ABSTRACT
Proteins such as aminopeptidases and alkaline phosphatases, both glycosyl-phosphatidyl-inositol (GPI) anchored proteins, were previously identified as Cry1Ac binding proteins in the Heliothis virescens midgut. To identify additional toxin binding proteins, brush border membrane vesicles from H. virescens larvae were treated with phosphatidyl inositol phospholipase C, and released proteins were resolved by two-dimensional electrophoresis. Protein spots selected by their ability to bind Cry1Ac were identified by MALDI-TOF mass spectrometry coupled to peptide mass fingerprinting (PMF) and database searching. As in previous studies, H. virescens alkaline phosphatase was identified as a Cry1Ac binding protein. V-ATP synthase subunit A and actin were identified as novel Cry1Ac binding proteins in H. virescens. Additional toxin-binding proteins were predicted based on MS/MS fragmentation and de novo sequencing, providing amino acid sequences that were used in database searches to identify a phosphatase and a putative protein of the cadherin superfamily as additional Cry1Ac binding proteins.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Endotoxins/metabolism , Hemolysin Proteins/metabolism , Insect Proteins/chemistry , Microvilli/chemistry , Moths/chemistry , Proteomics , Receptors, Cell Surface/chemistry , Animals , Bacillus thuringiensis Toxins , Electrophoresis, Gel, Two-Dimensional , Gastrointestinal Tract/chemistry , Gastrointestinal Tract/cytology , Glycosylphosphatidylinositols/metabolism , Mass Spectrometry , Peptide MappingSubject(s)
Basophils/pathology , Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , Leukemia, Myeloid/genetics , Leukemia, Myeloid/pathology , Translocation, Genetic , Acute Disease , Bone Marrow Cells/pathology , Child, Preschool , Core Binding Factor Alpha 2 Subunit , Female , Humans , Oncogene Proteins, Fusion/metabolism , RUNX1 Translocation Partner 1 Protein , Transcription Factors/metabolismABSTRACT
Immunocompromised children, including those undergoing chemotherapy treatment of malignant disease, are at particular risk for infection with parvovirus B19. However, these patients' attenuated immune responses may obscure the serologic and clinical manifestations of the infection. The authors describe a patient undergoing induction therapy for acute lymphoblastic leukemia whose parvovirus B19 infection was identified by the incidental detection of giant pronormoblasts and absence of normal mature erythroid precursors, characteristic of parvovirus infection, on a routine bone marrow examination. Intravenous immunoglobulin was administered and the patient's aplastic anemia resolved completely within 3 weeks. This highlights the importance of alertness to the possibility of parvovirus infection in children with cancer.
Subject(s)
Anemia, Aplastic/etiology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Burkitt Lymphoma/complications , Parvoviridae Infections/diagnosis , Parvovirus B19, Human/isolation & purification , Asparaginase/administration & dosage , Blood Transfusion , Burkitt Lymphoma/drug therapy , Child , Combined Modality Therapy , Cytarabine/administration & dosage , Daunorubicin/administration & dosage , Etoposide/administration & dosage , Female , Humans , Immunocompromised Host , Immunoglobulins, Intravenous/therapeutic use , Methotrexate/administration & dosage , Parvoviridae Infections/complications , Parvoviridae Infections/therapy , Prednisone/administration & dosage , Remission Induction , Vincristine/administration & dosageABSTRACT
Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.
Subject(s)
Bacillus Phages/genetics , Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/toxicity , Bacterial Toxins , Endotoxins/genetics , Endotoxins/toxicity , Insecticides , Peptide Library , Amino Acid Sequence , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacteriophage lambda/genetics , Endotoxins/chemistry , Endotoxins/metabolism , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Engineering , Genetic Vectors , Hemolysin Proteins , Immunoblotting , Molecular Sequence Data , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/toxicityABSTRACT
Erythroid Krüppel-like factor (EKLF) is a trans-acting factor that binds specifically to the beta-globin promoter CACCC box. EKLF is essential for adult erythroid development in mice. We have cloned and sequenced a cDNA encoding the human homologue of this gene, which shares 69% identity with the mEKLF protein. The expression of hEKLF is restricted to fetal liver and adult bone marrow. The genomic locus of hEKLF was mapped to chromosomal band 19p13.12-p13.13, using fluorescence in situ hybridization.
Subject(s)
Chromosomes, Human, Pair 19 , DNA-Binding Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Humans , In Situ Hybridization, Fluorescence , Kruppel-Like Transcription Factors , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Prediction of airborne concentrations of enviornmentally important contaminants is supported by field test data on tobacco smoke. The analysis discussed herein is completely general in nature, and the contaminants can be gases, vapors, liquid droplets, and solid particulates, including microorganisms and pollens. Several engineering controls can be applied to practical environnmental systems to reduce and control undesirable contaminants in normally occupied structures. The use of the method is an important research tool to more accurately quantify airborne contaminant levels in enviornmental medicine experiments with animals or human subjects. It is difficult and expensive to control inside environmental contaminant levels at values less than 20% to 25% of those occurring outside. The same is true for internally generated contaminants.
