An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.
Previously, we reported autocrine involvement of Sarcophaga lectin in the development of Sarcophaga imaginal discs (Kawaguchi et al., Dev. Biol. 144, 86–93 (1991)). In this study, we purified Sarcophaga lectin binding protein from the membrane fraction of cultured embryonic cells of Sarcophaga to near homogeneity and raised a monoclonal antibody against it. Histochemical analysis using the monoclonal antibody revealed that this binding protein is distributed heterogeneously on the surface of leg imaginal discs. This binding protein was especially clearly localized in the central region of the basal side of leg discs which forms the junction between the leg and body, suggesting the participation of Sarcophaga lectin in morphogenesis of the basal region of the developing leg.
When Sarcophaga lectin (from the flesh fly, Sarcophaga peregrina), an insect humoral lectin, was eluted from a column of DEAE-cellulose in the presence of galactose (a hapten sugar of this lectin), it emerged at a lower salt concentration than when galactose was absent. In the presence of galactose the lectin was, in addition, more susceptible to trypsin digestion. The lectin was found to have an affinity for basic proteins such as histone H3 and sarcotoxin IA, but this property was lost in the presence of galactose. These results suggested that the lectin changes its conformation on interaction with galactose. This change is suggested to result in the exposure of some hidden lysine and/or arginine residues.
Cell growth switches are engineered signaling molecules that can trigger cell growth in response to artificial ligands such as chemical inducers of dimerization (CIDs). We have previously shown that a cell growth switch comprised of the intracellular portion of the thrombopoietin receptor, Mpl, allows for the CID-dependent, in vivo expansion of genetically modified primary hematopoietic cells in mouse and dog models. Here we report the application of this approach to the in vivo expansion of genetically modified primary human hematopoietic cells using an immune deficient mouse model. A lentivirus vector encoding a CID-activatible deriviative of Mpl (F36VMpl) and a green fluorescent protein (GFP) reporter was used to transduce human cord blood CD34+ cells. Transduced human cord blood CD34+ cells expanded 347–495 fold in cultures containing no added growth factors and 100 nM of AP20187. We proceeded to test CID-responsiveness following transplantation into NOD-SCID-beta 2 microglobulin null mice. Since significant human red cell engraftment persists for only a few weeks post transplantation in this model, mice were evaluated at 3 weeks post transplantation, following a 2 week course of treatment with either CID (AP20187 10 mg/kg/day) or control vehicle alone (without CID). In 2 experiments totalling 42 mice, CID-administration resulted in a significant rise in GFP positive human cells, with the predominant response occuring among human erythroid cells (exp.1: 4.0 fold, P = 0.0003, exp.2: 12.7 fold, P = 0.038). An increase in transduced human erythroid progenitor cells was observed in the spleens (but not the femurs) of CID treated mice. Effects on other hematopoietic lineages were minor and variable. The effect of CID treatment was no longer evident five weeks after the last dose. The restriction of CID-induced cell growth to the erythroid lineage may make this approach well suited for gene therapy applications in sickle cell anemia and beta thalassemia.
Hematopoietic cells can respond differently to a mitogenic signal depending on whether the signal is delivered in vitro or in vivo. Results presented here suggest that the compartment in which a human hematopoietic progenitor resides can influence its response to a proliferative stimulus. An ectopically expressed conditional derivative of the thrombopoietin receptor (F36VMpl) in human cord blood CD34+ cells induces, upon provision of an agonist ligand, the differentiation (but not expansion) of transduced human burst forming units erythroid (BFUe) in culture. Here we show that upon transplantation into immune deficient mice, F36VMpl-expressing human BFUe and multipotential mixed progenitors (CFU-mix) decline in the marrows of ligand-exposed mice, replaced by a 12–17 fold expansion of differentiated erythroid precursors. In contrast, F36VMpl signaling dramatically expands BFUe and CFUmix in the spleen. Clonal analysis is consistent with the interpretation that F36Vmpl signaling induces an in situ expansion of BFUe and CFUmix in the spleen. In aggregate, these findings suggest that differences in environmental signals present in the marrow versus spleen can regulate the differentiation versus expansion of BFUe and CFU-mix.
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