Effect of pH and Ionic Strength on the Cytolytic Toxin Cyt1A: A Fluorescence Spectroscopy Study

Cyt1A is a cytolytic toxin produced by Bacillus thuringiensis var. israelensis. Due to its toxicity in vivo against mosquitoes and black flies, it is used as an environmentally friendly insecticide, although its mode of action is not completely understood. The toxin is membrane-active, but its membrane-bound conformation is unknown. In the absence of direct structural data, fluorescence spectroscopy was used to obtain indirect information on Cyt1A conformation changes in the environment mimicking the vicinity of the lipid membrane (lower pH and increased ionic strength). With decreasing pH, Cyt1A\u27s surface hydrophobicity increased, which is consistent with an increased interaction with model membranes at low pH values, as observed previously. The pKa value of this conformation change is 4.4±0.1. Intrinsic tryptophan fluorescence decreased with decreasing pH, and the pKa value was the same as the one determined with synthetic probes. The protein has two types of hydrophobic binding sites, and at low pH these sites bind more probe molecules (bis-ANS) with a higher affinity than at pH 7.4. When bound to the lipid, the toxin exhibited conformation similar to the molten-globule state and showed some characteristics also observed at low pH. However, the conformation of the lipid-bound toxin did not depend on pH. Neutral salts like NaCl and KCl induced conformational changes at neutral pH, but not at low pH. These changes were most probably due to specific interactions of the salt ions with the charged amino acids on the protein surface rather than due to general effects such as Hofmeister and Debye–Hückel. Our results might contribute to elucidating the mode of action of Cyt1A, and perhaps also to improving the formulation of the insecticidal preparations

A Detergent-like Mechanism of Action of the Cytolytic Toxin Cyt1A from \u3ci\u3eBacillus thuringiensis\u3c/i\u3e var. \u3ci\u3eisraelensis\u3c/i\u3e

The cytolytic δ-endotoxin Cyt1A from Bacillus thuringiensis var. israelensis is used in commercial preparations of environmentally safe insecticides. The current hypothesis on its mode of action is that the toxin self-assembles into well-defined cation-selective channels or pores, which results in colloid-osmotic lysis of the cell. Recently, a new hypothesis has been put forward suggesting that Cyt1A rather nonspecifically aggregates on the membrane surface and acts in a detergent-like manner. To distinguish between these two hypotheses, we investigated whether in the presence of lipid Cyt1A self-assembles into stoichiometric oligomers, which are characteristic of pores or channels, or aggregates into nonstoichiometric complexes, which would support the detergent-like model. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis revealed that in the presence of lipid Cyt1A forms protein aggregates with a broad range of molecular weights, some being too large to enter the gel. Cyt1A tryptophan (Trp) fluorescence in the presence of lipid exhibited a decrease in anisotropy and quantum yield, but an unchanged lifetime, which is consistent with the presence of toxin aggregates in the membrane. Electrostatic interactions between the charged amino acid residues and the lipid headgroups are responsible for bringing the protein to the membrane surface, while hydrophobic and/or van der Waals interactions make the membrane binding irreversible. Fluorescence photobleaching recovery, a technique that measures the diffusion coefficient of fluorescently labeled particles, and epifluorescence microscopy revealed that upon addition of Cyt1A lipid vesicles were broken into smaller, faster diffusing objects. Since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes, our results support the detergent-like mode of action of Cyt1A

A Comparison of CA242 With Twelve Other Tumor Antigens for the Serodiagnosis of Pancreatic, Gastric, and Other Gastrointestinal Cancers

The objective of this study was the comparison of CA 242 with twelve other cancer antigens for its usefulness in the diagnosis of pancreatic, gastric, and other gastrointestinal cancers. Sera from 554 patients (16 pancreatic cancer, 12 gastric cancer, 116 other gastrointestinal cancer, 215 other cancer, and 195 non-cancer) seen in a local hospital were assayed for carcinoembryonic antigen (CEA), CA 19-9, CA 195, CA 50, CA 242, CA 72-4, ferritin, CA 125, CA 15-3, CA 27.29, alpha fetoprotein (AFP), Cyfra 21-1, and neuron specific enolase (NSE). Diagnostic sensitivities for pancreatic and gastric cancers respectively were: CEA (37.5%, 50.0%), CA 19-9 (66.7%, 63.6%) , CA 195 (100%, 58.3%), CA 50 (66.7%, 70.0%), CA 242 (66.7%, 70.0%), CA 72-4 (31.3%, 27.3%), ferritin (50.0%, 11.1% ), CA 125 (40.0%, 40.0%), CA 15-3 (26.7%, 45.5%), CA 27.29 (40.0%, 30.0%), AFP (18.2%, 22.2%), Cyfra 21-1 (26.7%, 9.1%), and NSE (0.0%, 0.0)%). Diagnostic specificities and efficiencies were above 74% for all antigens and both cancers. Especially noteworthy was the fact that 9/16 pancreatic cancer and 6/12 gastric cancer patients had a CA 195 concentration which was greater than 20x the upper limit of normal (ULN). Two of the pancreatic cancer patients had CA 195 concentrations above 1000x ULN prior to their diagnosis by conventional methods (imaging and biopsy). CA 242 and CA 50 were superior to the other markers for the detection of gastric cancer. CA 195 proved the best with CA 19-9, CA 50, and CA 242 also proving excellent for the detection of pancreatic cancer

Evaluation of TB activity in immunized mouse sera by SMFA: one vaccine dose.

a<p>The proportion (percentage) of mosquitoes infected.</p>b<p>Median number of oocysts per mosquito (range).</p>c<p>% reduction = (mean control oocyst – mean test oocyst) ÷ mean control oocyst)*100.</p>d<p>Control.</p>*<p>CP – coat protein.</p

Pfs25-CP VLP particle analysis.

<p>(A) Negative stain transmission electron micrograph of Pfs25-CP VLPs shows highly uniform particles of 19.3±2.4 nm in diameter. (B) Transmission electron micrograph of Pfs25-CP VLPs labeled with anti-Pfs25 and gold-labeled anti-mouse antibodies confirms the presence of Pfs25 on the particles. (C) DLS histogram showing a narrow size distribution for Pfs25-CP VLPs. The average hydrodynamic radius is ∼14 nm with a polydispersity of <15%. (D) Analytical SEC showing a single, major eluting species confirmed by Western blot analysis (not shown) to be Pfs25-CP VLP. The void volume of the SRT 1000 column (range 7.5 MDa –50 kDa) is indicated by (a) molecular weight standards indicated by (b) for thyroglobulin, (c) for BSA and (d) for uracil.</p