Apoptosis during preimplantationdevelopment has received much interest because of its potential role in eliminating defective cells. Although development in humans is characterised by a high degree of genetic abnormality, little is known of the regulation of apoptosis in embryos. By PolyA PCR we analysed expression of 11 BCL-2 genes in individual human embryos representative of normal development and in severely fragmented embryos. We demonstrate constitutive expression of BAX in virtually all embryos at all stages of development, and variable expression of BCL2, BCL-XL, BCL-W, MCL-1 BAK, BAD, BOKL, BID,BIK, and BCL-XS. The frequency of expression of pro- and anti-apoptotic BCL-2 members was similar throughout development, except at the two-cell stage where pro-apoptotic genes predominated. Protein expression was confirmed for BCL-2, MCL-1, BCL-X,BAX, BAD, and activated caspase 3. BCL-2 protein was associated with mitochondria but expressed inconsis- tently in the blastocyst inner cell mass. Consistent differences between morphologically intact and frag- mented embryos included the expression of BAK in fragmented but not intact four-cell embryos. Our study addresses the importance of examining single human embryos representative of the viable population for a large number of genes, in order to establish meaningful expression profiles and provide information on over-lapping function in a large gene family

Formation of a fully differentiated, implantation competent blastocyst requires the expression of a complex repertoire of molecules. However, the events that drive morphogenesis are poorly elucidated in the human embryo. In this work, we describe the amplification of representative cDNAs from morphologically and developmentally normal, individual human embryos at all stages from pronucleate to blastocyst. These cDNAs were probed to reveal the temporal expression pattern of cell adhesion molecules thought to play a key role in murine preimplantation embryo development. We demonstrated constitutive expression of beta actin, beta 1 and alpha 6 integrins, ZO-1 and E-cadherin, as shown previously in mouse embryos. No expression of beta 3, alpha 2, alpha 3 or alpha 7 integrins nor of L or P selectin was detected at any stage of preimplantation development. beta 5 integrin showed a regulated pattern of expression and was not expressed in blastocysts, while desmocollin-2 could only be detected at the blastocyst stage. Expression and localization of beta 1, beta 5 and alpha 6 integrins and ZO-1 and E-cadherin proteins was confirmed in blastocyst stage embryos by immunocytochemistry. We have identified differences in the expression of integrin molecules between mouse and human embryos, and propose a role for alpha v beta 5 and alpha 6 beta 1 integrin dimers in the human embryo at implantation.

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells. Reproduction (2008) 135 635-647

The 'H NMR spectrum of chlorpromazine hydrochloride was fully assigned at 400 MHz Similarly, the "C NMR spectrum was assigned unambiguously using two-dimensional NMR. Measurements of chemical shift as a function of concentration in D,O showed appreciable changes of shift of both protons and carbons which were apparent even at solution concentrations two orders of magnitude lower than the critical micelle concentration (CMC). The relative magnitude of the shifts of the aromatic protons and carbons on dilution below the CMC were compatible with vertical stacking of the molecules in an off-set manner such that maximum overlap of the chlorinated rings occurred. Proton chemical shift data were interpreted using a stepwise association model to quantify the extent of association in the pre-CMC region.

Aims:The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria. This required the preparation of deuterium-tolerant bacterial strains and an understanding of the physiological mechanisms of acquisition of deuterium tolerance. Methods and Results: We report here improved methods for the cultivation of Escherichia coli on fully deuteriated minimal medium. A multi-stage adaptation protocol was developed; this included repeated plating and selection of colonies and resulted in highly deuterium-tolerant cell cultures. Three E. coli strains, JM109, MRE600 and MRE600Rif, were adapted to growth on deuteriated succinate medium. This is the first report of JM109 being adapted to deuteriated minimal media. The adapted strains showed good, consistent growth rates and were capable of being transformed with plasmids. Expression of heterologous proteins in these strains was reliable and yields were consistently high (100-200 mg l )1 ). We also show that all E. coli cells are inherently capable of growth on deuteriated media. Conclusions: We have developed a new adaptation protocol that resulted in three highly deuterium-tolerant E. coli strains. Deuterium-adapted cultures produced good yields of a deuteriated recombinant protein. We suggest that E. coli cells are inherently capable of growth on deuteriated media, but that non-specific mutations enhance deuterium tolerance. Thus plating and selection of colonies leads to highly deuterium-tolerant strains. Significance and Impact of Study: An understanding of the mechanism of adaptation of E. coli to growth on deuteriated media allows strategies for the development of disabled deuterium-tolerant strains suitable for high-level production of deuteriated recombinant proteins and other biomolecules. This is of particular importance for nuclear magnetic resonance and neutron scattering studies of biomolecules.