We report the crystal structure of a fragment of Sinorhizobium meliloti DctD, a bacterial enhancer binding protein, at 1.7 Å. The fragment contains the protein's two‐component receiver module and adjacent linker, which in the native protein joins the receiver domain to a σ54‐dependent ATPase domain. The structure reveals a novel dimerization surface, which sequence analysis indicates is common to 4.5% of the known two‐component receiver domains. Genetic, biochemical, and structural data for amino acid substitution variants indicate that the dimer is necessary to inhibit the basal activity of the ATPase domain. The dimerization element is thus needed to maintain the “off” state, and changes within it may signal activation. Analytical ultracentrifugation data for the phosphorylated fragment of DctD appear to rule out the simple model that signaling is mediated via monomerization of the receiver domain.
Insulin-like growth factor system components are synthesized and secreted by mammary epithelial cells and multiple IGF binding proteins (IGFBP) are found in milk of various species. This study was conducted to identify the IGFBP in bovine milk, to compare them with those found in blood, and to identify the cell(s) responsible for mammary IGFBP synthesis. Bovine blood, milk, and cell culture-conditioned media were analyzed and characterized with Western ligand blot procedures for specific IGFBP. Electrophoresis and [125I]IGF-II ligand blot analyses of the samples indicated that, unlike serum and mammary primary cell culture-conditioned media, milk required removal of casein in order to accurately disclose all IGFBP. Immunoprecipitation studies identified IGFBP-2, -3, -4, and -5 in blood, milk, and primary cell culture conditioned media. The IGFBP were present at higher concentrations in serum than in milk, and milk concentrations were greater than that shown in conditioned media from primary cultures of bovine mammary cells. Northern analysis detected IGFBP-3 messenger RNA in extracts from fresh tissue and cells in culture, and in situ hybridization studies with fresh tissue utilizing probes for IGFBP-3 and alphaS1-casein showed that the mRNA for IGFBP-3 is predominant in the secretory epithelial cells, when compared to other tissue cell types.
Newborn rat pups were artificially reared by the pup in cup (PIC) method to determine whether dietary long arginine3 IGF-I (long R3 IGF-I), an IGF-I analog with high receptor affinity and low IGF binding protein (IGFBP) affinity, had efficacy on intestinal growth. IGF effects are mediated by IGFBP and receptor interactions, hence dietary-induced changes in intestinal IGF-II receptor patterns and IGFBP-3 message levels were investigated. Intestinal micrographs of pups fed rat milk replacer (RMR) for 3 d showed flattened villi with low cell counts and appeared similar to newborn intestines. Mother-fed (MF) controls and long R3 IGF-I-fed pups showed increased villi height and cell counts when compared with RMR pups, with long R3 IGF-I fed pups showing the greatest increase. At birth IGF-II-specific binding was not uniform in the intestine; specific binding was higher in the proximal intestinal section than in the distal intestinal section. However, after 3 d of MF treatment, specific binding had reversed and the distal section showed higher IGF-II-specific binding. Three days of RMR feeding did not change IGF-II-specific binding from that of the newborn pup. An IGFBP-3 message was identified in intestinal epithelium by in situ hybridization. Northern analysis of IGFBP-3 message showed a decline over time, but the change was not influenced by dietary treatments. In summary, milk-borne growth factors have the potential to affect intestinal growth within 3 d of treatment.
A hallmark of mammary cell differentiation is the induction of beta-casein mRNA expression. A mouse mammary epithelial cell line (COMMA-1D) was treated with insulin, hydrocortisone (HC), and prolactin (Prl) at concentrations (50, 500, and 20 ng/ml, respectively) that resulted in less than half-maximal beta-casein mRNA expression. The cells secreted insulin-like growth factor (IGF)-II (106 pg/ml per 24 h) in the condition media under these conditions. Replacement of insulin with rhIGF-II (150 ng/ml) resulted in significantly less beta-casein mRNA expression. Long-Arg IGF-I (50 ng/ml) was similar to insulin in terms of its ability to induce differentiation, but its activity differed from that of insulin in that it also induced cell proliferation. When the two receptor-specific IGF-II analogs, Arg54,55IGF-II and Leu27IGF-II, were used in studies, only at high concentrations (150 ng/ml) was either analog capable of stimulating any beta-casein mRNA expression. When autocrine IGF-II was immuno-neutralized or bound by the addition of rhIGF binding protein-3 (IGFBP-3)beta-casein mRNA expression was enhanced seven-fold and three-fold, respectively. Exogenous application of IGF-II to counteract the IGF-II mAb stimulation resulted in increased cellular growth and reduced differentiation. We conclude that autocrine IGF-II inhibits mammary cell differentiation and that the blockage of autocrine IGF-II benefits mammary cell differentiation.
The Rhizobium meliloti DctD two-component receiver domain was expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using the hanging-drop vapor-diffusion geometry with ammonium phosphate as the precipitant. The crystals diffract to 2.3 A and exhibit the symmetry of space group I222 or I212121. The unit-cell dimensions are a = 59.0, b = 58.6 and c = 169.8 A. The asymmetric unit contains a dimer and the crystals have a Vm of 2.16 A3 Da-1.
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