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Sample records for recombinant anti-mesothelin immunotoxin

  1. 77 FR 8263 - Prospective Grant of Exclusive License: The Development of Anti-mesothelin Targeted Immunotoxins...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-14

    .... 209(c)(1) and 37 CFR Part 404.7(a)(1)(i), that the National Institutes of Health, Department of Health.../00224 entitled ``Mesothelin Antigen and Methods and Kits for Targeting It'' , U.S. Patent 5,747,654... immunotoxins for the treatment of mesothelin-expressing cancers, wherein the immunotoxins have: (1) A...

  2. Generation of active immunotoxins containing recombinant restrictocin.

    PubMed

    Rathore, D; Batra, J K

    1996-05-01

    Restrictocin, a toxin produced by the fungus Aspergillus restrictus, is a potent inhibitor of eukaryotic protein synthesis. Recombinant restrictocin was made in Escherichia coli and purified to homogeneity in large amounts. The recombinant protein was found to be poorly immunogenic in mice with low toxicity, when injected intraperitoneally. Two immunotoxins were constructed by coupling the recombinant restrictocin to an antibody to the human transferrin receptor, using a cleavable and a stable linkage. The immunotoxins so generated showed specific cytotoxic activity toward receptor bearing cells in tissue culture. Immunotoxin with a cleavable linkage, however, was more active than that containing a stable linkage. Restrictocin appears to be a promising candidate to be developed as a chimeric toxin for targeted therapy. PMID:8630074

  3. Recombinant Immunotoxins Containing Truncated Bacterial Toxins for the Treatment of Hematologic Malignancies

    PubMed Central

    Kreitman, Robert J.

    2009-01-01

    Immunotoxins are molecules that contain a protein toxin and a ligand that is either an antibody or a growth factor. The ligand binds to a target cell antigen, and the target cell internalizes the immunotoxin, allowing the toxin to migrate to the cytoplasm where it can kill the cell. In the case of recombinant immunotoxins, the ligand and toxin are encoded in DNA that is then expressed in bacteria, and the purified immunotoxin contains the ligand and toxin fused together. Among the most active recombinant immunotoxins clinically tested are those that are targeted to hematologic malignancies. One agent, containing human interleukin-2 and truncated diphtheria toxin (denileukin diftitox), has been approved for use in cutaneous T-cell lymphoma, and has shown activity in other hematologic malignancies, including leukemias and lymphomas. Diphtheria toxin has also been targeted by other ligands, including granulocyte-macrophage colony-stimulating factor and interleukin-3, to target myelogenous leukemia cells. Single-chain antibodies containing variable heavy and light antibody domains have been fused to truncated Pseudomonas exotoxin to target lymphomas and lymphocytic leukemias. Recombinant immunotoxins anti-Tac(Fv)-PE38 (LMB-2), targeting CD25, and RFB4(dsFv)-PE38 (BL22, CAT-3888), targeting CD22, have each been tested in patients. Major responses have been observed after failure of standard chemotherapy. The most successful application of recombinant immunotoxins today is in hairy cell leukemia, where BL22 has induced complete remissions in most patients who were previously treated with optimal chemotherapy. PMID:19344187

  4. Development of a diphtheria toxin based antiporcine CD3 recombinant immunotoxin.

    PubMed

    Wang, Zhirui; Duran-Struuck, Raimon; Crepeau, Rebecca; Matar, Abraham; Hanekamp, Isabel; Srinivasan, Srimathi; Neville, David M; Sachs, David H; Huang, Christene A

    2011-10-19

    Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models. PMID:21866954

  5. Effect of Antigen Shedding on Targeted Delivery of Immunotoxins in Solid Tumors from a Mathematical Model

    PubMed Central

    Pak, Youngshang; Pastan, Ira; Kreitman, Robert J.; Lee, Byungkook

    2014-01-01

    Most cancer-specific antigens used as targets of antibody-drug conjugates and immunotoxins are shed from the cell surface (Zhang & Pastan (2008) Clin. Cancer Res. 14: 7981-7986), although at widely varying rates and by different mechanisms (Dello Sbarba & Rovida (2002) Biol. Chem. 383: 69–83). Why many cancer-specific antigens are shed and how the shedding affects delivery efficiency of antibody-based protein drugs are poorly understood questions at present. Before a detailed numerical study, it was assumed that antigen shedding would reduce the efficacy of antibody-drug conjugates and immunotoxins. However, our previous study using a comprehensive mathematical model showed that antigen shedding can significantly improve the efficacy of the mesothelin-binding immunotoxin, SS1P (anti-mesothelin-Fv-PE38), and suggested that receptor shedding can be a general mechanism for enhancing the effect of inter-cellular signaling molecules. Here, we improved this model and applied it to both SS1P and another recombinant immunotoxin, LMB-2, which targets CD25. We show that the effect of antigen shedding is influenced by a number of factors including the number of antigen molecules on the cell surface and the endocytosis rate. The high shedding rate of mesothelin is beneficial for SS1P, for which the antigen is large in number and endocytosed rapidly. On the other hand, the slow shedding of CD25 is beneficial for LMB-2, for which the antigen is small in number and endocytosed slowly. PMID:25343405

  6. Bortezomib Reduces Preexisting Antibodies to Recombinant Immunotoxins in Mice*

    PubMed Central

    Manning, Michael L.; Mason-Osann, Emily; Onda, Masanori; Pastan, Ira

    2014-01-01

    Recombinant immunotoxin (RIT) therapy is limited in patients by neutralizing antibody responses. Ninety percent of patients with normal immune systems make neutralizing antibodies after one cycle of RIT, preventing repeated dosing. Furthermore, some patients have preexisting antibodies from environmental exposure to Pseudomonas exotoxin, the component of the RIT that elicits the neutralizing antibody response. Bortezomib (B) is an FDA-approved proteasome inhibitor which selectively targets and kills plasma cells which are necessary for the neutralizing antibody response. We hypothesized B may abrogate neutralizing antibody levels, making dosing of RIT possible in mice already immune to RIT. We immunized BALB/c mice with multiple doses of SS1P, a RIT whose antibody portion targets mesothelin. Mice with elevated antibody levels were separated into groups to receive saline, B, the pentostatin/cyclophosphamide (PC) regimen, or the bortezomib/pentostatin/cyclophosphamide (BPC) combination regimen. Four weeks after finishing therapy plasma antibody levels were assayed and bone marrow was harvested. The B and PC regimens both significantly reduced antibody levels, and we observed fewer plasma cells in the bone marrow of B treated mice, but not in PC treated mice. The BPC combination regimen nearly eliminated antibodies and further reduced plasma cells in the bone marrow. The BPC combination regimen is more effective than individual regimens and may reduce antibody levels in patients with preexisting neutralizing antibodies to Pseudomonas exotoxin allowing RIT treatment. PMID:25560410

  7. Development of a recombinant hCG-specific single chain immunotoxin cytotoxic to hCG expressing cancer cells.

    PubMed

    Nand, Kripa N; Gupta, Jagdish C; Panda, A K; Jain, S K

    2015-02-01

    A large number of cancers express human chorionic gonadotropin (hCG) or its subunits ectopically. Patients harboring such cancers have poor prognosis and adverse survival. PiPP is a monoclonal antibody of high affinity and specificity for hCGβ/hCG. Work was carried out to develop a PiPP based recombinant immunotoxin for the immunotherapy of hCG expressing cancers. Recombinant PiPP antibody was constructed in scFv format in which gene encoding the VH and VL domains were joined through a linker. This scFv gene was fused to the gene expressing Pseudomonas exotoxin (PE38), and cloned in a Escherichia coli based expression vector under the control of strong bacteriophage T7 promoter. Immunotoxin conjugating scFv(PiPP) and PE38, was expressed in E. coli as recombinant protein. Recombinant PiPP immunotoxin was purified from the bacterial cell lysate and tested for binding and killing of hCGβ expressing lymphoma, T-lymphoblastic leukemia and lung carcinoma cells in vitro. Immunotoxin showed nearly 90% killing on the cells. This is the first ever report on recombinant immunotoxin for binding and cytotoxicity to hCG expressing cancer cells, and thus can be a potential candidate for the immunotherapy of hCG expressing cells. PMID:25448825

  8. In vitro and in vivo effects of a recombinant anti-PSMA immunotoxin in combination with docetaxel against prostate cancer

    PubMed Central

    Michalska, Marta; Schultze-Seemann, Susanne; Bogatyreva, Lioudmila; Hauschke, Dieter; Wetterauer, Ulrich; Wolf, Philipp

    2016-01-01

    Docetaxel (DOC) is used for the first-line treatment of castration resistant prostate cancer (CPRC). However, the therapeutic effects are limited, only about one half of patients respond to the therapy and severe side effects possibly lead to discontinuation of treatment. Therefore, actual research is focused on the development of new DOC-based combination treatments. In this study we investigated the antitumor effects of a recombinant immunotoxin targeting the prostate specific membrane antigen (PSMA) in combination with DOC in vitro and in vivo. The immunotoxin consists of an anti-PSMA single chain antibody fragment (scFv) as binding and a truncated form of Pseudomonas aeruginosa Exotoxin A (PE40) as toxin domain. The immunotoxin induced apoptosis and specifically reduced the viability of androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cells. A synergistic cytotoxic activity was observed in combination with DOC with IC50 values in the low picomolar or even femtomolar range. Moreover, combination treatment resulted in an enhanced antitumor activity in a C4-2 SCID mouse xenograft model. This highlights the immunotoxin as a promising therapeutic agent for a future DOC-based combination therapy of CPRC. PMID:26968813

  9. Bortezomib reduces pre-existing antibodies to recombinant immunotoxins in mice.

    PubMed

    Manning, Michael L; Mason-Osann, Emily; Onda, Masanori; Pastan, Ira

    2015-02-15

    Recombinant immunotoxin (RIT) therapy is limited in patients by neutralizing Ab responses. Ninety percent of patients with normal immune systems make neutralizing Abs after one cycle of RIT, preventing repeated dosing. Furthermore, some patients have pre-existing Abs from environmental exposure to Pseudomonas exotoxin, the component of the RIT that elicits the neutralizing Ab response. Bortezomib is an U.S. Food and Drug Administration-approved proteasome inhibitor that selectively targets and kills plasma cells that are necessary for the neutralizing Ab response. We hypothesized that bortezomib may abrogate neutralizing Ab levels, making dosing of RIT possible in mice already immune to RIT. We immunized BALB/c mice with multiple doses of SS1P, a RIT whose Ab portion targets mesothelin. Mice with elevated Ab levels were separated into groups to receive saline, bortezomib, the pentostatin/cyclophosphamide (PC) regimen, or the bortezomib/PC (BPC) combination regimen. Four weeks after finishing therapy, plasma Ab levels were assayed, and bone marrow was harvested. The bortezomib and PC regimens significantly reduced Ab levels, and we observed fewer plasma cells in the bone marrow of bortezomib-treated mice but not in PC-treated mice. The BPC combination regimen almost completely eliminated Abs and further reduced plasma cells in the bone marrow. This regimen is more effective than individual regimens and may reduce Ab levels in patients with pre-existing neutralizing Abs to Pseudomonas exotoxin, allowing RIT treatment. PMID:25560410

  10. Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity.

    PubMed

    Onda, M; Nagata, S; Tsutsumi, Y; Vincent, J J; Wang, Q; Kreitman, R J; Lee, B; Pastan, I

    2001-07-01

    Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity. PMID:11431343

  11. Fusogenics: a recombinant immunotoxin-based screening platform to select internalizing tumor-specific antibody fragments.

    PubMed

    Cizeau, Jeannick; Torres, Marianne G P; Cowling, Sharla G; Stibbard, Stacy; Premsukh, Arjune; Entwistle, Joycelyn; MacDonald, Glen C

    2011-01-01

    Antibody-based therapeutics play a vital role in the treatment of certain cancers; however, despite commercial success, various strategies are being pursued to increase their potency and hence improve patient outcomes. The use of antibodies to deliver a cytotoxic payload offers a promising alternative for more efficacious therapies. Immunotoxins are composed of an internalizing antibody fragment linked to a bacterial or plant toxin. Once internalized, the payload, such as Pseudomonas exotoxin A (PE), blocks protein synthesis and induces apoptosis. Typically, immunotoxins are developed by first isolating a tumor-specific antibody, which is then either chemically linked to a toxin or reengineered as a fusion protein. Here, the authors describe the development of Fusogenics, an immunotoxin-based screening method that selects internalizing tumor-specific antibodies using a functional assay. Selected immune library clones were characterized and shown to be selective against normal tissues and specific to tumor tissues. In summary, the Fusogenics immunotoxin platform represents a unique, single-step selection approach combining specificity and functionality to isolate novel internalizing tumor-specific antibody fragments with potential for direct clinical application in the treatment of cancer. PMID:21131595

  12. Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes.

    PubMed

    Mazor, Ronit; Eberle, Jaime A; Hu, Xiaobo; Vassall, Aaron N; Onda, Masanori; Beers, Richard; Lee, Elizabeth C; Kreitman, Robert J; Lee, Byungkook; Baker, David; King, Chris; Hassan, Raffit; Benhar, Itai; Pastan, Ira

    2014-06-10

    Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins. PMID:24799704

  13. Targeted therapy against human lung cancer in nude mice by high-affinity recombinant antimesothelin single-chain Fv immunotoxin.

    PubMed

    Fan, Dominic; Yano, Seiji; Shinohara, Hisashi; Solorzano, Carmen; Van Arsdall, Melissa; Bucana, Corazon D; Pathak, Sen; Kruzel, Ewa; Herbst, Roy S; Onn, Amir; Roach, Jennifer S; Onda, Masanori; Wang, Qing-cheng; Pastan, Ira; Fidler, Isaiah J

    2002-06-01

    Several tumors, including mesothelioma and ovarian cancer, can overexpress mesothelin, a glycosylphosphatidylinositol-linked differentiation glycoprotein. The membrane-bound type of mesothelin is found in the blood of cancer patients at a very low level, which makes mesothelin a good candidate for targeted therapy of certain cancers. An antimesothelin disulfide-linked Fv (SS1 Fv) was fused to a truncated mutant of Pseudomonas exotoxin A to produce the recombinant immunotoxin SS1(dsFv)-PE38, which has a high binding affinity to mesothelin (Kd = 0.7 nM). Our studies in vitro showed that SS1(dsFv)-PE38 is significantly more cytotoxic to the high-mesothelin-producing NCI-H226 human non-small cell lung cancer cells than to human lung adenocarcinoma PC14PE6 cells, which do not express mesothelin. When administered at a nontoxic dose of 500 microg/kg on days 7, 9, and 11 to nude mice injected i.v. with the two human lung cancer cell lines, SS1(dsFv)-PE38 selectively inhibited experimental lung metastases produced by the mesothelin-producing NCI-H226 cells. Our data indicate that mesothelin-producing squamous cell carcinoma of the lung may be a good target for this immunotoxin. PMID:12479219

  14. Dual B- and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity.

    PubMed

    Mazor, Ronit; Onda, Masanori; Park, Dong; Addissie, Selamawit; Xiang, Laiman; Zhang, Jingli; Hassan, Raffit; Pastan, Ira

    2016-05-24

    Recombinant immunotoxins (RITs) are genetically engineered proteins being developed to treat cancer. They are composed of an Fv that targets a cancer antigen and a portion of a protein toxin. Their clinical success is limited by their immunogenicity. Our goal is to produce a new RIT that targets mesothelin and is non-immunogenic by combining mutations that decrease B- and T-cell epitopes. Starting with an immunotoxin that has B-cell epitopes suppressed, we added mutations step-wise that suppress T-cell epitopes. The final protein (LMB-T14) has greatly reduced antigenicity as assessed by binding to human anti-sera and a greatly decreased ability to activate helper T-cells evaluated in a T-cell activation assay. It is very cytotoxic to mesothelioma cells from patients, and to cancer cell lines. LMB-T14 produces complete remissions of a mesothelin expressing cancer (A431/H9) xenograft. The approach used here can be used to de-immunize other therapeutic foreign proteins. PMID:27167198

  15. Recombinant anti-monkey CD3 immunotoxin depletes peripheral lymph node T lymphocytes more effectively than rabbit anti-thymocyte globulin in naïve baboons

    PubMed Central

    Wamala, Isaac; Matar, Abraham J.; Farkash, Evan; Wang, Zhirui; Huang, Christene A.; Sachs, David H.

    2014-01-01

    T cell depletion is an important procedure for both experimental and therapeutic immune modulation. Rabbit anti-thymocyte globulin (ATG), which is a commonly used T cell depletion antibody in clinical organ and cell transplantation protocols, is effective in temporarily depleting peripheral blood T lymphocytes but only moderately effective in depleting peripheral lymph node T cells which comprise the majority of T lymphocytes. A recombinant anti-CD3 immunotoxin, A-dmDT390-scfbDb (C207), has been developed and shown in an initial study to retain the lymph node depleting properties of conjugated CD3 immunotoxin. This agent could potentially be used synergistically with or as a replacement for rabbit ATG in preclinical primate models of transplantation. We directly compared the peripheral blood and lymph node depleting abilities of this recombinant anti-CD3 immunotoxin and rabbit ATG in naïve animals at clinically tolerated doses. Baboons were treated with a full course of either rabbit ATG (n = 2) or CD3 immunotoxin (n = 3). Peripheral blood and lymph node T lymphocytes were measured before and following treatment. Peripheral blood CD3+ cells fell below 100 cells/μL in every animal. In the two animals receiving ATG, CD3+ cells represented 53% and 68% of lymph node cells two days following a full course of rabbit ATG. In contrast, CD3+ cells represented 3%, 5%, and 38% in lymph nodes following a full course of CD3-IT. Thus, recombinant anti-monkey CD3 immunotoxin showed improved peripheral lymph node T lymphocyte depletion to rabbit ATG and spared other immune cells. PMID:24157659

  16. Anticancer Effects of Mesothelin-Targeted Immunotoxin Therapy Are Regulated by Tyrosine Kinase DDR1.

    PubMed

    Ali-Rahmani, Fatima; FitzGerald, David J; Martin, Scott; Patel, Paresma; Prunotto, Marco; Ormanoglu, Pinar; Thomas, Craig; Pastan, Ira

    2016-03-15

    Recombinant immunotoxins (RIT) have been highly successful in cancer therapy due, in part, to the high cancer-specific expression of cell surface antigens such as mesothelin, which is overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancers, but is limited in normal cells. RG7787 is a clinically optimized RIT consisting of a humanized anti-mesothelin Fab fused to domain III of Pseudomonas exotoxin A, in which immunogenic B-cell epitopes are silenced. To enhance the therapeutic efficacy of RITs, we conducted a kinome RNAi sensitization screen, which identified discoidin domain receptor 1 (DDR1), a collagen-activated tyrosine kinase, as a potential target. The collagen/DDR1 axis is implicated in tumor-stromal interactions and potentially affects tumor response to therapy. Therefore, we investigated the effects of DDR1 on RIT. Knockdown of DDR1 by siRNA or treatment with inhibitor, 7rh, greatly enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed DDR1 silencing was associated with decreased expression of several ribosomal proteins and enhanced inhibition of protein synthesis. Conversely, induction of DDR1 expression or collagen-stimulated DDR1 activity protected cancer cells from RG7787 killing. Moreover, the combination of RG7787 and DDR1 inhibitor caused greater shrinkage of tumor xenografts than either agent alone. These data demonstrate that DDR1 is a key modulator of RIT activity and represents a novel therapeutic strategy to improve targeting of mesothelin-expressing cancers. PMID:26719540

  17. Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein.

    PubMed

    Chatterjee, Deboeeta; Chandran, Bala; Berger, Edward A

    2012-01-01

    Kaposi sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) is etiologically associated with three neoplastic syndromes: Kaposi sarcoma and the uncommon HIV-associated B-cell lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. The incidence of the latter B-cell pathology has been increasing in spite of antiretroviral therapy; its association with lytic virus replication has prompted interest in therapeutic strategies aimed at this phase of the virus life cycle. We designed and expressed a recombinant immunotoxin (2014-PE38) targeting the gpK8.1A viral glycoprotein expressed on the surface of the virion and infected cells. We show that this immunotoxin selectively kills KSHV-infected cells in dose-dependent fashion, resulting in major reductions of infectious virus release. The immunotoxin and ganciclovir, an inhibitor of viral DNA replication, showed marked reciprocal potentiation of antiviral activities. These results suggest that the immunotoxin, alone or in combination, may represent a new approach to treat diseases associated with KSHV lytic replication. PMID:22377676

  18. Resimmune, an anti-CD3ε recombinant immunotoxin, induces durable remissions in patients with cutaneous T-cell lymphoma

    PubMed Central

    Frankel, Arthur E.; Woo, Jung H.; Ahn, Chul; Foss, Francine M.; Duvic, Madeleine; Neville, Paul H.; Neville, David M.

    2015-01-01

    Resimmune is a second-generation recombinant immunotoxin composed of the catalytic and translocation domains of diphtheria toxin fused to two single chain antibody fragments reactive with the extracellular domain of CD3ε. We gave intravenous infusions of Resimmune 2.5 – 11.25 μg/kg over 15 minutes to 30 patients (25 with cutaneous T-cell lymphoma, 3 with peripheral T-cell lymphoma, 1 with T-cell large granular lymphocytic leukemia and 1 with T-cell prolymphocytic leukemia) in an inter-patient dose escalation trial. The most common adverse events were fever, chills, hypotension, edema, hypoalbuminemia, hypophosphatemia, and transaminasemia. Among the 25 patients with cutaneous T-cell lymphoma, there were nine responses for a response rate of 36% (95% CI, 18%–57%) including four complete remissions (16%, 95% CI, 5%–36%). The durations of the complete remissions were 72+, 72+, 60+ and 38+ months. There were five partial remissions lasting 3, 3, 3+, 6+ and 14 months. Of 17 patients with a modified skin weighted assessment tool score <50, 17 patients with stage IB/IIB, and 11 patients with both a score <50 and stage IB/IIB, nine (53%), eight (47%), and eight (73%) had responses, respectively. Further studies of Resimmune in patients with low tumor burden, stage IB-IIB cutaneous T-cell lymphoma are warranted. This trial is registered at clinicaltrials.gov as #NCT00611208. PMID:25795722

  19. A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model.

    PubMed

    Gattenlöhner, S; Jörissen, H; Huhn, M; Vincent, A; Beeson, D; Tzartos, S; Mamalaki, A; Etschmann, B; Muller-Hermelink, H K; Koscielniak, E; Barth, S; Marx, A

    2010-01-01

    Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) gamma-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms. PMID:20204062

  20. A Human Recombinant Autoantibody-Based Immunotoxin Specific for the Fetal Acetylcholine Receptor Inhibits Rhabdomyosarcoma Growth In Vitro and in a Murine Transplantation Model

    PubMed Central

    Gattenlöhner, S.; Jörißen, H.; Huhn, M.; Vincent, A.; Beeson, D.; Tzartos, S.; Mamalaki, A.; Etschmann, B.; Muller-Hermelink, H. K.; Koscielniak, E.; Barth, S.; Marx, A.

    2010-01-01

    Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) γ-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms. PMID:20204062

  1. Non-invasive tumor detection in small animals using novel functional Pluronic nanomicelles conjugated with anti-mesothelin antibody

    NASA Astrophysics Data System (ADS)

    Ding, Hong; Yong, Ken-Tye; Law, Wing-Chueng; Roy, Indrajit; Hu, Rui; Wu, Fang; Zhao, Weiwei; Huang, Kun; Erogbogbo, Folarin; Bergey, Earl J.; Prasad, Paras N.

    2011-04-01

    In this study QDs were encapsulated in carboxylated PluronicF127 (F127COOH) triblock polymeric micelles and conjugated with anti-mesothelin antibody for the purpose of alleviating potential toxicity, enhancing the stability and improving targeting efficiency of CdTe/ZnS quantum dots (QDs) in tumors. The amphiphilic triblock polymer of F127COOH contains hydrophilic carboxylated poly(ethylene oxide) (PEO) and hydrophobic poly(propylene oxide) (PPO) units. After encapsulating QDs into carboxylated F127 (F127COOH-QD) micelles, the particles were conjugated with anti-mesothelin antibodies to allow targeting of cancerous areas. The size of the monodispersed spherical QD-containing micelles was determined to be ~120 nm by dynamic light scattering (DLS). The critical micelle concentration (CMC) was estimated to be 4.7 × 10-7 M. In an in vitro study, the anti-methoselin antibody conjugated F127COOH (Me-F127COOH-QD) nanomicelles showed negligible cytotoxicity to pancreatic cancer cells (Panc-1). Confocal microscopy demonstrated that the Me-F127COOH-QD nanomicelles were taken up more efficiently by Panc-1cells, due to antibody mediated targeting. An in vivo imaging study showed that Me-F127COOH-QD nanomicelles accumulated at the pancreatic tumor site 15 min after intravenous injection. In addition, the low in vivo toxicity of the nanomicellar formulation was evaluated by pathological assays. These results suggest that anti-mesothein antibody conjugated carboxylated F127 nanomicelles may serve as a promising nanoscale platform for early human pancreatic cancer detection and targeted drug delivery.

  2. Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins.

    PubMed Central

    Deonarain, M. P.; Epenetos, A. A.

    1998-01-01

    Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia coli (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins. Images Figure 2 PMID:9484808

  3. Gigantoxin-4-4D5 scFv is a novel recombinant immunotoxin with specific toxicity against HER2/neu-positive ovarian carcinoma cells.

    PubMed

    Lv, Xinxin; Zhang, Jian; Xu, Rui; Dong, Yuguo; Sun, Aiyou; Shen, Yaling; Wei, Dongzhi

    2016-07-01

    Immunotoxins are a new class of antibody-targeted therapy in clinical development. Traditional immunotoxins that are constructed from the toxins of plants or bacteria need to be internalized to the cytoplasm and thus have limited antitumor efficacy. In the present study, we combined a recently reported sea anemone cytolysin Gigantoxin-4 with an anti-HER2/neu single-chain variable fragment 4D5 scFv to construct a novel immunotoxin. We fused a SUMO tag to the N-terminus of Gigantoxin-4-4D5 scFv and it was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. After purification, the purity of Gigantoxin-4-4D5 scFv reached 96 % and the yield was 14.3 mg/L. Our results demonstrated that Gigantoxin-4-4D5 scFv exerted a highly cytotoxic effect on the HER2/neu-positive ovarian carcinoma SK-OV-3 cell line. And the hemolytic activity was weaker, making it safe for normal cells. The results of immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition of human embryonic kidney 293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane. Moreover, it could also mediate apoptosis of SK-OV-3 cells. PMID:27063011

  4. Humanization of immunotoxins.

    PubMed

    Rybak, S M; Hoogenboom, H R; Meade, H M; Raus, J C; Schwartz, D; Youle, R J

    1992-04-15

    The construction and expression of a chimeric gene encoding a mouse/human antibody to the human transferrin receptor fused to the gene for angiogenin, a human homolog of pancreatic RNase, are described. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human angiogenin to a chimeric anti-transferrin receptor heavy chain gene. The antibody-enzyme fusion gene was introduced into a transfectoma that secretes the chimeric light chain of the same antibody, and cell lines were cloned that synthesize and secrete the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/ml. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis of K562 cells that express the human transferrin receptor but not toward a non-human-derived cell line that lacks this receptor. Whereas excess antibody to the same receptor did not itself inhibit protein synthesis, it was able to completely prevent the protein synthesis inhibition caused by the fusion protein. These results indicate that the cytotoxicity is due to a transferrin receptor-mediated mechanism involving the angiogenin portion of the fusion protein and demonstrate the feasibility of constructing recombinant antibody-RNase molecules capable of killing tumor cells bearing the transferrin receptor. The significance of the acquired cytotoxicity of a mouse/human chimeric antibody linked to a human protein may bear importantly in human therapeutic strategies that use mouse antibodies linked to toxins from plants or bacteria to target tumor cells. It is expected that the humanization of immunotoxins will lead to less toxicity and immunogenicity than currently available reagents. PMID:1565609

  5. Affinity-matured recombinant immunotoxin targeting gangliosides 3′-isoLM1 and 3′,6'-isoLD1 on malignant gliomas

    PubMed Central

    Piao, Hailan; Kuan, Chien-Tsun; Chandramohan, Vidya; Keir, Stephen T; Pegram, Charles N; Bao, Xuhui; Månsson, Jan-Eric; Pastan, Ira H; Bigner, Darell D

    2013-01-01

    About 60 percent of glioblastomas highly express the gangliosides 3′-isoLM1 and 3′,6′-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3′-isoLM1 and 3′,6′-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3′-isoLM1 and 3′,6′-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3′-isoLM1 and 3′,6′-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the KD of DmAb14m-IT for gangliosides 3′-isoLM1 and 3′,6′-isoLD1 was 2.6 × 10−9M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3′-isoLM1 and 3′,6′-isoLD1. The DmAb14m-IT IC50 was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3′-isoLM1 and 3′,6′-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3′-isoLM1 and 3′,6′-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and

  6. In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

    PubMed Central

    Liu, Fang; Le, Wenjun; Mei, Tianxiao; Wang, Tiegong; Chen, Luguang; Lei, Yi; Cui, Shaobin; Chen, Bingdi; Cui, Zheng; Shao, Chengwei

    2016-01-01

    Pancreatic cancer is a highly malignant disease with a 5-year survival rate <5% mainly due to lack of early diagnosis and effective therapy. In an effort to improve the early diagnostic rate of pancreatic cancer, a nanoprobe Fe3O4@SiO2 modified with anti-mesothelin antibody (A-MFS) was prepared to target cells and tumor tissues highly expressing mesothelin in vitro (human pancreatic cancer cell line SW1990) and in vivo (subcutaneously transplanted tumors) studies. The A-MFS probe was successfully prepared and was spherical and uniform with a hydrodynamic diameter between 110 and 130 nm. Cell Counting Kit-8 testing indicated that A-MFS was nontoxic in vitro and in vivo studies. The in vitro study showed that the A-MFS probe specifically targeted SW1990 cells with high mesothelin expression. The in vivo study was conducted in Siemens 3.0 T magnetic resonance imaging. The average T2-weighted signal values of the xenografts were 966.533±31.56 before injecting A-MFS and 691.133±56.84 before injecting saline solution. After injection of 0.1 mL A-MFS via nude mouse caudal vein for 2.5 hours, the average T2-weighted signal of the xenograft decreased by 342.533±42.6. The signal value decreased by −61.233±33.9 and −58.7±19.4 after injection of the saline and Fe3O4@SiO2. The decrease of tumor signal by A-MFS was much more significant than that by saline and Fe3O4@SiO2 (P<0.05). The results demonstrated the high stability and nontoxicity of A-MFS, which effectively targeted pancreatic cancer in vitro and in vivo. A-MFS is a promising agent for diagnosis of pancreatic cancer. PMID:27274243

  7. In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody.

    PubMed

    Liu, Fang; Le, Wenjun; Mei, Tianxiao; Wang, Tiegong; Chen, Luguang; Lei, Yi; Cui, Shaobin; Chen, Bingdi; Cui, Zheng; Shao, Chengwei

    2016-01-01

    Pancreatic cancer is a highly malignant disease with a 5-year survival rate <5% mainly due to lack of early diagnosis and effective therapy. In an effort to improve the early diagnostic rate of pancreatic cancer, a nanoprobe Fe3O4@SiO2 modified with anti-mesothelin antibody (A-MFS) was prepared to target cells and tumor tissues highly expressing mesothelin in vitro (human pancreatic cancer cell line SW1990) and in vivo (subcutaneously transplanted tumors) studies. The A-MFS probe was successfully prepared and was spherical and uniform with a hydrodynamic diameter between 110 and 130 nm. Cell Counting Kit-8 testing indicated that A-MFS was nontoxic in vitro and in vivo studies. The in vitro study showed that the A-MFS probe specifically targeted SW1990 cells with high mesothelin expression. The in vivo study was conducted in Siemens 3.0 T magnetic resonance imaging. The average T2-weighted signal values of the xenografts were 966.533±31.56 before injecting A-MFS and 691.133±56.84 before injecting saline solution. After injection of 0.1 mL A-MFS via nude mouse caudal vein for 2.5 hours, the average T2-weighted signal of the xenograft decreased by 342.533±42.6. The signal value decreased by -61.233±33.9 and -58.7±19.4 after injection of the saline and Fe3O4@SiO2. The decrease of tumor signal by A-MFS was much more significant than that by saline and Fe3O4@SiO2 (P<0.05). The results demonstrated the high stability and nontoxicity of A-MFS, which effectively targeted pancreatic cancer in vitro and in vivo. A-MFS is a promising agent for diagnosis of pancreatic cancer. PMID:27274243

  8. Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin.

    PubMed

    Chang, Chien-Hsing; Sapra, Puja; Vanama, Sailaja S; Hansen, Hans J; Horak, Ivan D; Goldenberg, David M

    2005-12-15

    Ranpirnase (Rap) is a cytotoxic ribonuclease (RNase) isolated from frog oocytes. Here we describe high antitumor activity of a novel immunotoxin, 2L-Rap-hLL1-gamma4P, composed of 2 Rap molecules, each fused to the N terminus of the light chain of hLL1, an internalizing anti-CD74 humanized antibody. To reduce unwanted side effects, the constant region of hLL1 was changed from gamma1 to gamma4 and further to gamma4P by replacing serine228 to proline to prevent the formation of a half immunoglobulin G (IgG) common for IgG4. In vitro, 2L-Rap-hLL1-gamma4P retained RNase activity, specific binding to CD74, and was significantly more potent against CD74+ cell lines (Daudi, Raji, and MC/CAR) than naked hLL1. In vivo, the pharmacokinetic profile of 2L-Rap-hLL1-gamma4P was similar to that of naked hLL1. The maximum tolerated dose of 2L-Rap-hLL1-gamma4P in severe combined immunodeficient mice (SCID) or BALB/c mice was 50 microg per mouse. In Raji and Daudi Burkitt lymphoma xenograft models, treatment with a single 5 to 50 microg dose of 2L-Rap-hLL1-gamma4P, given as early or delayed treatment, resulted in cures of most animals. Treatment with 2L-Rap-hLL1-gamma4P was significantly better than all controls, including saline, naked hLL1, and nonspecific immunotoxin. In conclusion, 2L-Rap-hLL1-gamma4P demonstrated excellent in vitro and in vivo efficacy and thus merits further consideration as a therapeutic for CD74+ tumors. PMID:16109781

  9. Immune Depletion to Enhance Immunotoxin Therapy

    Cancer.gov

    Patients with advanced mesothelioma who have not benefited from previous chemotherapy will receive pentostatin and cyclophosphamide to prevent an anti-immunotoxin immune response prior to and in conjunction with SS1P immunotoxin therapy.

  10. Scintigraphic monitoring of immunotoxins using radionuclides and heterobifunctional chelators

    SciTech Connect

    Reardan, D.; Bernhard, S.

    1991-10-22

    This patent describes a method for in vivo radioimmunodetection of cytotoxic immunotoxin. It comprises administering internally to a mammal a radio-labeled immunotoxin, wherein a heterobifunctional chelating agent provides a chemical bridge between a radiolabel and a cytotoxic component bound to the antigen-binding component of the immunotoxin, and detecting externally the distribution of the immunotoxin in the mammal.

  11. Protection of the Furin Cleavage Site in Low-Toxicity Immunotoxins Based on Pseudomonas Exotoxin A

    PubMed Central

    Kaplan, Gilad; Lee, Fred; Onda, Masanori; Kolyvas, Emily; Bhardwaj, Gaurav; Baker, David; Pastan, Ira

    2016-01-01

    Recombinant immunotoxins (RITs) are fusions of an Fv-based targeting moiety and a toxin. Pseudomonas exotoxin A (PE) has been used to make several immunotoxins that have been evaluated in clinical trials. Immunogenicity of the bacterial toxin and off-target toxicity have limited the efficacy of these immunotoxins. To address these issues, we have previously made RITs in which the Fv is connected to domain III (PE24) by a furin cleavage site (FCS), thereby removing unneeded sequences of domain II. However, the PE24 containing RITs do not contain the naturally occurring disulfide bond around the furin cleavage sequence, because it was removed when domain II was deleted. This could potentially allow PE24 containing immunotoxins to be cleaved and inactivated before internalization by cell surface furin or other proteases in the blood stream or tumor microenvironment. Here, we describe five new RITs in which a disulfide bond is engineered to protect the FCS. The most active of these, SS1-Fab-DS3-PE24, shows a longer serum half-life than an RIT without the disulfide bond and has the same anti-tumor activity, despite being less cytotoxic in vitro. These results have significance for the production of de-immunized, low toxicity, PE24-based immunotoxins with a longer serum half-life. PMID:27463727

  12. Immunotoxin Therapies for the Treatment of Epidermal Growth Factor Receptor-Dependent Cancers

    PubMed Central

    Simon, Nathan; FitzGerald, David

    2016-01-01

    Many epithelial cancers rely on enhanced expression of the epidermal growth factor receptor (EGFR) to drive proliferation and survival pathways. Development of therapeutics to target EGFR signaling has been of high importance, and multiple examples have been approved for human use. However, many of the current small molecule or antibody-based therapeutics are of limited effectiveness due to the inevitable development of resistance and toxicity to normal tissues. Recombinant immunotoxins are therapeutic molecules consisting of an antibody or receptor ligand joined to a protein cytotoxin, combining the specific targeting of a cancer-expressed receptor with the potent cell killing of cytotoxic enzymes. Over the decades, many bacterial- or plant-based immunotoxins have been developed with the goal of targeting the broad range of cancers reliant upon EGFR overexpression. Many examples demonstrate excellent anti-cancer properties in preclinical development, and several EGFR-targeted immunotoxins have progressed to human trials. This review summarizes much of the past and current work in the development of immunotoxins for targeting EGFR-driven cancers. PMID:27153091

  13. Efficacy of an immunotoxin to folate receptor beta in the intra-articular treatment of antigen-induced arthritis

    PubMed Central

    2012-01-01

    Introduction We previously demonstrated that synovial sublining macrophages express folate receptor beta (FRβ). The aim of this study was to evaluate the efficacy of intra-articular administration of a recombinant immunotoxin to FRβ for treating rat antigen-induced arthritis. Methods A monoclonal antibody (mAb) to rat FRβ was produced by immunizing mice with B300-19 cells (murine pre-B cells) transfected with the rat FRβ gene. Recombinant immunotoxin was prepared by conjugating the Fv portion of the anti-rat FRβ mAb heavy chain with a truncated Pseudomonas exotoxin A and the Fv portion of the anti-rat FRβ mAb light chain. Antigen-induced arthritis was induced through intra-articular injection of methylated bovine serum albumin (mBSA) after two subcutaneous injections of mBSA and complete Freund's adjuvant. Immunotoxin was intra-articularly injected into the arthritis joint every other day for seven days after arthritis onset. Joint swelling was measured and histological scores of inflammation, synovial thickness, cartilage, and bone destruction were determined. Immunohistochemistry was performed to detect osteoclast and osteoclast precursor FRβ-expressing macrophages and cathepsin K-positive cells on day 21. Results Intra-articular administration of the immunotoxin attenuated joint swelling (61% suppression; P < 0.01 compared to the control on day 21) and improved histological findings, particularly cartilage and bone destruction (scores of rats treated with control versus the immunotoxin: 2.2 versus 0.5; P < 0.01), by reducing the number of FRβ-expressing macrophages and cathepsin K-positive cells. Conclusions Intra-articular administration of an immunotoxin to FRβ is effective for improving rat antigen-induced arthritis. PMID:22551402

  14. Immunotoxin Therapy for Relapsed Hairy Cell Leukemia

    Cancer.gov

    In this trial, patients with hairy cell leukemia who have relapsed multiple times or not responded to prior chemotherapy will be treated with an experimental immunotoxin called moxetumomab pasudotox given intravenously on days 1, 3, and 5 of 28-day cycles

  15. New Life for Immunotoxin Cancer Therapy.

    PubMed

    Hassan, Raffit; Alewine, Christine; Pastan, Ira

    2016-03-01

    Immunotoxins are targeted anticancer therapeutics that kill cancer cells using a cytotoxic bacterial toxin payload. Their development for use in solid tumor malignancies was delayed due to issues with their immunogenicity and limited therapeutic window. However, new research has rejuvenated the field. Coadministration with a lymphocyte-depleting regimen of pentostatin and cyclophosphamide can delay antidrug antibody formation, increasing the number of treatment cycles that patients can receive and resulting in durable responses in heavily pretreated patients. In addition, a new generation of immunotoxin molecules with reduced immunogenicity and nonspecific toxicity has been developed through protein engineering techniques, and one has recently entered the clinic. In preclinical studies in mouse models, these new agents are effective against many tumor types as single agents, and also produce synergistic antitumor responses in combination with chemotherapy. These new immunotoxins have renewed excitement in the field and may prove a promising addition to the targeted therapy repertoire. Clin Cancer Res; 22(5); 1055-8. ©2015 AACR. PMID:26463707

  16. Antitumor effects of immunotoxins are enhanced by lowering HCK or treatment with SRC kinase inhibitors.

    PubMed

    Liu, Xiu-Fen; Xiang, Laiman; FitzGerald, David J; Pastan, Ira

    2014-01-01

    Recombinant immunotoxins (RIT) are agents being developed for cancer treatment. They are composed of an Fv that binds to a cancer cell, fused to a 38-kDa fragment of Pseudomonas exotoxin A. SS1P is a RIT that targets mesothelin, a protein expressed on mesothelioma as well as pancreatic, ovarian, lung, and other cancers. Because the protein tyrosine kinase family regulates a variety of cellular processes and pathways, we hypothesized that tyrosine kinases might regulate susceptibility to immunotoxin killing. To investigate their role, we used siRNAs to lower the level of expression of the 88 known tyrosine kinases. We identified five tyrosine kinases, INSR, HCK, SRC, PDGFRβ, and BMX that enhance the activity of SS1P when their level of expression is lowered by siRNAs. We further investigated the Src family member HCK in this study. Knocking down of SRC slightly increased SS1P killing in A431/H9 cells, but knocking down HCK substantially enhanced killing by SS1P. We investigated the mechanism of enhancement and found that HCK knockdown enhanced SS1P cleavage by furin and lowered levels of Mcl-1 and raised Bax. We then found that Src inhibitors mimic the stimulatory effect of HCK knockdown; both SU6656 and SKI-606 (bosutinib) enhanced immunotoxin killing of mesothelin-expressing cells by SS1P and CD22-expressing cells by HA22 (moxetumomab pasudotox). SU6656 also enhanced the antitumor effects of SS1P and HA22 in mouse xenograft tumor models. Our data suggest that the combination of immunotoxin with tyrosine kinase inhibitors may be an effective way to treat some cancers. PMID:24145282

  17. T lymphocyte killing by a xanthine-oxidase-containing immunotoxin.

    PubMed

    Battelli, M G; Abbondanza, A; Tazzari, P L; Bolognesi, A; Lemoli, R M; Stirpe, F

    1992-01-01

    We report on the preparation of an immunotoxin consisting of xanthine oxidase, a free-radical-producing enzyme, covalently linked to an anti-CD3 monoclonal antibody. The immunotoxin retained both enzymic and immunological properties and its toxicity to target cells (a) was greater than that of the free enzyme, (b) was proportional to the enzyme concentration, and (c) was reduced either in the absence of hypoxanthine or by an excess of free anti-CD3 monoclonal antibody. The cytotoxicity and selectivity of the hypoxanthine/conjugated xanthine oxidase system were potentiated by the addition of chelated iron and by washing away the unbound immunotoxin prior to the addition of substrate. The same system was not toxic to bone marrow progenitor cells. A possible use of this immunotoxin for the ex vivo purging of organs to be transplanted from T lymphocytes, to avoid the graft-versus-host reaction, is suggested. PMID:1394345

  18. Comparison of the potency and therapeutic efficacy of the anti-CD7 immunotoxin HB2-saporin constructed with one or two saporin moieties per immunotoxin molecule.

    PubMed Central

    Flavell, D. J.; Boehm, D. A.; Noss, A.; Flavell, S. U.

    1997-01-01

    Immunotoxins that carry two toxin molecules to the target cell should in theory have a greater anti-tumour effect than those that carry just one. We have investigated the therapeutic efficacy of two anti-CD7-saporin immunotoxins constructed with one saporin (HB2-Sap 1-mer) or two saporin molecules (HB2-Sap 2-mer) per immunotoxin molecule. In vitro, the 2-mer immunotoxin was 5.6 times more effective than the 1-mer immunotoxin at inhibiting protein synthesis in the CD7+ human T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2 and was also more effective at inhibiting HSB-2 cell proliferation. Flow cytometry revealed that the 2-mer immunotoxin had a reduced binding capacity to HSB-2 cells compared with the 1-mer immunotoxin or native HB2 antibody. In therapy studies in SCID mice with disseminated HSB-2 human leukaemia, the 2-mer immunotoxin performed marginally better than the 1-mer immunotoxin, but log-rank analysis did not reveal any significant differences between the two therapy groups. We therefore conclude that, although the 2-mer immunotoxin performed better than the 1-mer immunotoxin against target HSB-2 cells in vitro, this improved performance was not reflected as an improved in vivo therapeutic outcome in the SCID mouse model. Images Figure 2 PMID:9083340

  19. HER2-targeted immunotoxins with low nonspecific toxicity and immunogenicity.

    PubMed

    Guo, Rui; Cao, Li; Guo, Wenjun; Liu, Hui; Xu, Hua; Fang, Qi; Hong, Zhangyong

    2016-06-17

    Immunotoxins have efficient anti-tumor activity due to their extreme potency. However, dose-limiting off-target toxicity and immunogenicity are the critical barriers for these immunotoxins to be used in a clinical setting. In this study, we designed a Pseudomonas exotoxin A (PE)-based human epidermal growth factor receptor-2 (HER2)-specific immunotoxin HER2-PE25-X7 by deleting most of domain II and introducing seven point mutations into domain III of the PE38 toxin. The anti-cancer activity, off-target toxicity and immunogenicity of this immunotoxin were carefully evaluated in vitro and in vivo. This new construct maintained the therapeutic potency of the original PE38-based immunotoxin HER2-PE38, with a greatly reduced off-target toxicity and immunogenicity. To compare with HER2-PE38, which resulted in the death of most of the mice after a single dose of 1.0 mg/kg, the new construct was completely tolerated at a dose of 10 mg/kg by the mice and almost completely depleted the tumor after treatment with five doses of 5 mg/kg of the immunotoxin. This work demonstrates a potentially attractive therapeutic modality for HER2-specific cancer treatment. PMID:27178207

  20. Targeted introduction of a diphtheria toxin resistant mutation into the chromosomal EF-2 locus of Pichia pastoris and expression of immunotoxin in the EF-2 mutants.

    PubMed

    Liu, Yuan Yi; Woo, Jung Hee; Neville, David M

    2003-08-01

    In an attempt to increase the production of a diphtheria toxin (DT) based immunotoxin by Pichia pastoris, we have created DT-resistant mutants that contain a substitution of arginine for glycine at position 701 in elongation factor 2 (EF-2). To achieve this, we first cloned and characterized the EF-2 gene (PEF1), and then made a construct pBLURA-Delta5'mutEF-2 that efficiently introduces specific mutations into the chromosomal EF-2 gene in P. pastoris by in vivo homologous recombination. pBLURA-Delta5(')mutEF-2 contains a selection marker URA3 and a 5' truncated form of the P. pastoris PEF1 that had been modified in vitro to carry the nucleotide mutations for the Gly(701) to Arg transition. Unlike the non-mutated strains, the EF-2 mutants are resistant to high-level intracellular expression of DT A chain that can catalyze the ADP-ribosylation. When used to express the secreted bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), the EF-2 mutant strains showed increased viability compared to the non-mutated strains. However, they did not show an advantage over the non-mutated expressing strain in the production of the immunotoxin. Western blotting analysis revealed that although the EF-2 mutants did not increase the accumulation of intact A-dmDT390-bisFv(G4S) in the culture medium, they generated larger amounts of degraded products found in both the medium and cell pellets compared to the non-mutant expressing clone. In addition, double copy expression resulted in greater amounts of intact immunotoxin being retained within cellular compartments as well as degraded products. Based on these findings, we suggest that the secretory capacity may be rate limiting for divalent immunotoxin production in P. pastoris. PMID:12880776

  1. Fusion of an albumin-binding domain extends the half-life of immunotoxins.

    PubMed

    Guo, Rui; Guo, Wenjun; Cao, Li; Liu, Hui; Liu, Jieyu; Xu, Hua; Huang, Weiqiang; Wang, Fengwei; Hong, Zhangyong

    2016-09-10

    Immunotoxins have documented potential as a cancer treatment due to their extreme potency; a single toxin molecule delivered to the cytosol may be sufficient to kill a cell. However, their short half-life in the circulatory system may be one of the key problems associated with the clinical use of immunotoxins and may continue to limit their therapeutic activity. Herein, we genetically fused an albumin-binding domain (ABD) to the human epidermal growth factor receptor 2 (HER2)-specific immunotoxin ZHER2-PE38 to extend the circulation time and thus improve the therapeutic outcome of this immunotoxin. Furthermore, the fusion of an ABD to the immunotoxin was found to promote non-covalent interactions between the immunotoxin and serum albumin, which rescue the immunotoxin from lysosomal degradation through a serum albumin-mediated interaction with the neonatal Fc receptor (FcRn). This manuscript reports the construction, purification, and characterization of the ABD-fused HER2-specific immunotoxin, ABD-ZHER2-PE38, both in vitro and in vivo. Compared with non-fused ZHER2-PE38, this new construct exhibits a clearly increased half-life in plasma (330.8 versus 13.5min, approximately 24.4-fold extension) and remarkably improved antitumor effects in an NCI-N87 subcutaneous xenograft model. Therefore, the new construct represents a potentially attractive therapeutic modality, and the proposed strategy may also have useful applications for current immunotoxin designs. PMID:27457423

  2. A comparison of anti-lymphocyte immunotoxins containing different ribosome-inactivating proteins and antibodies.

    PubMed Central

    Bolognesi, A; Tazzari, P L; Tassi, C; Gromo, G; Gobbi, M; Stirpe, F

    1992-01-01

    Immunotoxins were prepared with several single-chain ribosome-inactivating proteins (RIPs type 1) and with the A-chain of ricin linked to the F(ab')2 fragment of sheep anti-mouse IgG. The cytotoxic activity of these conjugates was tested on human lymphocytes pretreated with an anti-CD3 murine MoAb. The immunotoxins inhibited DNA synthesis in phytohaemagglutinin (PHA)-stimulated lymphocytes with IC50S (concentrations causing 50% inhibition) ranging from 8.9 x 10(-13) to 5.7 x 10(-11) M (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP-S), bryodin, momordin, momorcochin, and trichokirin), 1 x 10(-8) M (immunotoxin containing gelonin) and 5 x 10(-9) M (immunotoxin containing ricin A-chain). The immunotoxin containing saporin linked to the anti-mouse IgG F(ab')2 fragment was also highly toxic to human lymphocytes pretreated with anti-CD2, -CD3, -CD5 and -CD45 MoAbs, with IC50S less than or equal to 10(-11) M. Immunotoxins were prepared also with saporin linked to MoAbs against various CD antigens. The immunotoxin prepared with the anti-CD3 antibody had the highest specific cytotoxicity to human lymphocytes. Images Fig. 2 PMID:1516253

  3. Naptumomab estafenatox: targeted immunotherapy with a novel immunotoxin.

    PubMed

    Eisen, Tim; Hedlund, Gunnar; Forsberg, Göran; Hawkins, Robert

    2014-02-01

    Improvement of cancer therapy by introducing new concepts is still urgent even though there have been major advancements lately. Immunotherapy is well on the way to becoming an established tool in the cancer treatment armory. It seems that a combination of (1) activation of immune effector cells and selective targeting of them to tumors and (2) the inhibition of immune suppression often induced by the tumor itself are necessary to achieve the therapeutic goal. The immunotoxin naptumomab estafenatox was developed in an effort to activate and target the patient's own T cells to their tumor, by fusing a superantigen (SAg) variant that activates T lymphocytes to the Fab moiety of a tumor-reactive monoclonal antibody. Naptumomab estafenatox targets the 5T4 tumor antigen, a 72-kDa oncofetal trophoblast protein expressed on many carcinomas, including renal cell carcinoma. The therapeutic effect is associated with activation of SAg-binding T cells. The SAg-binding T lymphocytes expand, differentiate to effector cells, and infiltrate the tumor. The therapeutic efficacy is most likely related to the dual mechanism of tumor cell killing: (1) direct lysis by cytotoxic T lymphocytes of tumor cells expressing the antigen recognized by the antibody moiety of the fusion protein and (2) secretion of cytokines eliminating antigen-negative tumor cell variants. Naptumomab estafenatox has been clinically tested in a range of solid tumors with focus on renal cell carcinoma. This review looks at the clinical experience with the new immunotoxin and its potential. PMID:24445502

  4. Prevention of immunotoxin-mediated vascular leak syndrome in rats with retention of antitumor activity.

    PubMed

    Siegall, C B; Liggitt, D; Chace, D; Tepper, M A; Fell, H P

    1994-09-27

    Immunotoxins are hybrid molecules composed of a cell-surface binding domain and a protein toxin moiety that together target specific cell populations for elimination. These agents represent a promising approach for the treatment of many human diseases, most notably cancer. However, it has recently become clear that many immunotoxins when used in human clinical trials induce vascular leak syndrome (VLS), restricting the administration of doses necessary to achieve good therapeutic responses. The lack of an appropriate animal model has hindered efforts to understand and prevent immunotoxin-induced VLS. We have found that in rats, intravenous administration of the single-chain immunotoxin BR96 sFv-PE40 results in symptoms that closely resemble VLS seen in human immunotoxin trials. A large fluid accumulation in the thoracic cavity was observed, along with an increase in hematocrit and body weight and a decrease in serum albumin. The VLS was apparent within 24 hr after administration of immunotoxin and was seen in both immunocompetent and athymic rats. Similar symptoms were not found in mice even at lethal doses. Prophylactic administration of the corticosteroid dexamethasone resulted in prevention of VLS and survival of rats injected with what would otherwise be lethal doses of BR96 sFv-PE40. Prophylactic treatment with dexamethasone in rats xenografted with human tumors either did not inhibit or minimally inhibited the antitumor activity of BR96 sFv-PE40. The use of prophylactic corticosteroids should be considered for immunotoxin clinical trials, since it may improve therapeutic efficacy by decreasing the dose-limiting toxicity of VLS. PMID:7937798

  5. Preparation of an engineered safer immunotoxin against colon carcinoma based on the ribotoxin hirsutellin A.

    PubMed

    Tomé-Amat, Jaime; Herrero-Galán, Elías; Oñaderra, Mercedes; Martínez-Del-Pozo, Álvaro; Gavilanes, José G; Lacadena, Javier

    2015-06-01

    Immunotoxins are chimeric proteins composed of an antibody domain that specifically directs the action of the toxic domain, resulting in the death of the targeted cells. Over recent years, immunotoxins have been widely studied and the number of different constructions has increased exponentially. Protein engineering has allowed the design of optimized versions of immunotoxins with an improved tumor binding affinity, stability or cytotoxic efficacy, although sometimes this has compromised the safety of the patient in terms of undesirable adverse secondary reactions. A triple mutant at three Trp residues (HtA3ΔW) of the ribotoxin hirsutellin A retains its specific ribonucleolytic activity, although cell internalization capacity is lacking. This toxin variant has been fused to the single chain variable fragment A33 (scFvA33). This immunoconjugate (IMTXA33HtA3ΔW) was produced in the methylotrophic yeast Pichia pastoris and purified using nickel-nitrilotriacetic acid affinity chromatography. Both target and toxic domains were characterized. The immunotoxin showed an exquisite specific binding against GPA33-positive culture cells, which results in the death of the targeted cells because of specific ribonucleolytic activity against ribosomes of the engineered hirsutellin A variant. IMTXA33HtA3ΔW represents a promising structure in the search for an improved immunotoxin without compromising the safety of patients. PMID:25752204

  6. Separation of bivalent anti-T cell immunotoxin from Pichia pastoris glycoproteins by borate anion exchange.

    PubMed

    Woo, Jung Hee; Neville, David M

    2003-08-01

    A major problem encountered in the large-scale purification of the bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), from Pichia pastoris supernatants was the presence of host glycoproteins exhibiting similar charge, size, and hydrophobicity characteristics. We overcame this problem by employing borate anion exchange chromatography. The borate anion has an affinity for carbohydrates and imparts negative charges to these structures. We found that at a concentration of sodium borate between 50 and 100 mM, the nonglycosylated immunotoxin did not bind to Poros 50 HQ anion exchanger resin, but glycoproteins, including aggregates related to the immunotoxin, did. By using this property of the immunotoxin in the presence of sodium borate, we successfully developed a 3-step purification procedure: (i) Butyl-650M hydrophobic interaction chromatography, (ii) Poros 50 HQ anion exchange chromatography in the presence of borate, and (iii) HiTrap Q anion exchange chromatography. The final preparation exhibited a purity of greater than 98% and a yield of greater than 50% from the supernatant. Previously, boronic acid resins have been used to separate glycoproteins from proteins. However, combining borate anion with conventional anion exchange resins accomplishes the separation of the immunotoxin from glycoproteins and eliminates the need to evaluate nonstandard resins with respect to good manufacturing practice guidelines. PMID:12951782

  7. Depletion of Alloreactive T-Cells by Anti-CD137-Saporin Immunotoxin.

    PubMed

    Lee, Sang C; Seo, Kwang W; Kim, Hye J; Kang, Sang W; Choi, Hye-Jeong; Kim, Ansuk; Kwon, Byoung S; Cho, Hong R; Kwon, Byungsuk

    2015-01-01

    Depletion of alloreactive T-lymphocytes from allogeneic bone marrow transplants may prevent graft-versus-host disease (GVHD) without impairing donor cell engraftment, immunity, and the graft-versus-leukemia (GVL) effect. Alloreactive T-cells may be identified by their expression, upon activation, of CD137, a costimulatory receptor and putative surrogate marker for antigen-specific effector T-cells. In this context, we tested the use of anti-CD137-saporin immunotoxin to selectively deplete mouse and human alloreactive T-cells. Anti-CD137 antibodies were internalized by cells within 4 h of binding to the cell surface CD137, and anti-CD137-saporin immunotoxin effectively killed polyclonally activated T-cells or antigen-stimulated T-cells. Transfer of donor T-cells after allodepletion with anti-CD137-saporin immunotoxin failed to induce any evident expression of GVHD; however, a significant GVL effect was observed. Targeting of CD137 with an immunotoxin was also effective in killing polyclonally activated or alloreactive human T-cells. Our results indicate that anti-CD137-saporin immunotoxin may be used to deplete alloreactive T-cells prior to bone marrow transplantation and thereby prevent GVHD and the relapse of leukemia. PMID:24594433

  8. Protein Kinase Inhibitor H89 Enhances the Activity of Pseudomonas Exotoxin A-Based Immunotoxins.

    PubMed

    Liu, Xiufen; Müller, Fabian; Wayne, Alan S; Pastan, Ira

    2016-05-01

    HA22 (Moxetumomab pasudotox) is a recombinant immunotoxin (RIT), composed of an anti-CD22 Fv fused to a truncated portion of Pseudomonas exotoxin A. HA22 is in clinical trials to treat patients with hairy cell leukemia and acute lymphoblastic leukemia (ALL). LMB-11 is an improved variant of HA22 with reduced immunogenicity, has a longer half-life in the blood and high activity in vitro and in a Burkitt lymphoma model in vivo Searching for RIT enhancing combination therapies, we found the protein kinase A inhibitor H89 to enhance LMB-11 and HA22 activity 5- to 10-fold on ALL cell lines and on patient-derived ALL samples. In addition, H89 increased the activity of mesothelin-targeting RITs SS1P (38-fold) and RG7787 (7-fold) against the cervical cancer cell line KB31. Unexpectedly we found that the enhancement by H89 was not because of inhibition of protein kinase A; it was partially recapitulated by inhibition of S6K1, which led to inactivation of its downstream targets rpS6 and GSK3β, resulting in a fall in MCL1 levels. H89 increased the rate of ADP-ribosylation of eukaryotic elongation factor 2, enhancing the arrest of protein synthesis and the reduction of MCL1 in synergy with the RIT. In summary, H89 increased RIT activity by enhancing the two key events: ADP-ribosylation of eEF2 and reduction of MCL1 levels. Significant enhancement was seen with both CD22- and mesothelin-targeting RITs, indicating that H89 might be a potent addition to RIT treatment of CD22-positive ALL and mesothelin-expressing solid tumors. Mol Cancer Ther; 15(5); 1053-62. ©2016 AACR. PMID:26939705

  9. Noradrenergic lesioning with an anti-dopamine beta-hydroxylase immunotoxin

    NASA Technical Reports Server (NTRS)

    Picklo, M. J.; Wiley, R. G.; Lappi, D. A.; Robertson, D.

    1994-01-01

    Sympathectomy has been achieved by a variety of methods but each has its limitations. These include lack of tissue specificity, incomplete lesioning, and the age range of susceptibility to the lesioning. To circumvent these drawbacks, an immunotoxin was constructed using a monoclonal antibody against the noradrenergic specific enzyme dopamine beta-hydroxylase (D beta H) coupled via a disulfide bond to saporin, a ribosomal inactivating protein. Three days after intravenous injection of the anti-D beta H immunotoxin (50 micrograms) into adult Sprague-Dawley rats, 66% of neurons in the superior cervical ganglia were chromatolytic. Superior cervical ganglia neurons were poisoned in 1 day old and 1 week old (86% of neurons) neonatal rats following subcutaneous injection of 3.75 and 15 micrograms, respectively. The anti-D beta H immunotoxin will be a useful tool in the study of the peripheral noradrenergic system in adult and neonatal animals.

  10. Properties of immunotoxins against a glycolipid antigen associated with Burkitt's lymphoma.

    PubMed

    Wiels, J; Junqua, S; Dujardin, P; Le Pecq, J B; Tursz, T

    1984-01-01

    A monoclonal immunoglobulin M (IgM) antibody (38-13) which recognizes Burkitt's lymphoma (BL) cells, by reacting with the neutral glycolipid Gal alpha 1 leads to 4-Gal beta 1 leads to 4-Glc beta 1 leads to 1-ceramide, was recently characterized. This monoclonal IgM was coupled to either ricin A chain or gelonin. The two different immunotoxins obtained retained the apparent immunological specificity of 38-13 IgM, as shown by flow cytofluorometry analysis and complement-dependent cytotoxicity test. The BL Ramos cells and the apparently irrelevant Epstein-Barr virus-containing lymphoblastoid Priess cells were used as targets in in vitro assays of the cytotoxic properties of the two immunotoxins by measuring the inhibition of protein synthesis. Isolated ricin A chain, gelonin, and 38-13 IgM exhibited very low intrinsic cytotoxicity on both target cells. 38-13 ricin A chain and 38-13 gelonin conjugates exerted toxic effects on both target cells which were about 6000-fold and 3000-fold higher than uncoupled ricin A chain and gelonin, respectively. The toxicity of these conjugates almost reached that of intact ricin. On Ramos BL cells, the kinetics of action of the 38-13 ricin A chain conjugate was almost as fast as that of intact ricin, because 50% protein synthesis inhibition was reached after 3 hr. In contrast, the kinetics of action in the non-BL Priess was much slower (50% protein synthesis inhibition after 10 hr). An obviously irrelevant immunotoxin (anti-trinitrophenol IgM-ricin A chain) had no significant cytotoxic effect on BL Ramos and non-BL Priess cells. An excess of D-galactose was shown previously to inhibit the 38-13 IgM from binding to the reactive glycolipid antigen bearing a terminal galactose. An excess of D-galactose (0.1 M) inhibited the cytotoxic effect of the two 38-13 immunotoxins, whereas it did not prevent the cytotoxic effect of the anti-trinitrophenol immunotoxin on the same trinitrophenol labeled target cells. These data suggest that the

  11. GUCY2C lysosomotropic endocytosis delivers immunotoxin therapy to metastatic colorectal cancer

    PubMed Central

    Marszalowicz, Glen P.; Snook, Adam E.; Magee, Michael S.; Merlino, Dante; Lisa, D. Berman-Booty; Waldman, Scott A.

    2014-01-01

    The emergence of targeted cancer therapy has been limited by the paucity of determinants which are tumor-specific and generally associated with disease, and have cell dynamics which effectively deploy cytotoxic payloads. Guanylyl cyclase C (GUCY2C) may be ideal for targeting because it is normally expressed only in insulated barrier compartments, including intestine and brain, but over-expressed by systemic metastatic colorectal tumors. Here, we reveal that GUCY2C rapidly internalizes from the cell surface to lysosomes in intestinal and colorectal cancer cells. Endocytosis is independent of ligand binding and receptor activation, and is mediated by clathrin. This mechanism suggests a design for immunotoxins comprising a GUCY2C-directed monoclonal antibody conjugated through a reducible disulfide linkage to ricin A chain, which is activated to a potent cytotoxin in lysosomes. Indeed, this immunotoxin specifically killed GUCY2C-expressing colorectal cancer cells in a lysosomal- and clathrin-dependent fashion. Moreover, this immunotoxin reduced pulmonary tumors >80% (p<0.001), and improved survival 25% (p<0.001), in mice with established colorectal cancer metastases. Further, therapeutic efficacy was achieved without histologic evidence of toxicity in normal tissues. These observations support GUCY2C-targeted immunotoxins as novel therapeutics for metastatic tumors originating in the GI tract, including colorectum, stomach, esophagus, and pancreas. PMID:25294806

  12. Anti-Human Endoglin (hCD105) Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    PubMed Central

    Barriuso, Begoña; Antolín, Pilar; Arias, F. Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-01-01

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M. PMID:27294959

  13. Antiviral activity of a single-domain antibody immunotoxin binding to glycoprotein D of herpes simplex virus 2.

    PubMed

    Geoghegan, Eileen M; Zhang, Hong; Desai, Prashant J; Biragyn, Arya; Markham, Richard B

    2015-01-01

    Despite years of research dedicated to preventing the sexual transmission of herpes simplex virus 2 (HSV-2), there is still no protective vaccine or microbicide against one of the most common sexually transmitted infections in the world. Using a phage display library constructed from a llama immunized with recombinant HSV-2 glycoprotein D, we identified a single-domain antibody VHH, R33, which binds to the viral surface glycoprotein D. Although R33 does not demonstrate any HSV-2 neutralization activity in vitro, when expressed with the cytotoxic domain of exotoxin A, the resulting immunotoxin (R33ExoA) specifically and potently kills HSV-2-infected cells, with a 50% neutralizing dilution (IC50) of 6.7 nM. We propose that R33ExoA could be used clinically to prevent transmission of HSV-2 through killing of virus-producing epithelial cells during virus reactivation. R33 could also potentially be used to deliver other cytotoxic effectors to HSV-2-infected cells. PMID:25385102

  14. Antiviral Activity of a Single-Domain Antibody Immunotoxin Binding to Glycoprotein D of Herpes Simplex Virus 2

    PubMed Central

    Geoghegan, Eileen M.; Zhang, Hong; Desai, Prashant J.; Biragyn, Arya

    2014-01-01

    Despite years of research dedicated to preventing the sexual transmission of herpes simplex virus 2 (HSV-2), there is still no protective vaccine or microbicide against one of the most common sexually transmitted infections in the world. Using a phage display library constructed from a llama immunized with recombinant HSV-2 glycoprotein D, we identified a single-domain antibody VHH, R33, which binds to the viral surface glycoprotein D. Although R33 does not demonstrate any HSV-2 neutralization activity in vitro, when expressed with the cytotoxic domain of exotoxin A, the resulting immunotoxin (R33ExoA) specifically and potently kills HSV-2-infected cells, with a 50% neutralizing dilution (IC50) of 6.7 nM. We propose that R33ExoA could be used clinically to prevent transmission of HSV-2 through killing of virus-producing epithelial cells during virus reactivation. R33 could also potentially be used to deliver other cytotoxic effectors to HSV-2-infected cells. PMID:25385102

  15. Anti-dopamine beta-hydroxylase immunotoxin-induced sympathectomy in adult rats

    NASA Technical Reports Server (NTRS)

    Picklo, M. J.; Wiley, R. G.; Lonce, S.; Lappi, D. A.; Robertson, D.

    1995-01-01

    Anti-dopamine beta-hydroxylase immunotoxin (DHIT) is an antibody-targeted noradrenergic lesioning tool comprised of a monoclonal antibody against the noradrenergic enzyme, dopamine beta-hydroxylase, conjugated to saporin, a ribosome-inactivating protein. Noradrenergic-neuron specificity and completeness and functionality of sympathectomy were assessed. Adult, male Sprague-Dawley rats were given 28.5, 85.7, 142 or 285 micrograms/kg DHIT i.v. Three days after injection, a 6% to 73% decrease in the neurons was found in the superior cervical ganglia of the animals. No loss of sensory, nodose and dorsal root ganglia, neurons was observed at the highest dose of DHIT. In contrast, the immunotoxin, 192-saporin (142 micrograms/kg), lesioned all three ganglia. To assess the sympathectomy, 2 wk after treatment (285 micrograms/kg), rats were anesthetized with urethane (1 g/kg) and cannulated in the femoral artery and vein. DHIT-treated animals' basal systolic blood pressure and heart rate were significantly lower than controls. Basal plasma norepinephrine levels were 41% lower in DHIT-treated animals than controls. Tyramine-stimulated release of norepinephrine in DHIT-treated rats was 27% of controls. Plasma epinephrine levels of DHIT animals were not reduced. DHIT-treated animals exhibited a 2-fold hypersensitivity to the alpha-adrenergic agonist phenylephrine. We conclude that DHIT selectively delivered saporin to noradrenergic neurons resulting in destruction of these neurons. Anti-dopamine beta-hydroxylase immunotoxin administration produces a rapid, irreversible sympathectomy.

  16. Immunotoxin Against a Donor MHC Class II Molecule Induces Indefinite Survival of Murine Kidney Allografts.

    PubMed

    Brown, K; Nowocin, A K; Meader, L; Edwards, L A; Smith, R A; Wong, W

    2016-04-01

    Rejection of donor organs depends on the trafficking of donor passenger leukocytes to the secondary lymphoid organs of the recipient to elicit an immune response via the direct antigen presentation pathway. Therefore, the depletion of passenger leukocytes may be clinically applicable as a strategy to improve graft survival. Because major histocompatibility complex (MHC) class II(+) cells are most efficient at inducing immune responses, selective depletion of this population from donor grafts may dampen the alloimmune response and prolong graft survival. In a fully MHC mismatched mouse kidney allograft model, we describe the synthesis of an immunotoxin, consisting of the F(ab')2 fragment of a monoclonal antibody against the donor MHC class II molecule I-A(k) conjugated with the plant-derived ribosomal inactivating protein gelonin. This anti-I-A(k) gelonin immunotoxin depletes I-A(k) expressing cells specifically in vitro and in vivo. When given to recipients of kidney allografts, it resulted in indefinite graft survival with normal graft function, presence of Foxp3(+) cells within donor grafts, diminished donor-specific antibody formation, and delayed rejection of subsequent donor-type skin grafts. Strategies aimed at the donor arm of the immune system using agents such as immunotoxins may be a useful adjuvant to existing recipient-orientated immunosuppression. PMID:26799449

  17. High in Vitro Anti-Tumor Efficacy of Dimeric Rituximab/Saporin-S6 Immunotoxin

    PubMed Central

    Bortolotti, Massimo; Bolognesi, Andrea; Battelli, Maria Giulia; Polito, Letizia

    2016-01-01

    The anti-CD20 mAb Rituximab has revolutionized lymphoma therapy, in spite of a number of unresponsive or relapsing patients. Immunotoxins, consisting of toxins coupled to antibodies, are being investigated for their potential ability to augment Rituximab efficacy. Here, we compare the anti-tumor effect of high- and low-molecular-weight Rituximab/saporin-S6 immunotoxins, named HMW-IT and LMW-IT, respectively. Saporin-S6 is a potent and stable plant enzyme belonging to ribosome-inactivating proteins that causes protein synthesis arrest and consequent cell death. Saporin-S6 was conjugated to Rituximab through an artificial disulfide bond. The inhibitory activity of HMW-IT and LMW-IT was evaluated on cell-free protein synthesis and in two CD20+ lymphoma cell lines, Raji and D430B. Two different conjugates were separated on the basis of their molecular weight and further characterized. Both HMW-IT (dimeric) and LMW-IT (monomeric) maintained a high level of enzymatic activity in a cell-free system. HMW-IT, thanks to a higher toxin payload and more efficient antigen capping, showed stronger in vitro anti-tumor efficacy than LMW-IT against lymphoma cells. Dimeric HMW-IT can be used for lymphoma therapy at least for ex vivo treatments. The possibility of using HMW-IT augments the yield in immunotoxin preparation and allows the targeting of antigens with low internalization rates. PMID:27338475

  18. High in Vitro Anti-Tumor Efficacy of Dimeric Rituximab/Saporin-S6 Immunotoxin.

    PubMed

    Bortolotti, Massimo; Bolognesi, Andrea; Battelli, Maria Giulia; Polito, Letizia

    2016-01-01

    The anti-CD20 mAb Rituximab has revolutionized lymphoma therapy, in spite of a number of unresponsive or relapsing patients. Immunotoxins, consisting of toxins coupled to antibodies, are being investigated for their potential ability to augment Rituximab efficacy. Here, we compare the anti-tumor effect of high- and low-molecular-weight Rituximab/saporin-S6 immunotoxins, named HMW-IT and LMW-IT, respectively. Saporin-S6 is a potent and stable plant enzyme belonging to ribosome-inactivating proteins that causes protein synthesis arrest and consequent cell death. Saporin-S6 was conjugated to Rituximab through an artificial disulfide bond. The inhibitory activity of HMW-IT and LMW-IT was evaluated on cell-free protein synthesis and in two CD20⁺ lymphoma cell lines, Raji and D430B. Two different conjugates were separated on the basis of their molecular weight and further characterized. Both HMW-IT (dimeric) and LMW-IT (monomeric) maintained a high level of enzymatic activity in a cell-free system. HMW-IT, thanks to a higher toxin payload and more efficient antigen capping, showed stronger in vitro anti-tumor efficacy than LMW-IT against lymphoma cells. Dimeric HMW-IT can be used for lymphoma therapy at least for ex vivo treatments. The possibility of using HMW-IT augments the yield in immunotoxin preparation and allows the targeting of antigens with low internalization rates. PMID:27338475

  19. A potent immunotoxin targeting fibroblast activation protein for treatment of breast cancer in mice.

    PubMed

    Fang, Jinxu; Xiao, Liang; Joo, Kye-Il; Liu, Yarong; Zhang, Chupei; Liu, Shuanglong; Conti, Peter S; Li, Zibo; Wang, Pin

    2016-02-15

    Fibroblast activation protein (FAP) is highly expressed in the tumor-associated fibroblasts (TAFs) of most human epithelial cancers. FAP plays a critical role in tumorigenesis and cancer progression, which makes it a promising target for novel anticancer therapy. However, mere abrogation of FAP enzymatic activity by small molecules is not very effective in inhibiting tumor growth. In this study, we have evaluated a novel immune-based approach to specifically deplete FAP-expressing TAFs in a mouse 4T1 metastatic breast cancer model. Depletion of FAP-positive stromal cells by FAP-targeting immunotoxin αFAP-PE38 altered levels of various growth factors, cytokines, chemokines and matrix metalloproteinases, decreased the recruitment of tumor-infiltrating immune cells in the tumor microenvironment and suppressed tumor growth. In addition, combined treatment with αFAP-PE38 and paclitaxel potently inhibited tumor growth in vivo. Our findings highlight the potential use of immunotoxin αFAP-PE38 to deplete FAP-expressing TAFs and thus provide a rationale for the use of this immunotoxin in cancer therapy. PMID:26334777

  20. Immunotoxin Against a Donor MHC Class II Molecule Induces Indefinite Survival of Murine Kidney Allografts

    PubMed Central

    Brown, K.; Nowocin, A. K.; Meader, L.; Edwards, L. A.; Smith, R. A.

    2016-01-01

    Rejection of donor organs depends on the trafficking of donor passenger leukocytes to the secondary lymphoid organs of the recipient to elicit an immune response via the direct antigen presentation pathway. Therefore, the depletion of passenger leukocytes may be clinically applicable as a strategy to improve graft survival. Because major histocompatibility complex (MHC) class II+ cells are most efficient at inducing immune responses, selective depletion of this population from donor grafts may dampen the alloimmune response and prolong graft survival. In a fully MHC mismatched mouse kidney allograft model, we describe the synthesis of an immunotoxin, consisting of the F(ab′)2 fragment of a monoclonal antibody against the donor MHC class II molecule I‐Ak conjugated with the plant‐derived ribosomal inactivating protein gelonin. This anti–I‐Ak gelonin immunotoxin depletes I‐Ak expressing cells specifically in vitro and in vivo. When given to recipients of kidney allografts, it resulted in indefinite graft survival with normal graft function, presence of Foxp3+ cells within donor grafts, diminished donor‐specific antibody formation, and delayed rejection of subsequent donor‐type skin grafts. Strategies aimed at the donor arm of the immune system using agents such as immunotoxins may be a useful adjuvant to existing recipient‐orientated immunosuppression. PMID:26799449

  1. Selective cytotoxic activity of immunotoxins composed of a monoclonal anti-Thy 1.1 antibody and the ribosome-inactivating proteins bryodin and momordin.

    PubMed Central

    Stirpe, F.; Wawrzynczak, E. J.; Brown, A. N.; Knyba, R. E.; Watson, G. J.; Barbieri, L.; Thorpe, P. E.

    1988-01-01

    The ribosome-inactivating proteins, bryodin, from Bryonia dioica, and momordin, from Momordica charantia, were coupled by a disulphide bond to a monoclonal anti-Thy 1.1 antibody (OX7). Both immunotoxins were specifically cytotoxic to the Thy 1.1-expressing mouse lymphoma cell line AKR-A in vitro. The OX7-bryodin immunotoxins were the more powerfully toxic and reduced protein synthesis in AKR-A cells by 50% at a concentration of 1-4 x 10(-11) M as compared with 1 x 10(-9) M for the OX7-momordin immunotoxins. Neither of the immunotoxins was toxic to mouse lymphoma EL4 cells, which lack the Thy 1.1 antigen, at concentrations up to 3 x 10(-8) M. Further, bryodin and momordin immunotoxins made from an antibody (R10) of irrelevant specificity were without effect on AKR-A cells. PMID:3265330

  2. Selective cytotoxic activity of immunotoxins composed of a monoclonal anti-Thy 1.1 antibody and the ribosome-inactivating proteins bryodin and momordin.

    PubMed

    Stirpe, F; Wawrzynczak, E J; Brown, A N; Knyba, R E; Watson, G J; Barbieri, L; Thorpe, P E

    1988-11-01

    The ribosome-inactivating proteins, bryodin, from Bryonia dioica, and momordin, from Momordica charantia, were coupled by a disulphide bond to a monoclonal anti-Thy 1.1 antibody (OX7). Both immunotoxins were specifically cytotoxic to the Thy 1.1-expressing mouse lymphoma cell line AKR-A in vitro. The OX7-bryodin immunotoxins were the more powerfully toxic and reduced protein synthesis in AKR-A cells by 50% at a concentration of 1-4 x 10(-11) M as compared with 1 x 10(-9) M for the OX7-momordin immunotoxins. Neither of the immunotoxins was toxic to mouse lymphoma EL4 cells, which lack the Thy 1.1 antigen, at concentrations up to 3 x 10(-8) M. Further, bryodin and momordin immunotoxins made from an antibody (R10) of irrelevant specificity were without effect on AKR-A cells. PMID:3265330

  3. Efficacy of RG7787, a next-generation mesothelin-targeted immunotoxin, against triple-negative breast and gastric cancers.

    PubMed

    Alewine, Christine; Xiang, Laiman; Yamori, Takao; Niederfellner, Gerhard; Bosslet, Klaus; Pastan, Ira

    2014-11-01

    The RG7787 mesothelin-targeted recombinant immunotoxin (RIT) consists of an antibody fragment targeting mesothelin (MSLN) fused to a 24-kD fragment of Pseudomonas exotoxin A for cell killing. Compared with prior RITs, RG7787 has improved properties for clinical development including decreased nonspecific toxicity and immunogenicity and resistance to degradation by lysosomal proteases. MSLN is a cell surface glycoprotein highly expressed by many solid tumor malignancies. New reports have demonstrated that MSLN is expressed by a significant percentage of triple-negative breast and gastric cancer clinical specimens. Here, panels of triple-negative breast and gastric cancer cell lines were tested for surface MSLN expression, and for sensitivity to RG7787 in vitro and in animal models. RG7787 produced >95% cell killing of the HCC70 and SUM149 breast cancer cell lines in vitro with IC50 < 100 pmol/L. RG7787 was also effective against gastric cancer cell lines MKN28, MKN45, and MKN74 in vitro, with subnanomolar IC50s. In a nude mouse model, RG7787 treatment (2.5 mg/kg i.v. qod ×3-4) resulted in a statistically significant 41% decrease in volumes of HCC70 xenograft tumors (P < 0.0001) and an 18% decrease in MKN28 tumors (P < 0.0001). Pretreatment with paclitaxel (50 mg/kg i.p.) enhanced efficacy, producing 88% and 70% reduction in tumor volumes for HCC70 and MKN28, respectively, a statistically significant improvement over paclitaxel alone (P < 0.0001 for both). RG7787 merits clinical testing for triple-negative breast and gastric cancers. PMID:25239937

  4. B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

    PubMed Central

    Vooijs, W. C.; Otten, H. G.; van Vliet, M.; van Dijk, A. J.; de Weger, R. A.; de Boer, M.; Bohlen, H.; Bolognesi, A.; Polito, L.; de Gast, G. C.

    1997-01-01

    In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin's disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration. Images Figure 1 PMID:9365164

  5. Complete suppression of in vivo growth of human leukemia cells by specific immunotoxins: nude mouse models

    SciTech Connect

    Hara, H.; Seon, B.K.

    1987-05-01

    In this study, immunotoxins containing monoclonal anti-human T-cell leukemia antibodies are shown to be capable of completely suppressing the tumor growth of human T-cell leukemia cells in vivo without any overt undersirable toxicity. These immunotoxins were prepared by conjugating ricin A chain (RA) with our monoclonal antibodies, SN1 and SN2, directed specifically to the human T-cell leukemia cell surface antigens TALLA and GP37, respectively. The authors have shown that these monoclonal antibodies are highly specific for human T-cell leukemia cells and do not react with various normal cells including normal T and B cells, thymocytes, and bone marrow cells. Ascitic and solid human T-cell leukemia cell tumors were generated in nude mice. The ascitic tumor was generated by transplanting Ichikawa cells (a human T-cell leukemia cell) i.p. into nude mice, whereas the solid tumor was generated by transplanting s.c. MOLT-4 cells (a human T-cell leukemia cell line) and x-irradiated human fibrosarcoma cells into x-irradiated nude mice. To investigate the efficacy of specific immunotoxins in suppression the in vivo growth of the ascitic tumor, they divided 40 nude mice that were injected with Ichikawa cells into four groups. None of the mice in group 4 that were treated with SN1-RA and SN2-RA showed any signs of a tumor or undesirable toxic effects for the 20 weeks that they were followed after the transplantation. Treatment with SN1-RA plus SN2-RA completely suppressed solid tumor growth in 4 of 10 nude mice carrying solid tumors and partially suppressed the tumor growth in the remaining 6 nude mice. These results strongly suggest that SN1-RA and SN2-RA may be useful for clinical treatment.

  6. A novel bispecific immunotoxin delivered by human bone marrow-derived mesenchymal stem cells to target blood vessels and vasculogenic mimicry of malignant gliomas

    PubMed Central

    Zhang, Yonghong; Sun, Xinlin; Huang, Min; Ke, Yiquan; Wang, Jihui; Liu, Xiao

    2015-01-01

    Background In previous years, immunotoxins have been shown to be a greatly promising therapeutic tool for brain malignancies, such as gliomas. Human mesenchymal stem cells (hMSCs) exhibit tropism to tumor tissue. However, the effect of bispecific immunotoxins in malignant gliomas is still unknown. The aim of this study was to investigate the function of bispecific immunotoxins in human malignant gliomas. Materials and methods In the present study, the bispecific immunotoxin VEGF165-ephrin A1-PE38KDEL was established using deoxyribonucleic acid shuffling and cloning techniques. The VEGF165-ephrin A1-PE38KDEL was delivered by hMSCs to mouse malignant gliomas. The effects of the bispecific immunotoxins on glioma-derived blood vessels and vasculogenic mimicry to elucidate the molecular mechanisms underlying the antitumorigenic effects of immunotoxins were examined in vivo. Results In vitro, transfected hMSCs significantly inhibited the cell viability of gliomas cell lines U87 and U251 in a dose-dependent manner compared with untransfected hMSCs (P<0.01). In vivo, the intratumoral injection of engineered hMSCs was effective at inhibiting tumor growth in a malignant glioma tumor model. Conclusion The bispecific immunotoxin secreted from hMSCs acts as a novel strategy for improving treatment options for malignant gliomas in the clinic. PMID:26089644

  7. Immunotoxins, ligand-toxin conjugates and molecular targeting.

    PubMed

    Soria, M

    1989-01-01

    Biotechnology provides tools for therapeutic exploitation following advances in the elucidation of protein-to-cell and cell-to-cell interactions. Molecular targeting of bacterial and plant toxins to the desired district of action can be achieved through effector molecules like monoclonal antibodies or protein ligands. Biochemical conjugation of these effectors to SO-6, a single-chain Ribosome Inactivating Protein from Saponaria officinalis, yielded powerful cytotoxic agents that are attractive candidates for therapeutic evaluation. Cloning of the gene for this plant toxin has been achieved. Technologies for expression of protein ligands, such as apolipoproteins or several growth factors, are available in recombinant microorganisms, providing adequate partners for the assembly of targeted chimaeras. Domain engineering of structural and functional regions in effector proteins is now possible and will be carried out with the available technologies to improve existing therapy. PMID:2698471

  8. Cell-targeting fusion constructs containing recombinant gelonin.

    PubMed

    Lyu, Mi-Ae; Cao, Yu Joshua; Mohamedali, Khalid A; Rosenblum, Michael G

    2012-01-01

    Therapeutic agents capable of targeting tumor cells present as established tumors and micrometastases have already demonstrated their potential in clinical trials. Immunotoxins targeting hematological malignancies and solid tumors have additionally demonstrated excellent clinical activity. This review focuses on our design and characterization studies of constructs composed of recombinant gelonin toxin fused to either growth factors or single-chain antibodies targeting solid tumor cells, tumor vasculature or hematological malignancies. These agents demonstrate cytotoxicity at nanomolar or sub-nanomolar levels. All of these constructs display impressive selectivity and specificity for antigen-bearing target cells in vitro and in vivo and are excellent clinical trial candidates. PMID:22208986

  9. Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression.

    PubMed

    Bogner, Christian; Dechow, Tobias; Ringshausen, Ingo; Wagner, Michaela; Oelsner, Madlen; Lutzny, Gloria; Licht, Thomas; Peschel, Christian; Pastan, Ira; Kreitman, Robert J; Decker, Thomas

    2010-01-01

    Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti- CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl-2 expression (NCEB-1, JeKo-1 and JVM-2) compared to Granta-519 cells with high Bcl-2 expression. While the expression of the antiapoptotic protein Mcl-1 declined (NCEB-1, Granta-519), Bcl-2 levels remained unchanged in Granta-519 cells. However transfection of BCL2 cDNA into NCEB-1, JeKo-1 and JVM-2 cells significantly reduced BL22-mediated toxicity. Accordingly we examined the effects of Bcl-2 inactivation in Granta-519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta-519 cells with silenced Bcl-2. Our results were confirmed in freshly isolated MCL-cells from patients with leukaemic MCL. We conclude that Bcl-2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells. PMID:19821820

  10. The novel immunotoxin HM1.24-ETA′ induces apoptosis in multiple myeloma cells

    PubMed Central

    Staudinger, M; Glorius, P; Burger, R; Kellner, C; Klausz, K; Günther, A; Repp, R; Klapper, W; Gramatzki, M; Peipp, M

    2014-01-01

    Despite new treatment modalities, the clinical outcome in a substantial number of patients with multiple myeloma (MM) has yet to be improved. Antibody-based targeted therapies for myeloma patients could make use of the HM1.24 antigen (CD317), a surface molecule overexpressed on malignant plasma cells and efficiently internalized. Here, a novel immunotoxin, HM1.24-ETA′, is described. HM1.24-ETA′ was generated by genetic fusion of a CD317-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). HM1.24-ETA′ inhibited growth of interleukin 6 (IL-6)-dependent and -independent myeloma cell lines. Half-maximal growth inhibition was observed at concentrations as low as 0.3 nM. Target cell killing occurred via induction of apoptosis and was unaffected in co-culture experiments with bone marrow stromal cells. HM1.24-ETA′ efficiently triggered apoptosis of freshly isolated/cryopreserved cells of patients with plasma cell leukemia and MM and was active in a preclinical severe combined immunodeficiency (SCID) mouse xenograft model. Importantly, HM1.24-ETA′ was not cytotoxic against CD317-positive cells from healthy tissue (monocytes, human umbilical vein endothelial cells). These results indicate that CD317 may represent a promising target structure for specific and efficient immunotoxin therapy for patients with plasma cell tumors. PMID:24927408

  11. Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation.

    PubMed

    Premsukh, Arjune; Lavoie, Joelle M; Cizeau, Jeannick; Entwistle, Joycelyn; MacDonald, Glen C

    2011-07-01

    VB4-845 is a recombinant immunotoxin comprised of an anti-epithelial cell adhesion molecule (EpCAM) scFv fused to a truncated form of the bacterial toxin, Pseudomonas exotoxin A. VB4-845, purified from TB fed-batch fermentation, showed clinical efficacy when administered locally to treat non-muscle invasive bladder cancer (NMIBC) and squamous cell carcinomas of the head and neck (SCCHN). Here, we describe the implementation of an Escherichia coli high cell density (HCD) cultivation and purification process for VB4-845. HCD cultivation was a prerequisite for achieving higher yields necessary for Phase III clinical trials and commercialization. Using this process, the VB4-845 titer in the supernatant was increased by 30-fold over the original TB fed-batch cultivation. To obtain clinical grade material, a process involving a five-step column purification procedure was implemented and led to an overall recovery of ∼ 40%. VB4-845 purity of >97% was achieved after the first three columns following the removal of low-molecular weight product-related impurities and aggregates. Endotoxins were effectively separated from VB4-845 on the Q-columns and by washing the Ni-column with a detergent buffer while host cell proteins were removed using ceramic hydroxyapatite. Comparability studies demonstrated that the purified product from the Phase III process was identical to the Phase II reference standard produced using TB fed-batch fermentation. PMID:21421055

  12. Diphtheria-toxin based anti-human CCR4 immunotoxin for targeting human CCR4(+) cells in vivo.

    PubMed

    Wang, Zhaohui; Wei, Min; Zhang, Huiping; Chen, Hongyuan; Germana, Sharon; Huang, Christene A; Madsen, Joren C; Sachs, David H; Wang, Zhirui

    2015-08-01

    CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4(+) tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 immunotoxins bound to the human CCR4(+) tumor cell line as well as CCR4(+) human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4(+) tumor cells compared to the monovalent anti-human CCR4 immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4(+) tumor-bearing mouse model. The immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ(-/-) (NSG) mice injected with human CCR4(+) acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 immunotoxin is a promising drug candidate for targeting human CCR4(+) tumor cells and Tregs in vivo. PMID:25958791

  13. 77 FR 5036 - Prospective Grant of Exclusive License: The Development of Human Anti-Mesothelin Monoclonal...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-01

    ... entitled ``Human Monoclonal Antibody Against Mesothelin'' , Australian patent application AU 2009228361 entitled ''Human Monoclonal Antibody Against Mesothelin'' , Canadian patent application CA 2718321 entitled... ``Human Monoclonal Antibody Against Mesothelin'' , U.S. patent application 12/ 934,060 entitled...

  14. Anti-Mesothelin Monoclonal Antibodies for the Treatment of Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute, Laboratory of Molecular Biology is seeking parties interested in collaborative research to further co-develop monoclonal antibodies for the treatment of mesothelin-expressing cancers.

  15. 76 FR 63317 - Prospective Grant of Exclusive License: The Development of Human Anti-Mesothelin Monoclonal...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-12

    ...This is notice, in accordance with 35 U.S.C. 209(c)(1) and 37 CFR part 404.7(a)(1)(i), that the National Institutes of Health, Department of Health and Human Services, is contemplating the grant of an exclusive patent license to practice the inventions embodied in U.S. Patent Application 61/040,005 entitled ``Human Monoclonal Antibodies Specific for Mesothelin'' [HHS Ref. E-079-2008/0-US-01],......

  16. High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries

    PubMed Central

    Hou, Shin-Chen; Chen, Hong-Sen; Lin, Hung-Wei; Chao, Wei-Ting; Chen, Yao-Sheng; Fu, Chi-Yu; Yu, Chung-Ming; Huang, Kai-Fa; Wang, Andrew H.-J.; Yang, An-Suei

    2016-01-01

    Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates. PMID:27550798

  17. High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries.

    PubMed

    Hou, Shin-Chen; Chen, Hong-Sen; Lin, Hung-Wei; Chao, Wei-Ting; Chen, Yao-Sheng; Fu, Chi-Yu; Yu, Chung-Ming; Huang, Kai-Fa; Wang, Andrew H-J; Yang, An-Suei

    2016-01-01

    Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates. PMID:27550798

  18. GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials.

    PubMed

    Woo, Jung Hee; Liu, Jen-Sing; Kang, Soo Hyun; Singh, Ravibhushan; Park, Seong Kyu; Su, Yunpeng; Ortiz, Janelle; Neville, David M; Willingham, Mark C; Frankel, Arthur E

    2008-03-01

    The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical grade bivalent anti-T cell immunotoxin for phase I/II clinical trials, a single batch of 120 L bioreactor culture was performed using the Pichia pastoris mutEF2JC307-8(2) strain expressing the bivalent anti-T cell immunotoxin. After 162 h induction of the culture by methanol, the culture medium was harvested by a 0.1 microm hollow-fiber microfiltration step. The recombinant protein was purified by a 3-step purification procedure (Butyl 650 M capturing step, borate anion exchange step and final Poros anion exchange step). The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Expression level was 207 mg/L of culture supernatant and the final production yield was 69.6% or 144.2mg/L of culture supernatant. The final product was characterized by multiple assays. Vialed product was sterile. The drug concentration was 0.8 mg/mL in 150 mM NaCl, 5% glycerol, 1mM EDTA, and 5mM Tris (pH 8.0). Purity by SDS-PAGE was 98%. Aggregates by Superdex 200 HPLC were <1%. Potency revealed a 20 h IC(50) of 17f M on Jurkat cells. Endotoxin level was 0.02 U/mg. Chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The drug did not react with tested frozen human tissue sections except for T cells. LD(10) in mice was between 500 and 75 0microg/kg. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 1.5 year at -80 degrees C. The scalable synthesis of this protein drug should be useful for production for phase I/II clinical trials and can be applicable for other diphtheria toxin fusion drugs for clinical development. PMID:18160309

  19. Construction of an immunotoxin by linking a monoclonal antibody against the human epidermal growth factor receptor and a hemolytic toxin.

    PubMed

    Avila, Ana D; Calderón, Carlos F; Pérez, Rita M; Pons, Carmen; Pereda, Celia M; Ortiz, Ana R

    2007-01-01

    Hybrid molecules obtained through conjugation of monoclonal antibodies and toxins constitute an approach under exploration to generate potential agents for the treatment of cancer and other diseases. A frequently employed toxic component in the construction of such immunotoxins is ricin, a plant toxin which inhibits protein synthesis at ribosomal level and so requires to be internalized by the cell. A hemolytic toxin isolated from the sea anemone Stichodactyla helianthus, which is active at the cell membrane level, was linked through a disulfide bond to the anti-epidermal growth factor receptor monoclonal antibody ior egf/r3. The resulting immunotoxin did not exhibit hemolytic activity except under reducing conditions. It was toxic for H125 cells that express the human epidermal growth factor receptor, but non-toxic for U1906 cells that do not express this receptor. PMID:18064354

  20. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin.

    PubMed

    Palchaudhuri, Rahul; Saez, Borja; Hoggatt, Jonathan; Schajnovitz, Amir; Sykes, David B; Tate, Tiffany A; Czechowicz, Agnieszka; Kfoury, Youmna; Ruchika, Fnu; Rossi, Derrick J; Verdine, Gregory L; Mansour, Michael K; Scadden, David T

    2016-07-01

    Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45-saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45-SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases. PMID:27272386

  1. Targeted Delivery of Immunotoxin by Antibody to Ganglioside GD3: A Novel Drug Delivery Route for Tumor Cells

    PubMed Central

    Torres Demichelis, Vanina; Vilcaes, Aldo A.; Iglesias-Bartolomé, Ramiro; Ruggiero, Fernando M.; Daniotti, Jose L.

    2013-01-01

    Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1GD3+ cells cultured in attachment-free conditions. A drastic growth inhibition (>80–90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer. PMID:23383146

  2. Cytotoxic effect of the immunotoxin constructed of the ribosome-inactivating protein curcin and the monoclonal antibody against Her2 receptor on tumor cells.

    PubMed

    Jaramillo-Quintero, Lidia Patricia; Contis Montes de Oca, Arturo; Romero Rojas, Andrés; Rojas-Hernández, Saúl; Campos-Rodríguez, Rafael; Martínez-Ayala, Alma Leticia

    2015-01-01

    The toxicity of the curcin on cancer cells allows to consider this protein as the toxic component of an immunotoxin directed to Her2, which is associated with cancer. Reductive amination was proposed to conjugate curcin and an anti-Her2; the binding was tested using Polyacrylamide gel electrophoresis, western blot, and immunocytochemistry. The in vitro cytotoxicity of curcin and the immunotoxin was assessed on breast cancer cell lines SK-BR-3 (Her2(+)) and MDA-MB-231 (Her2(-)). IC50 values for curcin were 15.5 ± 8.3 and 18.6 ± 2.4 μg/mL, respectively, statistically equivalent (p < 0.05). While to the immunotoxin was 2.2 ± 0.08 for SK-BR-3 and 147.6 ± 2.5 μg/mL for MDA-MB-231. These values showed that the immunotoxin was seven times more toxic to the SK-BR-3 than curcin and eight times less toxic to the MDA-MB-231. The immunotoxin composed of curcin and an antibody against Her2 and constructed by reductive amination could be a therapeutic candidate against Her2(+) cancer. PMID:25704287

  3. Facilitation of allogeneic bone marrow transplantation by a T cell-specific immunotoxin containing daunomycin

    SciTech Connect

    Xie, S.S.; Inazawa, M.; Sinha, N.; Sawada, S.; Vergidis, R.; Diener, E.

    1987-12-01

    Daunomycin coupled via an acid-sensitive spacer to monoclonal Thy-1.2-specific antibody was used to purge T lymphocytes from a 1:1 mixture of murine C57BL/6J bone marrow and spleen cells prior to engraftment in fully allogeneic, irradiated BALB/c recipients. Treatment of bone marrow with the immunotoxin at a concentration used for purging had no effect on the viability of committed hematopoietic progenitor or multipotent stem cells. All of the recipients of purged bone marrow were at least 80% chimeric for donor peripheral blood cells and none developed graft-versus-host disease. Out of 50 chimeras, 49 were still alive more than 200 days posttransplantation. The chimeras were shown to be tolerant to donor tissue as tested by mixed lymphocyte reactivity, cell-mediated cytotoxicity, and skin grafting. The same tests revealed full immunocompetence of chimeras to third-party alloantigens. In vivo IgM and IgG antibody responses to sheep red blood cells were similar in magnitude in allogeneically and syngeneically reconstituted mice.

  4. Role of monensin PLGA polymer nanoparticles and liposomes as potentiator of ricin A immunotoxins in vitro.

    PubMed

    Ferdous, A J; Stembridge, N Y; Singh, M

    1998-01-01

    Monensin is a carboxylic ionophore which can potentiate the activity of ricin based immunotoxins (IT) in vitro and in vivo against a variety of human tumours. Monensin was encapsulated into nanoparticles (NP) by using biodegradable poly(DL-lactide-co-glycolide) (PLGA, 50:50). The NP were prepared by modified emulsification-solvent evaporation method. High shear homogenization followed by simultaneous stirring and bath sonication were used for preparing NP. The size of NP was determined by photon correlation spectroscopy using a BI 90 particle sizer (Brookhaven Instruments). The average size of NP could be decreased from 567 nm to 163 nm by increasing the concentration of polyvinyl alcohol from 10% to 100% of PLGA. The NP were spherical in shape as observed by Atomic Force Microscopy. The concentration of monensin in the NP was analyzed by HPLC and the entrapment efficiency was found to be more than 12%. The zeta potential of NP was -25.8 (+/- 1.3) mv, which did not change significantly after resuspension of the freeze dried sample. The NP were tested against HL-60 and HT-29 human tumour cell lines in vitro. Monensin NP potentiated the activity of IT by 40 to 50 times against these cell lines. There was however, no difference between the NP and liposomes for their potentiating affect of IT against the two tumour cell lines. PMID:9685874

  5. λ Recombination and Recombineering.

    PubMed

    Murphy, Kenan C

    2016-05-01

    The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821

  6. Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins.

    PubMed

    Goyal, A; Batra, J K

    2000-01-15

    Chimaeric toxins have considerable therapeutic potential to treat various malignancies. We have previously used the fungal ribonucleolytic toxin restrictocin to make chimaeric toxins in which the ligand was fused at either the N-terminus or the C-terminus of the toxin. Chimaeric toxins containing ligand at the C-terminus of restrictocin were shown to be more active than those having ligand at the N-terminus of the toxin. Here we describe the further engineering of restrictocin-based chimaeric toxins, anti-TFR(scFv)-restrictocin and restrictocin-anti-TFR(scFv), containing restrictocin and a single chain fragment variable (scFv) of a monoclonal antibody directed at the human transferrin receptor (TFR), to enhance their cell-killing activity. To promote the independent folding of the two proteins in the chimaeric toxin, a linear flexible peptide, Gly-Gly-Gly-Gly-Ser, was inserted between the toxin and the ligand to generate restrictocin-linker-anti-TFR(scFv) and anti-TFR(scFv)-linker-restrictocin. A 12-residue spacer, Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu, containing the recognition site for the protease furin, was incorporated between the toxin and the ligand to generate restrictocin-spacer-anti-TFR(scFv) and anti-TFR(scFv)-spacer-restrictocin. The incorporation of the proteolytically cleavable spacer enhanced the cell-killing activity of both constructs by 2-30-fold depending on the target cell line. However, the introduction of linker improved the cytotoxic activity only for anti-TFR(scFv)-linker-restrictocin. The proteolytically cleavable spacer-containing chimaeric toxins had similar cytotoxic activities irrespective of the location of the ligand on the toxin and they were found to release the restrictocin fragment efficiently on proteolysis in vitro. PMID:10620501

  7. Effectiveness of HB2 (anti-CD7)--saporin immunotoxin in an in vivo model of human T-cell leukaemia developed in severe combined immunodeficient mice.

    PubMed Central

    Morland, B. J.; Barley, J.; Boehm, D.; Flavell, S. U.; Ghaleb, N.; Kohler, J. A.; Okayama, K.; Wilkins, B.; Flavell, D. J.

    1994-01-01

    The transplantation of the human T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2 into severe combined immunodeficient (SCID) mice was found to produce a disseminated pattern of leukaemia similar to that seen in man. The intravenous injection of 10(7) HSB-2 cells was associated with a universally fatal leukaemia. Histopathological examination of animals revealed the spread of leukaemia initially from bone marrow to involve all major organs including the meninges. An immunotoxin (HB2-Sap) was constructed by conjugating the anti-CD7 MAb HB2 to the ribosome-inactivating protein saporin. An in vitro protein synthesis inhibition assay revealed specific delivery of HB2-Sap immunotoxin (IT) to CD7+ HSB-2 target cells with an IC50 of 4.5 pM. When SCID mice were injected with 10(6) HSB-2 cells and then treated 8 days later with a single intravenous dose of 10 micrograms of immunotoxin there was a significant therapeutic effect evidenced by the numbers of animals surviving in the therapy group compared with untreated controls (chi 2 = 5.348, P = 0.021). These results demonstrate the useful application of human leukaemia xenografts in SCID mice and the potential therapeutic effect of an anti-CD7 immunotoxin in human T-ALL. Images Figure 1 PMID:7507691

  8. A multi-antigen vaccine in combination with an immunotoxin targeting tumor-associated fibroblast for treating murine melanoma

    PubMed Central

    Fang, Jinxu; Hu, Biliang; Li, Si; Zhang, Chupei; Liu, Yarong; Wang, Pin

    2016-01-01

    A therapeutically effective cancer vaccine must generate potent antitumor immune responses and be able to overcome tolerance mechanisms mediated by the progressing tumor itself. Previous studies showed that glycoprotein 100 (gp100), tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) are promising immunogens for melanoma immunotherapy. In this study, we administered these three melanoma-associated antigens via lentiviral vectors (termed LV-3Ag) and found that this multi-antigen vaccine strategy markedly increased functional T-cell infiltration into tumors and generated protective and therapeutic antitumor immunity. We also engineered a novel immunotoxin, αFAP-PE38, capable of targeting fibroblast activation protein (FAP)-expressing fibroblasts within the tumor stroma. When combined with αFAP-PE38, LV-3Ag exhibited greatly enhanced antitumor effects on tumor growth in an established B16 melanoma model. The mechanism of action underlying this combination treatment likely modulates the immune suppressive tumor microenvironment and, consequently, activates cytotoxic CD8+ T cells capable of specifically recognizing and destroying tumor cells. Taken together, these results provide a strong rationale for combining an immunotoxin with cancer vaccines for the treatment of patients with advanced cancer. PMID:27119119

  9. Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy

    PubMed Central

    Gilabert-Oriol, Roger; Weng, Alexander; von Mallinckrodt, Benedicta; Melzig, Matthias F; Fuchs, Hendrik; Thakur, Mayank

    2014-01-01

    The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success. PMID:25341935

  10. The Development and Characterization of a Human Mesothelioma In Vitro 3D Model to Investigate Immunotoxin Therapy

    PubMed Central

    Feng, Mingqian; Nagashima, Kunio; Zhang, Jingli; Broaddus, V. Courtney; Hassan, Raffit; FitzGerald, David; Ho, Mitchell

    2011-01-01

    Background Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates. Tumor microenvironments, however, are difficult to study in vitro. Cells cultured as monolayers exhibit less resistance to therapy than those grown in vivo and an alternative research model more representative of the in vivo tumor is more desirable. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing clinical trials in mesothelioma. Methodology/Principal Findings Here, we examined how the tumor microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D) spheroid model cultured in vitro using the human mesothelioma cell line (NCI-H226) and two primary cell lines isolated from the ascites of malignant mesothelioma patients. Mesothelioma cells grown as monolayers or as spheroids expressed comparable levels of mesothelin; however, spheroids were at least 100 times less affected by SS1P. To understand this disparity in cytotoxicity, we made fluorescence-labeled SS1P molecules and used confocal microscopy to examine the time course of SS1P penetration within spheroids. The penetration was limited after 4 hours. Interestingly, we found a significant increase in the number of tight junctions in the core area of spheroids by electron microscopy. Expression of E-Cadherin, a protein involved in the assembly and sealing of tight junctions and highly expressed in malignant mesothelioma, was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting E-Cadherin could enhance SS1P immunotoxin therapy in vitro. Conclusion/Significance This work is one of the first to investigate immunotoxins in 3D tumor spheroids in vitro. This initial description of an in vitro tumor model may offer a simple and more representative model of in vivo tumors and will allow for

  11. Use of ribosome-inactivating proteins from Sambucus for the construction of immunotoxins and conjugates for cancer therapy.

    PubMed

    Ferreras, José M; Citores, Lucía; Iglesias, Rosario; Jiménez, Pilar; Girbés, Tomás

    2011-05-01

    The type 2 ribosome-inactivating proteins (RIPs) isolated from some species belonging to the Sambucus genus, have the characteristic that although being even more active than ricin inhibiting protein synthesis in cell-free extracts, they lack the high toxicity of ricin and related type 2 RIPs to intact cells and animals. This is due to the fact that after internalization, they follow a different intracellular pathway that does not allow them to reach the cytosolic ribosomes. The lack of toxicity of type 2 RIPs from Sambucus make them good candidates as toxic moieties in the construction of immunotoxins and conjugates directed against specific targets. Up to now they have been conjugated with either transferrin or anti-CD105 to target either transferrin receptor- or endoglin-overexpressing cells, respectively. PMID:22069717

  12. Purification and characterization of a novel type i ribosome inactivating protein, pachyerosin, from Pachyrhizus erosus seeds, and preparation of its immunotoxin against human hepatoma cells.

    PubMed

    Guo, Jin-Lin; Cheng, Yuan-Liu; Qiu, Yi; Shen, Cai-Hong; Yi, Bin; Peng, Cheng

    2014-07-01

    Pachyrhizus erosus seeds have a high protein content and are used in China due to their cytotoxic effect. Here we report the biological and pharmacological activity of the protein extracts from P. erosus seeds. A novel ribosome-inactivating protein, pachyerosin, from P. erosus seeds was successively purified to homogeneity using ammonium sulfate precipitation, DEAE-sepharose FF, and Sephacryl S-200. Pachyerosin showed to be a type I ribosome-inactivating protein with a molecular mass of 29 kDa and an isoelectric point of 9.19. It strongly inhibited protein synthesis of rabbit reticulocyte lysate with an IC50 of 0.37 ng/mL and showed N-glycosidase activity on rat liver ribosomes with an EC50 of 85.9 pM. The N-terminal 27 amino acids of pachyerosin revealed a 60.71% sequence identity with abrin A from the seeds of Abrus precatorius. With the aim of targeting the delivery of pachyerosin, immunotoxin was prepared by conjugating pachyerosin with anti-human AFP monoclonal antibodies SM0736. The immunotoxin pachyerosin-SM0736 efficiently inhibited the growth of the human hepatoma cell line HuH-7 with an IC50 of 0.050 ± 0.004 nM, 2360 times lower than that of pachyerosin and 430 times lower than that of the immunotoxin against human gastric cancer cell line SGC7901. These results imply that pachyerosin may be used as a new promising anticancer agent. PMID:25029173

  13. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  14. Cosmological Recombination

    NASA Astrophysics Data System (ADS)

    Wong, Wan Yan

    2008-11-01

    In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

  15. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  16. Constitutive expression and anticancer potency of a novel immunotoxin onconase-DV3.

    PubMed

    Sun, Miaonan; Tang, Huichun; Gao, Yan; Dai, Xinxuan; Yuan, Yue; Zhang, Chunmei; Sun, Dejun

    2016-04-01

    Onconase is an RNase of the ribonuclease A superfamily that is purified from the Northern leopard frog (Rana pipiens). It targets several types of malignant tumors, digests cytoplasmic transfer RNA (tRNA), and causes tumor cell apoptosis. Onconase has been employed in clinical trials as an antitumor drug, and has revealed its valuable clinical activity in several types of tumors, particularly pleural mesothelioma. However, its inefficiency in targeting tumor cells and its non‑specific toxicity in normal tissues have diminished its clinical benefits. Furthermore, cyclization of the N-terminal glutamine residue (Gln), possesses more RNase activity than the structure of Met ahead of Glu in the N-terminal (99:1), which is more difficult for producing onconase by Pichia pastoris. Under the guidance of α-mating factor-pre (α-MF-pre) secretion signal, the secretion of the recombinant protein can reach a high level. In the present study, we constructed a constitutive expression vector for onconase-(DV3)2 (Onc-DV3) production in yeast Pichia pastoris with the GAP promoter, in which the Onc-DV3 gene is inserted downstream of the truncated Saccharomyces cerevisiae α-mating factor-pre (α-MF-pre) secretion signal. The immuno-RNase Onc-DV3 expressed a high level of production and bioactivity and possessed enhanced capability to deliver the Onc molecule to tumor cell monomeric counterparts. Notably, Onc-DV3 showed strong cytotoxicity to highly metastatic tumor cells, weak cytotoxicity to lowly metastatic tumor cells and no toxicity to normal cells. These results demonstrate that the specific toxicity to highly metastatic tumor cells has made Onc-DV3 a promising antitumor drug by using two copies of DV3 for the targeted delivery of onconase. PMID:26782924

  17. Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis.

    PubMed

    Bolognesi, A; Barbieri, L; Carnicelli, D; Abbondanza, A; Cenini, P; Falasca, A I; Dinota, A; Stirpe, F

    1989-12-01

    A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells. PMID:2597699

  18. Phase II clinical trial of amatuximab, a chimeric anti-mesothelin antibody with pemetrexed and cisplatin in advanced unresectable pleural mesothelioma

    PubMed Central

    Hassan, Raffit; Kindler, Hedy L.; Jahan, Thierry; Bazhenova, Lyudmila; Reck, Martin; Thomas, Anish; Pastan, Ira; Parno, Jeff; O’Shannessy, Daniel J.; Fatato, Penny; Maltzman, Julia D.; Wallin, Bruce A.

    2014-01-01

    Purpose Amatuximab is a chimeric monoclonal antibody to mesothelin, a cell surface glycoprotein highly expressed in malignant pleural mesothelioma (MPM). Based on its synergy with chemotherapy in pre-clinical studies, we evaluated the antitumor activity of amatuximab plus pemetrexed and cisplatin in patients with unresectable MPM. Experimental Design In a single-arm phase II study, amatuximab 5 mg/kg was administered on days 1 and 8 with pemetrexed, 500 mg/m2 and cisplatin, 75 mg/m2 on day 1 of 21-day cycles for up to 6 cycles. Patients with response or stable disease received amatuximab maintenance until disease progression. Primary endpoint was progression-free survival (PFS) at 6 months. Secondary endpoints were overall survival (OS), response rate and safety. Results Eighty nine patients were enrolled at 26 centers. Median of five cycles (range 1–6) of combination treatment was administered and 56 (63%) patients received amatuximab maintenance. Combination therapy resulted in no overlapping toxicities. Eleven patients (12.4%) had amatuximab-related hypersensitivity reactions. Responses included partial responses in 33 (40%) and stable disease in 42 (51%). Six month-PFS rate was 51% (95% CI: 39.1, 62.3), median PFS 6.1 months (95% CI: 5.8, 6.4) and median OS 14.8 months (95% CI: 12.4, 18.5) with 29 patients alive at data cut-off. Conclusions Amatuximab with pemetrexed and cisplatin was well-tolerated with objective tumor response or stable disease rate of 90% by independent radiological review. Although PFS was not significantly different from historical controls, the median OS was 14.8 months with a third of patients alive and 5 continuing to receive amatuximab at the time of analysis. PMID:25231400

  19. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  20. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  1. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  2. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  3. Combination of the PI3K inhibitor ZSTK474 with a PSMA-targeted immunotoxin accelerates apoptosis and regression of prostate cancer.

    PubMed

    Baiz, Daniele; Hassan, Sazzad; Choi, Young A; Flores, Anabel; Karpova, Yelena; Yancey, Dana; Pullikuth, Ashok; Sui, Guangchao; Sadelain, Michel; Debinski, Waldemar; Kulik, George

    2013-10-01

    The phosphoinositide 3-kinase (PI3K) pathway is activated in most advanced prostate cancers, yet so far treatments with PI3K inhibitors have been at best tumorostatic in preclinical cancer models and do not show significant antitumor efficacy in clinical trials. Results from tissue culture experiments in prostate cancer cells suggest that PI3K inhibitors should be combined with other cytotoxic agents; however, the general toxicity of such combinations prevents translating these experimental data into preclinical and clinical models. We investigated the emerging concept of tumor-targeted synthetic lethality in prostate cancer cells by using the pan-PI3K inhibitor ZSTK474 and the immunotoxin J591PE, a protein chimera between the single-chain variable fragment of the monoclonal antibody J591 against the prostate-specific membrane antigen (PSMA) and the truncated form of the Pseudomonas aeruginosa exotoxin A (PE38QQR). The combination of ZSTK474 and J591PE increased apoptosis within 6 hours and cell death (monitored at 24-48 hours) in the PSMA-expressing cells LNCaP, C4-2, and C4-2Luc but not in control cells that do not express PSMA (PC3 and BT549 cells). Mechanistic analysis suggested that induction of apoptosis requires Bcl-2-associated death promoter (BAD) dephosphorylation and decreased expression of myeloid leukemia cell differentiation protein 1 (MCL-1). A single injection of ZSTK474 and J591PE into engrafted prostate cancer C4-2Luc cells led to consistent and stable reduction of luminescence within 6 days. These results suggest that the combination of a PI3K inhibitor and a PSMA-targeted protein synthesis inhibitor toxin represents a promising novel strategy for advanced prostate cancer therapy that should be further investigated. PMID:24204196

  4. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  5. Recombination in electron coolers

    NASA Astrophysics Data System (ADS)

    Wolf, A.; Gwinner, G.; Linkemann, J.; Saghiri, A. A.; Schmitt, M.; Schwalm, D.; Grieser, M.; Beutelspacher, M.; Bartsch, T.; Brandau, C.; Hoffknecht, A.; Müller, A.; Schippers, S.; Uwira, O.; Savin, D. W.

    2000-02-01

    An introduction to electron-ion recombination processes is given and recent measurements are described as examples, focusing on low collision energies. Discussed in particular are fine-structure-mediated dielectronic recombination of fluorine-like ions, the moderate recombination enhancement by factors of typically 1.5-4 found for most ion species at relative electron-ion energies below about 10 meV, and the much larger enhancement occurring for specific highly charged ions of complex electronic structure, apparently caused by low-energy dielectronic recombination resonances. Recent experiments revealing dielectronic resonances with very large natural width are also described.

  6. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  7. Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin by photochemical internalization - A minimally invasive cancer stem cell-targeting strategy.

    PubMed

    Bostad, Monica; Olsen, Cathrine Elisabeth; Peng, Qian; Berg, Kristian; Høgset, Anders; Selbo, Pål Kristian

    2015-05-28

    The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin (PCIAC133-saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133-saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133-saporin. PCI of picomolar levels of AC133-saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133-saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133-saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133-saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133-saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133-saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133+ cells in localized primary tumors or metastasis. PMID:25758331

  8. Preclinical toxicity evaluation of a novel immunotoxin, D2C7-(scdsFv)-PE38KDEL, administered via intracerebral convection-enhanced delivery in rats.

    PubMed

    Bao, Xuhui; Chandramohan, Vidyalakshmi; Reynolds, Randall P; Norton, John N; Wetsel, William C; Rodriguiz, Ramona M; Aryal, Dipendra K; McLendon, Roger E; Levin, Edward D; Petry, Neil A; Zalutsky, Michael R; Burnett, Bruce K; Kuan, Chien-Tsun; Pastan, Ira H; Bigner, Darell D

    2016-04-01

    D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel immunotoxin that reacts with wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFRvIII proteins overexpressed in glioblastomas. This study assessed the toxicity of intracerebral administration of D2C7-IT to support an initial Food and Drug Administration Investigational New Drug application. After the optimization of the formulation and administration, two cohorts (an acute and chronic cohort necropsied on study days 5 and 34) of Sprague-Dawley (SD) rats (four groups of 5 males and 5 females) were infused with the D2C7-IT formulation at total doses of 0, 0.05, 0.1, 0.4 μg (the acute cohort) and 0, 0.05, 0.1, 0.35 μg (the chronic cohort) for approximately 72 h by intracerebral convection-enhanced delivery using osmotic pumps. Mortality was observed in the 0.40 μg (5/10 rats) and 0.35 μg (4/10 rats) high-dose groups of each cohort. Body weight loss and abnormal behavior were only revealed in the rats treated with high doses of D2C7-IT. No dose-related effects were observed in clinical laboratory tests in either cohort. A gross pathologic examination of systemic tissues from the high-dose and control groups in both cohorts exhibited no dose-related or drug-related pathologic findings. Brain histopathology revealed the frequent occurrence of dose-related encephalomalacia, edema, and demyelination in the high-dose groups of both cohorts. In this study, the maximum tolerated dose of D2C7-IT was determined to be between 0.10 and 0.35 μg, and the no-observed-adverse-effect-level was 0.05 μg in SD rats. Both parameters were utilized to design the Phase I/II D2C7-IT clinical trial. PMID:26728879

  9. [Recombinant antibodies against bioweapons].

    PubMed

    Thullier, Philippe; Pelat, Thibaut; Vidal, Dominique

    2009-12-01

    The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies. PMID:20035695

  10. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  11. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  12. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  13. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  14. Recombineering Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  15. Recombinant renewable polyclonal antibodies

    PubMed Central

    Ferrara, Fortunato; D’Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew RM

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. PMID:25530082

  16. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  17. Oligonucleotide recombination in bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Today, there are more than 1,500 completed or draft bacterial genome sequences available for public access. To functionally analyze these genomes and to test the hypotheses that are generated from the sequence information we require new and generically useful tools. Recombineering (genetic engineer...

  18. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  19. Recombinant vaccines against leptospirosis.

    PubMed

    Dellagostin, Odir A; Grassmann, André A; Hartwig, Daiane D; Félix, Samuel R; da Silva, Éverton F; McBride, Alan J A

    2011-11-01

    Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine. PMID:22048111

  20. Recombinant influenza vaccines.

    PubMed

    Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L

    2012-10-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377

  1. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  2. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  3. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    PubMed

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528

  4. Dielectronic recombination theory

    SciTech Connect

    LaGattuta, K.J.

    1991-12-31

    A theory now in wide use for the calculation of dielectronic recombination cross sections ({sigma}{sup DR}) and rate coefficients ({alpha}{sup DR}) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of {sigma}{sup DR} have been described by Fano and by Seaton. We will not consider those theories here. Calculations of {alpha}{sup DR} have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of {sigma}{sup DR}. While the measurements of {sigma}{sup DR} for {delta}n {ne} 0 excitations have tended to agree very well with calculations, the case of {delta}n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.

  5. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  6. Did the universe recombine?

    NASA Technical Reports Server (NTRS)

    Bartlett, James G.; Stebbins, Albert

    1991-01-01

    The Zel'dovich-Sunyaev model-independent arguments for the existence of a neutral hydrogen phase is reviewed in light of new limits on the Compton y parameter from COBE. It is concluded that with baryon densities compatible with standard cosmological nucleosynthesis, the universe could have remained fully ionized throughout its history without producing a detectable spectral distortion. It is argued that it is unlikely that spectral observations of the cosmic microwave background will ever require the universe to have recombined for flat cosmologies.

  7. Recombinant factor VIIa.

    PubMed

    Aitken, Michael G

    2004-01-01

    Human coagulation factor (F) VII is a single chain protease that circulates in the blood as a weakly active zymogen at concentrations of approximately 10 nmol/L. When converted to the active 2 chain form (FVIIa), it is a powerful initiator of haemostasis. Recombinant factor VIIa (rFVIIa, eptacog alfa, NovoSeven) is a genetically engineered product that was first introduced in 1988 for the treatment of patients with haemophilia A and B with high inhibitory antibody titres to factors VIII and IX. Recent reports in the form of case studies and series, and early trial data, have suggested a role for rFVIIa across a diverse range of indications including bleeding associated with trauma, surgery, thrombocytopaenia, liver disease and oral anticoagulant toxicity. This review describes the physiology of the coagulation pathway and in particular the role of recombinant factor VIIa. It will also focus on the emerging role of rFVIIa in both trauma and non-trauma bleeding and its potential use in the ED. PMID:15537408

  8. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  9. Unraveling recombination rate evolution using ancestral recombination maps

    PubMed Central

    Munch, Kasper; Schierup, Mikkel H; Mailund, Thomas

    2014-01-01

    Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it. PMID:25043668

  10. Algebraic theory of recombination spaces.

    PubMed

    Stadler, P F; Wagner, G P

    1997-01-01

    A new mathematical representation is proposed for the configuration space structure induced by recombination, which we call "P-structure." It consists of a mapping of pairs of objects to the power set of all objects in the search space. The mapping assigns to each pair of parental "genotypes" the set of all recombinant genotypes obtainable from the parental ones. It is shown that this construction allows a Fourier decomposition of fitness landscapes into a superposition of "elementary landscapes." This decomposition is analogous to the Fourier decomposition of fitness landscapes on mutation spaces. The elementary landscapes are obtained as eigenfunctions of a Laplacian operator defined for P-structures. For binary string recombination, the elementary landscapes are exactly the p-spin functions (Walsh functions), that is, the same as the elementary landscapes of the string point mutation spaces (i.e., the hypercube). This supports the notion of a strong homomorphism between string mutation and recombination spaces. However, the effective nearest neighbor correlations on these elementary landscapes differ between mutation and recombination and among different recombination operators. On average, the nearest neighbor correlation is higher for one-point recombination than for uniform recombination. For one-point recombination, the correlations are higher for elementary landscapes with fewer interacting sites as well as for sites that have closer linkage, confirming the qualitative predictions of the Schema Theorem. We conclude that the algebraic approach to fitness landscape analysis can be extended to recombination spaces and provides an effective way to analyze the relative hardness of a landscape for a given recombination operator. PMID:10021760

  11. Recombinant Human Erythropoietin

    PubMed Central

    Bartels, Claudia; Späte, Kira; Krampe, Henning

    2008-01-01

    Treatment of multiple sclerosis (MS) is still unsatisfactory and essentially non-existing for the progressive course of the disease. Recombinant human erythropoietin (EPO) may be a promising neuroprotective/neuroregenerative treatment of MS. In the nervous system, EPO acts anti-apoptotic, antioxidative, anti-inflammatory, neurotrophic and plasticity-modulating. Beneficial effects have been shown in animal models of various neurological and psychiatric diseases, including different models of experimental autoimmune encephalomyelitis. EPO is also effective in human brain disease, as shown in double-blind placebo-controlled clinical studies on ischemic stroke and chronic schizophrenia. An exploratory study on chronic progressive MS yielded lasting improvement in motor and cognitive performance upon high-dose long-term EPO treatment. PMID:21180577

  12. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  13. The recombination of genetic material

    SciTech Connect

    Low, K.B.

    1988-01-01

    Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.

  14. Coalescent Simulation of Intracodon Recombination

    PubMed Central

    Arenas, Miguel; Posada, David

    2010-01-01

    The coalescent with recombination is a very useful tool in molecular population genetics. Under this framework, genealogies often represent the evolution of the substitution unit, and because of this, the few coalescent algorithms implemented for the simulation of coding sequences force recombination to occur only between codons. However, it is clear that recombination is expected to occur most often within codons. Here we have developed an algorithm that can evolve coding sequences under an ancestral recombination graph that represents the genealogies at each nucleotide site, thereby allowing for intracodon recombination. The algorithm is a modification of Hudson's coalescent in which, in addition to keeping track of events occurring in the ancestral material that reaches the sample, we need to keep track of events occurring in ancestral material that does not reach the sample but that is produced by intracodon recombination. We are able to show that at typical substitution rates the number of nonsynonymous changes induced by intracodon recombination is small and that intracodon recombination does not generally result in inflated estimates of the overall nonsynonymous/synonymous substitution ratio (ω). On the other hand, recombination can bias the estimation of ω at particular codons, resulting in apparent rate variation among sites and in the spurious identification of positively selected sites. Importantly, in this case, allowing for variable synonymous rates across sites greatly reduces the false-positive rate and recovers statistical power. Finally, coalescent simulations with intracodon recombination could be used to better represent the evolution of nuclear coding genes or fast-evolving pathogens such as HIV-1.We have implemented this algorithm in a computer program called NetRecodon, freely available at http://darwin.uvigo.es. PMID:19933876

  15. Delayed recombination and standard rulers

    SciTech Connect

    De Bernardis, Francesco; Melchiorri, Alessandro; Bean, Rachel; Galli, Silvia; Silk, Joseph I.; Verde, Licia

    2009-02-15

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  16. A Phase I study of an intravesically administered immunotoxin targeting EpCAM for the treatment of nonmuscle-invasive bladder cancer in BCGrefractory and BCG-intolerant patients

    PubMed Central

    Kowalski, Mark; Entwistle, Joycelyn; Cizeau, Jeannick; Niforos, Demi; Loewen, Shauna; Chapman, Wendy; MacDonald, Glen C

    2010-01-01

    Purpose A Phase I dose-escalation study was performed to determine the maximum tolerated dose (MTD) of the immunotoxin VB4-845 in patients with nonmuscle-invasive bladder cancer (NMIBC) refractory to or intolerant of bacillus Calmette–Guerin (BCG). Secondary objectives included evaluation of the safety, tolerability, pharmacokinetics, immunogenicity, and efficacy of VB4-845. Patients and methods Sixty-four patients with Grade 2 or 3, stage Ta or T1 transitional cell carcinoma or in situ carcinoma, either refractory to or intolerant of BCG therapy, were enrolled. Treatment was administered in ascending dose cohorts ranging from 0.1 to 30.16 mg. After receiving weekly instillations of VB4-845 to the bladder via catheter for 6 consecutive weeks, patients were followed for 4–6 weeks post-therapy and assessed at week 12. Results An MTD was not determined, as a dose-limiting toxicity was not identified over the dose range tested. VB4-845 therapy was safe and well tolerated with most adverse events reported as mild; as a result, no patients were removed from the study in response to toxicity. By the end of the study, the majority of patients had developed antibodies to the exotoxin portion of VB4-845. A complete response was achieved in 39% of patients at the 12-week time point. Conclusions VB4-845 dosed on a weekly basis for 6 weeks was very well tolerated at all dose levels. Although an MTD was not determined at the doses administered, VB4-845 showed evidence of an antitumor effect that warrants further clinical investigation for the treatment of NMIBC in this patient population. PMID:21151619

  17. Expression and characterization of recombinant human eosinophil-derived neurotoxin and eosinophil-derived neurotoxin-anti-transferrin receptor sFv.

    PubMed

    Newton, D L; Nicholls, P J; Rybak, S M; Youle, R J

    1994-10-28

    The gene for the human recombinant eosinophil-derived neurotoxin (rEDN) was synthesized and fused to the gene encoding a single chain antibody (sFv) to the human transferrin receptor (EDNsFv). Both rEDN and EDNsFv were expressed as insoluble proteins in inclusion bodies in Escherichia coli BL21(DE3). Following denaturation and renaturation, EDN and EDNsFv were partially purified by chromatography on heparin-Sepharose. Final purification of EDN was achieved by Sephadex G-100, whereas EDNsFv which contained a 6-histidyl residue carboxyl terminus was highly purified using the metal chelate resin, Ni(2+)-nitriloacetic acid. Whereas the recombinant EDN had ribonuclease activity that was similar to the native protein, the fusion protein had enzymatic activity that was 6-13% that of native EDN. The fusion protein was able to bind to the human transferrin receptor. In contrast to rEDN that had no inherent cytotoxicity to human tumor cells, the EDNsFv fusion protein was cytotoxic to human leukemia cells that express the human transferrin receptor with an IC50, 0.2-1 nM. At 1.3 nM EDNsFv, no cytotoxicity was observed on cells that lack the human transferrin receptor. Free antibody to the human transferrin receptor, E6, inhibited the cytotoxicity of the EDNsFv. Human enzymes may be engineered to acquire cytotoxic properties by fusing them to antibodies. Thus, they may be candidates for the construction of immunofusion proteins that may be less immunogenic than immunotoxins containing bacterial- or plant-derived toxin moieties. PMID:7929408

  18. Controlled Release from Recombinant Polymers

    PubMed Central

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  19. Recombinant DNA means and method

    SciTech Connect

    Alford, B.L.; Mao, J.I.; Moir, D.T.; Taunton-Rigby, A.; Vovis, G.F.

    1987-05-19

    This patent describes a transformed living cell selected from the group consisting of fungi, yeast and bacteria, and containing genetic material derived from recombinant DNA material and coding for bovine rennin.

  20. Three Decades of Recombinant DNA.

    ERIC Educational Resources Information Center

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  1. Recombination device for storage batteries

    DOEpatents

    Kraft, H.; Ledjeff, K.

    1984-01-01

    A recombination device including a gas-tight enclosure connected to receive the discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  2. Recombination device for storage batteries

    DOEpatents

    Kraft, Helmut; Ledjeff, Konstantin

    1985-01-01

    A recombination device including a gas-tight enclosure connected to receive he discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  3. Stable recombination hotspots in birds.

    PubMed

    Singhal, Sonal; Leffler, Ellen M; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M; Strand, Alva I; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N; Griffith, Simon C; McVean, Gil; Przeworski, Molly

    2015-11-20

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but it appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking the gene that encodes PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species: the zebra finch, Taeniopygia guttata, and the long-tailed finch, Poephila acuticauda. We found that both species have recombination hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, most hotspots are shared between the two species, and their conservation seems to extend over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757

  4. [Antithrombotic recombinant antibodies].

    PubMed

    Muzard, Julien; Loyau, Stéphane; Ajzenberg, Nadine; Billiald, Philippe; Jandrot-Perrus, Martine

    2006-01-01

    Coronary syndromes, stroke and other ischaemic arterial diseases are the leading cause of death in the world and will probably remain it at least until 2020. Cardiovascular diseases kill 17 million people each year with an expected increase to 20 million in 2020 and 24 million in 2030. The global impact of recurrence and death during the 6 months following an acute coronary syndrome remains at 8-15% in the present state of medical practice. Acute ischaemic syndromes have a common aetiology that is the formation of a platelet-rich clot at the site of severe coronary stenosis and of eroded atherosclerotic plaques. Therapy consists of medical treatments associating thrombolysis, antiplatelet drugs, and the re-opening of the coronary artery by angioplasty. But these treatments do not prevent morbidity and mortality reaching 15% at 6 months. Finally the treatment of stroke is very limited. There is thus a real clinical need to improve existing treatments and to discover new molecules. Platelet activation is a critical step in ischaemic cardiovascular diseases. This is the reason why antiplatelet drugs are most often prescribed in these cases. Currently, only one recombinant antithrombotic antibody is used in therapy. This is a chimeric Fab, c7E3 or abciximab, which inhibits the final phase of platelet aggregation. Abciximab is prescribed in acute coronary syndromes treated by angioplasty. However, treatment by abciximab can induce severe complications, principally, hemorrages and thrombopenia. Other platelet receptors involved in the earlier steps of platelet activation, such as the phases of contact with and of activation by the subendothelium matrix, have been identified as potential targets for the development of antithrombotic antibodies and are described in this revue. PMID:17652972

  5. Genetic recombination in Streptomyces griseus.

    PubMed Central

    Parag, Y

    1978-01-01

    Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described. PMID:415037

  6. [Vaccine application of recombinant herpesviruses].

    PubMed

    Yokoyama, N; Xuan, X; Mikami, T

    2000-04-01

    Recently, genetic engineering using recombinant DNA techniques has been applied to design new viral vaccines in order to reduce some problems which the present viral vaccines have. Up to now, many viruses have been investigated for development of recombinant attenuated vaccines or live viral vectors for delivery of foreign genes coding immunogenic antigens. In this article, we introduced the new vaccine strategy using genetically engineered herpesviruses. PMID:10774221

  7. Combinatorics in Recombinational Population Genomics

    NASA Astrophysics Data System (ADS)

    Parida, Laxmi

    The work that I will discuss is motivated by the need for understanding, and processing, the manifestations of recombination events in chromosome sequences. In this talk, we focus on two related problems. First, we explore the very general problem of reconstructability of pedigree history. How plausible is it to unravel the history of a complete unit (chromosome) of inheritance? The second problem deals with reconstructing the recombinational history of a collection of chromosomes.

  8. Ethanol production by recombinant hosts

    DOEpatents

    Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

    1995-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  9. Ethanol production by recombinant hosts

    DOEpatents

    Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  10. Delayed recombination and cosmic parameters

    NASA Astrophysics Data System (ADS)

    Galli, Silvia; Bean, Rachel; Melchiorri, Alessandro; Silk, Joseph

    2008-09-01

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, ns, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z*=1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: γα<0.39 and γi<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  11. Delayed recombination and cosmic parameters

    SciTech Connect

    Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

    2008-09-15

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n{sub s}, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z{sub *}=1078{+-}11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1{sigma} to R=1.734{+-}0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: {epsilon}{sub {alpha}}<0.39 and {epsilon}{sub i}<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  12. The cytotoxicity of anti-CD22 immunotoxin is enhanced by bryostatin 1 in B-cell lymphomas through CD22 upregulation and PKC-βII depletion

    PubMed Central

    Biberacher, Viola; Decker, Thomas; Oelsner, Madlen; Wagner, Michaela; Bogner, Christian; Schmidt, Burkhard; Kreitman, Robert J.; Peschel, Christian; Pastan, Ira; Meyer zum Büschenfelde, Christian; Ringshausen, Ingo

    2012-01-01

    Background In spite of potent first-line therapies for chronic lymphocytic leukemia, treatment remains palliative and all patients frequently relapse. Treatment options for these patients are more limited. BL22 is a recombinant protein composed of the variable region of a monoclonal antibody that binds to CD22 and of PE38, a truncated Pseudomonas exotoxin. BL22 is a very potent drug already used in patients with hairy cell leukemia, whereas in chronic lymphocytic leukemia its cytotoxicity is limited by a lower expression of CD22. Here we demonstrate that this limitation can be overcome by pre-activation of chronic lymphocytic leukemia cells with bryostatin 1. Design and Methods Primary malignant B cells from chronic lymphocytic leukemia and mantle cell lymphoma patients were used in vitro to assess the therapeutic impact of drug combinations using BL22 and bryostatin 1. Results We demonstrate that bryostatin 1 sensitizes chronic lymphocytic leukemia cells for the cytotoxic effects of BL22 through activation of protein kinase C and subsequently increased CD22 surface expression. Dose and time response analysis reveals that activation of protein kinase C further activates an autocrine feedback loop degrading protein kinase C-βII protein. Depletion of protein kinase C-βII and upregulation of CD22 persist for several days following pre-stimulation with bryostatin 1. Therefore, our data provide a rationale for the sequential administration of BL22 following bryostatin 1 treatment. In addition to primary chronic lymphocytic leukemia cells, bryostatin 1 also sensitizes diffuse large B-cell lymphoma and mantle cell lymphoma cells to BL22 induced apoptosis. Conclusions Our data suggest that the combination of bryostatin 1 with antibodies directed against CD22 is a potent drug combination for the treatment of low- and high-grade B-cell lymphoma. PMID:22180432

  13. Recombination Drives Vertebrate Genome Contraction

    PubMed Central

    Nam, Kiwoong; Ellegren, Hans

    2012-01-01

    Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process. PMID:22570634

  14. Recombination processes in ionised plasmas

    NASA Astrophysics Data System (ADS)

    Bastin, Robert

    The observational analysis of astrophysical plasmas relies on accurate calculations of the atomic processes involved. The recombination spectra of singly ionised oxygen (O il) and carbon (C il) present excellent tools for investigating regions such as planetary nebulae and H II regions. In this thesis, detailed treatments of the recombination processes of both O II and C II are presented. Using the R-matrix solution to the close coupling equations, I present the results of accurate photoionisation calculations. Bound state energy levels are determined and oscillator strengths calculated for both species. Recombination coefficients were evalu ated for low n and 1, for C II in LS-coupling, and 0 II in intermediate coupling, taking particular care to treat resonances effectively. Sample photoionisation cross-sections are presented for both species, and compared to previous work. A complete radiative-cascade model is treated for both species, in order to determine line emissivities under nebular conditions at a wide range of temperatures and densities. Collisional effects are treated for C II, along with, for the first time, the effects of high temperature dielectronic recombination, allowing the modelling of regions of much higher electron temperature than previous work. The O II calculations were performed under intermediate coupling for the first time, allowing the effects of non-statistical popula tions of the parent ion fine-structure levels and dielectronic recombination onto bound states within this fine-structure to be taken into account in line emissivities. Detailed comparison with previous theoretical work was made for both species. The application of the C II and 0 n recombination spectra to determining tempera ture and densities from the observed spectra of a number of ionised nebulae is considered. The potential for using the new recombination spectra as diagnostic tools to solve some of the key problems in the study of ionised nebulae is demonstrated.

  15. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  16. Recombinant allergens for specific immunotherapy.

    PubMed

    Cromwell, Oliver; Häfner, Dietrich; Nandy, Andreas

    2011-04-01

    Recombinant DNA technology provides the means for producing allergens that are equivalent to their natural counterparts and also genetically engineered variants with reduced IgE-binding activity. The proteins are produced as chemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and these provide the means for making a very detailed diagnosis of a patient's sensitization profile. Clinical development programs are now in progress to assess the suitability of recombinant allergens for both subcutaneous and sublingual immunotherapy. Recombinant hypoallergenic variants, which are developed with the aim of increasing the doses that can be administered while at the same time reducing the risks for therapy-associated side effects, are also in clinical trials for subcutaneous immunotherapy. Grass and birch pollen preparations have been shown to be clinically effective, and studies with various other allergens are in progress. Personalized or patient-tailored immunotherapy is still a very distant prospect, but the first recombinant products based on single allergens or defined mixtures could reach the market within the next 5 years. PMID:21377719

  17. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    SciTech Connect

    Moriya, Takashi J.

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  18. Current Drive in Recombining Plasma

    SciTech Connect

    P.F. Schmit and N.J. Fisch

    2012-05-15

    The Langevin equations describing the average collisional dynamics of suprathermal particles in nonstationary plasma remarkably admit an exact analytical solution in the case of recombining plasma. The current density produced by arbitrary particle fluxes is derived including the effect of charge recombination. Since recombination has the effect of lowering the charge density of the plasma, thus reducing the charged particle collisional frequencies, the evolution of the current density can be modified substantially compared to plasma with fixed charge density. The current drive efficiency is derived and optimized for discrete and continuous pulses of current, leading to the discovery of a nonzero "residual" current density that persists indefinitely under certain conditions, a feature not present in stationary plasmas.

  19. Stable recombination hotspots in birds

    PubMed Central

    Singhal, Sonal; Leffler, Ellen M.; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M.; Strand, Alva I.; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N.; Griffith, Simon C.; McVean, Gil; Przeworski, Molly

    2016-01-01

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species, the zebra finch Taeniopygia guttata and the long-tailed finch Poephila acuticauda. We find that both species have hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, the two species share most hotspots, with conservation seemingly extending over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757

  20. The Dissociative Recombination of OH(+)

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.

    1995-01-01

    Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.

  1. Recombinant snake venom prothrombin activators.

    PubMed

    Lövgren, Ann

    2013-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active γ-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318

  2. Selenium incorporation using recombinant techniques

    SciTech Connect

    Walden, Helen

    2010-04-01

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  3. Classifications and comparisons of multilocus recombination distributions

    PubMed Central

    Karlin, Samuel; Liberman, Uri

    1978-01-01

    Various classifications and representations of multilocus recombination structures are delineated based on generalized notions of linkage values and recombination rates. An important class of recombination distributions (called the count-location chiasma process) is parameterized by a distribution of the number of crossover events and, for each such crossover count, by a conditional distribution of crossover locations. A number of properties of this recombination structure are developed. A multilocus definition of a “natural” recombination range is set forth. Orderings among recombination distributions in the multilocus setting are also discussed. Comparisons are made in terms of complete linkage, free assortment and noninterference schemes serving as standards. PMID:16592601

  4. Inhomogeneous recombinations during cosmic reionization

    NASA Astrophysics Data System (ADS)

    Sobacchi, Emanuele; Mesinger, Andrei

    2014-05-01

    By depleting the ionizing photon budget available to expand cosmic H II regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a subgrid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parametrizing photoheating feedback on star formation, we present large-scale, seminumeric reionization simulations which self-consistently track the local (subgrid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large H II regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reionization. As a result, typical H II regions are smaller by factors of ˜2 to 3 throughout reionization. The large-scale (k ≲ 0.2 Mpc-1) ionization power spectrum is suppressed by factors of ≳2-3 in the second half of reionization. Therefore properly modelling recombinations is important in interpreting virtually all reionization observables, including upcoming interferometry with the redshifted 21cm line. Consistent with previous works, we find the clumping factor of ionized gas to be C H II ˜ 4 at the end of reionization.

  5. Improving recombinant protein purification yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  6. CRMAGE: CRISPR Optimized MAGE Recombineering

    PubMed Central

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O. A.; Nielsen, Alex Toftgaard

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering. PMID:26797514

  7. Recombinant DNA: History of the Controversy.

    ERIC Educational Resources Information Center

    Vigue, Charles L.; Stanziale, William G.

    1979-01-01

    The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

  8. Antibacterial activity of recombinant murine beta interferon.

    PubMed Central

    Fujiki, T; Tanaka, A

    1988-01-01

    Recombinant murine beta interferon was protective and therapeutic for mice against Listeria monocytogenes infection in vivo. The recombinant murine beta interferon caused enhanced H2O2 release by macrophages in vivo, but not in vitro. PMID:3343048

  9. [Recombination in Drosophila in space flight].

    PubMed

    Filatova, L P; Vaulina, E N; Lapteva, N Sh; Grozdova, T Ia

    1988-04-01

    An experiment with Drosophila melanogaster males was performed aboard the Artificial Satellite "Kosmos-1667". Mutagenic effects of a 7-day space flight on intergene recombination in chromosome 2 were studied. The space flight factors decreased the frequency of recombination. A model experiment on a laboratory centrifuge demonstrated insignificant increase in recombination frequency caused by acceleration. PMID:3135244

  10. Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15.

    PubMed

    Peraino, Jaclyn Stromp; Hermanrud, Christina E; Springett, Lauren; Zhang, Huiping; Li, Guoying; Srinivasan, Srimathi; Gusha, Ashley; Sachs, David H; Huang, Christene A; Wang, Zhirui

    2012-01-01

    The porcine CD3 specific monoclonal antibody 898H2-6-15 has been used in allo- and xeno-transplantation studies as a porcine CD3 marker and as an effective T cell depletion reagent when conjugated to the diphtheria toxin mutant, CRM9. A recombinant anti-porcine CD3 immuntoxin was recently developed using single-chain variable fragments (scFv) derived from 898H2-6-15. In this study, using published sequence data, we have expressed the porcine CD3 ectodomain molecules in E. coli through inclusion body isolation and in vitro refolding approach. The expressed and refolded porcine CD3 ectodomain molecules include CD3ε, CD3γ, CD3δ, CD3εγ heterodimer, CD3εδ heterodimer, CD3εγ single-chain fusion protein and CD3εδ single-chain fusion protein. These refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin Poros 50HQ. ELISA analysis demonstrated that only the porcine CD3εγ ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3εγ ectodomain single-chain fusion protein will allow screening for affinity matured variants of scFv derived from 898H2-6-15 to improve the recombinant anti-porcine CD3 immunotoxin. Porcine CD3εγ ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3εγ and porcine CD3 antibodies such as 898H2-6-15. PMID:22672968

  11. Surface recombination statistics at traps

    NASA Astrophysics Data System (ADS)

    Landsberg, P. T.; Abrahams, M. S.

    1983-09-01

    The Shockley-Read-Hall recombination statistics was recently generalised by Dhariwal, Kothari and Jain to include the effect of a finite time of relaxation before the captured carrier settles into its ground state, and by Landsberg to allow for Auger effects and so-called "extra" carriers supplied to the semiconductor from the outside. The combined result of these effects is studied here theoretically, together with the consideration of a simple distribution of trap states. It is found that the surface recombination velocity s has the usual minimum in the near intrinsic state and that s passes through a maximum as a function of excess electron concentration. Both extrema are enhanced if the trap states are distributed over an energy range. Experimental plots of s as a function of excess electron and hole concentrations should yield insight concerning the numerical importance of (a) Auger effects with the participation of traps and (b) relaxation times.

  12. Selection of Recombinant Human Antibodies.

    PubMed

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  13. Recombination Catalysts for Hypersonic Fuels

    NASA Technical Reports Server (NTRS)

    Chinitz, W.

    1998-01-01

    The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.

  14. Chemical kinetics of geminal recombination

    SciTech Connect

    Levin, P.P.; Khudyakov, I.V.; Brin, E.F.; Kuz'min, V.A.

    1988-09-01

    The kinetics of geminal recombination of triplet radical pairs formed in photoreduction of benzophenone by p-cresol in glycerin solution was studied by pulsed laser photolysis. The experiments were conducted at several temperatures and in a constant magnetic field of H = 0.34 T. The parameters in six kinetic equations describing geminal recombination were determined with a computer. The values of the sums of the squares of the residual deviations of the approximation were obtained. It was found that the kinetics are best described by the functions proposed by Noyes and Shushin. It was shown that it is necessary to use the mutual diffusion coefficient of the radicals, which is significantly smaller than the sum of the estimations of the experimental values of the radical diffusion coefficients, for describing the kinetics due to the correlations of the molecular motions of the radicals in the cage.

  15. Introductory experiments in recombinant DNA.

    PubMed

    Tait, R C

    2000-07-01

    Nine practical exercises demonstrate the basic principles in recombinant DNA. The exercises explain the principles that DNA equals genes and that changes in DNA cause changes in genetic properties. The aim is to provide a teaching resource that can be used to illustrate the theory and applications of molecular biology to highschool students, undergraduate students, medics, dentists, doctors, nurses, life scientists, and anyone learning the basics of DNA technology. PMID:11471559

  16. Recombinant Toxins for Cancer Treatment

    NASA Astrophysics Data System (ADS)

    Pastan, Ira; Fitzgerald, David

    1991-11-01

    Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.

  17. Recombinant vector and eukaryotic host transformed thereby

    SciTech Connect

    Sugden, W.M.

    1987-08-11

    A recombinant plasmid is described comprising: a segment from a first plasmid which is not a lymphotrophic herpes virus segment and which facilitates the replication of the recombinant plasmid in a prokaryotic host; a segment from a lymphotrophic herpes virus which is linked to the first plasmid segment such that is a capable of assisting in maintaining the recombinant plasmid as a plasmid if the recombinant plasmid is inserted into a eukaryotic host that has been transformed by the lymphotrophic herpes virus; and a foreign eukaryotic gene component linked as part of the recombinant plasmid.

  18. A coalescent model of recombination hotspots.

    PubMed Central

    Wiuf, Carsten; Posada, David

    2003-01-01

    Recent experimental findings suggest that the assumption of a homogeneous recombination rate along the human genome is too naive. These findings point to block-structured recombination rates; certain regions (called hotspots) are more prone than other regions to recombination. In this report a coalescent model incorporating hotspot or block-structured recombination is developed and investigated analytically as well as by simulation. Our main results can be summarized as follows: (1) The expected number of recombination events is much lower in a model with pure hotspot recombination than in a model with pure homogeneous recombination, (2) hotspots give rise to large variation in recombination rates along the genome as well as in the number of historical recombination events, and (3) the size of a (nonrecombining) block in the hotspot model is likely to be overestimated grossly when estimated from SNP data. The results are discussed with reference to the current debate about block-structured recombination and, in addition, the results are compared to genome-wide variation in recombination rates. A number of new analytical results about the model are derived. PMID:12750351

  19. Nondisjunction of chromosome 15: Origin and recombination

    SciTech Connect

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. ); Langlois, S. ); Morris, M.A.; Malcolm, S.

    1993-09-01

    Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N=27) and Angelman syndrome patients (N-5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, more paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. 33 refs., 1 fig., 7 tabs.

  20. Effect of gamma radiation on retroviral recombination.

    PubMed

    Hu, W S; Temin, H M

    1992-07-01

    To elucidate the mechanism(s) of retroviral recombination, we exposed virions to gamma radiation prior to infecting target cells. By using previously described spleen necrosis virus-based vectors containing multiple markers, recombinant proviruses were studied after a single round of retrovirus replication. The current models of retroviral recombination predict that breaking virion RNA should promote minus-strand recombination (forced copy-choice model), decrease or not affect plus-strand recombination (strand displacement/assimilation model), and shift plus-strand recombination towards the 3' end of the genome. However, we found that while gamma irradiation of virions reduced the amount of recoverable viral RNA, it did not primarily cause breaks. Thus, the frequency of selected recombinants was not significantly altered with greater doses of radiation. In spite of this, the irradiation did decrease the number of recombinants with only one internal template switch. As a result, the average number of additional internal template switches in the recombinant proviruses increased from 0.7 to 1.4 as infectivity decreased to 6%. The unselected internal template switches tended to be 5' of the selected crossover even in the recombinants from irradiated viruses, inconsistent with a plus-strand recombination mechanism. PMID:1602553

  1. Bacteriophage recombination systems and biotechnical applications.

    PubMed

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

  2. Recombinant erythropoietin in clinical practice

    PubMed Central

    Ng, T; Marx, G; Littlewood, T; Macdougall, I

    2003-01-01

    The introduction of recombinant human erythropoietin (RHuEPO) has revolutionised the treatment of patients with anaemia of chronic renal disease. Clinical studies have demonstrated that RHuEPO is also useful in various non-uraemic conditions including haematological and oncological disorders, prematurity, HIV infection, and perioperative therapies. Besides highlighting both the historical and functional aspects of RHuEPO, this review discusses the applications of RHuEPO in clinical practice and the potential problems of RHuEPO treatment. PMID:12897214

  3. Recombinant DNA technology in apple.

    PubMed

    Gessler, Cesare; Patocchi, Andrea

    2007-01-01

    This review summarizes the achievements of almost 20 years of recombinant DNA technology applied to apple, grouping the research results into the sections: developing the technology, insect resistance, fungal disease resistance, self-incompatibility, herbicide resistance, fire blight resistance, fruit ripening, allergens, rooting ability, and acceptance and risk assessment. The diseases fire blight, caused by Erwinia amylovora, and scab, caused by Venturia inaequalis, were and still are the prime targets. Shelf life improvement and rooting ability of rootstocks are also relevant research areas. The tools to create genetically modified apples of added value to producers, consumers, and the environment are now available. PMID:17522823

  4. Annealing Vs. Invasion in Phage λ Recombination

    PubMed Central

    Stahl, M. M.; Thomason, L.; Poteete, A. R.; Tarkowski, T.; Kuzminov, A.; Stahl, F. W.

    1997-01-01

    Genetic recombination catalyzed by λ's Red pathway was studied in rec(+) and recA mutant bacteria by examining both intracellular λ DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec(+) cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of λ catalyzes recombination primarily by annealing. PMID:9383045

  5. Specific Immunosuppression by Immunotoxins Containing Daunomycin

    NASA Astrophysics Data System (ADS)

    Diener, E.; Diner, U. E.; Sinha, A.; Xie, S.; Vergidis, R.

    1986-01-01

    Daunomycin, when conjugated with a targeting antigen by an acid-sensitive spacer, remains inactive at the intravascular pH of 7 but becomes active after cleavage within the acidic lysosomal environment of the target cell. This observation made it possible to construct cytocidal compounds that caused antigen-specific suppression of murine lymphocyte function. When daunomycin was coupled to the hapten conjugate of ovalbumin by an acid-sensitive cis-aconityl group, it caused hapten-specific impairment of immunocompetence in murine B lymphocytes in vitro and in vivo. Furthermore, the response by T lymphocytes to concanavalin A in vitro was selectively eliminated by a conjugate between daunomycin plus the acid-sensitive spacer and a monoclonal antibody specific for T cells.

  6. Recombinant DNA production of spider silk proteins

    PubMed Central

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  7. Recombinant bacteria for mosquito control.

    PubMed

    Federici, B A; Park, H-W; Bideshi, D K; Wirth, M C; Johnson, J J

    2003-11-01

    Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B. sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor. The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria. PMID:14506223

  8. Dissociative recombination in planetary ionospheres

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1993-01-01

    Ionization in planetary atmospheres can be produced by solar photoionization, photoelectron impact ionization, and, in auroral regions, by impact of precipitating particles. This ionization is lost mainly in dissociative recombination (DR) of molecular ions. Although atomic ions cannot undergo DR, they can be transformed locally through ion-molecule reactions into molecular ions, or they may be transported vertically or horizontally to regions of the atmosphere where such transformations are possible. Because DR reactions tend to be very exothermic, they can be an important source of kinetically or internally excited fragments. In interplanetary thermospheres, the neutral densities decrease exponentially with altitude. Below the homopause (or turbopause), the atmosphere is assumed to be throughly mixed by convection and/or turbulence. Above the homopause, diffusion is the major transport mechanism, and each species is distributed according to its mass, with the logarithmic derivative of the density with repect to altitude given approximately by -1/H, where H = kT/mg is the scale height. In this expression, T is the neutral temperature, g is the local acceleratiion of gravity, and m is the mass of the species. Thus lighter species become relatively more abundant, and heavier species less abundant, as the altitude increases. This variation of the neutral composition can lead to changes in the ion composition; furthermore, as the neutral densities decrease, dissociative recombination becomes more important relative to ion-neutral reactions as a loss mechanism for molecular ions.

  9. Human Insulin from Recombinant DNA Technology

    NASA Astrophysics Data System (ADS)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  10. Modeling Interference in Genetic Recombination

    PubMed Central

    McPeek, M. S.; Speed, T. P.

    1995-01-01

    In analyzing genetic linkage data it is common to assume that the locations of crossovers along a chromosome follow a Poisson process, whereas it has long been known that this assumption does not fit the data. In many organisms it appears that the presence of a crossover inhibits the formation of another nearby, a phenomenon known as ``interference.'' We discuss several point process models for recombination that incorporate position interference but assume no chromatid interference. Using stochastic simulation, we are able to fit the models to a multilocus Drosophila dataset by the method of maximum likelihood. We find that some biologically inspired point process models incorporating one or two additional parameters provide a dramatically better fit to the data than the usual ``no-interference'' Poisson model. PMID:7713406

  11. Deleterious background selection with recombination

    SciTech Connect

    Hudson, R.R.; Kaplan, N.L.

    1995-12-01

    An analytic expression for the expected nucleotide diversity is obtained for a neutral locus in a region with deleterious mutation and recombination. Our analytic results are used to predict levels of variation for the entire third chromosome of Drosophila melanogaster. The predictions are consistent with the low levels of variation that have been observed at loci near the centromeres of the third chromosome of D. melanogaster. However, the low levels of variation observed near the tips of this chromosome are not predicted using currently available estimates of the deleterious mutation rate and of selection coefficients. If considerably smaller selection coefficients are assumed, the low observed levels of variation at the tips of the third chromosome are consistent with the background selection model. 33 refs., 4 figs., 1 tab.

  12. Dielectronic recombination of tungsten ions

    NASA Astrophysics Data System (ADS)

    Li, Bowen; O’Sullivan, Gerry; Dong, Chenzhong; Chen, Ximeng

    2016-08-01

    Ab initio calculations of dielectronic recombination rate coefficients of Ne-, Pd- and Ag-like tungsten have been performed. Energy levels, radiative transition probabilities and autoionization rates were calculated using the Flexible Atomic Code. The contributions from different channels to the total rate coefficients are discussed. The present calculated rate coefficients are compared with other calculations where available. Excellent agreement has been found for Ne-like W while a large discrepancy was found for Pd-like W, which implies that more ab initio calculations and experimental measurements are badly needed. Further calculations demonstrated that the influence of configuration interaction is small while nonresonant radiative stabilizing (NRS) contribution to doubly excited non-autoionizing states are vital. The data obtained are expected to be useful for modeling plasmas for fusion applications, especially for the ITER community, which makes experimental verification even more essential.

  13. Steric effects on geminate recombinations

    SciTech Connect

    Traylor, T.G.; Taube, D.; Jongeward, K.A.; Magde, D. )

    1990-09-12

    Steric effects on the binding of isonitrile ligands to iron(II) porphyrins were investigated by picosecond flash photolysis. Two different types of steric effects were distinguished and characterized: (1) steric restrictions to porphyrin planarity and (2) blocking of the pathway for ligand approach. Heme planarity was restricted by coordinating 1,2-dimethylimidazole trans to the ligand binding site being investigated. Blocking of the binding site was explored by using adamantane heme 6,6-cyclophane, in which the adamantane moiety forms a cap over the binding site. Results of picosecond kinetic measurements demonstrate that the first effect, heme nonplanarity or trans strain, influences the bond-making step, whereas the second effect, ligand blocking, involves a conformational preequilibrium prior to bond making. Relevance of these findings for contact pair recombination, in general, and for heme protein ligation, in particular, are discussed.

  14. Recombinant prolylcarboxypeptidase activates plasma prekallikrein.

    PubMed

    Shariat-Madar, Zia; Mahdi, Fakhri; Schmaier, Alvin H

    2004-06-15

    The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment. PMID:14996700

  15. Fundamental Studies of Recombinant Hydrogenases

    SciTech Connect

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  16. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

    PubMed Central

    Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

    2015-01-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

  17. RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

    PubMed Central

    Liao, C L; Lai, M M

    1992-01-01

    Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur

  18. Oligonucleotide recombination in gram negative bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...

  19. Dielectronic recombination lines of C{sup +}

    SciTech Connect

    Sochi, Taha Storey, Peter J.

    2013-11-15

    The present paper presents atomic data generated to investigate the recombination lines of C II in the spectra of planetary nebulae. These data include energies of bound and autoionizing states, oscillator strengths and radiative transition probabilities, autoionization probabilities, and recombination coefficients. The R-matrix method of electron scattering theory was used to describe the C{sup 2+} plus electron system.

  20. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    NASA Astrophysics Data System (ADS)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  1. Oligo Recombination in Gram Negative Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homologous recombination is important for bacterial survival because it simultaneously provides genomic stability as well as genomic plasticity. Of the mechanistic pathways for homologous recombination, those mediated by RecA are the most thoroughly characterized and are understood to be structural...

  2. Dissociative recombination of highly symmetric polyatomic ions.

    PubMed

    Douguet, Nicolas; Orel, Ann E; Greene, Chris H; Kokoouline, Viatcheslav

    2012-01-13

    A general first-principles theory of dissociative recombination is developed for highly symmetric molecular ions and applied to H(3)O(+) and CH(3)(+), which play an important role in astrophysical, combustion, and laboratory plasma environments. The theoretical cross sections obtained for the dissociative recombination of the two ions are in good agreement with existing experimental data from storage ring experiments. PMID:22324682

  3. High efficiency recombineering in lactic acid bacteria

    PubMed Central

    van Pijkeren, Jan-Peter; Britton, Robert A.

    2012-01-01

    The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to Gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lactic acid bacteria. Mutations were incorporated in the chromosome of Lactobacillus reuteri and Lactococcus lactis without selection at frequencies ranging between 0.4% and 19%. Whole genome sequence analysis showed that ssDNA recombineering is specific and not hypermutagenic. To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold. By creating a single amino acid change in the d-Ala-d-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration. Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry. We expect that this work will serve as a blueprint for the adaptation of ssDNA recombineering to other Gram-positive bacteria. PMID:22328729

  4. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  5. Recombinant Swinepox Virus for Veterinary Vaccine Development.

    PubMed

    Fan, Hong-Jie; Lin, Hui-Xing

    2016-01-01

    Poxvirus-vectors have been widely used in vaccine development for several important human and animal diseases; some of these vaccines have been licensed and used extensively. Swinepox virus (SPV) is well suited to develop recombinant vaccines because of its large packaging capacity for recombinant DNA, its host range specificity, and its ability to induce appropriate immune responses. PMID:26458836

  6. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  7. V(D)J recombination deficiencies.

    PubMed

    de Villartay, Jean-Pierre

    2009-01-01

    V(D)J recombination not only comprises the molecular mechanism that insures diversity of the immune system but also constitutes a critical checkpoint in the developmental program of B- and T-lymphocytes. The analysis of human patients with Severe Combined Immune Deficiency (SCID) has contributed to the understanding of the biochemistry of the V(D)J recombination reaction. The molecular study V(D)J recombination settings in humans, mice and in cellular mutants has allowed to unravel the process of Non Homologous End Joining (NHEJ), one of the key pathway that insure proper repair of DNA double strand breaks (dsb), whether they occur during V(D)J recombination or secondary to other DNA injuries. Two NHEJ factors, Artemis and Cernunnos, were indeed discovered through the study of human V(D)J recombination defective human SCID patients. PMID:19731800

  8. Biochemistry of Meiotic Recombination: Formation, Processing, and Resolution of Recombination Intermediates

    PubMed Central

    Ehmsen, Kirk T.

    2009-01-01

    Meiotic recombination ensures accurate chromosome segregation during the first meiotic division and provides a mechanism to increase genetic heterogeneity among the meiotic products. Unlike homologous recombination in somatic (vegetative) cells, where sister chromatid interactions prevail and crossover formation is avoided, meiotic recombination is targeted to involve homologs, resulting in crossovers to connect the homologs before anaphase of the first meiotic division. The mechanisms responsible for homolog choice and crossover control are poorly understood, but likely involve meiosis-specific recombination proteins, as well as meiosis-specific chromosome organization and architecture. Much progress has been made to identify and biochemically characterize many of the proteins acting during meiotic recombination. This review will focus on the proteins that generate and process heteroduplex DNA, as well as those that process DNA junctions during meiotic recombination, with particular attention to how recombination activities promote crossover resolution between homologs. PMID:20098639

  9. GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION

    EPA Science Inventory

    Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...

  10. Charge recombination and thermoluminescence in photosystem II.

    PubMed

    Rappaport, Fabrice; Cuni, Aude; Xiong, Ling; Sayre, Richard; Lavergne, Jérôme

    2005-03-01

    In the recombination process of Photosystem II (S(2)Q(A)(-)-->S(1)Q(A)) the limiting step is the electron transfer from the reduced primary acceptor pheophytin Ph(-) to the oxidized primary donor P(+) and the rate depends on the equilibrium constant between states S(2)PPhQ(A)(-) and S(1)P(+)Ph(-)Q(A). Accordingly, mutations that affect the midpoint potential of Ph or of P result in a modified recombination rate. A strong correlation is observed between the effects on the recombination rate and on thermoluminescence (TL, the light emission from S(2)Q(A)(-) during a warming ramp): a slower recombination corresponds to a large enhancement and higher temperature of the TL peak. The current theory of TL does not account for these effects, because it is based on the assumption that the rate-limiting step coincides with the radiative process. When implementing the known fact that the radiative pathway represents a minor leak, the modified TL theory readily accounts qualitatively for the observed behavior. However, the peak temperature is still lower than predicted from the temperature-dependence of recombination. We argue that this reflects the heterogeneity of the recombination process combined with the enhanced sensitivity of TL to slower components. The recombination kinetics are accurately fitted as a sum of two exponentials and we show that this is not due to a progressive stabilization of the charge-separated state, but to a pre-existing conformational heterogeneity. PMID:15653722

  11. Electron Recombination in a Dense Hydrogen Plasma

    SciTech Connect

    Jana, M.R.; Johnstone, C.; Kobilarcik, T.; Koizumi, G.M.; Moretti, A.; Popovic, M.; Tollestrup, A.V.; Yonehara, K.; Leonova, M.A.; Schwarz, T.A.; Chung, M.; /Unlisted /IIT, Chicago /Fermilab /MUONS Inc., Batavia /Turin Polytechnic

    2012-05-01

    A high pressure hydrogen gas filled RF cavity was subjected to an intense proton beam to study the evolution of the beam induced plasma inside the cavity. Varying beam intensities, gas pressures and electric fields were tested. Beam induced ionized electrons load the cavity, thereby decreasing the accelerating gradient. The extent and duration of this degradation has been measured. A model of the recombination between ionized electrons and ions is presented, with the intent of producing a baseline for the physics inside such a cavity used in a muon accelerator. Analysis of the data taken during the summer of 2011 shows that self recombination takes place in pure hydrogen gas. The decay of the number of electrons in the cavity once the beam is turned off indicates self recombination rather than attachment to electronegative dopants or impurities. The cross section of electron recombination grows for larger clusters of hydrogen and so at the equilibrium of electron production and recombination in the cavity, processes involving H{sub 5}{sup +} or larger clusters must be taking place. The measured recombination rates during this time match or exceed the analytic predicted values. The accelerating gradient in the cavity recovers fully in time for the next beam pulse of a muon collider. Exactly what the recombination rate is and how much the gradient degrades during the 60 ns muon collider beam pulse will be extrapolated from data taken during the spring of 2012.

  12. Intraspecific variation of recombination rate in maize

    PubMed Central

    2013-01-01

    Background In sexually reproducing organisms, meiotic crossovers ensure the proper segregation of chromosomes and contribute to genetic diversity by shuffling allelic combinations. Such genetic reassortment is exploited in breeding to combine favorable alleles, and in genetic research to identify genetic factors underlying traits of interest via linkage or association-based approaches. Crossover numbers and distributions along chromosomes vary between species, but little is known about their intraspecies variation. Results Here, we report on the variation of recombination rates between 22 European maize inbred lines that belong to the Dent and Flint gene pools. We genotype 23 doubled-haploid populations derived from crosses between these lines with a 50 k-SNP array and construct high-density genetic maps, showing good correspondence with the maize B73 genome sequence assembly. By aligning each genetic map to the B73 sequence, we obtain the recombination rates along chromosomes specific to each population. We identify significant differences in recombination rates at the genome-wide, chromosome, and intrachromosomal levels between populations, as well as significant variation for genome-wide recombination rates among maize lines. Crossover interference analysis using a two-pathway modeling framework reveals a negative association between recombination rate and interference strength. Conclusions To our knowledge, the present work provides the most comprehensive study on intraspecific variation of recombination rates and crossover interference strength in eukaryotes. Differences found in recombination rates will allow for selection of high or low recombining lines in crossing programs. Our methodology should pave the way for precise identification of genes controlling recombination rates in maize and other organisms. PMID:24050704

  13. Advances in recombinant antibody manufacturing.

    PubMed

    Kunert, Renate; Reinhart, David

    2016-04-01

    Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today. PMID:26936774

  14. Recombinant protein production and streptomycetes.

    PubMed

    Anné, Jozef; Maldonado, Bárbara; Van Impe, Jan; Van Mellaert, Lieve; Bernaerts, Kristel

    2012-04-30

    The biopharmaceutical market has come a long way since 1982, when the first biopharmaceutical product, recombinant human insulin, was launched. Just over 200 biopharma products have already gained approval. The global market for biopharmaceuticals which is currently valued at over US$99 billion has been growing at an impressive compound annual growth rate over the previous years. To produce these biopharmaceuticals and other industrially important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All expression systems have some advantages as well as some disadvantages that should be considered in selecting which one to use. Choosing the best one requires evaluating the options--from yield to glycosylation, to proper folding, to economics of scale-up. No host cell from which all the proteins can be universally expressed in large quantities has been found so far. Therefore, it is important to provide a variety of host-vector expression systems in order to increase the opportunities to screen for the most suitable expression conditions or host cell. In this overview, we focus on Streptomyces lividans, a Gram-positive bacterium with a proven excellence in secretion capacity, as host for heterologous protein production. We will discuss its advantages and disadvantages, and how with systems biology approaches strains can be developed to better producing cell factories. PMID:21777629

  15. Bacterial genome remodeling through bacteriophage recombination.

    PubMed

    Menouni, Rachid; Hutinet, Geoffrey; Petit, Marie-Agnès; Ansaldi, Mireille

    2015-01-01

    Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes. PMID:25790500

  16. Dissociative Recombination without a Curve Crossing

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.

    1994-01-01

    Ab initio calculations show that a curve crossing is not always needed for a high dissociative- recombination cross section. For HeH(+), in which no neutral states cross the ion potential curve, dissociative recombination is driven by the nuclear kinetic-energy operator on adiabatic potential curves. The kinetic-energy derivative operator allows for capture into repulsive curves that are outside of the classical turning points for the nuclear motion. The dominant dissociative route is the C (2)Sigma(+) state leading to H(n = 2) atoms. An analogous mechanism is proposed for the dissociative recombination of H3(+).

  17. Recombination of N4(+) ions with electrons

    NASA Technical Reports Server (NTRS)

    Cao, Y. S.; Johnsen, R.

    1991-01-01

    Using a modified high-pressure-afterglow/mass spectrometer apparatus similar to that described by Lee and Johnsen (1989), spectroscopic observations of afterglow helium plasmas, with N2 as a minor additive, were carried out in order to verify the mechanism suggested by Bates (1991) for dissociative recombination of electrons with N4(+) ions. It was found that dissociative recombination of electrons with N4(+) ions results in the formation of N2 molecules in the C 3Pi(u) (v = 0,1) state, with the recombination rate coefficient of (2.6 +/- 0.3) x 10 exp -6 cu cm/sec at 300 K.

  18. Spacecraft thermal energy accommodation from atomic recombination

    NASA Technical Reports Server (NTRS)

    Carleton, Karen L.; Marinelli, William J.

    1991-01-01

    Measurements of atomic recombination probabilities important in determining energy release to reusable spacecraft thermal protection surfaces during reentry are presented. An experimental apparatus constructed to examine recombination of atomic oxygen from thermal protection and reference materials at reentry temperatures is described. The materials are examined under ultrahigh vacuum conditions to develop and maintain well characterized surface conditions that are free of contamination. When compared with stagnation point heat transfer measurements performed in arc jet facilities, these measurements indicate that a significant fraction of the excess energy available from atom recombination is removed from the surface as metastable O2.

  19. The Kinetics of Nitrogen Atom Recombination

    ERIC Educational Resources Information Center

    Brown, G. Ronald; Winkler, C. A.

    1977-01-01

    Describes a study of the kinetics of the recombination of nitrogen atoms in which concentration-time relations are determined directly by utilizing visual observations of emissions to make gas phase titrations of N atoms with NO. (MLH)

  20. Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation.

    PubMed

    He, Yan; Wang, Minghui; Sun, Qi; Pawlowski, Wojciech P

    2016-01-01

    Genome-wide maps of recombination sites provide valuable information not only on the recombination pathway itself but also facilitate the understanding of genome dynamics and evolution. Here, we describe a chromatin immunoprecipitation (ChIP) protocol to map the sites of recombination initiation in plants with maize used as an example. ChIP is a method that allows identification of chromosomal sites occupied by specific proteins. Our protocol utilizes RAD51, a protein involved in repair of double-strand breaks (DSBs) that initiate meiotic recombination, to identify DSB formation hotspots. Chromatin is extracted from meiotic flowers, sheared and enriched in fragments bound to RAD51. Genomic location of the protein is then identified by next-generation sequencing. This protocol can also be used in other species of plants, animals, and fungi. PMID:27511175

  1. Constraints from jet calculus on quark recombination

    SciTech Connect

    Jones, L.M.; Lassila, K.E.; Sukhatme, U.; Willen, D.

    1981-02-01

    Within the quantum-chromodynamic jet-calculus formalism, we deduce an equation describing recombination of quarks and antiquarks into mesons within a quark or gluon jet. This equation relates the recombination function R(x/sub 1/,x/sub 2/,x) used in current literature to the fragmentation function for producing that same meson out of the parton initiating the jet. We submit currently used recombination functions to our consistency test, taking as input mainly the u-quark fragmentation ''data'' into ..pi../sup +/ mesons. The qq-bar..--> pi.. recombination functions popular in the literature are consistent with measured fragmentation functions, but they must be supplemented by other contributions to provide the full D..pi../sup +//sub u/. We also discuss the Q/sup 2/ dependence of the resulting fragmentation functions.

  2. Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  3. Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  4. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    Cancer.gov

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  5. Chemical recombination in an expansion tube

    NASA Technical Reports Server (NTRS)

    Bakos, Robert J.; Morgan, Richard G.

    1994-01-01

    The note describes the theoretical basis of chemical recombination in an expansion tube which simulates energy, Reynolds number, and stream chemistry at near-orbital velocities. Expansion tubes can satisfy ground-based hypersonic propulsion and aerothermal testing requirements.

  6. Fermentations with new recombinant organisms

    SciTech Connect

    Bothast, R.J.; Nichols, N.N.; Dien, B.S.

    1999-10-01

    US fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. Most of this ethanol was produced from over 550 million bushels of corn. Expanding fuel ethanol production will require developing lower-cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. Major technical hurdles to converting lignocellulose to ethanol include the lack of low-cost efficient enzymes for saccharification of biomass to fermentable sugars and the development of microorganisms for the fermentation of these mixed sugars. To date, the most successful research approaches to develop novel biocatalysts that will efficiently ferment mixed sugar syrups include isolation of novel yeasts that ferment xylose, genetic engineering of Escherichia coli and other gram negative bacteria for ethanol production, and genetic engineering of Saccharomyces cerevisiae and Zymomonas mobilis for pentose utilization. The authors have evaluated the fermentation of corn fiber hydrolyzates by the various strains developed. E. coli K011, E. coli SL40, E. coli FBR3, Zymomonas CP4 (pZB5), and Saccharomyces 1400 (pLNH32) fermented corn fiber hydrolyzates to ethanol in the range of 21--34 g/L with yields ranging from 0.41 to 0.50 g of ethanol per gram of sugar consumed. Progress with new recombinant microorganisms has been rapid and will continue with the eventual development of organisms suitable for commercial ethanol production. Each research approach holds considerable promise, with the possibility existing that different industrially hardened strains may find separate applications in the fermentation of specific feedstocks.

  7. Recombination walking: Genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination

    SciTech Connect

    Miller, A.M.; Savinelli, E.A.; Couture, S.M.; Hannigan, G.M.; Han, Z.; Selden, R.F.; Treco, D.A. )

    1993-09-01

    Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unorderd) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. 29 refs., 4 figs., 1 tab.

  8. Tunnel surface recombination in optoelectronic device modeling

    NASA Astrophysics Data System (ADS)

    Ptashchenko, Alexander A.; Ptashchenko, Fedor A.

    1997-08-01

    The rate of tunnel surface recombination (TSR) in a p-n structure has been calculated as a function of the excitation level and temperature in a semiclassical approximation under the assumption that the excess energy of a recombining electron is transferred to phonons or to a photon. The approximating analytical expressions obtained are applied in calculations of the effect of TSR on the characteristics of photodiodes, solar cells, light-emitting diodes and diode lasers.

  9. Recombination-deficient mutant of Streptococcus faecalis

    SciTech Connect

    Yagi, Y.; Clewell, D.B.

    1980-08-01

    An ultraviolet radiation-sensitive derivative of Streptococcus faecalis strain JH2-2 was isolated and found to be deficient in recombination, using a plasmid-plasmid recombination system. The strain was sensitive to chemical agents which interact with deoxyribonucleic acid and also underwent deoxyribonucleic acid degradation after ultraviolet irradiation. Thus, the mutant has properties similar to those of recA strains of Escherichia coli.

  10. Recombination-generation currents in degenerate semiconductors

    NASA Technical Reports Server (NTRS)

    Von Roos, O.

    1978-01-01

    The classical Shockley-Read-Hall theory of free carrier recombination and generation via traps is extended to degenerate semiconductors. A concise and simple expression is found which avoids completely the concept of a Fermi level, a concept which is alien to nonequilibrium situations. Assumptions made in deriving the recombination generation current are carefully delineated and are found to be basically identical to those made in the original theory applicable to nondegenerate semiconductors.

  11. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  12. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  13. Recombination hotspots: Models and tools for detection.

    PubMed

    Paul, Prosenjit; Nag, Debjyoti; Chakraborty, Supriyo

    2016-04-01

    Recombination hotspots are the regions within the genome where the rate, and the frequency of recombination are optimum with a size varying from 1 to 2kb. The recombination event is mediated by the double-stranded break formation, guided by the combined enzymatic action of DNA topoisomerase and Spo 11 endonuclease. These regions are distributed non-uniformly throughout the human genome and cause distortions in the genetic map. Numerous lines of evidence suggest that the number of hotspots known in humans has increased manifold in recent years. A few facts about the hotspot evolutions were also put forward, indicating the differences in the hotspot position between chimpanzees and humans. In mice, recombination hot spots were found to be clustered within the major histocompatibility complex (MHC) region. Several models, that help explain meiotic recombination has been proposed. Moreover, scientists also developed some computational tools to locate the hotspot position and estimate their recombination rate in humans is of great interest to population and medical geneticists. Here we reviewed the molecular mechanisms, models and in silico prediction techniques of hot spot residues. PMID:26991854

  14. Biochemistry of homologous recombination in Escherichia coli.

    PubMed Central

    Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M

    1994-01-01

    Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921

  15. Detecting the cosmological recombination signal from space

    NASA Astrophysics Data System (ADS)

    Desjacques, Vincent; Chluba, Jens; Silk, Joseph; de Bernardis, Francesco; Doré, Olivier

    2015-08-01

    Spectral distortions of the cosmic microwave background (CMB) have recently experienced an increased interest. One of the inevitable distortion signals of our cosmological concordance model is created by the cosmological recombination process, just a little before photons last scatter at redshift z ≃ 1100. These cosmological recombination lines, emitted by the hydrogen and helium plasma, should still be observable as tiny deviation from the CMB blackbody spectrum in the cm-dm spectral bands. In this paper, we present a forecast for the detectability of the recombination signal with future satellite experiments. We argue that serious consideration for future CMB experiments in space should be given to probing spectral distortions and, in particular, the recombination line signals. The cosmological recombination radiation not only allows determination of standard cosmological parameters, but also provides a direct observational confirmation for one of the key ingredients of our cosmological model: the cosmological recombination history. We show that, with present technology, such experiments are futuristic but feasible. The potential rewards won by opening this new window to the very early universe could be considerable.

  16. Multiplex PCR Method for Identifying Recombinant Vaccine-Related Polioviruses

    PubMed Central

    Kilpatrick, David R.; Ching, Karen; Iber, Jane; Campagnoli, Ray; Freeman, Christopher J.; Mishrik, Nada; Liu, Hong-Mei; Pallansch, Mark A.; Kew, Olen M.

    2004-01-01

    The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR. PMID:15365031

  17. Oligonucleotide recombination enabled site-specific mutagenesis in bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligo recombination is one type of recombineering that uses ssDNA olig...

  18. The Saccharomyces cerevisiae recombination enhancer biases recombination during interchromosomal mating-type switching but not in interchromosomal homologous recombination.

    PubMed Central

    Houston, Peter; Simon, Peter J; Broach, James R

    2004-01-01

    Haploid Saccharomyces can change mating type through HO-endonuclease cleavage of an expressor locus, MAT, followed by gene conversion using one of two repository loci, HML or HMR, as donor. The mating type of a cell dictates which repository locus is used as donor, with a cells using HML and alpha cells using HMR. This preference is established in part by RE, a locus on the left arm of chromosome III that activates the surrounding region, including HML, for recombination in a cells, an activity suppressed by alpha 2 protein in alpha cells. We have examined the ability of RE to stimulate different forms of interchromosomal recombination. We found that RE exerted an effect on interchromosomal mating-type switching and on intrachromosomal homologous recombination but not on interchromosomal homologous recombination. Also, even in the absence of RE, MAT alpha still influenced donor preference in interchromosomal mating-type switching, supporting a role of alpha 2 in donor preference independent of RE. These results suggest a model in which RE affects competition between productive and nonproductive recombination outcomes. In interchromosome gene conversion, RE enhances both productive and nonproductive pathways, whereas in intrachromosomal gene conversion and mating-type switching, RE enhances only the productive pathway. PMID:15082540

  19. DNA RECOMBINATION IN EUCARYOTIC CELLS BY THE BACTERIOPHAGE PHIC31 RECOMBINATION SYSTEM",

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the ph:C31 integrase, that can mediate recombination between th...

  20. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  1. Graded recombination layers for multijunction photovoltaics.

    PubMed

    Koleilat, Ghada I; Wang, Xihua; Sargent, Edward H

    2012-06-13

    Multijunction devices consist of a stack of semiconductor junctions having bandgaps tuned across a broad spectrum. In solar cells this concept is used to increase the efficiency of photovoltaic harvesting, while light emitters and detectors use it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron current from the next cell. We recently reported a tandem solar cell in which the recombination layer was implemented using a progression of n-type oxides whose doping densities and work functions serve to connect, with negligible resistive loss at solar current densities, the constituent cells. Here we present the generalized conditions for design of efficient graded recombination layer solar devices. We report the number of interlayers and the requirements on work function and doping of each interlayer, to bridge an work function difference as high as 1.6 eV. We also find solutions that minimize the doping required of the interlayers in order to minimize optical absorption due to free carriers in the graded recombination layer (GRL). We demonstrate a family of new GRL designs experimentally and highlight the benefits of the progression of dopings and work functions in the interlayers. PMID:22554234

  2. Recombination in Eukaryotic Single Stranded DNA Viruses

    PubMed Central

    Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-01-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

  3. Perinatal induction of Cre recombination with tamoxifen.

    PubMed

    Lizen, Benoit; Claus, Melissa; Jeannotte, Lucie; Rijli, Filippo M; Gofflot, Françoise

    2015-12-01

    Temporal control of site-specific recombination is commonly achieved by using a tamoxifen-inducible form of Cre or Flp recombinases. Although powerful protocols of induction have been developed for gene inactivation at adult stages or during embryonic development, induction of recombination at late gestational or early postnatal stages is still difficult to achieve. In this context, using the ubiquitous CMV-CreER(T2) transgenic mice, we have tested and validated two procedures to achieve recombination just before and just after birth. The efficiency of recombination was evaluated in the brain, which is known to be more problematic to target. For the late gestation treatment with tamoxifen, different protocols of complementary administration of progesterone and estrogen were tested. However, delayed delivery and/or mortality of pups due to difficult delivery were always observed. To circumvent this problem, pups were collected from tamoxifen-treated pregnant dams by caesarian section at E18.5 and given to foster mothers. For postnatal treatment, different dosages of tamoxifen were administered by intragastric injection to the pups during 3 or 4 days after birth. The efficiency of these treatments was analyzed at P7 using a transgenic reporter line. They were also validated with the Hoxa5 conditional allele. In conclusion, we have developed efficient procedures that allow achieving efficient recombination of floxed alleles at perinatal stages. These protocols will allow investigating the late/adult functions of many developmental genes, whose characterization has been so far restricted to embryonic development. PMID:26395370

  4. Recombination and collisionally excited Balmer lines

    NASA Astrophysics Data System (ADS)

    Raga, A. C.; Castellanos-Ramírez, A.; Esquivel, A.; Rodríguez-González, A.; Velázquez, P. F.

    2015-10-01

    We present a model for the statistical equilibrium of the levels of H, considering recombinations to excited levels, collisional excitations up from the ground state and spontaneous radiative transitions. This problem has a simple "cascade matrix" solution, describing a cascade of downwards spontaneous transitions fed by both recombinations and collisional excitations. The resulting predicted Balmer line ratios show a transition between a low temperature and a high temperature regime (dominated by recombinations and by collisional excitations, respectively), both with only a weak line ratio vs. temperature dependence. This clear characteristic allows a direct observational identification of regions in which the Balmer lines are either recombination or collisionally excited transitions. We find that for a gas in coronal ionization equilibrium the Halpha and Hbeta lines are collisionally excited for all temperatures. In order to have recombination Halpha and Hbeta it is necessary to have higher ionization fractions of H than the ones obtained from coronal equilibrium (e.g., such as the ones found in a photoionized gas).

  5. Jet fragmentation via recombination of parton showers

    NASA Astrophysics Data System (ADS)

    Han, Kyong Chol; Fries, Rainer J.; Ko, Che Ming

    2016-04-01

    We propose to model hadronization of parton showers in QCD jets through a hybrid approach involving quark recombination and string fragmentation. This is achieved by allowing gluons at the end of the perturbative shower evolution to undergo a nonperturbative splitting into quark and antiquark pairs, then applying a Monte Carlo version of instantaneous quark recombination, and finally subjecting remnant quarks (those which have not found a recombination partner) to Lund string fragmentation. When applied to parton showers from the pythia Monte Carlo event generator, the final hadron spectra from our calculation compare quite well to pythia jets that have been hadronized with the default Lund string fragmentation. Our new approach opens up the possibility to generalize hadronization to jets embedded in a quark gluon plasma.

  6. Meiotic recombination and genome evolution in plants.

    PubMed

    Melamed-Bessudo, Cathy; Shilo, Shay; Levy, Avraham A

    2016-04-01

    Homologous recombination affects genome evolution through crossover, gene conversion and point mutations. Whole genome sequencing together with a detailed epigenome analysis have shed new light on our understanding of how meiotic recombination shapes plant genes and genome structure. Crossover events are associated with DNA sequence motifs, together with an open chromatin signature (hypomethylated CpGs, low nucleosome occupancy or specific histone modifications). The crossover landscape may differ between male and female meiocytes and between species. At the gene level, crossovers occur preferentially in promoter regions in Arabidopsis. In recent years, there is rising support suggesting that biased mismatch repair during meiotic recombination may increase GC content genome-wide and may be responsible for the GC content gradient found in many plant genes. PMID:26939088

  7. Dielectronic recombination of xenonlike tungsten ions

    SciTech Connect

    Schippers, S.; Bernhardt, D.; Mueller, A.; Krantz, C.; Grieser, M.; Repnow, R.; Wolf, A.; Lestinsky, M.; Hahn, M.; Novotny, O.; Savin, D. W.

    2011-01-15

    Dielectronic recombination (DR) of xenonlike W{sup 20+} forming W{sup 19+} has been studied experimentally at a heavy-ion storage ring. A merged-beams method has been employed for obtaining absolute rate coefficients for electron-ion recombination in the collision-energy range 0-140 eV. The measured rate coefficient is dominated by strong DR resonances even at the lowest experimental energies. At plasma temperatures where the fractional abundance of W{sup 20+} is expected to peak in a fusion plasma, the experimentally derived plasma recombination rate coefficient is over a factor of 4 larger than the theoretically calculated rate coefficient which is currently used in fusion plasma modeling. The largest part of this discrepancy stems most probably from the neglect in the theoretical calculations of DR associated with fine-structure excitations of the W{sup 20+}([Kr]4d{sup 10} 4f{sup 8}) ion core.

  8. Transposon-specified site-specific recombination.

    PubMed Central

    Kitts, P; Symington, L; Burke, M; Reed, R; Sherratt, D

    1982-01-01

    Cointegrate DNA molecules containing two copies of a transposable element appear to be intermediates in the transposition process. These structures are resolved by site-specific recombination to yield the normal end products of transposition. The transposable element gamma delta (Tn1000) synthesizes a product interchangeable with the Tn1/3tnpR protein in promoting Tn1/3 site-specific recombination. These data support the hypothesis that cointegrates containing directly repeated copies of Tn1/3 are obligatory intermediates in interreplicon transposition of Tn1/3. In addition, we show here that the reaction is independent of the element-encoded tnpA gene product. Tn501, which specifies mercury resistance, also produces cointegrates as intermediates in interreplicon transposition. The appearance of Tn501-specified recombination activity that can act on these cointegrates requires growth of cells in the presence of Hg2+. Images PMID:6275390

  9. Recombination and DNA Repair in Helicobacter pylori

    PubMed Central

    Dorer, Marion S.; Sessler, Tate H.; Salama, Nina R.

    2013-01-01

    All organisms have pathways that repair the genome, ensuring their survival and that of their progeny. But these pathways also serve to diversify the genome, causing changes on the level of nucleotide, whole gene, and genome structure. Sequencing of bacteria has revealed wide allelic diversity and differences in gene content within the same species, highlighting the importance of understanding pathways of recombination and DNA repair. The human stomach pathogen Helicobacter pylori is an excellent model system for studying these pathways. H. pylori harbors major recombination and repair pathways and is naturally competent, facilitating its ability to diversify its genome. Elucidation of DNA recombination, repair, and diversification programs in this pathogen will reveal connections between these pathways and their importance to infection. PMID:21682641

  10. Transcription and Recombination: When RNA Meets DNA

    PubMed Central

    Aguilera, Andrés; Gaillard, Hélène

    2014-01-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910