Quality sample collection, handling, and preservation for an effective microbial forensics program.
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Bioterrorism↗
Biomedical subjects
Martha L Hale
Publications and source records attributed to Martha L Hale.
High-performance liquid chromatography-mass selective detection assay for adenine released from a synthetic RNA substrate by ricin A chain.
High-performance liquid chromatography (HPLC) and selected ion monitoring mass spectrometry (MS) were used to develop a quantitative assay for adenine released from a synthetic RNA substrate by ricin A chain, which contains the toxin's N-glycosidase activity. Because ricin and ricin A chain have potential applications as biotherapeutics and bioweapons, assays are needed to evaluate potency and potential inhibitors of activity. The detection limit for adenine was 0.02 microM (2.4 ng/ml), and the standard curve was linear up to 27.3 microM. The lower limit of quantitation was 0.27 microM and was reproducible throughout this range. Reaction characterization showed that most adenine was released by 5h and that the reaction could not be fully stopped with formic acid concentrations up to 0.75 mM (the maximum typically used for HPLC-MS). Injections were made at 2-min intervals, 10 injections could be performed before the column was backflushed, and no ricin A chain was observed in the column effluent. This assay would also be useful for ricin since ricin A chain did not pass through the HPLC column. With minor modifications to this system, the assay should provide rapid, sensitive, selective, and quantitative assessment of the activity of most ribosome-inactivating proteins. In addition, further chromatographic and mass spectrometric improvements could reduce sample requirements and analysis times.
Adenine↗
Proteomic analysis of detergent-resistant membrane rafts.
A combined, detergent- and organic solvent-based proteomic method for the analysis of detergent-resistant membrane rafts (DRMR) is described. These specialized domains of the plasma membrane contain a distinctive and dynamic protein and/or lipid complement, which can be isolated from most mammalian cells. Lipid rafts are predominantly involved in signal transduction and adapted to mediate and produce different cellular responses. To facilitate a better understanding of their biology and role, DRMR were isolated from Vero cells as a Triton X-100 insoluble fraction. After detergent removal, sonication in 60% buffered methanol was used to extract, solubilize and tryptically digest the resulting protein complement. The peptide digestate was analyzed by microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. Gas-phase fractionation in the mass-to-charge range was employed to broaden the selection of precursor ions and increase the number of identifications in an effort to detect less abundant proteins. A total of 380 proteins were identified including all known lipid raft markers. A total of 91 (24%) proteins were classified as integral alpha-helical membrane proteins, of which 51 (56%) were predicted to have multiple transmembrane domains.
Amino Acid Sequence↗
Detergent-resistant membrane microdomains facilitate Ib oligomer formation and biological activity of Clostridium perfringens iota-toxin.
Clostridium perfringens iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na(+)/K(+)-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol. Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown. In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100. Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types. The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions. Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation. While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-beta-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity. These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.
ADP Ribose Transferases↗
Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex.
Clostridium perfringens type E iota toxin consists of two unlinked proteins designated as iota a (Ia; molecular mass approximately 47 kDa), an ADP-ribosyltransferase and iota b (Ib; molecular mass approximately 81 kDa) which binds to the cell surface and facilitates Ia entry into the cytosol. By Western-blot analysis, Ib incubated with Vero cells at 37 degrees C generated a cell-associated, SDS-insoluble oligomer of Ib (molecular mass>220 kDa) within 15 s, which was still evident 110 min after washing cells. Ib oligomerization was temperature, but not pH, dependent and was facilitated by a cell-surface protein(s). Within 5 min at 37 degrees C, cell-bound Ib generated Na(+)/K(+) permeable channels that were blocked by Ia. However, Ib-induced channels or oligomers were not formed at 4 degrees C. Two monoclonal antibodies raised against Ib that recognize unique, neutralizing epitopes within residues 632-655 either inhibited Ib binding to cells and/or oligomerization, unlike a non-neutralizing monoclonal antibody that binds within Ib residues 28-66. The Ib protoxin (molecular mass approximately 98 kDa), which does not facilitate iota cytotoxicity but binds to Vero cells, did not oligomerize or form ion-permeable channels on cells, and neither trypsin nor chymotrypsin treatment of cell-bound Ib protoxin induced large complex formation. The link between Ib oligomers and iota toxicity was also apparent with a resistant cell line (MRC-5), which bound to Ib with no evidence of oligomerization. Overall, these studies revealed that the biological activity of iota toxin is dependent on a long-lived, cell-associated Ib complex that rapidly forms ion-permeable channels in cell membranes. These results further reveal the similarities of C. perfringens iota toxin with other bacterial binary toxins produced by Bacillus anthracis and C. botulinum.
ADP Ribose Transferases↗
Clostridium botulinum C2 toxin: binding studies with fluorescence-activated cytometry.
Clostridium botulinum C2 enterotoxin consists of two unlinked proteins designated as C2II, which recognizes a cell-surface glycoprotein and translocates an ADP-ribosyltransferase, C2I, into the cytosol of a targeted cell. Fluorescence-activated cytometry was used to study the cellular interactions of Alexa488-labeled C2I (C2I-A488) and proteolytically activated C2II (C2IIa-A488). The binding of C2IIa-A488 (4 degrees C/10 min) to Chinese hamster ovary (CHO) and African green monkey kidney (Vero) cells yielded a signal/noise ratio of 7:1 and 4:1, respectively. C2I-A488 binding required C2IIa and resulted in a 4:1 (CHO) and 10:1 (Vero) signal/noise ratio that was readily competed by unlabeled C2I. Neither C2I nor C2IIa bound to a CHO line (RK14), lacking the receptor for C2IIa. C2I-A488 did not dock with the heterologous cell-binding component (iota b) of Clostridium perfringens iota toxin, a binary toxin closely related to C2. Pretreatment of wild-type CHO or Vero cells with pronase or papain (37 degrees C/30 min) prevented a cell-associated C2IIa-specific signal. However, CHO and Vero cells pretreated with papain at 25 degrees C had a 1.5- to 2.3-fold increase in C2IIa-specific fluorescence versus untreated cells incubated with C2IIa-A488. Overall, these studies further demonstrated the utility of fluorescence-activated cytometry for studying the binding characteristics of bacterial binary toxins like C2.
Animals↗
Pirfenidone blocks the in vitro and in vivo effects of staphylococcal enterotoxin B.
Pirfenidone [5-methyl-1-phenyl-2-(1H)-pyridone] down-regulates expression of cytokines and other mediators involved in the onset and development of pulmonary fibrosis. Pirfenidone also inhibits production of tumor necrosis factor alpha (TNF-alpha) from macrophages incubated with endotoxin and protects mice against endotoxin shock. Pirfenidone's ability to reduce cytokine expression in these disorders led us to investigate the drug's effect on another cytokine anomaly, superantigen-induced shock. BALB/c mice were exposed to staphylococcal enterotoxin B (SEB) either systemically or by aerosol and subsequently potentiated with a sublethal dose of lipopolysaccharide. In these experiments, pirfenidone given 2 to 4.25 h after SEB resulted in 80 to 100% survival versus only 0 to 10% survival among untreated control animals. Relative to serum cytokine levels from controls given toxin but no drug, there was a 35 to 80% decrease in TNF-alpha, interleukin 1, and other proinflammatory cytokines. In vitro experiments with human peripheral blood lymphocytes revealed that pirfenidone reduced SEB-induced cytokine levels 50 to 80% and inhibited 95% of SEB-induced T-cell proliferation. Overall, these studies demonstrated the potential utility of pirfenidone as a therapeutic against septic shock and the biological effects of SEB.
Animals↗
Quantitative profiling of the detergent-resistant membrane proteome of iota-b toxin induced vero cells.
Enzyme-mediated 18O/16O differential labeling of proteome samples often suffers from incomplete exchange of the carboxy-terminus oxygen atoms, resulting in ambiguity in the measurable abundance differences. In this study, an 18O/16O labeling strategy was optimized for and applied to the solution-based comparative analysis of the detergent-resistant membrane proteome (DRMP) of untreated and Iota-b (Ib)-induced Vero cells. Solubilization and tryptic digestion of the DRMP was conducted in a buffer containing 60% methanol. Unfortunately, the activity of trypsin is attenuated at this methanol concentration hampering the ability to obtain complete oxygen atom turnover. Therefore, the incorporation of the 18O atoms was decoupled from the protein digestion step by carrying out the trypsin-mediated heavy atom incorporation in a buffer containing 20% methanol; a concentration at which trypsin activity is enhanced compared to purely aqueous conditions. After isotopic labeling, the samples were combined, fractionated by strong cation exchange and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. In total, over 1400 unique peptides, corresponding to almost 600 proteins, were identified and quantitated, including all known caveolar and lipid raft marker proteins. The quantitative profiling of Ib-induced DRMP from Vero cells revealed several proteins with altered expression levels suggesting their possible role in Ib binding/uptake.
ADP Ribose Transferases↗