Bacterial Toxin Research Citations

Materials:

Tetanus toxin was provided by List Biological Laboratories Inc. (Campbell, CA, USA). …

Results:

… As can be seen in Fig. 1, treatment with a moderate TeTx concentration of 1.0 nM was able to block the specific [3H]5-HT uptake in P2 fractions. On the other hand, the growth factors used, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), which are endogenous agonists of some tyrosine-kinase receptors, were unable to produce the same effect at the indicated concentrations in Fig. 1. On the contrary, the growth factors tested produced significant increases in [3H]5-HT uptake (127.2% of Control with 200 nM EGF, 151.2% with 200 nM bFGF, and 138.5% with 200 nM NGF). Co-application of the neurotoxin (1 nM) with each one of the indicated growth factors was able to produce a progressive, dose-dependent reversion of the TeTx effect. …

• Product #190 – Tetanus Toxin from Clostridium tetani

PA-Binding Studies:

PA binding to CHO-R1.1 cells that expressed CMG2⁴⁸⁹-EGFP or ATR/TEM8 sv2-EGFP was assessed by flow-cytometric analysis; 10⁶ cells of each type were incubated with 50 nM PA (1 h, 4C), washed with PBS, incubated with a polyclonal PA-specific rabbit antiserum (9) (1:500 dilution, 1 h, 4C), washed with PBS again, and then incubated with an allophycocyanin-conjugated anti-rabbit antibody (Molecular Probes; 1:2,000 dilution, 1 h, 4C). ELISAs were performed by binding 0.3 g of PA83 (List Biological Laboratories, Campbell, CA)/100 l of TBS in a well of a MaxiSorp plate (Nalge). Samples were then treated with TBST solution (TBS containing 3% BSA and 0.05% Tween 20), washed with TBST, and incubated with 0300 ng of purified CMG2^VWA/I-MycHis/100 l TBST in the absence or presence of 1 mM MgCl₂, MnCl₂, CaCl₂, or ZnCl₂. The samples were then washed with TBST and incubated with a horseradish peroxidase-conjugated anti-His antibody (1:2,000 dilution; Santa Cruz Biotechnology). All incubations for ELISAs were performed for 1 h at room temperature. The levels of bound antibody were then measured by using Supersignal ELISA Pico chemiluminescent substrate (Pierce) and a luminometer (Victor V, Wallac).

Intoxication Assays:

Cell viability assays (WST-1 assay; Roche Molecular Biochemicals) were performed in triplicate by incubating 5,000 cells of each type for 30 h with 2 10¹⁰ M LF_N-diptheria toxin A chain (DTA) and varying concentrations of PA (10¹² to 10⁸ M; List Biological Laboratories) or no PA (100% viability control). The inhibition assays were performed as above with different amounts of CMG2^VWA/I-MycHis (01,000 ng/100 l) added to 10⁹ M PA and 10¹⁰ M LF_N-DTA before this mixture was added to cells. The IC₅₀ was determined by regression analysis (prism, GraphPad, San Diego).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Materials:

… The purified recombinant anthrax lethal factor (LF) and both MAPKKide substrates are products of List Biological Laboratories.

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

Materials:

ReLPS from Salmonella Minessota Re595 was purchased from List Labs (CA, USA). …

Repurification of LPS prepration:

Commercial LPS was resuspended in endotoxin-free water containing 0.2% triethylamine followed by vortexing. LPS was repurified using a modified phenol-water extraction procedure followed by ethanol precipitation as described previously ( Tapping et al., 2000; Hirschfeld et al., 2000; Manthey and Vogel, 1994). Recovery of LPS was determined by 3-deoxy-D-manno-octulosonic acid assay.

TNF induction:

Human monocytes (510⁵) were mixed with 50 l of serial dilutions of LPS in the presence of 1% HPS in serum free medium. After 2.5 hours, the supernatant was collected and analysed for TNF- using an enzyme-linked immunosorbent assay (Research Diagnostics Inc). For inhibition experiments, cells were treated with either 60 g/ml nystatin or 10 mM MCD for 10 minutes prior to LPS stimulation.

• Product #304 – LPS from Salmonella minnesota R595 (Re)

Immunization protocol:

A standard nasal immunization protocol was used in this study [12]. C57BL/6 mice were nasally immunized to both nostrils on days 0, 7 and 14 with 25 g of diphtheria toxoid (DT) (1050 Lfu/mg) kindly provided by the Biken Foundation (Osaka University) either alone or combined with 5g of the LPS-free form of mCT or 0.5g of nCT (List Biologic Laboratories Inc.) as mucosal adjuvants.

Detection of neutralizing Abs by use of diphtheria toxin-specific Vero cell assay:

The cytotoxicity induced by the diphtheria toxin (List Biological Laboratories Inc.) in Vero cell cultures was used to measure the toxin-specific neutralizing titers of the Abs induced by the nasal vaccine.

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae
• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

Materials:

TeTx was provided by List Biological Laboratories, Inc. (Campbell, CA, USA). …

Results:

• TeTx, like TPA, induces [3H]5-HT uptake inhibition in rat-brain synaptosomes
• TeTx induces dose-dependent translocation of PKC isoforms
• TeTx induces both PLC activation and PLCQ1 phosphorylation at Tyr residues via tyrosine kinase receptors (TyrKRs)
• TeTx, like TPA, induces protein phosphorylations in Ser residues and specic SERT phosphorylation
• Bisindolylmaleimide-1 (BIM-1) blocks the TeTx and TPA induction of SERT phosphorylation

• Product #190 – Tetanus Toxin from Clostridium tetani

Reagents:

LPS from the LCD 25 strain of Escherichia coli K12 (Rb-LPS mutant) and LPS of E. coli 0111:B4 (S-LPS) were purchased from List Biological Laboratories (Campbell, CA).

Microtiter Plate LPS Binding Assay:

Immulon-2 96-well microtiter plates were coated with LCD 25 E. coli K 12 LPS or 0111:B4 E. coli LPS (5 g/well), AP-SP-A (positive control for SP-D binding) and myosin (positive control for SP-A binding [550 ng/well]) or nothing (BSA) in 0.1 M Na₂CO₃, 20 mM EDTA, pH 9.6, for 3 h at 37C. After incubation, the LPS solution was removed, and the plates were air-dried overnight. …

• Product #314 – LPS from Escherichia coli K12 strain LCD25
• Product #201 – Escherichia coli O111:B4

Mice and reagents:

Female C3H/HeJ mice 5 weeks of age were purchased from the Jackson Laboratory (Bar Harbor, Me) and maintained on peanut-free chow under specific pathogen-free conditions. …

Freshly ground whole peanut was used as the antigen. Crude peanut extracts (CPEs), Ara h 1 and Ara h 2, were prepared as described previously.^12, 13 Cholera toxin was purchased from List Biological Laboratories, Inc (Campbell, Calif). …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae