Cellular interactions between 3T3 cells and interleukin-3-dependent multipotent haemopoietic cells: a model system for stromal-cell-mediated haemopoiesis

With the aid of a multipotent stem cell line (FDCP-mix cells) co-cultured with either normal or irradiated Swiss 3T3, cellular interactions between stromal cells and haemopoietic stem cells were studied by electron microscopy and time-lapse video microscopy. When cultured in the presence of interleukin 3 (IL-3) but in the absence of stromal cells, the FDCP-mix cells have a characteristic blast morphology. In the absence of IL-3, the cells die unless they are co-cultured with marrow stromal cells or 3T3 cells. In the latter case, they attach, proliferate, and differentiate on both normal and irradiated Swiss 3T3 cell layers without the addition of extrinsic growth factor (IL-3). At the initial attachment sites of these two cell lines, cellular recognition seemed to be mediated by the formation of microvillus cytoplasmic projections and extracellular matrix. These areas may well be the sites of plasma-membrane-bound signalling/adhesional molecules between the interacting cells.     

Bactericidal activity of peripheral blood neutrophils during the oestrous cycle and early pregnancy in the mare

The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of 10 mg oestradiol-17 beta and 150 mg progesterone given i.m. for 10 days. On the 10th day, 10-15 mg prostaglandin F-2 alpha was administered i.m. to induce oestrus. Neutrophils were isolated from jugular blood on the 2nd or 3rd day of oestrus, Days 5 and 7 after ovulation or during early pregnancy (Days 18-34 of pregnancy). Neutrophils were challenged with Staphylococcus aureus and their bactericidal activity examined after 30 and 120 min of incubation for a reduction of colony forming units. Bactericidal activity increased with the time of incubation (P less than 0.01) but did not differ for the oestrous cycle or pregnancy (P greater than 0.05).     

Mechanics of root growth

Summary A model is developed for the rate of elongation of a root tip in terms of the balance of pressures acting on the root. Differentials of this equation give expressions for the changes in root elongation rate with respect to soil water potential and soil mechanical resistance. The model predicts that root cells osmoregulate against both water stress and soil mechanical resistance with predicts that root cells osmoregulate against both water stress and soil mechanical resistance with similar efficiencies which are less than 100%. Analysis of published data leads to the conclusion that root tips of pea osmoregulate with 70% efficiency. A working equation is developed for the elongation rate of roots in conditions of combined water stress and mechanical resistance.     

Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells

When highly enriched multipotential spleen colony forming cells (CFU-S) obtained following fluorescence activated cell sorting (FACS-CFU-S) are cultured on marrow stromal cells, they undergo proliferation and development to produce mature haemopoietic cells (Spooncer et al., Nature, 316:62-64, 1985). We now show that FACS-CFU-S behave in a similar way when cultured on monolayers of 3T3 cells, indicating that the 3T3 cells can supply at least part of the environment which is representative of marrow stromal cells and provide, therefore, a system for studying stromal cell: haemopoietic cell interactions. We also demonstrate that IL-3-dependent multipotential stem cell lines (FDCP-Mix), but not a variety of other "committed" IL-3-dependent cell lines, resemble FACS-CFU-S in terms of their ability to proliferate and differentiate when cultured on 3T3 cells in the absence of IL-3. In this system, attachment of the FDCP-Mix to the 3T3 cells is critical for the subsequent maintenance of viability and stimulation of development of the cells. When the FDCP-Mix cells are physically separated from the 3T3 cells, they die and their death cannot be prevented by using 3T3-cell-conditioned medium. The extracellular matrix generated by 3T3 cells is not sufficient for promoting attachment or viability of the FDCP-Mix cells, indicating the importance of integral membrane components. However, attachment and development of FDCP-Mix cells occurs on 3T3 cells that have been lightly fixed with glutaraldehyde indicating that active metabolism is not essential for the effects promoted by the 3T3 cells. We suggest that the ability of FACS-CFU-S and FDCP-Mix cells to respond to 3T3 cells involves specific ligand/receptor interactions.     

Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.     

Studies on the Tissue Distribution of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases

An antiserum generated to the soluble form of the rat brain puromycin-sensitive enkephalin-degrading aminopeptidase was used to determine the tissue distribution of the soluble and membrane-associated forms of this enzyme. All tissues examined contained significant levels of the soluble enzyme form, with this enzyme accounting for greater than 90% of the arylamidase activity in brain, heart, and skeletal muscle. Native gel electrophoresis coupled with activity staining as well as inhibition studies were used to confirm the presence of this enzyme in various tissues. Serum was found not to contain this particular aminopeptidase. In contrast to the results obtained with the soluble enzyme form, brain was the only tissue found to contain the membrane-associated enzyme form. Although all tissues contained membrane-associated aminopeptidase activity only the brain enzyme could be maintained in solution in the absence of detergent. In addition, the brain membrane-associated enzyme could be distinguished from the membrane-associated aminopeptidase activity in other tissues on the basis of its sensitivity to inhibition by puromycin.     

Glyphosate Induces 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Potato (Solanum tuberosum L.) Cells Grown in Suspension Culture

The activity of the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, varies during the growth cycle of Solanum tuberosum L. cv superior cells in suspension culture. Maximum specific enzyme activity was observed midway through the linear phase of growth. When mid-log phase cells are exposed to glyphosate, the specific activity of the enzyme increases severalfold within 24 hours. The glyphosate-induced increase in enzyme activity is due to an increase in the amount of enzyme as determined by immunoblotting. Glyphosate (up to 2 millimolar) has no effect on the enzyme activity in vitro. Dehydroquinate synthase, the second enzyme of the shikimate pathway, is not induced by glyphosate.