scholarly journals Inhibition of Fungal β-1,3-Glucan Synthase and Cell Growth by HM-1 Killer Toxin Single-Chain Anti-Idiotypic Antibodies

2006 ◽  
Vol 50 (9) ◽  
pp. 3090-3097 ◽  
Author(s):  
Dakshnamurthy Selvakumar ◽  
Masahiko Miyamoto ◽  
Yasuhiro Furuichi ◽  
Tadazumi Komiyama
Keyword(s):  
Recombinant Dna ◽  
Killer Toxin ◽  
Binding Assays ◽  
Single Chain ◽  
Recombinant Dna Technology ◽  
Surface Plasmon Resonance Analysis ◽  
Glucan Synthase ◽  
Recombinant Scfv ◽  
Neutralizing Monoclonal Antibody

ABSTRACT Single-chain variable-fragment (scFv) anti-idiotypic antibodies of an HM-1 killer toxin (HM-1) from the yeast Williopsis saturnus var. mrakii IFO 0895 have been produced by recombinant DNA technology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a neutralizing monoclonal antibody (nMAb-KT). The fungicidal activity of scFv anti-idiotypic antibodies against the isolates of four Candida species was assessed by MIC analysis. scFv antibodies were fungicidal at concentrations of 1.56 to 12.5 μg/ml in vitro against four Candida species. The scFv antibodies exerted a strong candidacidal activity in vitro, with 50% inhibitory concentration (IC50) values ranging from 7.3 × 10−8 to 16.0 × 10−8 M, and were neutralized by adsorption with nMAb-KT. Furthermore, all scFv antibodies effectively inhibited fungal β-1,3-glucan synthase activity in vitro, with IC50 values ranging from 2.0 × 10−8 to 22.7 × 10−8 M, values which almost coincide with the values that are inhibitory to the growth of fungal cells. Binding assays showed that the scFv antibodies specifically bind to nMAb-KT, and this binding pattern was confirmed by surface plasmon resonance analysis. The binding ability was further demonstrated by the competition observed between scFv antibodies and HM-1 to bind nMAb-KT. To the best of our knowledge, this is the first study to show that an antifungal anti-idiotypic antibody, in the form of recombinant scFv, potentially inhibits β-1,3-glucan synthase activity.

scholarly journals The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential.

1996 ◽  
Vol 133 (2) ◽  
pp. 359-369 ◽  
Author(s):  
N A Chitaev ◽  
R E Leube ◽  
R B Troyanovsky ◽  
L G Eshkind ◽  
W W Franke ◽  
...  
Keyword(s):  
Recombinant Dna ◽  
Chimeric Protein ◽  
Binding Assays ◽  
Recombinant Dna Technology ◽  
Desmosomal Cadherins ◽  
Binding Domains ◽  
Transmembrane Glycoprotein ◽  
E Cadherin

Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C-domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so-called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.

Interferons in the treatment of human cancer.

1984 ◽  
Vol 2 (4) ◽  
pp. 336-352 ◽  
Author(s):  
J M Kirkwood ◽  
M S Ernstoff
Keyword(s):  
Recombinant Dna ◽  
Human Cancer ◽  
Clinical Results ◽  
Common Species ◽  
Current Phase ◽  
Recombinant Dna Technology ◽  
Disease States ◽  
Wide Range

The interferons are the best known of biologic antineoplastic agents. Progress with the clinical application of interferons to cancer has been slow and complicated by the need for attention to a new spectrum of therapeutic and toxic effects manifest by the interferons. This summary of current phase I and II trial results with the interferons establishes their clinical potential. The maximally tolerated dosages of the most common species of interferon alpha produced in eukaryotic cells as well as by recombinant DNA technology in bacteria are now described in a variety of different disease states. "Naturally" produced eukaryotic as well as bacterially synthesized interferons have a similar, wide range of biologic effects in vitro and in vivo. Antiviral, antiproliferative, immunologic, and enzymologic functions of the interferons relevant to antineoplastic functions are under study. Knowledge of these mechanisms should improve the clinical results obtained in human cancer. Species and subspecies differences in the activity of interferons may lead to selective use of the pure interferon subspecies, alone or in combination. The use of the interferons and other antineoplastic biologics, such as antibody or chemotherapy, are subsequent goals that are now on the horizon.

scholarly journals Therapeutic Activity of an Engineered Synthetic Killer Antiidiotypic Antibody Fragment against Experimental Mucosal and Systemic Candidiasis

2003 ◽  
Vol 71 (11) ◽  
pp. 6205-6212 ◽  
Author(s):  
Luciano Polonelli ◽  
Walter Magliani ◽  
Stefania Conti ◽  
Luisa Bracci ◽  
Luisa Lozzi ◽  
...  
Keyword(s):  
Wide Spectrum ◽  
Model Organism ◽  
Killer Toxin ◽  
Antibody Fragment ◽  
Systemic Infection ◽  
Vaginal Candidiasis ◽  
Single Chain ◽  
Significant Prolongation ◽  
Antiidiotypic Antibody

ABSTRACT Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exerted a strong candidacidal activity in vitro, with a 50% inhibitory concentration of 0.056 μM, and was therefore designated killer peptide (KP). Its activity was neutralized by laminarin, a β1-3 glucan molecule, but not by pustulan, a β1-6 glucan molecule. KP also competed with the binding of a KT-like monoclonal IdAb to germinating cells of the fungus. In a rat model of vaginal candidiasis, local, postchallenge administration of KP was efficacious in rapidly abating infections caused by fluconazole-susceptible or -resistant C. albicans strains. In systemic infection of BALB/c or SCID mice preinfected intravenously with a lethal fungal load, KP caused a highly significant prolongation of the median survival time, with >80% of the animals still surviving after >60 days, whereas >90% of control mice died within 3 to 5 days. KP is therefore the first engineered peptide derived from a recombinant IdAb retaining KT microbicidal activity, probably through the interaction with the β-glucan KT receptor on target microbial cells.

scholarly journals Bone morphogenetic protein 2: heterologous expression and potential in bone regeneration

2021 ◽  
Vol 43 (114) ◽  
Author(s):  
Andrea Mesa Restrepo ◽  
Juan Fernando Alzate ◽  
Edwin Bairon Patiño Gonzalez
Keyword(s):  
Heterologous Expression ◽  
Bone Morphogenetic Protein ◽  
Recombinant Dna ◽  
Redox System ◽  
Bone Morphogenetic Protein 2 ◽  
Recombinant Dna Technology ◽  
Morphogenetic Protein ◽  
Alkaline Phosphate ◽  
Heterologous Expression Systems

Currently, bone morphogenetic protein 2 (BMP-2) is one of the two osteoinductive growth factors used in medical devices to promote bone formation. Typically, this protein is bought from commercial houses at high rates and in small quantities that are not enough to cover clinical needs. Because of this, it has been proposed that research centers use their own heterologous expression systems to have a constant supply of BMP-2. The aim of this study was to standardize the heterologous expression of BMP-2 and evaluate its osteoinductive activity in vitro. Our procedure for expression and purification was based on recombinant DNA technology using the plasmid pET-28 and IPTG as inductor. After extracting the protein from inclusion bodies, folding it and modifying it via a redox system, we observed via electrophoresis a 26 kDa dimer. We evaluated its osteoinductive activity in myoblastic C2C12 by quantifying enzymatically the activity of alkaline phosphate (ALP) and staining mineralization nodules. ALP activity is proportional to BMP-2 concentration, increasing 90% at 3 µg/mL. These cells form calcium nodules, mineralizing 50% of the area.

scholarly journals James Watson’s genius (dedicated to the 90th birth anniversary)

2018 ◽  
Vol 22 ◽  
pp. 363-367
Author(s):  
H. B. Humeniuk ◽  
M. Z. Mosula ◽  
I. B. Chen ◽  
N. M. Drobyk
Keyword(s):  
Molecular Biology ◽  
Nobel Prize ◽  
Cell Biology ◽  
Recombinant Dna ◽  
Double Helix ◽  
X Rays ◽  
Recombinant Dna Technology ◽  
Molecular Oncology ◽  
Double Helix Structure

The scientific and organizational activities of the worldwide known scientist in the field of molecular biology James Dewey Watson were described in this article. 55 years ago James Watson and Francis Crick made one of the key discoveries of the twentieth century. They have found that DNA has a double helix structure. This discovery was based on the X-rays patterns obtained by Maurice Wilkson and Rosalind Franklin. Subsequently, this DNA model had been proved, and J. Watson and F. Crick were awarded with the Nobel Prize in Physiology or Medicine in 1962. Since, our knowledge of the main molecule of life has been greatly expanded. A significant flowering of molecular genetics has began: synthesis of RNA and DNA in vitro, decoding of genetic code, recombinant DNA technology, genetic engineering, sequencing of genomes and post genomic technologies. James Watson is one of the authors of the cell biology classic textbook “Molecular Biology of the Cell”. In addition, he has developed the current areas of molecular biology such as  molecular oncology and molecular neurobiology. Today genomes of different animals and humans have been decoded and the functions of many genes have been determined. But at present still unknown how the DNA starts and how it affects the work of the organs and the organism as a system. Keywords: James Watson, DNA structure, Nobel Prize in physiology or medicine, Molecular Biology of the Cell.

scholarly journals Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts

2021 ◽  
Vol 22 (19) ◽  
pp. 10740
Author(s):  
Magdalena Klimek-Chodacka ◽  
Miron Gieniec ◽  
Rafal Baranski
Keyword(s):  
Polyethylene Glycol ◽  
Dna Cleavage ◽  
Gene Editing ◽  
Recombinant Dna ◽  
Recombinant Dna Technology ◽  
Culture Methods ◽  
Target Dna ◽  
Fluorescent Pcr ◽  
Carrot Protoplasts

The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33–152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.

In Vitro Carboxylation of a Blood Coagulation Factor IX Precursor Produced by Recombinant-DNA Technology

1989 ◽  
Vol 61 (02) ◽  
pp. 238-242 ◽  
Author(s):  
Berry A M Soute ◽  
Alain Balland ◽  
Thérèse Faure ◽  
Henri de la Salle ◽  
Cees Vermeer
Keyword(s):  
Blood Coagulation ◽  
Recombinant Dna ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Recombinant Dna Technology ◽  
Coagulation Factor Ix ◽  
Recombinant Factor Ix

SummaryBlood coagulation factor IX (Christmas factor) is a plasma protein which is required for normal haemostasis. A functional deficiency of factor IX results in haemophilia B, a bleeding disorder which is generally treated by infusions of factor IX concentrates prepared from pooled human plasma. The use of human blood products is connected with the risk of transmitting viral agents responsible for diseases such as hepatitis B and AIDS. Recombinant DNA techniques may provide the means to produce the required proteins without exposing the patients to these risks and at lower costs. One of the problems which has to be overcome before recombinant factor IX can be used for therapeutical purposes is related to the vitamin K-dependent carboxylation of its 12 NH2-terminal glutamate residues. In cell cultures this carboxylation, which is required to render the protein its procoagulant activity, is far from complete, especially at high expression levels. In this paper we describe the in vitro carboxylation of non and/or partly carboxylated recombinant factor IX produced by transformed Chinese hamster ovary cells. The identity of the newly formed Gla residues was verified and it could be demonstrated that all carboxyl groups had been incorporated into the recombinant factor IX.

The Parkes lecture: controlled ovarian stimulation in women

Reproduction ◽  
2000 ◽  
pp. 201-210 ◽  
Author(s):  
SG Hillier
Keyword(s):  
Ovarian Stimulation ◽  
Ovarian Function ◽  
Recombinant Dna ◽  
New Drugs ◽  
Controlled Ovarian Stimulation ◽  
Recombinant Dna Technology ◽  
Vitro Fertilization ◽  
Ovarian Folliculogenesis

Recent advances in knowledge of the endocrine and paracrine mechanisms that regulate human ovarian folliculogenesis have been parallelled by the introduction into clinical practice of new drugs that can be used safely and effectively to stimulate ovarian function in infertile women. Most notably, recombinant DNA technology has been applied to the production of molecularly pure forms of the gonadotrophins, FSH and LH, opening the way to the development of improved strategies for manipulating the ovarian paracrine system. The clinical objectives of controlled ovarian stimulation fall into two categories, depending on patient needs: (1) induction of multiple follicles from which mature oocytes can be harvested for use in assisted reproduction protocols such as in vitro fertilization and embryo transfer; or (2) induction of spontaneous ovulation of a single mature follicle so that conception might occur in vivo. This review summarizes the physiological principles upon which the use of gonadotrophins for clinical purposes is based, highlighting new opportunities for improved treatment as a result of the availability of recombinant FSH and LH.

scholarly journals Immunoregulatory role of transforming growth factor beta (TGF-beta) in development of killer cells: comparison of active and latent TGF-beta 1.

1990 ◽  
Vol 172 (6) ◽  
pp. 1777-1784 ◽  
Author(s):  
S C Wallick ◽  
I S Figari ◽  
R E Morris ◽  
A D Levinson ◽  
M A Palladino
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Recombinant Dna ◽  
Chinese Hamster ◽  
Tgf Beta ◽  
Recombinant Dna Technology ◽  
Growth Factor Beta

Using recombinant DNA technology, we have generated Chinese hamster ovary (CHO) cell lines that synthesize latent transforming growth factor beta 1 (TGF-beta 1) to study immune regulation by TGF-beta 1. In vitro, latent TGF-beta 1 synthesized by transfectants or added exogenously as a purified complex after activation inhibited CTL generation to a similar extent as seen with acid-activated recombinant human (rHu) TGF-beta 1. In vivo, serum from nu/nu mice bearing CHO/TGF-beta 1 tumors contained significant levels of latent TGF-beta 1 in addition to depressed natural killer (NK) activity in spleens which paralleled that seen in C3H/HeJ mice treated with acid-activated rHuTGF-beta 1. rHuTGF-beta 1 treatment of mice receiving heart allografts resulted in significant enhancement of organ graft survival. Because of possible regulated tissue-specific activation, administration of latent rather than active TGF-beta may provide a better route to deliver this powerful immunosuppressive agent in vivo.

Sign in / Sign up

Export Citation Format

Share Document