Deidre Patterson

Detection of Escherichia coli O157:H7 using immunomagnetic capture and luciferin-luciferase ATP measurement.

Abstract A procedure for rapid assay (about 30 min) of viable Escherichia coli O157:H7 in solutions is described. The bacteria, captured by specific immunomagnetic beads, were lysed by commercially available reagents. The released cellular ATP was determined by the chemiluminescence method using firefly lantern extracts. The ATP content of the E. coli capable of consuming oxygen was influenced by the presence of glucose. The immunomagnetic procedure was applied to determine the presence of E. coli O157:H7 in beef hamburger spiked with the bacteria. After a 6-h enrichment at 37°C, significant ATP luciferin-luciferase bioluminescence was detected from beef hamburger suspensions spiked with less than one CFU/ml of the E. coli . Possible utilization of the developed procedure for practical application is discussed.

The interaction of phospholipids in monolayers with bovine serum albumin and α-lactalbumin adsorbed from solution

Abstract The interaction of phospholipids in monolayers with bovine serum albumin (BSA) and bovine α-lactalbumin (ALA) adsorbed from solution has been studied by ultraviolet and circular dichroism spectroscopy of films transferred from the air—water interface of a Langmuir film balance. The lipid monolayers were mixtures of palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG). The subphase pHs studied (4.4–7.0) covered the range of acidities found in milk and other dairy products. Both BSA and ALA exhibited considerable adsorption to monolayers of POPC POPG ( 65 35 mol%) at pH 4.4, but only a small amount of either protein was detected in the mixed films when the subphase pH was 7. At a subphase pH of 4.4, a 50% reduction in BSA—lipid binding was observed with about 0.5 mM calcium in the subphase, whereas less than 25 μM calcium was required when the subphase pH was 5.2. At pH 4.4, reduction of ALA—lipid binding by 50% required only 25 μM calcium. The results with BSA suggest an electrostatic mechanism for lipid—protein interaction where the protein is tightly bound to the lipid at pHs below the isoionic point of the protein while the lipid—protein interaction is weaker at the isoionic pH and essentially negligible at pHs above the isoionic point. The results with ALA are consistent with this mechanism.

Detection of immunomagnetically captured Escherichia coli O157:H7 by antibody-conjugated alkaline phosphatase

A rapid and sensitive detection process for Escherichia coli O157:H7 was developed using alkaline phosphatase (APase)-labeled anti-E. coli O157 antibodies to tag the targeted bacteria. Immunomagnetic beads or antibody-labeled streptavidin-coated magnetic beads were then used to capture the APase-tagged E. coli. Immunomagnetically captured bacteria were washed and distributed into microplates or optical cuvettes. The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed. Less than 1000 cfu/ml of E. coli O157:H7 could be detected. This approach was applied to detect the bacteria artificially spiked in beef hamburgers. Less than 1 cfu/g of E. coli O157:H7 produced a significant response after cultural enrichment for 4–6 h at 37°C. Journal of Industrial Microbiology & Biotechnology (2001) 26, 345–349.

Unique properties of cell wall-associated β-glucosidases

Abstract Two maize ( Zea mays L.) root cell wall-associated β-glucosidases were compared to two cellular forms. The ionically bound cell wall enzyme was more difficult to dissociate from the cell wall compared to artifactually bound cytosolic β-glucosidase activity. The ionically bound cell wall enzyme also had an isoelectric pH of 6.8–7.0 compared to the acid p I of the cytosolic enzyme (4.8–5.2). A second cell wall enzyme was very tightly bound to the cell wall and could not be extracted with 3 M NaCl or LiCl. Both the ionically bound cell wall β-glucosidase and the tightly bound β-glucosidase were more thermostable at 40°C and 50°C compared to the cytosolic enzyme and the particulate intracellular isozyme. The tightly bound cell wall β-glucosidase was the only thermostable enzyme at 60°C.

Aluminum inhibition of NADH-linked electron transfer by corn root plasma membranes

Abstract Vesicles enriched in right‐side‐out plasma membranes were isolated from corn roots by a modified two‐phase partition method. Using reduced nicotinamide adenine dinucleotide as electron donor, isolated vesicles were capable of reducing both ferricyanide and oxygen. At pH 6.0, the presence of Al inhibited both reduction processes to a similar extent. This result indicates that these two reduction processes share at least one common step that is sensitive to Al. Inhibition was not associated with a change in the structure of the membrane domain, as revealed by fluorescence polarization of membrane incorporated probes. These results are the first indications that the electron transfer processes of plasma membranes are sensitive to the presence of Al.

Partial purification and characterization of various β-glucosidases associated with cellular components of corn roots

Abstract β-Glucosidases were isolated from various cellular components from corn root cortical tissue. The subcellular components were separated and the enzymes were partially purified and characterized. The pH-activity profile of the cytosolic enzyme and the unidentified particulate-associated β-glucosidase were identical. Further kinetic analyses of these two enzymes at pH 5.5 and 7.5 suggests that the unidentified particulate-associated β-glucosidase is the bound from of the cytosolic enzyme. The ionically bound cell wall enzyme (in muro) had a pH optimum of 5.5 while the free from (salt solubilized) had a pH optimum of 5.0. The enzyme kinetics of the extracellular enzymes were compared to the intracellular forms with particular reference to bound versus free enzymes. The K m values and the shape of the kinetic curves of free enzymes were not changed when enzymes were assayed in the bound state. The tightly bound cell wall enzyme had a very broad pH-activity profile and had a K m value approximately one-half the K m value of the other β-glucosidases.

Effects of divalent cations on nadh‐linked electron transfer in corn root plasma membrane

The effects of calcium (Ca 2+ ), cadmium (Cd 2+ ), and copper (Cu 2+ ) cations on NADH-linked electron transfer in corn root plasma membrane vesicles were investigated. The reduction of both cytochrome c and ferricyanide were slightly stimulated by Ca 2+ but not significantly affected by Cd 2+ . However, Cd 2+ induced a redox-linked increase in light scattering suggesting an increase in the size/volume of the vesicles. The presence of micromolar levels of Cu 2+ decreased the reduction rates of both cytochrome c and ferricyanide. However, in contrast to ferricyanide reduction, Cu inhibition to the cytochrome c reduction was more effective and was less sensitive to ionic strength. Copper inhibition changed the Michaelis-Menten dependence of the ferricyanide reduction but not that of cytochrome c. These results suggest that the reduction of cytochrome c and ferricyanide must occur at different membrane sites