Ex vivo expansion and differentiation of hematopoietic stem cells.

Hematopoietic stem cells are phenotypically very heterogeneous, probably reflecting the degree of activation and/or differentiation. This cell population is capable of high-level proliferative activity and multilineage differentiation. Despite its potential for self-renewal, the hematopoietic stem cell exists in a quiescent state for prolonged periods of time. The mechanism(s) involved in triggering these cells to enter the cell cycle is/are not totally clear; however, cytokines (both positive and negative regulators) are implicated. Most, if not all known cytokines that interact at the stem cell level do so not only by inducing proliferation but also differentiation. The ability to maintain a population of truly primitive stem cells for extended periods of time in vitro is currently under investigation by many research groups.

Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.

We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth.

Multilevel control of extracellular sucrose metabolism in Streptococcus salivarius by sucrose.

Standardized experimental conditions were established to test the role of sucrose in the regulation of control of its metabolism in Streptococcus salivarius. A fresh isolate of S. salivarius was used. The extracellular dextranase activity of cells grown on sucrose was 10-fold higher than that of cells grown on glucose, fructose or galactose. This activity increased in less than 5 min following the addition of sucrose to galactose-grown cells, a phenomenon which was affected by neither rifampicin nor chloramphenicol which inhibit transcription and translation, respectively. Extracellular fructanase activity was 2-fold higher when cells were grown on sucrose than when they were grown on the other sugars. This increase also occurred within 5 min, but was diminished by transcriptional and translational inhibitors. De novo synthesis was required for the production of extracellular glucosyltransferase (GTF) activity which, upon the addition of sucrose, became associated with the cell surface. Conversely, cell-associated fructosyltransferase (FTF) activity appeared to require genetic induction for its production and cell-surface association, but required sucrose for its release from the surface framework. Versatility in the control mechanisms of this complex set of enzymes allows their expression and function to be regulated at several widely separated stages in the life histories of these proteins.