Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, shortterm co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced targetindependent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

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The present study was undertaken to explore the protective effect of melatonin against isoproterenol bitartrate (ISO)-induced myocardial injury in rat. Treatment of rats with ISO increased the level of lipid peroxidation products and decreased the reduced glutathione levels in cardiac tissue indicating that this synthetic catecholamine induces oxidative damage following oxidative stress. Pretreatment of ISO-injected rats with melatonin at a dose of 10 mg/kg body weight, i.p. prevented these changes. Additionally, melatonin also restored the activities and the levels of antioxidant enzymes which were found to be altered by ISO treatment. Treatment of rats with ISO resulted into an increased generation of hydroxyl radicals with melatonin pretreatment significantly reducing their production. Finally, treatment of rats with ISO caused a lowering of systolic pressure with reduced cardiac output and diastolic dysfunction whereas melatonin pretreatment significantly restored many of these parameters to normal. The findings document melatonin's ability to provide cardio protection at a low pharmacological dose. Melatonin has virtually no toxicity which raises the possibility of this indole being a therapeutic treatment for ischemic heart disease.

The fluorescence emission maximum of a polar fluorophore in viscous medium often shows a dependence on excitation wavelength, a phenomenon which is named red edge excitation shift (REES). We have found that the fluorescence spectra of the tubulin tryptophans exhibit a REES of about 7 nm. Also, their steady state fluorescence polarization and mean lifetimes show a dependence on both excitation and emission wavelengths. These results indicate that the average tryptophan environment in tubulin is motionally restricted. Although the tryptophan(s) responsible for the observed REES effect could not be localized, it could be concluded from energy transfer experiments with the tubulin-colchicine complex that the tryptophan(s) participating in energy transfer with bound colchicine probably does not contribute to the REES. A REES of 7 nm was also observed in the case of colchicine complexed with tubulin. However, such a REES was not seen in similar studies with the B-ring analogs of colchicine, viz. 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (called AC because it lacks the B ring of colchicine) and deacetamidocolchicine (which lacks the acetamido substituent at the C-7 position of the B ring). There may be two possible reasons to explain these data. (1) Structural differences between colchicine and its analogs may give rise to differences in their excited state dipole moments which will directly affect the extent of REES, and (2) The B-ring substituent, hanging outside the colchicine binding site on the beta-subunit of the tubulin dimer, probably makes contact with the alpha-subunit of tubulin and imparts a rigidity to that region of the protein, which facilitates the REES.