Calcium-sensing receptor (CaSR), which is better known for its action as regulating calcium homeostasis, can bind various ligands. To facilitate research on CaSR and understand the receptor's function further, an in silico designed truncated protein was developed. The resulting protein folding indicated that 99% of predicted three dimensional (3D) structure residues are located in favored and allowed Ramachandran plots. However, it was found that such protein does not fold properly when expressed in prokaryotic host cells. Thioredoxin (Trx) tag was conjugated to increase the final protein's solubility, which could help obtain the soluble antigen with better immunogenic properties. The truncated recombinant proteins were expressed and purified in two forms (Trx-CaSR: RR19 and CaSR: RRJ19). The polyclonal antibody was induced by the rabbit immunization with the form of RR19. Western blot on mouse kidney lysates evidenced the proper immune recognition of the receptor by the produced antibody. The specificity and sensitivity of antibodies were also assayed by immunohistofluorescence. These experiments affirmed antibody's ability to indicate the receptor on the cell surface in native form and the possibility of applying such antibodies in further cellular and tissue assays.
相似文献Although the biology of adrenocorticotropic hormone (ACTH) protein has already been scrutinized, some functional aspects of its biology are yet to be elucidated in the context of immunological disorders. In this regard, virtual screening of a compound library was performed against the structure of Cytotoxic T-Lymphocyte Associated Protein-4 (CTLA-4) (assessed both spatially and energetically) to discover novel biological functions for ACTH. The results of virtual screening and the MD simulation demonstrated that DB01284 has high binding energy along with proper interaction orientation against CTLA-4 (FG loop) by a clamp like structure. The employed methodology was checked using confirmatory control analyses. Intriguingly, DB01284 belongs to Tetracosactide (already prescribed protein drug for clinical conditions) which is the N-terminal region of ACTH. This is the first study to reveal that ACTH protein binds to the same amino acids of CTLA-4 (FG-loop) as B7 and anti-CTLA-4 antibody binds. In light of this finding, the molecular mechanism of ACTH function in patients suffering from Cushing’s Syndrom and the immunological bases for ACTH therapy of multiple sclerosis (MS) patients could be further delineated. Moreover, this finding suggests that ACTH could also act to block CTLA-4 in the context of anticancer immune check point blockade.
相似文献Many periodontal patients may need orthodontic treatment. Alterations in oral environment particularly the reduction of pH in periodontal patients could affect metal ion release from orthodontic appliances. However, there is no study on metal ion release in periodontal patients. The aim of this preliminary study was to comparatively evaluate, for the first time, salivary levels of nickel and chromium in periodontal patients (versus healthy controls) under orthodontic treatment for 2 months. In this in vivo study, 40 subjects were evaluated. Patient selection and standardization of orthodontic treatment protocols were prospectively designed and performed. Two groups of n = 20 each (control: healthy orthodontic patients, cohort: orthodontic patients with periodontitis) underwent similar protocols of fixed orthodontic treatment for 2 months. After 2 months, salivary nickel and chromium concentrations of the case and cohort groups were measured using inductively coupled plasma mass spectrometry (ICP-MS). The values were compared between the two groups using t test. There were 10 men and 10 women in each group. The mean age of patients was 34.6 ± 3.6 years old. The salivary level of nickel was 338.2 ± 235.5 ng/ml and 182.8 ± 116.5 ng/ml in the cohort and control groups, respectively (P = 0.0118). The salivary level of chromium was 7.4 ± 3.15 ng/ml in the cohort and 6.35 ± 2.39 ng/ml in the control group (P = 0.2214). Salivary level of nickel might be considerably higher in periodontal patients undergoing 2 months of orthodontic treatment compared to orthodontic patients with healthy gingivae.
相似文献