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Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.

Clinical application of PD-1 and PD-L1 monoclonal antibodies (mAbs) is hindered by their relatively low response rates and the occurrence of drug resistance. Co-expression of B7-H3 with PD-L1 has been found in various solid tumors, and combination therapies that target both PD-1/PD-L1 and B7-H3 pathways may provide  additional therapeutic benefits. Up to today, however, no bispecific antibodies targeting both PD-1 and B7-H3 have reached the clinical development stage. In this study, we generated a stable B7-H3×PD-L1 bispecific antibody (BsAb) in IgG1-VHH format by coupling a humanized IgG1 mAb against PD-L1 with a humanized camelus variable domain of the heavy-chain of heavy-chain antibody (VHH) against human B7-H3. The BsAb exhibited favorable thermostability, efficient T cell activation, IFN-γ production, and antibody-dependent cell-mediated cytotoxicity (ADCC). In a PBMC humanized A375 xenogeneic tumor model, treatment with BsAb (10 mg/kg, i.p., twice a week for 6 weeks) showed enhanced antitumor activities compared to monotherapies and, to some degree, combination therapies. Our results suggest that targeting both PD-1 and B7-H3 with BsAbs increases their specificities to B7-H3 and PD-L1 double-positive tumors and induces a synergetic effect. We conclude that B7-H3×PD-L1 BsAb is favored over mAbs and possibly combination therapies in treating B7-H3 and PD-L1 double-positive tumors.

The three-dimensional (3D) model of the human acidic mammalian chitinase (hAMCase) was constructed based on the crystal structure of the human chitotriosidase (EC 3.2.1.44, PDB code 1HKK) by using InsightII/Homology module. With the aid of molecular mechanics and molecular dynamics methods, the last refined model was obtained and further assessed by Profile-3D and Procheck, which confirms that the refined model is reliable. Furthermore, the docking results of the ligands (allosamidin and NAG 2 ) into the active site of hAMCase indicate that allosamidin is a more preferred ligand than NAG 2 , and that Glu119 forms hydrogen bond with allosamidin, which is in good agreement with the experimental results. From the docking studies, we also suggest that Trp10, Glu49, Asp192, and Glu276 in hAMCase are four important determinant residues in binding as they have strong van-der-Waals and electrostatic interactions with the ligand, respectively.

In this study, the role of two conversed tyrosines (Tyr5 and Tyr42) from the scorpion toxin BmK AGP-SYPU1 was investigated with an effective Escherichia coli expression system. Site-directed mutagenesis was used to individually substitute Tyr5 and Tyr42 with hydrophobic or hydrophilic amino acids, and the extent to which these scorpion toxin BmK AGP-SYPU1 tyrosines contribute to analgesic activity was evaluated. The results of the mouse-twisting test showed that Tyr5 and Tyr42 are associated with the analgesic activity of the toxin because the analgesic activities of Y5F and Y42F were significantly increased compared with the rBmK AGP-SYPU1; however, the Y5W had decreased activity. The results of molecular simulation reveal the following: (1) for analgesic activity, the core domain of the scorpion toxin BmK AGP-SYPU1 is key and (2) for pharmacological function, Tyr42 is most likely involved when the core domain conformation is altered. These studies identify a new relationship between the structure and analgesic activity of the scorpion toxin BmK AGP-SYPU1 and are significant for further research and the application of analgesic peptides.

In this study, we investigated the functional role of arginines in the C-terminal (65–67) of BmK AGP-SYPU1, an analgesic peptide from the Chinese scorpion Buthus martensii Karsch. Using site-directed mutagenesis, arginines at the C-terminal (65–66) were deleted or added to the C-terminal (67). The genes for three mutants of BmK AGP-SYPU1 were obtained by PCR. An analgesic activity assay was used to evaluate the role of arginine residues in the analgesic activity. The three-dimensional structure of BmK AGP-SYPU1 was established by homology modeling. As a result, we showed that the arginines in the C-terminal are crucial for the analgesic activity and may be located at analgesic functional sites. Our work has implications for further modification of scorpion toxins to obtain new analgesic peptides with enhanced activity.

Elucidating structural determinants in the functional regions of toxins can provide useful knowledge for designing novel analgesic peptides. Glycine residues at the C-terminal region of the neurotoxin BmK AGP-SYPU2 from the scorpion Buthus martensii Karsch (BmK) have been shown to be crucial to its analgesic activity. However, there has been no research on the structure–function relationship between the C-terminal segment of this toxin and its analgesic activity. To address this issue, we performed three MD simulations: one on the native structure and the other two on mutants of that structure. Results of these calculations suggest that the existence of glycine residues at the C-terminal segment stabilizes the protruding topology of the NC domain, which is considered an important determinant of the analgesic activity of BmK AGP-SYPU2.

With homology modeling techniques, molecular mechanics and molecular dynamics methods, a 3D structure model of N-acetylneuraminate lyase from human (hNAL, EC 4.1.3.3) was created and refined. This model was further assessed by Profile-3D and PROCHECK, which confirms that the refined model is reliable. Furthermore, the docking results of the substrates (sialic acid and KDO) into the active site of hNAL indicate that hNAL can cleave the sialic acid and KDO. Thr51 and Tyr143 may be the key amino acids residues as they have strong hydrogen bonding interactions with the substrates, which is in good agreement with the experimental results by Izard et al. (Structure 2:361–369. doi:10.1016/S0969-2126(00)00038-1 (1994)). From the docking studies, we also suggest that Asp176 and Ser218 only form hydrogen bonds with sialic acid, therefore, they may help sialic acid interact with hNAL steadly.

Congenital disorder of glycosylation type 1a (CDG-1a) which is a congenital disease, is caused by mutations in α-Phosphomannomutase1. The reaction mechanism of the α-phosphomannomutase1 enzyme has been investigated by means of density functional theory using the hybrid functional B3LYP. The α-phosphomannomutase1 catalyzes the interconversion of the α-D-mannose 1-phosphate to D-mannose 6-phosphate via a mannose-1,6-(bis) phosphate intermediate. The quantum chemical models, which were chosen in protonated/deprotonated states models, were built on the basis of the docking result. The process of the phosphoryl group transferred from Asp19 to the mannose 6-phosphate is in different steps in the two states, but are both coupled with the protons transfer. Our computational results support the hypothesis that the Asp19 as a nucleophile plays an important role in the α-phosphomannomutase1 biology function, and indicate Gln62 could help to stabilize the phosphoryl group and the structure of the substrate. In addition, we can conjecture that the deprotonated state is more suitable for product release.

Recent studies have shown that induced expression of endogenous antioxidative enzymes thr- ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 （Nrf2） pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 ktM curcumin or post-treated with 5 pM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thi- oredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neu- roprotection after cerebral ischemia.

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.