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ABSTRACT: The RadA/Sms protein facilitates DNA repair in Escherichia coli cells damaged by UV radiation, X-rays, and chemical agents. However, the precise mechanism by which RadA/Sms aids DNA repair is unknown. Here we report the production of monoclonal antibodies (MAbs) specific for RadA/Sms for use in biochemical and physiological investigations. Histidine-tagged RadA/Sms (RadA-6xHis) was overproduced in E. coli BL21 cells transformed with the radA/sms coding region in plasmid pRSET A and purified by nickel affinity chromatography. Splenocytes from female BALB/c mice hyperimmunized with the purified protein were fused to SP2/0-Ag14 myeloma cells, and the resultant hybridomas were selected in HAT medium. MAbs were detected in hybridoma culture supernatants by indirect ELISA and Western blot analysis against purified RadA-6xHis. MAbs from four cell lines were further evaluated by Western blotting against peptide maps generated by endoproteinase Glu-C digestion of RadA-6xHis. Each of the four MAbs recognized a unique epitope on the fusion protein. Two of the MAbs (6F5 and 2A2) also detected wild-type (tagless) RadA/Sms produced from the pJS003 plasmid in E. coli K-12 cells. We anticipate that these antibodies will prove useful for the detection, isolation, and functional analysis of RadA/Sms.
Hybridoma (2005) 02/2012; 31(1):25-31. · 0.42 Impact Factor
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ABSTRACT: Small-group case presentation exercises (CPs) were created to increase course relevance for medical students taking Medical Microbiology (MM) and Infectious Diseases (ID) METHODS: Each student received a unique paper case and had 10 minutes to review patient history, physical exam data, and laboratory data. Students then had three minutes to orally present their case and defend why they ruled in or out each of the answer choices provided, followed by an additional three minutes to answer questions.
Exam scores differed significantly between students who received the traditional lecture-laboratory curriculum (Group I) and students who participated in the CPs (Group II). In MM, median unit exam and final exam scores for Group I students were 84.4% and 77.8%, compared to 86.0% and 82.2% for Group II students (P<0.018; P<0.001; Mann-Whitney Rank Sum Test). Median unit and final ID exam scores for Group I students were 84.0% and 80.0%, compared to 88.0% and 86.7% for Group II students (P<0.001; P<0.001).
Students felt that the CPs improved their critical thinking and presentation skills and helped to prepare them as future physicians.
Medical Education Online 01/2012; 17.
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ABSTRACT: Monoclonal antibody (Mab) 8B7 was shown in a previous study to inhibit protein translation in lysates of Sf21 cells. The antibody was thought to be specific for a 60-kDa form of elongation factor-1 alpha (EF-1alpha), primarily because the antigen immunoprecipitated by Mab 8B7 cross-reacted with Mab CBP-KK1, an antibody generated to EF-1alpha from Trypanosoma brucei. The purpose of the current study was to investigate further the antigenic specificity of Mab 8B7. The concentration of the 60-kDa antigen relative to total cellular protein proved insufficient for its definitive identification. However, subcellular fractionation of Sf21 cells yielded an additional protein of 37 kDa in the cytosolic and microsomal fractions that was reactive with Mab 8B7. The 37-kDa protein could be easily visualized by colloidal Coomassie Blue G-250 staining as a series of pI 6.9-8.4 spots on two-dimensional gels. Excision of an abundant immunoreactive spot enabled identification of the protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and protein database searching. Subsequent immunoblotting of purified rabbit skeletal muscle GAPDH with Mab 8B7 confirmed the antibody's specificity for GAPDH. Besides the pivotal role GAPDH plays in glycolysis, the enzyme has a number of noncanonical functions, including binding to mRNA and tRNA. The ability of Mab 8B7 to disrupt these lesser-known functions of GAPDH may account for the antibody's inhibitory effect on in vitro translation.
Archives of Insect Biochemistry and Physiology 11/2008; 69(3):107-17. · 1.36 Impact Factor
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ABSTRACT: Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain alpha-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12 degrees C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.
Applied and environmental microbiology 09/2008; 74(19):5882-90. · 3.69 Impact Factor
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ABSTRACT: Transient fluctuations in immune function after heavy exercise have been linked to an increased incidence of infection in athletes. Several parameters of immunity, including salivary immunoglobulin A (IgA), are affected by heavy exercise in the laboratory setting. However, few observations have been made during true competition. We tested the hypothesis that salivary IgA levels will be decreased after a collegiate rugby game. Saliva samples obtained from 16 men's college rugby players before and after an 80-minute regulation rugby game were analyzed for total volume, IgA, total protein content, and osmolality. Salivary IgA was expressed relative to secretion rate (s-IgA), osmolality (IgA-Osm), and total protein (IgA-Pro). No significant pregame-postgame changes in salivary IgA were observed (s-IgA: -13%, IgA-Osm: -16%, IgA-Pro: +10%). These data indicate that strenuous physical activity, such as a competitive rugby game, does not affect IgA levels. More study on the immune response to athletic competition is needed.
The Journal of Strength and Conditioning Research 03/2007; 21(1):86-90. · 1.83 Impact Factor
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ABSTRACT: Elongation factor-1alpha (EF-1alpha) promotes the delivery of aminoacyl-tRNA to the acceptor site of the ribosome during protein synthesis. The enzyme has a number of additional functions, including regulation of apoptosis and interaction with the cytoskeleton. We determined the distribution of EF-1alpha in larval tissues of the fall armyworm, Spodoptera frugiperda , with a monoclonal antibody generated to EF-1alpha from Sf21 cells, a cell line developed from ovarian tissue of S. frugiperda. Enzyme-linked immunosorbent assay showed that EF-1alpha comprised 1.9-9.9% of the total protein within the tissues that were examined, which included fat body, Malpighian tubules, midgut, muscle, salivary glands, trachea, and ventral nerve cord. To a certain extent, EF-1alpha concentrations reflected the expected metabolic activity level of each of the represented tissues. Closer examination by immunofluorescence microscopy revealed that EF-1alpha concentrations varied among different cell types within a given tissue, i.e. midgut columnar epithelial cells yielded strong signals, while goblet cells failed to react with the EF-1alpha-specific antibody.
Journal of Insect Science 02/2006; 6:1-9. · 0.95 Impact Factor
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ABSTRACT: Groups of female BALB/c mice infected by intravenous injection with 50 erythrocytes containing Plasmodium berghei Vincke et Lips, 1948 were sacrificed on days 3 through 12 after infection. Rheumatoid factor-like IgM (RF-IgM) and parasite-specific IgG levels were determined by enzyme-linked immunosorbent assay in serum specimens and in culture medium removed from spleen cell cultures established at sacrifice. All four mouse IgG subisotypes were recognized by RF-IgM molecules induced by Plasmodium berghei infection, and in this regard, the parasite-induced RF-IgM response resembled that induced by lipopolysaccharide polyclonal activation. Plasmodium berghei infection resulted in a biphasic RF-IgM response, with infected animals demonstrating significantly increased levels of RF-IgM early in the infection and significantly decreased levels late in the infection, compared to uninfected control mice. The decreased levels of RF-IgM observed late in infection correlated with increasing parasitaemia levels, and were primarily due to a decrease in RF-IgM specific for mouse IgG2a. Late infection levels of RF-IgM specific for IgGI, IgG2b, and IgG3 were not significantly different from those of control animals.
Folia parasitologica 10/2003; 50(3):176-82. · 1.81 Impact Factor
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ABSTRACT: We partially characterized the antigens recognized by monoclonal antibodies raised against eggs of Helicoverpa zea (Boddie) and purified vitellin of Heliothis virescens (F.). Through western blot analysis following pore-limiting electrophoresis, both antibodies were shown to recognize vitellin specifically. Western blots generated following SDS-PAGE indicated that the determinant for the antibody produced to H. virescens vitellin (HVE-1) was found exclusively on the large molecular weight apoprotein of vitellin, apoVn-I. The determinant for the antibody produced to H. zea eggs (HZE-1) was not detected by western blot analysis following SDS-PAGE, indicating that its determinant was disrupted by protein denaturation. Electrophoretic analyses also indicated that the apparent molecular weights of native heliothine vitellins ranged from 472,000 to 522,000 and those of their apoproteins ranged from 164,000 to 169,000 (apoVn-I) and from 45,000 to 47,000 (apoVn-II). The species- and stage-specificities of the antibodies also was determined. The HZE-1 antibody was found to be specific for vitellin of H. zea and 2 Old World relatives, Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren), but did not recognize vitellins of H. virescens, Heliothis subflexa (Grote), or 3 genera of nonheliothine noctuids. The HVE-1 antibody recognized vitellins of all Helicoverpa-Heliothis species tested, but not those of the nonheliothine noctuids. Neither antibody cross-reacted with native proteins from homogenates or hemolymph of H. zea or H. virescens larvae or pupae. Western blots of adult hemolymph indicated that the antibodies also recognized vitellogenin, vitellin's precursor.
Annals of the Entomological Society of America 12/1996; 90(1):83-90. · 1.32 Impact Factor
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Alternative therapies in health and medicine 10(4):74-6. · 1.77 Impact Factor
