A proposed design for the cryopreservation of intact and adherent human embryonic stem cell colonies.

Recently, it was demonstrated that the application of slow-cooling cryopreservation protocols to adherent human embryonic stem (hES) cell colonies, cultured on matrigel or murine embryonic fibroblast feeder layers, resulted in marked improvement in postthaw viability and reduction in cell differentiation. However, the use of commercially available culture plates for this purpose presents several limitations. Most obviously, these plates are not designed for cryopreservation or to withstand the low temperatures encountered during liquid nitrogen cryopreservation, or both. The physical storage of cryopreserved plates is another consideration, in addition to difficulty in maintaining sterile conditions in liquid nitrogen storage and during the thaw phase in a water bath. Hence, a redesign of the cell culture plate for the cryopreservation of adherent hES cell colonies is proposed. In this model, a culture plate made of synthetic materials resistant to storage at -196 degrees C of liquid nitrogen is designed, with readily attachable screw-cap culture wells that function as a replacement for cryovial storage. The detachable wells facilitate storage and after thawing can easily be reattached to a specially designed holding plate. Currently, there are no commercially available cell culture plates using this design concept. The proposed design is envisioned to facilitate the cryopreservation of intact adherent hES cell colonies that could assist the development of automated systems for handling bulk quantities of cells.

Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source.

Bacillus subtilis can utilize the purine bases adenine, hypoxanthine and xanthine as nitrogen sources. The utilization of guanine as a nitrogen source is reported here. The first step is the deamination of guanine to xanthine catalysed by guanine deaminase (GDEase). To isolate mutants defective in GDEase activity, a collection of mutant strains was screened for strains unable to use guanine as a nitrogen source. The strain BFA1819 (yknA) showed the expected phenotype and no GDEase activity could be detected in this strain. A new name for yknA, namely gde, is proposed. The gde gene encodes a 156 amino acid polypeptide and was preceded by a promoter sequence that is recognized by the sigma(A) form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular mass inducer molecule. The level of GDEase was found to be subjected to global nitrogen control exerted by the GlnA/TnrA-dependent signalling pathway. The two regulatory proteins of this pathway, TnrA and GlnR, indirectly and positively affected gde expression. This is the first instance of a gene whose expression is positively regulated by GlnR. The GDEase amino acid sequence shows no homology with the mammalian enzyme. In agreement with this are the different physiological roles for the two enzymes.