Melissa K. Stuart

Insertional Inactivation of Branched-Chain α-Keto Acid Dehydrogenase in Staphylococcus aureus Leads to Decreased Branched-Chain Membrane Fatty Acid Content and Increased Susceptibility to Certain Stresses

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 α-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°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.

Immunological identification of Trogoderma granarium Everts (Coleoptera: Dermestidae)

Abstract A monoclonal antibody-based enzyme-linked immunosorbant assay specific for Trogoderma granarium Everts was developed. The assay could rapidly and accurately distinguish T. granarium adults, pupae and larvae from six other Trogoderma species found within the United States.

Utilization of case presentations in medical microbiology to enhance relevance of basic science for medical students

Abstract Background : 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. Results : 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). Conclusion : Students felt that the CPs improved their critical thinking and presentation skills and helped to prepare them as future physicians.

Changes in Cytokines, Sensory Tests, and Self-Reported Pain Levels After Manual Treatment of Low Back Pain.

Study Design: Unbalanced 3-factor design with repeated measures on 1 factor. Objective: To determine the effect of manual treatment (MT) on cytokine and pain sensations in those with and without low back pain (LBP). Summary of Background Data: Evidence suggests that MT reduces LBP but by unknown mechanisms. Certain cytokines have been elevated in patients with LBP and may be affected by MT. Methods: Participants aged 20–60 years with chronic LBP or without LBP were recruited and randomly assigned to MT, sham ultrasound treatment, or no treatment groups. Venous blood samples were collected and pain levels assessed at baseline, 1 hour later, and 24 hours later. Blood was analyzed for interleukin (IL)-1&bgr;, IL-6, tumor necrosis factor-&agr;, and C-reactive protein. Pain levels were measured by pressure pain threshold (PPT), mechanical detection threshold (MDT), dynamic mechanical allodynia, and self-report. Results: Forty (30 women, age 36±11 y) participants completed the study, 33 with LBP (13 MT, 13 sham ultrasound treatment, and 7 no treatment) and 7 without LBP. Participants with or without LBP could not be differentiated on the basis of serum cytokine levels, PPT, or MDT (P≥0.08). There were no significant differences between the groups at 1 hour or 24 hours on serum cytokines, PPT, or MDT (P≥0.07). There was a significant decrease from baseline in IL-6 for the no treatment (LBP) group (P=0.04), in C-reactive protein for the sham ultrasound treatment group (P=0.03), in MDT for all 3 LBP groups (P⩽0.02), and in self-reported pain for the MT and sham ultrasound treatment groups (P=0.03 and 0.01). Conclusions: Self-reported pain was reduced with MT and sham ultrasound treatment 24 hours after treatment, but inflammatory markers within venous circulation and quantitative sensory tests were unable to differentiate between study groups. Therefore, we were unable to characterize mechanisms underlying chronic LBP.

A Monoclonal Antibody That Inhibits Translation in Sf21 Cell Lysates Is Specific for Glyceraldehyde-3-Phosphate Dehydrogenase

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 antibodys 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 antibodys inhibitory effect on in vitro translation.

An antibody diagnostic for hymenopteran parasitism is specific for a homologue of elongation factor‐1α

An enzyme-linked immunosorbent assay (ELISA) useful for identifying noctuid pests parasitized by hymenopteran endoparasitoids was recently described. The ELISA employed a monoclonal antibody (MAb 9A5) that appeared highly polyspecific for parasitoid antigens, yielding banding patterns more typical of a polyclonal antiserum than of a monoclonal antibody in immunoblots of parasitoid homogenates subjected to SDS-PAGE. Although MAb 9A5 appeared capable of binding to dozens of parasitoid antigens, no cross-reactivity for noctuid antigens was evident by either immunoblotting or ELISA. In the study described here, immunoprecipitation, SDS-PAGE, and N-terminus amino acid sequencing were used to identify the protein recognized by MAb 9A5 as a homologue of elongation factor-1 alpha (EF-1 alpha). The propensity for EF-1 alpha to bind to cytoskeletal components, the additional subunits of EF-1, and other proteins may account for the apparent polyspecificity of MAb 9A5 in immunoblots of whole-body parasitoid homogenates. The presence of a unique hymenopteran epitope suggests that EF-1 alpha molecules from other insect groups could similarly express novel determinants. These determinants may prove useful not only for insect detection, but also as targets for selective insecticides that act by inhibiting protein synthesis.

Distribution of elongation factor-1α in larval tissues of the fall armyworm, Spodoptera frugiperda

Abstract Elongation factor-1α (EF-1α) 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-1α in larval tissues of the fall armyworm, Spodoptera frugiperda, with a monoclonal antibody generated to EF-1α from Sf21 cells, a cell line developed from ovarian tissue of S. frugiperda. Enzyme-linked immunosorbent assay showed that EF-1α 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-1α concentrations reflected the expected metabolic activity level of each of the represented tissues. Closer examination by immunofluorescence microscopy revealed that EF-1α 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-1α -specific antibody.

Monoclonal antibodies against the Escherichia coli DNA repair protein RadA/Sms.

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.

Monoclonal antibody 10A5 recognizes an antigen unique to the water-insoluble 25/45 membrane fraction of the rat ocular lens

The water-insoluble 25/45 fraction and non-sedimenting membrane fraction (NSMF) are two membrane preparations isolated from the ocular lens. The fractions are postulated to represent distinct subdomains of the lens with unique functions. However, attempts to distinguish between the two fractions by detecting proteins present in one fraction but absent from other have been unsuccessful. In this study, we exploited the ability of the mouse immune system to detect antigenic differences between the 25/45 fraction and NSMF isolated from the lenses of 20-day-old rats. We generated a monoclonal antibody (MAb 10A5) that reacts with a ganglioside-like antigen that is present in the 25/45 fraction but absent from the NSMF. Restriction of the antigen to the 25/45 fraction in 20-day-old animals supports the hypothesis that the 25/45 fraction and NSMF represent different subdomains within the ocular lens.