Amino acid residue E543 in JAK2 C618R is a potential therapeutic target for myeloproliferative disorders caused by JAK2 C618R mutation.

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays a key role in the hematopoietic and immune responses. The acquired JAK2 C618R somatic mutation is detected in a subset of myeloproliferative disorders (MPDs) patients and presumed to be a biomarker for MPDs. However, how JAK2 C618R mutation causes MPDs is still unclear. Our results indicate that the amino acid residue E543 in JAK2 C618R is indispensable for its constitutive activation. When the glutamic acid at this position was mutated to alanine (E543A) in the JAK2 C618R, its activity significantly decreased. However when the glutamic acid was mutated to the acidic amino acid, aspartic acid, JAK2 C618R activity changed little. These results suggest that there is an interaction between the amino acid residue R618 and E543, and that this interaction is crucial to sustain the constitutive activation of JAK2 C618R. More importantly, the E543 single mutation had no effects on the function of wild type JAK2 (WT JAK2). This study suggests that the amino acid residue E543 might be a potential target for specific inhibitors to treat MPDs caused by the JAK2 C618R mutation.

Escopoletin treatment induces apoptosis and arrests cell cycle at G0/G1 phase in the oral squamous cancer cell lines.

This study was aimed to investigate the antiproliferative effect of escopoletin on CAL 27 and CAL 33 oral squamous cancer cell lines. MTT assay and flow cytometry were used for the analysis of escopoletin effect on cell viability and apoptosis, respectively. Western blot analysis was used for the examination of cyclin and cyclin-dependent kinase expression after treatment of CAL 33 cells with escopoletin. The results showed a marked decrease in the viability of CAL 27 and CAL 33 cell lines after 48 h of escopoletin treatment. Treatment of CAL 33 cells with escopoletin led to the induction of apoptosis and arrest of cell cycle at G0/G1 phase. In the cells treated with escopoletin cyclin D1 and E expression was reduced and CDK1 expression was inactivated. The above findings suggest that escopoletin exhibits inhibitory effect on the oral squamous cancer through induction of apoptosis and arrest of cell cycle. Therefore escopoletin can be a promising candidate for the prevention of oral squamous cancer.

T273 plays an important role in the activity and structural stability of arginine kinase.

Arginine kinase (AK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphoarginine to ADP in invertebrates. The amino acid residue C271 is involved in keeping AK's activity and constraining the orientation of the substrate arginine. However, the roles of the C271 interaction amino acid residues in AK's substrate synergism, activity and structural stability are still unclear. The crystal structure of AK implied that the amino acid residue T273 interacted with the residue C271 and might play vital roles in keeping AK's activity, substrate synergism and structural stability. The mutations T273G and T273A led to significantly loss of activity, obviously decreased of substrate synergism and structural stability. Furthermore, spectroscopic experiments indicated that mutations T273G and T273A impaired the structure of AK and led them to a partially unfolded state. The inability to fold to the functional state made the mutations prone to aggregate under environmental stresses. Moreover, the mutations T273S and T273D almost had no effects on AK's activity and structural stability. This study herein indicated that the residue T273 played key roles in AK's activity, substrate synergism and structural stability.

Disruption of E627 and R683 interaction is responsible for B-cell acute lymphoblastic leukemia caused by JAK2 R683G(S) mutations.

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in hematopoietic and immune responses. The acquired JAK2 R683G(S) somatic mutations are detected in 15% of patients with B-cell acute lymphoblastic leukemia (B-ALL) and are presumed to be a biomarker for B-ALL. However, how JAK2 R683G(S) mutations lead to B-ALL is still unclear. Our results indicated that the E627 and R683 interaction played a vital role in JAK2 autoinhibition. Mutations (R683S, R683G and E627A) disrupting this interaction led to JAK2 constitutive activation, while mutations (R683K, E627D) restoring this interaction decreased its activity. Furthermore, spectroscopy experiments implied that disruption of the E627 and R683 interaction abolished JH1/JH2 domain interactions and forced the JH1 domain into the open, active conformation. Mutations abolishing this interaction promoted the proliferation of Ba/F3 cells. The results herein may provide clues to understanding the mechanism of JAK2 R683G(S) mutation-associated B-ALL.

Disrupting of E79 and K138 interaction is responsible for human muscle creatine kinase deficiency diseases.

Creatine kinase (CK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphocreatine to ADP in vertebrates. Due to its important physiological functions, the acquired CK somatic mutations are closely correlated to diseases. In this study, the E79G point mutation was identified in two acute myocardial infarction patients with muscle CK activity deficiency. The crystal structure of CK indicates that the E79 and K138 interaction plays key roles in sustaining the recognition between N-terminal and C-terminal domains. Mutations of these residues caused pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Moreover, spectroscopic spectra experiments suggested that mutations disrupting this hydrogen bond impaired the secondary and tertiary structure of CK. Meanwhile, protein folding experiments implied that mutations lead them to the partially unfolded state which made them easier to be inactivated and unfolded under environmental stresses. Furthermore, this partially unfolded state upon environmental stresses might gradually decrease the CK level in the patients. Thus, these results might provide clues in the mechanism of CK deficiency diseases.

The effects of R683S (G) genetic mutations on the JAK2 activity, structure and stability.

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in the hematopoietic and immune response. The acquired JAK2 R683S (G) mutations are presumed to be a biomarker for B-cell acute lymphoblastic leukemia (B-ALL). However, how these mutations leading to the B-ALL is still unclear. The crystal structure of JAK2 JH2 domain suggests that the residue R683 locating in the linker between the N and C lobes of JH2 domain is important for keeping the compact structure, activity and structural stability of this domain. Mutations R683S, R683G and R683E significantly increase JAK2 activity and decrease its structural stability. While the R683K and R683H mutations almost have no effects on the JAK2 activity and structural stability. Furthermore, the spectroscopic experiments imply that mutations R683S, R683G and R683E impair the structure of JAK2 JH2 domain, and lead JAK2 to partially unfolded state. It may be this partially unfolded state that caused JAK2 R683S (G) constitutive activation. This study provides clues in understanding the mechanism of JAK2 R683S (G) mutations caused B-ALL.

[The accuracy of linear measurements of jaw implant regions obtained by multi-slice spiral CT and dental software].

PURPOSE: To evaluate the measurement accuracy by multi-slice spiral CT(MSCT) and dental software as a tool for preoperative design before tooth implantations. METHODS: Twenty volunteers with marker template underwent MSCT. The diameter of the front teeth crowns in the bucco-lingual direction were measured by MSCT using a standard dental software package. Postoperatively, the same distances were clinically measured using a sliding caliper. The consistency between the two measurements was analyzed by paired t test using SPSS17.0 software package. RESULTS: The difference of measurements was -0.04-0.21mm. There was no significant difference between the CT and the manual measurement of the crown diameter in the bucco-lingual direction(P>0.05). CONCLUSIONS: The results demonstrate that MSCT and marker template promises to be a valuable method for the measurement of jaw implant regions.