DTT (Dithiothreitol)

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.caut362
Keyword(s):  
Dtt Dithiothreitol

scholarly journals Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 588
Author(s):  
Laura von Berg ◽  
Daniel Stern ◽  
Jasmin Weisemann ◽  
Andreas Rummel ◽  
Martin Bernhard Dorner ◽  
...  
Keyword(s):  
Design Of Experiments ◽  
Catalytic Efficiency ◽  
Taguchi Design ◽  
Systematic Evaluation ◽  
Tween 20 ◽  
Taguchi Design Of Experiments ◽  
Protein Receptor ◽  
Catalytically Active ◽  
Botulinum Neurotoxins ◽  
Dtt Dithiothreitol

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase: evidence for a wider role in plant Rubisco regulation

2012 ◽  
Vol 442 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Paul John Andralojc ◽  
Pippa J. Madgwick ◽  
Yong Tao ◽  
Alfred Keys ◽  
Jane L. Ward ◽  
...  
Keyword(s):  
Protein Sequence ◽  
French Bean ◽  
Phosphoglycerate Mutase ◽  
Bisphosphate Carboxylase ◽  
Progressive Decline ◽  
Oxygenase Activity ◽  
Dtt Dithiothreitol ◽  
2,3 Dpg ◽  
Active Site Sequence

The genes for CA1Pase (2-carboxy-D-arabinitol-1-bisphosphate phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical with the PGM (phosphoglycerate mutase) active site sequence [LIVM]-x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1Pase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-DPG (2,3-diphosphoglycerate) to 2-phosphoglycerate. DTT (dithiothreitol) activation and GSSG inactivation of this enzyme was pH-sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate that was present in the RuBP (ribulose-1,5-bisphosphate) preparation, and was found to be PDBP (D-glycero-2,3-pentodiulose-1,5-bisphosphate). The substrate specificity of the expressed protein indicates a role for CA1Pase in the removal of ‘misfire’ products of Rubisco.

scholarly journals Calebin A Potentiates the Effect of 5-FU and TNF-β (Lymphotoxin α) against Human Colorectal Cancer Cells: Potential Role of NF-κB

2020 ◽  
Vol 21 (7) ◽  
pp. 2393 ◽  
Author(s):  
Constanze Buhrmann ◽  
Ajaikumar Kunnumakkara ◽  
Bastian Popper ◽  
Muhammed Majeed ◽  
Bharat Aggarwal ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cells ◽  
Nuclear Translocation ◽  
Chemotherapeutic Agents ◽  
Colorectal Cancer Cells ◽  
Tumor Environment ◽  
Proliferation And Metastasis ◽  
Dtt Dithiothreitol ◽  
Apoptotic Effects ◽  
Lymphotoxin Α

Objective: The majority of chemotherapeutic agents stimulate NF-κB signaling that mediates cell survival, proliferation and metastasis. The natural turmeric non-curcuminoid derivate Calebin A has been shown to suppress cell growth, invasion and colony formation in colorectal cancer cells (CRC) by suppression of NF-κB signaling. Therefore, we hypothesized here that Calebin A might chemosensitize the TNF-β-treated tumor cells and potentiates the effect of 5-Fluorouracil (5-FU) in advanced CRC. Materials and Methods: CRC cells (HCT116) and their clonogenic 5-FU chemoresistant counterparts (HCT116R) were cultured in monolayer or alginate-based 3D tumor environment culture and were treated with/without Calebin A, TNF-β, 5-FU, BMS-345541 and DTT (dithiothreitol). Results: The results showed that TNF-β increased proliferation, invasion and resistance to apoptosis in chemoresistant CRC cells. Pretreatment with Calebin A significantly chemosensitized HCT116R to 5-FU and inhibited the TNF-β-induced enhanced efforts for survival, invasion and anti-apoptotic effects. We found further that Calebin A significantly suppressed TNF-β-induced phosphorylation and nuclear translocation of p65-NF-κB, similar to BMS-345541 (specific IKK inhibitor) and NF-κB-induced tumor-promoting biomarkers (NF-κB, β1-Integrin, MMP-9, CXCR4, Ki67). This was associated with increased apoptosis in HCT116 and HCT116R cells. Furthermore, blocking of p65-NF-κB stimulation by Calebin A was imparted through the downmodulation of p65-NF-κB binding to the DNA and this suppression was turned by DTT. Conclusion: Our findings indicate, for the first time, that Calebin A chemosensitizes human CRC cells to chemotherapy by targeting of the p65-NF-κB signaling pathway.

DTT (Dithiothreitol; 50 mM)

2009 ◽  
Vol 2009 (2) ◽  
pp. pdb.rec11687-pdb.rec11687
Keyword(s):  
Dtt Dithiothreitol
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