Free energy perturbation calculations on binding and catalysis after mutating Asn 155 in subtilisin

Site-directed mutagenesis is a very powerful approach to altering the biological functions of proteins, the structural stability of proteins and the interactions of proteins with other molecules. Several experimental studies in recent years have been directed at estimating the changes in catalytic properties, (rates of binding and catalysis) in site-directed mutants of enzymes compared to the native enzymes. Simulation approaches to the study of complex molecules have also become more powerful, in no small measure owing to the increase in computer power. These simulations have often allowed results of experiments to be rationalized and understood mechanistically. A new approach called the free-energy pertubation method, which uses statistical mechanics and molecular dynamics can often be used for quantitative calculation of free energy differences. We have applied such a technique to calculate the differential free energy of binding and free energy of activation for catalysis of a tripeptide substrate by native subtilisin and a subtilisin mutant (Asn 155----Ala 155). Our studies lead to a calculated difference in free energy of binding which is relatively small, but a calculated change in free energy of catalysis which is substantial. These energies are very close to those determined experimentally (J. A. Wells and D. A. Estell, personal communication), which were not known to us until the simulations were completed. This demonstrates the predictive power and utility of theoretical simulation methods in studies of the effects of site-specific mutagenesis on both enzyme binding and catalysis.     

Survival of rabbit embryos after rapid freezing and thawing

Summary Frozen storage of rabbit embryos at the 16-cell stage in 2.0 M dimethylsulfoxide (DMSO) in phosphate-buffered saline (PBS) was achieved by a 2-step procedure. After storage for 10 days at–196°C they were revived by rapidly thawing at 500°C/min. On transfer of these embryos to pseudopregnant foster mothers, 50% survived to term. The difference in in vivo survival between frozen-thawed and frozen-thawed-cultured embryos was not significant.Acknowledgment. The authors thank the Director for the facilities. VHR is supported by Council of Scientific and Industrial Research (India).     

Y－TZP和氮气压力对GPSSi_3N_4性能的影响

本文研究了Ｙ－ＴＺＰ（３ｍｏｌ％Ｙ２Ｏ３）和氮气压力对ＧＰＳＳｉ３Ｎ４陶瓷材料的烧结性能和力学性能、相组成及微观结构的影响，添加５ｗｔ％、１０ｗｔ％、１５ｗｔ％、２０ｗｔ％Ｙ－ＴＺＰ的氨化硅复合材料在１７７０～１８００℃，氮气压力分别为１ＭＰａ、２ＭＰａ、３ＭＰａ下烧成，获得相对密度＞９５％的烧结体。实验结果表明：添加＜１０ｗｔ％的Ｙ－ＴＺＰ及增大氮气压力有利于改善氰化硅陶瓷材料的烧结性能；Ｙ－ＴＺＰ可提高Ｓｉ３Ｎ４基体的断裂韧性，添加１５ｗｔ％ＴＺＰ的Ｓｉ３Ｎ４材料断裂韧性可达８．３３ＭＰａｍ１／２，与基体相比提高３０％，微裂纹增韧和第二相粒子增韧为主要增韧机理．     

低温腐蚀及防护

阐述了低温腐蚀的危害性,低温腐蚀的机理低温腐蚀的影响因素,以及露点的计算和腐蚀规律,并且提出了防止发生和减少低温腐蚀的具体措施。     

Movement and segregation of kinetochores experimentally detached from mammalian chromosomes

The kinetochore is a specialized structure at the centromere of eukaryotic chromosomes that attaches chromosomes to the mitotic spindle. Recently, several lines of evidence have suggested that kinetochores may have more than a passive role in the movement of chromosomes during mitosis and meiosis. Kinetochores seem to attract and 'capture' microtubules that grow from the spindle poles and microtubules may lengthen or shorten by the addition or subtraction of tubulin subunits at their kinetochore-associated ends. An attractive hypothesis is that kinetochores function as 'self-contained engines running on a microtubule track'. Here, we show that kinetochores can be experimentally detached from chromosomes when caffeine is applied to Chinese hamster ovary cells that are arrested in the G1/S phase of the cell cycle. The detached kinetochore fragments can still interact with spindle microtubules and complete all the mitotic movements in the absence of other chromosomal components. As these cells enter mitosis before DNA synthesis is completed, chromosome replication need not be a prerequisite for the pairing, alignment and segregation of kinetochores.     

Ciliary structures in the branchial unicellular glands of the grass shrimp,Palaemonetes pugio

Summary A unicellular exocrine gland possessing an epicuticular ductule occurs in grass shrimp gills. This gland displays ultrastructural changes in relation to the molt cycle. These changes include an increase in the quantity of secretory granules during late premolt, and the development of ciliary axonemes in relation to ductule formation.This investigation was supported in part by grant CR-807417 from the U.S. Environmental Protection Agency.     

Shoot regeneration fromAgrobacterium tumefaciens-induced tumors of a tropical timber tree,Fagraea fragrans

Summary Agrobacterium tumefaciens A208 with nopaline plasmid pTiT37 was used to obtain stem tumors on plantlets ofFagraea fragrans grown in vitro. Bacterial elimination and tissue proliferation were simultaneously achieved by growing tumors on cefatoxime medium. After some tissue growth the shoots regenerated. An examination of these showed the presence of nopaline, indicating genetic transformation by T-DNA.