Lymphoid precursor cells adhere to two different sites on fibronectin

Several precursor lymphoid cell lines, blocked at specific stages of differentiation, adhere specifically to fibronectin in vitro. Whereas the Ba F3 cell line, which has both immunoglobulin heavy- and light-chain genes in germline configuration, interacts with the arg-gly-asp-containing cell-binding domain of fibronectin, the B-committed line PD 31, which is undergoing rearrangement of immunoglobulin light-chain genes, does not. Accordingly the Ba F3, but not the putative PD 31 surface fibronectin receptor, binds to an affinity matrix containing the 115-kD cell-binding domain of fibronectin. PD 31 cells recognize a different domain of the fibronectin molecule, which is contained within the carboxy terminal segment possessing a high-affinity binding site for heparin. A polyclonal antibody raised against the fibronectin receptor of mouse erythroleukemic cells inhibits adhesion of these lymphoid lines to fibronectin. It precipitates two major species of 140 and 70 kD from surface-radioiodinated Ba F3 cells and species of 140 and 120 kD from PD 31 cells. We propose that the two types of cells express different fibronectin receptors mediating substrate adhesion, and suggest that receptor(s) with different specificity might be expressed in the course of B cell maturation. Because we show that these adhesion properties are shared by normal bone marrow lymphoid precursors, we infer that these receptors may play a role in normal lymphopoiesis.     

Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells

Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3–0.5 ng/10⁵ cells-hour when exposed to medium without secretagogues. When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10⁵ cells-hour while intracellular hGH declined sharply. Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial. In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted. When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles. The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur. Operational modifications that may improve the performance of cycling schemes are discussed.     

In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.     

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.     

Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.     

Signaling by chimeric erythropoietin-TGF-beta receptors: homodimerization of the cytoplasmic domain of the type I TGF-beta receptor and heterodimerization with the type II receptor are both required for intracellular signal transduction.

Transforming growth factor-beta (TGF-beta) affects multiple cellular functions through the type I and type II receptor Ser/Thr kinases (TbetaRI and TbetaRII). Analysis of TGF-beta signaling pathways has been hampered by the lack of cell lines in which both TbetaRI and TbetaRII are deleted, and by the inability to study signal transduction by TbetaRI independently of TbetaRII since TbetaRI does not bind TGF-beta directly. To overcome these problems, we constructed and expressed chimeric receptors with the extracellular domain of the erythropoietin receptor (EpoR) and the cytoplasmic domains of TbetaRI or TbetaRII. When expressed in Ba/F3 cells, which do not express EpoR, Epo induces the formation of a heteromeric complex between cell surface EpoR-TbetaRI and EpoR-TbetaRII chimeras. Neither the EpoR-TbetaRI nor the EpoR-TbetaRII chimera interacts with endogenous TGF-beta receptors. Ba/F3 cells expressing both EpoR-TbetaRI and EpoR-TbetaRII chimeras, but not EpoR-TbetaRI or EpoR-TbetaRII alone, undergo Epo-induced growth arrest. When expressed in Ba/F3 cells in the absence of the EpoR-TbetaRII chimera, EpoR-TbetaRI(T204D), a chimeric receptor with a point mutation in the GS domain of TbetaRI that is autophosphorylated constitutively, triggers growth inhibition in response to Epo. Thus, both homo- and heterodimerization of the cytoplasmic domain of the type I TGF-beta receptor are required for intracellular signal transduction leading to inhibition of cell proliferation. These chimeric receptors provide a unique system to study the function and signal transduction of individual TGF-beta receptor subunits independently of endogenous TGF-beta receptors.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.