of constructing scFv fragments for display on the surface of phage. However, mismatch annealing often occurs during in this step, leading to cloning and display of incomplete Fab or scFv fragments. To overcome this limitation, we developed a ligation-based two-fragment assembly (LTFA) protocol that involved separately cloning VH and Vκ fragments into the high-copy-number plasmid pUC18. The VH and Vκ fragments had randomized complementarity-determining region 3 (CDR3) and were joined with a peptidyl linker composed of (G4S)3. Using this approach, complete sequences of scFv fragments were successfully constructed, and the sequencing of 83 scFv clones revealed that none of the sequences, including the linker region, contained deletions or mutations. In contrast, linker sequences generated using a conventional two-fragment PCR assembly (TFPA) protocol often contained sequence anomalies, including large truncations. Using the LTFA protocol, a final library size of 1.0×10(8)cfu was achieved. Examination of the amino acid profiles of the generated scFv fragments within the randomized regions introduced using degenerate codons did not detect any bias from that expected based on stochastic distribution. After several cycles of panning with this library, antigen-specific scFvs against two reference antigens, hen egg lysozyme and streptavidin were detected. In addition, scFvs with specificity against peptidyl antigens in the loop region of the Medaka ortholog of human C6orf89, which encodes a histone deacetylase enhancer that interacts with the bombesin receptor, were also obtained. The LTFA protocol developed here is robust and allows for the easy construction of integral scFv fragments compared with conventional TFPA. Utilizing LTFA, other CDRs can be readily combined. This approach also allows for the in vitro maturation of scFv fragments by separately introducing randomization in CDRs or

  14. Design, synthesis and antibacterial activities of 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiol derivatives containing Schiff base formation as FabH inhibitory.

    PubMed

    Zhang, Fei; Wen, Qing; Wang, She-Feng; Shahla Karim, Baloch; Yang, Yu-Shun; Liu, Jia-Jia; Zhang, Wei-Ming; Zhu, Hai-Liang

    2014-01-01

    A series of novel schiff base derivatives (H(1)-H(20)) containing pyrazine and triazole moiety have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of β-ketoacyl-acyl carrier protein synthase III (FabH). These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Bacillus amyloliquefaciens and selected compounds among them were tested for their Escherichia coli FabH inhibitory activity. Based on the biological data, compound H(17) showed the most potent antibacterial activity with MIC values of 0.39-1.56μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC50 of 5.2μM, being better than the positive control Kanamycin B with IC50 of 6.3μM. Furthermore, docking simulation was performed to position compound H(17) into the E. coli FabH active site to determine the probable binding conformation. This study indicated that compound H(17) has demonstrated significant E. coli FabH inhibitory activity as a potential antibacterial agent and provides valuable information for the design of E. coli FabH inhibitors.

  15. Optimal design of Ig 5' primers for construction of diverse phage antibody library established to select anti-HAb18GEF and anti-DOTA-Y Fabs for hepatoma pretargeting RIT.

    PubMed

    Zhang, Sihe; Xing, Jinliang; Zhang, Qing; Song, Fei; Li, Yu; Yang, Xiangmin; Chen, Zhinan

    2006-05-01

    Phage antibody library yields antibodies with higher affinity against different antigens, if diverse IgV gene repertoires can be amplified. As the currently available Fab primer sets cannot guarantee efficient amplification with high diversity, and because rare cloning sites can be found in certain Ig genes, here, we present an optimal set of Ig 5' primers, compatible with Fd 5' clone site replaced pComb3 vector, for diverse Fab phage display library construction. These novel Fab primes designed based on the newly classified IgV families, not only have best match and highest coverage for IgV family with minimized N-terminal amino acid changes, but also present good amplification diversity and efficienc