The native sIGFIR decoy protein blocks IGF-I and IGF-II mediated functions in vitro

Summary Sporulation in Bacillus subtilis has long been a model of cellular differentiation, many aspects of which are well understood. The early stage of this process is of particular interest, especially the interrelationship of regulatory processes with metabolism in response to environmental changes. We analyzed cellular fatty acids as their methyl esters using capillary gas chromatography coupled to mass spectrometry during the transition from vegetative growth to early sporulation phase. Measurement of changes in the content of heavy fatty acid analogs in cultures supplemented with deuterium-labeled valine or methionine, or 13 C-labeld valine, showed that label was incorporated into the backbone of 12-methyltridecanoic and 14-methylpentadecanoic acid, in both sporulating and Δspo0A cultures. These fatty acids were formed starting with isobutyryl-CoA apparently originating only from L -valine-d 8 in cultures so supplemented. Our observations indicate that following vegetative growth a pathway exists from certain amino acids into fatty acid methylene groups, evidently passing through propionyl-CoA. This finding has the potential to deepen understanding of the metabolic basis of cellular differentiation and identify new targets for antibiotics. We also observed a significant, continuous increase in the proportion of 13-methyltetradecanoic acid in fatty acids during the same period in which the pre-spore membrane would be formed. Graphical abstract: Abbreviated Summary Early in at least some Bacillus subtilis differentiation scenarios, a straight-chain metabolite derived from propionyl-CoA is incorporated into fatty acids primed with isobutyryl-CoA possibly derived from cellular protein valine. Concurrently a leucine related fatty acid increases significantly, potentially comprising the predominant pre-spore septum fatty acid component. These processes occur in conjunction with the onset of fatty acid β-oxidation and bulk protein turnover.

Production and characterization of native, sIGFIR

This document contains Table S1 to S3

Plasma insulin levels are not markedly altered in IGF-Trap injected mice

The IGF-Trap blocks IGF-I and IGF-II mediated functions in vitro

Summary Sporulation in Bacillus subtilis has long been a model of cellular differentiation, many aspects of which are well understood. The early stage of this process is of particular interest, especially the interrelationship of regulatory processes with metabolism in response to environmental changes. We analyzed cellular fatty acids as their methyl esters using capillary gas chromatography coupled to mass spectrometry during the transition from vegetative growth to early sporulation phase. Measurement of changes in the content of heavy fatty acid analogs in cultures supplemented with deuterium-labeled valine or methionine, or 13C-labeld valine, showed that label was incorporated into the backbone of 12-methyltridecanoic and 14-methylpentadecanoic acid, in both sporulating and Δspo0A cultures. These fatty acids were formed starting with isobutyryl-CoA apparently originating only from L -valine-d8 in cultures so supplemented. Our observations indicate that following vegetative growth a pathway exists from certain amino acids into fatty acid methylene groups, evidently passing through propionyl-CoA. This finding has the potential to deepen understanding of the metabolic basis of cellular differentiation and identify new targets for antibiotics. We also observed a significant, continuous increase in the proportion of 13-methyltetradecanoic acid in fatty acids during the same period in which the pre-spore membrane would be formed. ![Figure][1] Abbreviated Summary Early in at least some Bacillus subtilis differentiation scenarios, a straight-chain metabolite derived from propionyl-CoA is incorporated into fatty acids primed with isobutyryl-CoA possibly derived from cellular protein valine. Concurrently a leucine related fatty acid increases significantly, potentially comprising the predominant pre-spore septum fatty acid component. These processes occur in conjunction with the onset of fatty acid β-oxidation and bulk protein turnover. ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Production and characterization of the IGF-Trap

Kinetics and specificity of ligand binding to rhIGFBP-1

This documents contains the materials and methods of experiments described in supplementary data

This documents contains the legends of Figures 1 to S6

AbstractThe IGFI receptor promotes malignant progression and has been recognized as a target for cancer therapy. Clinical trials with anti-IGFIR antibodies provided evidence of therapeutic efficacy but exposed limitations due in part to effects on, and the compensatory function of, the insulin receptor system. Here, we report on the production, characterization, and biologic activity of a novel, IGF-targeting protein (the IGF-Trap) comprising a soluble form of hIGFIR and the Fc portion of hIgG1. The IGF-Trap has a high affinity for hIGFI and hIGFII but low affinity for insulin, as revealed by surface plasmon resonance. It efficiently blocked IGFIR signaling in several carcinoma cell types and inhibited tumor cell proliferation, migration, and invasion in vitro. In vivo, the IGF-Trap showed favorable pharmacokinetic properties and could suppress the growth of established breast carcinoma tumors when administered therapeutically into tumor-bearing mice, improving disease-free survival. Moreover, IGF-Trap treatment markedly reduced experimental liver metastasis of colon and lung carcinoma cells, increasing tumor cell apoptosis and reducing angiogenesis. Finally, when compared with an anti-IGFIR antibody or IGF-binding protein-1 that were used at similar or higher concentrations, the IGF-Trap showed superior therapeutic efficacy to both inhibitors. Taken together, we have developed a targeted therapeutic molecule with highly potent anticancer effects that could address limitations of current IGFIR-targeting agents. Mol Cancer Ther; 14(4); 982–93. ©2015 AACR.

Thermodynamic analysis of hydrophobic interaction chromatography of amino acid methyl esters showed entropy-driven adsorption, consistent with solvophobic theory, except for phenyl ester on the Toyopearl resins. All esters adsorbed more strongly to the Toyopearl resins, including the polymethacrylate base matrix, than to Butyl Sepharose. Enthalpy changes were more favorable with the former, explaining the retention difference between Toyopearl Butyl and Butyl Sepharose. An enthalpy change versus heat capacity change plot showed Van der Waals interactions predominantly with the resin matrix. Literature data revealed the same effect for dansylamino acids, shown by isothermodynamic temperature analysis to adsorb more entropically than the esters.

A production process for B. thuringiensis (Bt) bioinsecticides was designed in detail, including alternative batch, low‐density fed‐batch (LDFB), and high‐density fed‐batch (HDFB) fermentation configurations. Capital and operating costs, as well as profitability based on simple rate of return, were performed using a purpose‐written FORTRAN program, explicitly analyzing production of a water‐based flowable product used in forestry applications.

The specific oxygen uptake rate (qO2, respiration rate) of Bacillus thuringiensis subsp. kurstaki HD‐1 was very high at inoculation and was found to decrease essentially monotonically throughout both vegetative growth phase and transition phase under different batch culture conditions. Average qO2 values decreased from 8–10 mmol/g h at 1 h after inoculation to less than 2 mmol/g h by the time growth ended. The results are shown to be consistent with the few previous reports on qO2 in B. thuringiensis in the literature but also novel in that this pattern of monotonic decline has not been described previously. Both pH control and EDTA in low concentration shortened the vegetative growth phase and reduced the 10 h biomass concentration. Using plots of qO2 versus specific growth rate, μ, biomass yield based on the oxygen used for growth, YmaxO2, was calculated for transition phase to be 0.041–0.047 g/mmol, consistent with literature values. The same plot also showed that the presence of EDTA resulted in an atypical qO2‐μ trajectory and apparently much higher biomass yield from the oxygen consumed.

In this study, a continuous protein recovery process using a Liquid-Solid Circulating Fluidized Bed (LSCFB) ion exchange system is described and a model with known kinetics has been developed. Experiments and computer simulations using Matlab(TM) are conducted at different operating conditions. The effects of hydrodynamic parameters and kinetic parameters on the performance of the LSCFB ion exchange system are discussed. The model is shown to be applicable tor the design of LSCFB ion exchange systems for protein recovery.

Bacillus thuringiensis kurstaki (HD-1) was grown on two different complex media to study its fatty acid composition during vegetative growth and sporulation. In contrast to literature results, iso-even branched-chain fatty acids were found to predominate after early vegetative growth and throughout sporulation.