Intact but not truncated insulin-like growth factor binding protein-3 (IGFBP-3) blocks IGF I-induced stimulation of osteoblasts: control of IGF signalling to bone cells by IGFBP-3-specific proteolysis?

IGFBP-3 is the predominant IGFBP in serum and the major IGFBP secreted by osteoblasts. Native and recombinant IGFBP-3 and a truncated form lacking the carboxyterminal third were tested for their effects on 2 osteoblastic cell lines. Intact but not truncated IGFBP-3 blocked IGF I-stimulated DNA and glycogen synthesis. Inhibition was dose-dependent and found whenever the concentration of intact IGFBP-3 exceeded the concentration of IGF I. Truncated IGFBP-3 appears to result from proteolytic cleavage and does occur in vivo. The loss of inhibition by IGFBP-3 may be regulated at the site of IGF target cells and thus be essential for IGF I-induced osteoblast growth.     

Synthesis and biological evaluation of a modified insulin incorporating the COOH-terminal hexapeptide (“D-region”) of insulin-like growth factor II

A modified insulin, in which the A chain moiety has been extended at the C-terminus with the “D region” of the insulin-like growth factor II, has been synthesized essentially by the procedures employed in this laboratory for the synthesis of insulin and analogues. This hybrid molecule displayed reduced insulin-like activities, 34.5% receptor binding, and 40.4% stimulation of lipogenesis relative to natural insulin. These findings suggest that the extension sequence (“D region”) attached at the C-terminus of the A chain may partially cover the putative receptor binding region of insulin, in support of speculations based on computer-generated models. These same models indicate that the extension peptide may interfere with one of the two regions implicated in insulin antibody recognition. In this regard, radioimmunoassay of the hybrid revealed potency even more reduced than biological activity: 18% relative to insulin. Growth factor assays of the hybrid (this laboratory, unpublished data) suggest that the “D region” of insulin-like growth factor II is not in itself the determinant of growth-promoting activity.     

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1–3)insulin-like growth factor I

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.     

A rapid method for the purification of β-lactamase from Bacillus cereus by affinity chromatography

A procedure for the purification of β-lactamase from Bacillus cereus in a single chromatographic step is described. The enzyme is isolated from the crude culture supernatant by affinity chromatography. An inhibitor, methicillin, was immobilised by covalent attachment to the insoluble column gel, Sepharose. The enzyme was adsorbed to the column ligand from the crude supernatant and was subsequently released by increasing the ionic strength of the eluting buffer. In this way the enzyme was selectively isolated from other proteins in the crude supernatant. About 98% of the original β-lactamase activity was recovered in the purified enzyme fraction.     

An alternate insulin-like growth factor I receptor signaling pathway for the progression of endothelial–mesenchymal transition

Emerging evidence suggests that endothelial-to-mesenchymal transition (EndoMT) is an important contributor to cardiovascular diseases and to vascular development and pathologies as well as in cancer progression. As in epithelial–mesenchymal transition (EMT), EndoMT may involve several regulated steps: disassembly of adherence junctions or loss of cell–cell contacts, cytoskeletal reorganization, proteases, cytokines and growth factor synthesis and secretion, extracellular matrix remodeling, membrane receptor expression, cell detachment and cell migration and differentiation. Loss of cell–cell contacts is a necessary and sufficient step in the progression of EndoMT. In endothelial cells, adherence junctions are composed of transmembrane adhesive proteins belonging to the cadherin family, with the VE-cadherin being the most important. This protein interacts with β-catenin, which links cadherin to the actin cytoskeleton. Tyrosine phosphorylation of both VE-cadherin and β-catenin is considered an important mechanism associated to the disassembly of adherence junctions or loss of cell–cell contacts. Insulin-like growth factor receptor I (IGFIR) is a transmembrane tyrosine kinase that has been involved in the alterations of cell–cell contacts and in the expression of some genes during cancer development and progression. Here, it is hypothesized that IGFIR autophosphorylation may initiate a signaling pathway that would lead to the loss of cell–cell contacts or adherence junctions, remarkable remodeling of the cytoskeleton, increased cell motility, and finally to the progressive transition towards a mesenchymal phenotype. Data supporting this hypothesis are presented here.     

Binding and degradation of soluble immunoglobulin aggregates by mouse mononuclear phagocytes—Stimulation by colony-stimulating factor

The binding and degradation of soluble guinea pig IgG₂ aggregates by murine mononuclear phagocytes were studied. Bone marrow mononuclear phagocytes cultured in the presence of an embryonic fibroblast-conditioned medium (CM) degraded the aggregates to a much greater degree than did resident peritoneal macrophages. Binding and degradation by resident peritoneal macrophages were enhanced by culture in the presence of CM.Freshly harvested thioglycollate-induced peritoneal macrophages bound and degraded the aggregates to the same degree as the cultured bone marrow mononuclear phagocytes did. However, the thioglycollate-induced macrophages lost most of these capacities when cultured in vitro without CM. When CM was added to these cultures, the capacity to bind and degrade was restored in a dose-dependent fashion. To obtain the maximum effect, exposure to CM must be maintained for more than 2 days. The effect of CM could be reproduced with purified CSF-1. Taken together the results of this study indicate that Fc receptor expression is modulated by CSF-1.     

RNA-Seq reveals placental growth factor regulates the human retinal endothelial cell barrier integrity by transforming growth factor (TGF-β) signaling

Molecular and Cellular Biochemistry - Previous studies have indicated that long non-coding RNAs (lncRNAs) were closely related to diabetes. In this study, we aimed to explore the possible role and...     

Are plasma insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) useful markers of prostate cancer?

Increased concentrations of insulin-like growth factor I (IGF-I) and decreased insulin-like growth factor binding protein 3 (IGFBP-3) in serum have been proposed as markers of prostate cancer (CaP). The evidence for this, however, is contradictory. We assayed serum for IGF-I, IGFBP-3 and prostate-specific antigen (PSA) in patients with CaP and benign prostatic hyperplasia (BPH) and in healthy controls (HC). The mean +/- SD concentration of IGF-I in CaP (98.3 +/- 39.3 ng/mL; n = 15) was lower than in BPH (119 +/- 31.1 ng/mL; n=24) and HC (119 +/- 36.1 ng/mL; n=46), but the differences between the three groups were not statistically significant (p > 0.05). The mean IGFBP-3 concentrations in CaP (2691 +/- 1105 ng/mL; n = 16; p = 0.029) and BPH (2618 +/- 816 ng/mL; n = 26; p = 0.006) patients were significantly lower than that of the HC (3119 +/- 618 ng/mL; n=59), but the difference between the two groups of patients was not significant (p > 0.05). PSA concentrations in CaP (median = 80.8 ng/mL; n = 25) were significantly higher than those in BPH (median = 8.6 ng/mL; n = 39) (p < 0.001). Ninety-six percent of CaP and 72% of BPH patients had PSA concentrations >4.0 ng/mL; the proportions of patients with concentrations exceeding 20 ng/mL were 76% and 10%, respectively. We conclude that IGF-I and IGFBP-3 are inferior to PSA for CaP detection.     

Binding of histamine to the H1 receptor—a molecular dynamics study

Binding of histamine to the G-protein coupled histamine H₁ receptor plays an important role in the context of allergic reactions; however, no crystal structure of the resulting complex is available yet. To deduce the histamine binding site, we performed unbiased molecular dynamics (MD) simulations on a microsecond time scale, which allowed to monitor one binding event, in which particularly the residues of the extracellular loop 2 were involved in the initial recognition process. The final histamine binding pose in the orthosteric pocket is characterized by interactions with Asp107^3.32, Tyr108^3.33, Thr194^5.43, Asn198^5.46, Trp428^6.48, Tyr431^6.51, Phe432^6.52, and Phe435^6.55, which is in agreement with existing mutational data. The conformational stability of the obtained complex structure was subsequently confirmed in 2 μs equilibrium MD simulations, and a metadynamics simulation proved that the detected binding site represents an energy minimum. A complementary investigation of a D107A mutant, which has experimentally been shown to abolish ligand binding, revealed that this exchange results in a significantly weaker interaction and enhanced ligand dynamics. This finding underlines the importance of the electrostatic interaction between the histamine ammonium group and the side chain of Asp107^3.32 for histamine binding.     

Secretory vesicle — Cytosol interactions in exocytosis: Isolation by Ca2+-dependent affinity chromatography of proteins that bind to the chromaffin granule membrane

Proteins from adrenal medullary cytosol that bind to chromaffin granule membranes in the presence of Ca²⁺ were isolated by affinity chromatography on granule membranes coupled to Sepharose 4B. Cytosol was applied to the affinity column in the presence of 2 mM free Ca²⁺. One group of proteins was eluted at 50 μM Ca²⁺ and had molecular weights of 60,000, 46,000, 36,000, 34,000, 32,000 and 26,000. At 0.1 μM Ca²⁺ additional proteins of molecular weights 70,000, 44,000 and 33,000 were eluted. Both groups of proteins aggregated isolated chromaffin granules in the presence of Ca²⁺. Since exocytosis involves cytosol-membrane interactions regulated by Ca²⁺, these proteins may have functional roles in this process. The term “chromobindins” is introduced to describe these proteins.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

Purification of complement components by hydrophobic affinity chromatography on phenyl—Sepharose: Purification of human C5

Human complement components C5 and C3 were purified with 41% and 20% yields, respectively, by euglobulin precipitation, DEAE—Sephacel ion-exchange chromatography and gel filtration. Phenyl—Sepharose chromatography allowed the complete separation of C3 and C5. C3 bound loosely on the resin whereas C5 bound firmly and was eluted with 50% glycerin solution. Gel filtration on Sephacryl S-300 allowed the depletion of C4bp and H that contaminated C5 preparations. Homogeneity of C5 and C3 preparations was demonstrated by SDS—PAGE and immunochemical analysis. C5 and C3 consisted of two chains (α, 110000; β, 75000) linked by disulfide bridges.     

Binding of congo red to amyloid protofibrils of the Alzheimer aβ(9-40) Peptide probed by molecular dynamics simulations

Congo red (CR) is a commonly used histological amyloid dye and a weak amyloid inhibitor. There is currently no experimentally available structure of CR bound to an amyloid fibril and the binding modes, and the mechanisms governing its inhibitory and optical properties are poorly understood. In this work, we present the first, to our knowledge, atomistically detailed picture of CR binding to protofibrils of the Alzheimer Aβ_9–40 peptide. We identify three major binding modes, with the primary mode residing in the grooves formed by the β-sheets, and observe a restriction of the torsional rotation of the CR molecule upon binding. Our simulations reveal a novel, to our knowledge, electrostatic steering mechanism that plays an important role in the initial recognition and binding of CR to the positively charged surface residues of the fibril. Our simulations provide new, to our knowledge, insights into the striking spectrophotometric and inhibitory properties of CR. In particular, we show that birefringence upon CR binding is due to the anisotropic orientation of the CR dipoles resulting from the spatial ordering of these molecules in the grooves along the fibril axis. The fluorescent enhancement of the bound CR, in turn, is associated with the torsional restriction of this molecule upon binding.     

Is quantity of protein in barley forms determined by proteins localized in the subaleurone layer?

Barley biotypes from the world collection differ in their storage protein content even till 200 %. This is the first report including results of the research, in which the structure of grains containing different amount of protein was tested to explain this difference. The endosperm was investigated using scanning electron microscopy. The structure of the aleurone layer, storing large quantities of protein, did not differ between the high- and low-protein forms of barley. It has been proven that the large quantities of kernel protein may be stored in some cells of the zone adjacent to the aleurone layer, defined as the subaleurone cells. It has been shown that morphologically uniform kernels of the same plant and even of the same ear can vary greatly with respect to the number of these subaleurone cells. The purpose of the study was an examination of variation in protein structure in single kernels of a fodder, a brewery and in an extra high-protein form of barley as well. Moreover the studies were aimed to detect qualitative differences in the subaleurone protein. Application of mass spectrometry made possible the identification of several kinds of proteins which were present in subaleurne layer of kernels. In the granule-bound protein fraction isolated from the subaleurone type kernels, a much stronger representation of some protein was found, with the molecular mass between 29 and 45 kDa, in comparison with the low-protein kernels. It is supposed, that these protein are isoforms of z-type serpin and B3-hordein.     

Estrogen receptor alpha is essential for the proliferation of prostatic smooth muscle cells stimulated by 17β-estradiol and insulin-like growth factor 1

Estradiol (E₂) and its receptor estrogen receptor alpha (ERα) are implicated in the pathology of stromal‐predominant benign prostatic hyperplasia (BPH). Insulin‐like growth factor 1(IGF1) is produced primarily by stromal cells in the prostate. Recent study showed that E₂‐mediated regulation of IGF1 in mouse uterus requires the DNA binding ability of ERα. However, the crosstalk between ERα and IGF1 in the prostate has not been addressed yet. Therefore, in this study we employed mouse prostatic smooth muscle cells (PSMCs) as a model to demonstrate that E₂ stimulated the proliferation of PSMCs and up‐regulated the expression of IGF1 and its receptor IGF1R as well as cyclin D1 in PSMCs, all of which could be inhibited by shRNA‐mediated knockdown of ERα. Furthermore, we found that exogenous IGF1 could not promote cell proliferation and cyclin D1 expression in PSMCs subjected to shRNA‐mediated knockdown of ERα. Interestingly, blockage of IGF1R by antibody could inhibit E₂‐stimulated PSMCs proliferation. Altogether our present study provides the first line of evidence that there is crosstalk between ERα and IGF1 signaling in PSMCs proliferation in which ERα up‐regulates the expression of IGF1 and IGF1R, and IGF1 signaling is indispensable to mediate downstream effects of E₂ and ERα. Copyright © 2011 John Wiley & Sons, Ltd.     

Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway

Transforming growth factor β (TGF-β) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-β on CTGF expression. We found that myoblasts treated with LPA and TGF-β1 produced an additive effect on CTGF expression. In the absence of TGF-β, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-β receptor type II (TGF-βRII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-βRI, suggesting that CTGF induction is dependent on LPA and requires active TGF-βRs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-βRI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-βRI-mediated activity. These novel results for the mechanism of action of LPA and TGF-β are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases.