IL‐1 signaling is critical for expansion but not generation of autoreactive GM‐CSF+ Th17 cells

Interleukin‐1 (IL‐1) is implicated in numerous pathologies, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which IL‐1 is involved in the generation of pathogenic T cells and in disease development remains largely unknown. We found that following EAE induction, pertussis toxin administration leads to IL‐1 receptor type 1 (IL‐1R1)‐dependent IL‐1β expression by myeloid cells in the draining lymph nodes. This myeloid‐derived IL‐1β did not vitally contribute to the generation and plasticity of Th17 cells, but rather promoted the expansion of a GM‐CSF⁺ Th17 cell subset, thereby enhancing its encephalitogenic potential. Lack of expansion of GM‐CSF‐producing Th17 cells led to ameliorated disease in mice deficient for IL‐1R1 specifically in T cells. Importantly, pathogenicity of IL‐1R1‐deficient T cells was fully restored by IL‐23 polarization and expansion in vitro. Therefore, our data demonstrate that IL‐1 functions as a mitogenic mediator of encephalitogenic Th17 cells rather than qualitative inducer of their generation.     

Microfabricated arrays of cylindrical wells facilitate single‐molecule enzymology of α‐chymotrypsin

Single‐molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single‐molecule enzymatic kinetics and provide comparisons to ensemble‐averaged kinetics. To isolate our model enzyme, α‐chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 μm in diameter and 1.35 μm in height. Inside the wells, a protease assay with a profluorescent substrate detects α‐chymotrypsin activity. We hold the concentration of α‐chymotrypsin at 0.39 nM in a given well with an enzyme‐to‐substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge‐coupled device. This method allows for the simultaneous real‐time characterization of hundreds of individual enzymes. We analyze single‐molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Synthesis of the kyotorphin precursor benzoyl‐L‐tyrosine‐L‐argininamide with immobilized α‐chymotrypsin in sequential batch with enzyme reactivation

α‐Chymotrypsin was immobilized in activated agarose support and the stability of the biocatalyst was assessed in three polar organic solvents, namely, ethanol, diglyme, and acetonitrile. Ethanol was the solvent in which the stability of the enzyme was higher and was then selected to perform the synthesis of the kyotorphin derivative benzoyl‐tyrosine argininamide, evaluating enzyme reactivation after synthesis. Substrates for reaction were benzoyl tyrosine ethyl ester and argininamide, the reaction being performed under kinetic control. High conversion yield (85%) was obtained and the immobilized enzyme was successfully used in sequential batch reactor operation with enzyme reactivation after three batches. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:54–59, 2016     

Oligoethylene glycols prevent thermal aggregation of α‐chymotrypsin in a temperature‐dependent manner: Implications for design guidelines

Protein aggregation is problematic in various fields, where aggregation can frequently occur during routine experiments. This study showed that tetraethylene glycol (TEG) and tetraethylene glycol dimethyl ether (TEGDE) act as aggregation suppressors that have different unique properties from typical additives to prevent protein aggregation, such as arginine (Arg) and NaCl. Thermal aggregation of α‐chymotrypsin was well suppressed with the addition of both TEG and TEGDE. Interestingly, the suppressive effects of Arg and NaCl on thermal aggregation were almost unchanged when temperature was shifted from 65°C to 85°C, whereas both TEG and TEGDE significantly decreased the aggregation rate with increasing temperature. Note that the effects of TEG and TEGDE were higher than Arg above 75°C. This temperature‐dependent behavior of TEG and TEGDE provides a novel design guideline to develop aggregation suppressors for use at high temperature, i.e., the importance of the ethylene oxide group. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1325–1330, 2013