Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, a chain), and CDlla (LFA-1, a chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect...Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, a chain), and CDlla (LFA-1, a chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophecto-derm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophecto-derm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CDlla was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo.展开更多
The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase I1 has been investigated using molec- ular mechanics and quantum mechanics methods. Molecular dynamics （MD） simulations were carri...The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase I1 has been investigated using molec- ular mechanics and quantum mechanics methods. Molecular dynamics （MD） simulations were carried out using the TIP3 water model and generalized solvent boundary potential （GSBP） by CHARMM based on the X-ray crystal structure. Two models of the ternary elongation complex were constructed based on CHARMM MD calculations. All the species including reactants, transition states, intermediates, and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2. Three pathways were explored using the DFT method. The most favorable reaction pathway involves indirect proton migration from the RNA primer 3＇-OH to the oxygen atom of a-phosphate via a solvent water mole- cule, proton rotation from the oxygen atom of a-phosphate to the 13-phosphate side, the RNA primer 3＇-O nucleophilic attack on the a-phosphorus atom, and P-O bond breakage. The corresponding reaction potential profile was obtained. The rate limit- ing step, with a barrier height of 21.5 kcal/mol, is the RNA primer 3＇-0 nucleophilic attack, rather than the commonly consid- ered proton transfer process. A high-resolution crystal structure including crystallographic water molecules is required for fur- ther studies.展开更多
文摘Expression of the adhesion molecules, ICAM-1, VCAM-1, NCAM, CD44, CD49d (VLA-4, a chain), and CDlla (LFA-1, a chain) on mouse oocytes, and pre- and peri-implantation stage embryos was examined by quantitative indirect immunofluorescence microscopy. ICAM-1 was most strongly expressed at the oocyte stage, gradually declining almost to undetectable levels by the expanded blastocyst stage. NCAM, also expressed maximally on the oocyte, declined to undetectable levels beyond the morula stage. On the other hand, CD44 declined from highest expression at the oocyte stage to show a second maximum at the compacted 8-cell/morula. This molecule exhibited high expression around contact areas between trophecto-derm and zona pellucida during blastocyst hatching. CD49d was highly expressed in the oocyte, remained significantly expressed throughout and after blastocyst hatching was expressed on the polar trophecto-derm. Like CD44, CD49d declined to undetectable levels at the blastocyst outgrowth stage. Expression of both VCAM-1 and CDlla was undetectable throughout. The diametrical temporal expression pattern of ICAM-1 and NCAM compared to CD44 and CD49d suggest that dynamic changes in expression of adhesion molecules may be important for interaction of the embryo with the maternal cellular environment as well as for continuing development and survival of the early embryo.
基金supported by the Natural Sciences and Engineering Research Council of Canada (10174)the Project-sponsored by SRF for ROCS,SEM
文摘The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase I1 has been investigated using molec- ular mechanics and quantum mechanics methods. Molecular dynamics （MD） simulations were carried out using the TIP3 water model and generalized solvent boundary potential （GSBP） by CHARMM based on the X-ray crystal structure. Two models of the ternary elongation complex were constructed based on CHARMM MD calculations. All the species including reactants, transition states, intermediates, and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2. Three pathways were explored using the DFT method. The most favorable reaction pathway involves indirect proton migration from the RNA primer 3＇-OH to the oxygen atom of a-phosphate via a solvent water mole- cule, proton rotation from the oxygen atom of a-phosphate to the 13-phosphate side, the RNA primer 3＇-O nucleophilic attack on the a-phosphorus atom, and P-O bond breakage. The corresponding reaction potential profile was obtained. The rate limit- ing step, with a barrier height of 21.5 kcal/mol, is the RNA primer 3＇-0 nucleophilic attack, rather than the commonly consid- ered proton transfer process. A high-resolution crystal structure including crystallographic water molecules is required for fur- ther studies.
