Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts.

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity.

Purification and characterization of Shiga toxin 2f, an immunologically unrelated subtype of Shiga toxin 2.

Background: Shiga-like toxin 2 (Stx2) is one of the most important virulence factors in enterohaemorrhagic Escherichia coli (E. coli) strains such as O157H7. Subtypes of Stx2 are diverse with respect to their sequence, toxicity, and distribution.

Oxidation of methionine 216 in sheep and elk prion protein is highly dependent upon the amino acid at position 218 but is not important for prion propagation.

We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to study the oxidation of a methionine at position 216 (Met216), because this oxidation (MetSO216) has been implicated in prion formation. Three polymorphisms (Ile218, Val218, and Thr218) of sheep recombinant prion protein were prepared.

Utility of mass spectrometry in the diagnosis of prion diseases.

We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the integrated MRM signals from selected analyte peptides and their oxidized analogues to their homologous stable isotope labeled internal standards were prepared. The limit of detection (LOD) and limit of quantitation (LOQ) for the synthetic peptides from human, sheep, deer, cow, and mouse PrP were determined to be below 100 amol.

Assessing the role of oxidized methionine at position 213 in the formation of prions in hamsters.

Prions are infectious proteins that are able to recruit a normal cellular prion protein and convert it into a prion. The mechanism of this conversion is unknown. Detailed analysis of the normal cellular prion protein and a corresponding prion has shown they possess identical post-translational modifications and differ solely in conformation.

Validation of a cell-free translation assay for detecting shiga toxin 2 in bacterial culture.

A cell-free translation (CFT) assay for detecting Shiga toxin (Stx) at different levels of purity has been validated. The limits of detection for pure Stx2 (PStx2) and partially pure Stx2 (PPStx2) in water reached 20 and 3.5 pg/microL, respectively, without the artificial process of proteolytic activation and reduction of the pro-toxin.

Effect of food matrices on the biological activity of ricin.

A cell-free translation assay was applied for the quick detection of ricin in food samples. Three economically important foods-ground beef, low-fat milk, and liquid chicken egg--were tested. The results indicated that ground beef had very little matrix effect on the assay, whereas low-fat milk and liquid chicken egg showed clear interference on the protein translation.

Prion infected meat-and-bone meal is still infectious after biodiesel production.

The epidemic of bovine spongiform encephalopathy (BSE) has led to a world-wide drop in the market for beef by-products, such as Meat-and-Bone Meal (MBM), a fat-containing but mainly proteinaceaous product traditionally used as an animal feed supplement. While normal rendering is insufficient, the production of biodiesel from MBM has been suggested to destroy infectivity from transmissible spongiform encephalopathies (TSEs). In addition to producing fuel, this method simultaneously generates a nutritious solid residue.

Detection of botulinum neurotoxin-A activity in food by peptide cleavage assay.

The World Health Organization (WHO) and U.S. Centers for Disease Control and Prevention (CDC) have labeled botulinum toxins as a high priority biological agent that may be used in terrorist attacks against food supplies.

B cell epitope mapping using synthetic peptides.

Synthetic peptides are used for identification of functional B cell epitopes in antibody preparations. ELISA-type assays are used to identify sequences of proteins comprising antibody-binding regions. This unit describes three peptide display formats.

Production of anti-peptide antisera.

The protocols described in this unit can be used for generating and testing anti-peptide antisera, assuming that a scientist has already selected and conjugated peptides for experimental vaccines. Described are immunization and bleeding of experimental animals, a simple ELISA for antiserum screening, and affinity purification of the serum, including preparation of columns.

Sonication induced intermediate in prion protein conversion.

We have observed that hamster prion protein (PrP(C)) undergoes conformational changes on exposure to heat or sonication. If a sonication induced new conformer is seeded with a small amount of its abnormal pathogenic isoform (PrP(Sc)) it undergoes a significant conversion to a proteinase-resistant isoform. This suggests the presence of a third stable PrP conformer, which may be intermediate in the conversion of PrP(C) to PrP(Sc).

Sensitive, preclinical detection of prions in brain by nanospray liquid chromatography/tandem mass spectrometry.

More sensitive detection of prions in brain is important because it would allow early detection of disease in young animals and assure a safer food supply. We have quantitated the amount of proteinase K-resistant prion protein (PrP 27-30) by use of nano-scale liquid chromatography coupled to tandem mass spectrometry using the multiple reaction monitoring mode of operation. We used a method based on the detection of VVEQMCTTQYQK (residues 209-220) obtained by reduction, alkylation and digestion with trypsin.

Application of a real time polymerase chain reaction method to detect castor toxin contamination in fluid milk and eggs.

The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was applied to the detection of castor contamination in milk and liquid egg samples. The targeting gene sequence of the primer set, Ricin-F4/R4, was not found in either the bovine or chicken genome.

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS.

Sensitive quantitation of prions in biological samples is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). At this time, there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans.

Detection of castor contamination by real-time polymerase chain reaction.

Due to the potential for intentional contamination of food with crude preparations containing ricin, a real-time PCR method was developed for the detection of castor plant material in ground beef. One primer pair was identified and confirmed to be castor-specific and efficient for amplification of ricin in DNA extracts from castor or beef matrices. Of three different DNA extraction protocols compared, the hexadecyltrimethylammonium bromide (CTAB) method yielded the highest quality of DNA for QPCR assay.

PV-1 labels trans-cellular openings in mouse endothelial cells and is negatively regulated by VEGF.

The PV-1 protein is endogenously expressed from a single mRNA in the mouse pancreatic MS-1 endothelial cell line as a 60-kDa N-glycosylated and 50-kDa non-glycosylated protein that form DTT sensitive oligomers. In the absence of cell permeabilization, PV-1 antibodies label transcellular openings of variable size, many that penetrate through the cytosol with circular openings on the free and attached surface of the plasma membrane. Intracellular PV-1 is localized in perinuclear aggregates that can extend as a fibrous network through the cytosol and often surround the nuclear compartment.