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September 1, 2008
List Labs POSTER - Presented at the 45th Annual Interagency Botulinum Research Coordinating Committee Meeting, September 2008 in Philadelphia, PA.
SNAPtidesubstrate #520 contains the FRET pair o-aminobenzoic acid/2,4 dinitrophenyl (o-Abz/Dnp) and SNAPtidesubstrate Mca/Dnp contains (7methoxy-coumarin-4-yl)acetyl/2,4 dinitrophenyl as its FRET pair. Botulinum Neurotoxin Type A (Product #130A) and the SNAPtidesubstrates are products of List Biological Laboratories.
... Escherichia coli LPS 0111:B4 was purchased from List Biological Laboratories, ...
... On day 4, the BM cells were treated with 0 or 50 mM EtOH for another 5 days. On day 9, the cell density was regulated to 1 106/ml/well in 24-well plates. After culturing overnight, medium was changed and cells were incubated for additional 6 h with 0 or 100 ng/ml LPS (E. coli 0111:B4; List Biological Laboratories). ...
TNF- mRNA half-life assay:
A total of 1 g/ml actinomycin was added to the culture at 0, 30, and 60 min after stimulation with LPS/PMA. Mono Mac 6 cells were collected at each time point and TNF- mRNA was measured by real-time PCR described previously.
Chromatin IP (ChIP) assay for human IRF-3:
Mono Mac 6 cells culture in control or EtOH conditions were plated to 24-well plate at a cell density of 2 106 of cells/ml/well, and stimulated with LPS and PMA for 1 h as stated above. Then the cells were crosslinked with 1% (v/v) formaldehyde at 37C for 20 min. ...
Cell transduction with lentivirus and luciferase assay:
... BM cells were transduced overnight by lentivirus with ISRE, AP-1 driving luciferase as a reporter on day 3 before treating EtOH, and followed with EtOH and LPS as described above. After stimulation by LPS/PMA (for Mono Mac 6 cells) or LPS (for BM cells) for 6 h, the cells were harvested and ISRE, AP-1, or TNF- promoters activities were analysis using the Luciferase Reporter Assay System (Promega). The luciferase activities were normalized by total cellular proteins. ...
New High Affinity Antibodies Against Botulinum Neurotoxin Type A:
… To test the affinity purified antibodies we determined the titer of the anti-HccA antibodies against Botulinum Neurotoxin Type A (BTA) holotoxin, List Product #130. The ability of the anti-HccA antibodies to capture BTA was also tested using a Sandwich ELISA.
Comparison of FRET Substrates for Botulinum Neurotoxin Type A:
A new FRET substrate for the zinc endoprotease activity of the 50 kDa light chain (LcA) of the type A Botulinum neurotoxin has been designed and evaluated. This new substrate, which contains the FRET pair Mca/Dnp was compared to the SNAPtide substrate (Product #520 and #521) FRET peptides, which contain the oAbz/Dnp and FITC/DABCYL FRET pairs, respectively …
LPS was from List Biological Laboratories (Campbell, CA) ...
Results - Monocytes of cHCV patients lack tolerance to TNF -inducing TLR ligands:
... Monocytes of controls and HCV patients were primed in vitro with LPS, a TLR4 ligand, and re-stimulated with LPS to assess homotolerance. In agreement with previous reports (8,9), we found that LPS priming lead to low TNF production in response to a re-challenge with LPS in normal monocytes (Fig 1A). However, homo-tolerance to TLR4 ligand was not found in monocytes from HCV patients (Fig 1B). We further identified that pretreatment with LPS induced hetero-tolerance to subsequent stimulation with TLR2 (PGN), TLR2/TLR6 (PAM3CSK4), TLR2/TLR1 (PAM2CSK), TLR3 (poly I:C) and TLR7/8 (Gardiquimod) ligands in controls (Fig 2A), and in patients with liver inflammation due to non-viral non-alcoholic steatohepatitis (NASH) (Fig 2C) but not in HCV-infected patients (Fig 2B). This data indicated that monocytes of HCV-infected patients have lost TLR tolerance.
Quantification of PA83bound to chimeras 206 and 264:
Recombinant PA83 (List Biological Laboratories) in 5 mM Hepes, 50 mM NaCl (pH 7.5) was mixed with purified chimeras 206 and 264 in 50 mM Hepes (pH 7.5) in a ratio of 180:1 (equimolar amounts of PA83and VWA domains). Following incubation for 20 min at room temprature, an aliquot from each of the samples was removed and stored at 20 C pending analysis. The remainder of the samples was transferred to an ultracentrifuge tube and underlayed with a 30% (w/w) sucrose cushion in 50 mM Hepes (pH 7.5). Complexes of chimeras decorated with PA83were pelleted by centrifugation at 200,000g for 45 min. ...
The retrogradely transported marker CTb (List Biological Laboratories, Campbell, CA, USA) was unilaterally injected by iontophoresis into the VTA of 16 rats. CTb is considered to be a very sensitive retrograde tracer when visualized by immunohistochemistry. ... A hand drill was used to expose the brain surface above the VTA and a borosilicate glass micropipette (tip diameter 510 m) filled with a low-salt CTb solution (0.5% made in 0.1 M phosphate buffer, pH 6.0) was lowered into the brain at the following stereotaxic coordinates: AP, 5.5 mm from bregma; L, 0.7 mm from the midline; and V 7.2 mm from the dural surface (Paxinos & Watson, 2005). CTb was delivered by applying positive pulses of 14 A (7 s on, 7 s off) for 10 min through a chlorinated silver wire placed in the micropipette and connected to a current generator (Bionic Instruments, France). The micropipette was withdrawn 10 min after the CTb ejection, the scalp incision was sutured and rats were returned to their home cages for recovery.