[Effect of ectodysplasin-A1 on proliferation and cell cycle of ameloblast-like cell].

Objective: To investigate the effects of ectodysplasin-A1 (EDA1) on the proliferation and cell cycle of ameloblast-like epithelial cells (LS8 cells). Methods: Wild EDA1 plasmid pCR3-Flag-EDA1-W (wild group), syndrome mutant EDA1 plasmid pCR3-Flag-EDA1-H252L (mutant group) and empty vector plasmid pCR3-Flag (control group) were transfected into LS8 cells. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay and cell cycle was detected by flow cytometry. All tests were repeated three times. Results: Compared with the control group (0.105±0.032), the proliferation activity of the wild group (0.201±0.009) was significantly higher after 72 h (P<0.05). Compared with the control group (0.168±0.054) and the mutant group (0.194±0.059), the proliferation activity of the wild group (0.386±0.066) was significantly higher after 96 h (P<0.05). There was no significant difference between the mutant group and the control group at all time points (P>0.05). In the G0/G1 phase, compared with the control group (65.4%±2.1%) and the mutant group (66.6%±3.1%), the cell distribution ratio of the wild group (51.2%±1.1%) was significantly lower (P<0.01). In the S phase, compared with the control group (23.1%±2.0%) and the mutant group (21.9%±1.8%), the cell distribution ratio of the wild type group (37.3%±2.4%) was significantly higher (P<0.01). There was no significant difference in cell cycle distribution between the mutant group and the control group (P<0.05). Conclusions: Wild EDA1 promotes the proliferation of LS8 cells and the transformation from G0/G1 to S phase. The syndrome mutant EDA1 (EDA1-H252L) loses its function of regulating the cell proliferation and cell cycle of LS8 cells.

Electrostatic fields at the active site of ribulose-1,5-bisphosphate carboxylase.

A macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose-1,5-bisphosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2-type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C-terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N-terminal domains have mainly negative potential. At the active site situated between the C-terminal domain of one subunit and the N-terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the epsilon-amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8-type Rubisco from spinach is, despite only 30% amino acid homology for the L-chains, strikingly similar to that of the L2-type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8-type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.

Crystal structure determination of desheptapeptide (B24--B30)-insulin.

Desheptapeptide (B24--B30)-insulin (DHPI), an essentially inactive insulin analog, is crystallized in space group P212121 with two molecules in an asymmetric unit. The orientations of the molecules in the crystal cell have been determined by using Patterson search method at 6 A resolution and the positions of the molecules are deduced from translation function calculation and R search at 3 A resolution. After using the rigid body refinement (CORELS) further to refine the orientational and positional parameters as well as the initial energy restrained refinement (EREF) for the model, the crystallographic R value is reduced to 0.384 at 3 A resolution. The initial Fourier map shows that the B-chain N-terminal (B1-B8) and C-terminal (B20-B22) segments, compared with the native 2 zinc insulin, exhibit drastic conformational changes, but the three helices of B- and A-chains and their relative arrangement are essentially kept in DHPI.