Derivation and characterisation of hESC lines from supernumerary embryos, experience from Odense, Denmark

The derivation and characterisation of human embryonic stem cells provides a source of pluripotent stem cells with potential for clinical applications. Utilising locally sourced embryos from two IVF clinics, we derived and characterised five new cell lines for use in a non-clinical setting. Analysis of clinical data showed that the majority of embryos (94.5%) failed to reach the blastocyst stage of development and of all embryos, regardless of developmental status, 248 embryos were needed to create one stem cell line. From the number of embryos (69) which developed to the blastocyst stage 8.7% developed into cell lines. Using outgrowth of the whole blastocyst, we derived five new, unreported cell lines in Odense, Denmark between 2005 and 2006. Characterisation was carried out using RT-PCR, staining, karyotyping, EB formation and teratoma formation. The KMEB hESC lines will, in the future, be made available through the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk/).     

Soluble rat adipocyte phosphatidate phosphatase activity: characterization and effects of fasting and various lipids.

Phosphatidate phosphatase (phosphatidate phosphohydrolase, EC 3.1.3.4) was present at very high specific activity in the soluble fraction of isolated rat adipocytes. Using phosphatidate in aqueous dispersion 90% of its hydrolysis depended on the presence of Mg2+. Mg2+ appeared to almost saturate the enzyme at 20-40 mM with no indication of an optimum. The substrate concentration was optimum at 1.2 mM and the pH at 6.8. Initial rates were linear for only 4-5 min at optimum conditions. Increasing inhibition occurred at high phosphatidate concentrations. At optimum conditions acid or alkaline phosphatase activity was not measurable. The Mg2+-dependent activity was enhanced by 3-sn-phophatidylcholine and inhibited by albumin, 3-sn-phosphatidyletanolamine, 3-sn-phosphatidylinositol, diacylglycerol, oleoyl-CoA, and oleate. Oleoyl-CoA was the most potent "effector". Fasting for 24, 48 and 72 h decreased the activity both relative to protein and to DNA. The activity thus decreased to about one-third of that of the fed rat during 72 h of fasting. The effects of Mg2+, various lipids, and fasting may indicate that some form of control of glyceride synthesis can be exerted through the soluble phosphatidate phosphatase.     

Purification and properties of 2,3-bisphosphoglycerate phosphatase-mutase from erythrocytes of day-old chicks.

After tryptic digestion of intact Staphylococcus aureus the residual portion of protein A that was still bound to the cell wall was cleaved off with lysostaphin. From the two digests Fc-binding fragments were isolated and the following characteristics observed. (a) There are four Fc-binding, highly homologous regions, each consisting of 58--62 amino acid residues. (b) These regions are consecutively arranged from the N-terminal part of the protein. (c) The residual C-terminal part, approximately 150-residues long, differs to a great extent with respect to primary and secondary structures from the four active regions. Furthermore, it is suggested that the protein is bound to the bacterial cell wall structure through this C-terminal part.     

Developmental changes in purine phosphoribosyltransferases in human and rat tissues.

1. The hypoxanthine/guanine and adenine phosphoribosyltransferase activities in a wide variety of human tissues were studied during their growth and development from foetal life onward. A wide range of activities develop after birth, with especially high values in the central nervous system and testes. 2. Postnatal development of hypoxanthine/guanine phosphoribosyltransferase was also defined in the rat. Although there were increases in the central nervous system and testes, there was also a rise in activity in the liver, which was less marked in man. 3. A sensitive radiochemical assay method, using dTTP to inhibit 5'-nucleotidase activity, suitable for tissue extracts, was developed. 4. No definite evidence of the existence of tissue-specific isoenzymes of hypoxanthine/guanine or adenine phosphoribosyltransferase was found. Hypoxanthine/guanine phosphoribosyltransferase in testes, however, had a significantly different thermal-denaturation rate constant. 5. The findings are discussed in an attempt to relate activity of hypoxanthine/guanine phosphoribosyltransferase to biological function. Growth as well as some developmental changes appear to be related to increase in the activity of this enzyme.     

Supramolecular structures of peptide assemblies in membranes by neutron off-plane scattering: method of analysis

In a previous paper (Yang et al., Biophys. J. 75:641-645, 1998), we showed a simple, efficient method of recording the diffraction patterns of supramolecular peptide assemblies in membranes where the samples were prepared in the form of oriented multilayers. Here we develop a method of analysis based on the diffraction theory of two-dimensional liquids. Gramicidin was used as a prototype model because its pore structure in membrane in known. At full hydration, the diffraction patterns of alamethicin and magainin are similar to gramicidin except in the scale of q (the momentum transfer of scattering), clearly indicating that both alamethicin and magainin form pores in membranes but of different sizes. When the hydration of the multilayer samples was decreased while the bilayers were still fluid, the in-plane positions of the membrane pores became correlated from one bilayer to the next. We believe that this is a new manifestation of the hydration force. The effect is most prominent in magainin patterns, which are used to demonstrate the method of analysis. When magainin samples were further dehydrated or cooled, the liquid-like diffraction turned into crystal-like patterns. This discovery points to the possibility of investigating the supramolecular structures with high-order diffraction.