The role of vitrification techniques of cryopreservation in reproductive medicine

Traditional cryopreservation methods allow ice to form and solute concentrations to rise during the preservation process: both ice and high solute concentrations can cause damage. Cryoprotectants are highly soluble, permeating compounds of low toxicity; they reduce the amount of ice that crystallises at any given temperature and thereby limit the solute concentration factor. Vitrification methods use cryoprotectant concentrations that are sufficient to prevent the crystallisation of ice altogether: the material solidifies as an amorphous glass and both ice and solute concentration are avoided. However, the concentrations of cryoprotectant required are very high indeed and therefore are potentially, and often actually, harmful to cells. Optimisation of the temperature and the rate of introduction and removal of such high cryoprotectant concentrations are critical. The necessary concentration can be lowered if very rapid cooling, and even more rapid warming, are used. This paper draws on experience in other fields of cryobiology to discuss these basic phenomena and to consider the place of vitrification techniques in the cryopreservation of human gametes, embryos and gonads.     

粒细胞对氯氮平药物敏感度的体外实验预测

目的:探讨精神病患者对服用氯氮平适应性的体外实验预测方法.方法:抽取600例受试者静脉血1 ml,注入含3.8%柠檬酸钠抗凝剂0.1 ml的试管内,加入10g/L氯氮平乙醇溶液100 ul,混匀,37℃温浴2h,制片,瑞氏染色.用显微镜观察粒细胞的结构变化.结果:在600例受试者中,585例粒细胞完整,结构无变化.15...     

Effect of alterations of key active site residues in O6-alkylguanine-DNA Alkyltransferase on its ability to modulate the genotoxicity of 1,2-dibromoethane

The production of mutations and the reduction in survival of cells treated with alpha,omega-dihaloalkanes is greatly enhanced by the presence of O6-alkylguanine-DNA alkyltransferase (AGT), a DNA repair protein that removes O6-alkylguanine adducts from DNA [Liu, L., Hachey, D. L., Valadez, G., Williams, K. M., Guengerich, F. P., Loktionova, N. A., Kanugula, S., and Pegg, A. E. (2004) J. Biol. Chem. 279, 4250-4259]. The effects of alterations to key residues in the active site of AGT were studied using AGTs with point mutations. It was found that mutants of AGT at positions Tyr114, Arg128, Pro140, Gly156, Gly160, and Tyr158 did not bring about the increase in genotoxicity of 1,2-dibromoethane seen with wild-type AGT, although these mutants, with the exception of those at Tyr114 and Arg128, are known to have sufficient AGT repair function to be able to protect cells from alkylating agents. The R128A mutant was able to react with 1,2-dibromoethane at the Cys145 acceptor site, but the resulting AGT-Cys145S-(CH2)2Br was much less able to produce a covalent adduct with DNA. This result is explained by the need for AGT to induce a structural change in the DNA "flipping" of a guanine nucleotide into the substrate binding pocket where Cys145 is located since the side chain of residue Arg128 plays a critical role in this reaction. Point mutations in AGT at the other sites (Y114A, P140K, and Y158H) reduced the ability of the protein to react with 1,2-dibromoethane as measured by the loss of activity. These results were confirmed by MS analysis of the tryptic peptide that contains the modified Cys145. There was no change in the stability of the AGT-Cys145S-(CH2)2Br intermediate formed in mutants Y158H and P140K. The reaction was studied in detail with mutant P140K using dihaloalkanes of different length; no effect of the mutations was seen with dibromomethane, but an enhanced difference was observed with 1,3-dibromopropane and 1,5-dibromopentane. These results show that even slight alterations in the active site pocket of AGT that do not prevent its ability to protect cells from alkylating agents can block the paradoxical enhancement of the genotoxicity of the larger alpha,omega-dihaloalkanes by reducing the reaction with Cys145.     

(ii) The role of imaging in follow-up of newly introduced implants

A wide variety of orthopaedic implants is in clinical use and many new devices are brought to the market each year. The vast majority of these devices have no published clinical data. The concept of phased introduction has been developed. The use of radiostereometry and cross-sectional imaging in the follow-up and phased introduction of new orthopaedic implants is discussed.     

The Sequence-Activity Relationship between Metallo-β-Lactamases IMP-1, IMP-6, and IMP-25 Suggests an Evolutionary Adaptation to Meropenem Exposure

Metallo-β-lactamases are important determinants of antibacterial resistance. In this study, we investigate the sequence-activity relationship between the closely related enzymes IMP-1, IMP-6, and IMP-25. While IMP-1 is the more efficient enzyme across the overall spectrum of tested β-lactam antibacterial agents, IMP-6 and IMP-25 seem to have evolved to specifically inactivate the newer carbapenem meropenem. Molecular modeling indicates that the G235S mutation distinguishing IMP-25 from IMP-1 and IMP-6 may affect enzyme activity via Asn233.