Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.     

Glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction

Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of homotypic CCI between “Os Fr.B” having 5–6 GlcNAc termini, vs. absence of such homotypic CCI between “Os Fr.1” having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3 ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using BAP conjugates of glycosyl epitopes.     

The extraction of a tissue collagenase associated with ovulation in the rat

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.