Jun Fujita

Presence of an activity indispensable for the granulocyte/macrophage colony-stimulating activity of interleukin 3 in bovine serum albumin.

Media conditioned by an interleukin 3 (IL-3)-producing T-cell line, STIL-3, as well as recombinant mouse IL-3 showed granulocyte/macrophage (GM) colony-stimulating activity in the semi-solid culture medium containing horse serum (HS) or bovine serum, but the activity was not apparent when fetal calf serum (FCS) was used. No such serum-dependency of GM colony formation was observed when abdominal wall conditioned medium or L-cell conditioned medium containing GM colony-stimulating factor was used. Although the levels of albumin and total protein were lower in FCS than HS, increase of FCS concentration did not affect the GM colony-stimulating activity of IL-3. Addition of bovine serum albumin (BSA) preparation to FCS, however, increased the number of GM colonies to the same level as that observed with HS. The levels of bacterial lipopolysaccharide (LPS) in sera and BSA and the effect on the bone marrow cells from LPS-nonresponsive C3H/HeJ mice indicated that the observed effect of BSA was not due to the contaminating LPS. The activity of BSA was not substituted by IL-1, IL-2, IL-4, IFN-gamma, TNF, NGF or erythropoietin. The present study suggests the presence in BSA of co-factor(s) of IL-3 in stimulating GM colony formation.

Detection of ras oncogenes by analysis of p21 proteins in human tumor cell lines

SummaryTo detect mutationally activated ras oncogenes, we analyzed electrophoretic mobilities of ras p21 proteins utilizing the fact that many ras oncogenes produce abnormal p21 proteins that migrate at SDS/polyacrylamide gel electrophoresis as a fast-moving or slow-moving species in comparison to a normal p21 depending on the kind of mutation. Of 18 human tumor cell lines analyzed, four (SW480, SW620 and SW403 colon cancers, and SW626 ovary cancer) produced p21 belonging to the slow-moving species, suggesting a point mutation within codon 12 of a member of the three ras genes, H-, Ki- and N-ras. Subsequent DNA transfection analysis using NIH/3T3 cells as recipients identified activated Ki-ras oncogenes in the same four but not in other 14 cell lines. Thus, the analysis of p21 might serve as a rapid primary method to screen a large number of tumor materials for the presence of certain types of mutationally activated ras oncogenes.

Production of interleukin-3 from a T-cell neoplasm

We have established a new T-cell line (CL-8313) that produces interleukin-3 from the spleen cells of mice with a radiation-induced myeloproliferative disorder. IL-3 activity was detected at an extremely high level in the culture medium of the CL-8313 cell line in the absence of any exogenous stimulator. A large amount of IL-3 transcript also was demonstrated in CL-8313 cells. BPA- and CSF-activity was detected at a high level in the culture medium of the CL-8313 cell line. Southern blot analysis of high molecular weight DNA from the CL-8313 cells showed they had unique rearrangement of the antigen receptor beta chain gene. Therefore, we concluded that CL-8313 cells belonged to T-lineage lymphocytes and constitutively produced a high level of IL-3.

Kidney Proximal Tubule Cells Originate from Approximately Four Progenitor Cells and Make Distinct Patches in Mouse Aggregation Chimeras

Giant lysosomal granules of bgJ/bgJ mutant mice were used as a marker to investigate the histological composition of kidney proximal tubule cells. Embryos of the bgJ/bgJ genotype and those of the +/+ genotype were aggregated, and lysosomes of their proximal tubule cells were histochemically stained with the β‐glucuronidase activity. Tubules composed of bgJ/bgJ‐type epithelial cells alone, tubules composed of +/+‐type epithelial cells alone and tubules containing both types of epithelial cells were observed in cross sections. When the long straight portion of proximal tubules was reconstructed from serial sections, most of the tubules were of the mixed type, and distinct patches of bgJ/bgJ‐type or +/+‐type epithelial cells were detectable. These patches appeared to extend to the longitudinal rather than the circumferential direction. This suggests the clonal proliferation followed the mixing of embryonic progenitor cells for proximal tubule cells. Estimation from proportions of bgJ/bgJ‐type proximal tubules in total examined proximal tubules gave a pool size of approximately four primordial precursor cells for proximal tubules in both right and left kindeys. The fact that the proportion of bgJ/bgJ‐type components was comparable between the right and left kidneys of each chimera suggests that all proximal tubule cells in both right and left kidneys may originate from common primordial precursor cells.