Regulation of insulin-like growth factor binding protein-1 expression during aging

Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is primarily produced in the liver during inflammation and regulates biological activities of IGF-I. Here we demonstrate that interleukin-1beta (IL-1beta) stimulates IGFBP-1 mRNA production in a dose-dependent manner in hepatocytes from Fisher 344 rats. Employment of c-Jun N-terminal kinase (JNK) inhibitor SP600125 resulted in 3-fold reduction of IGFBP-1 mRNA and protein levels, indicating that IL-1beta-induced IGFBP-1 production is mediated through JNK activation. We further show that hepatocytes from aged rats (20-22 mo), as compared to young (3-4 mo), exhibit up to 2-fold higher levels of IGFBP-1 in response to IL-1beta. IL-1beta-induced phosphorylation of JNK was also significantly higher in aged hepatocytes, and SP600125 treatment eliminated age-related differences in IGFBP-1 mRNA production. Moreover, glutathione depletion in hepatocytes from young rats potently activated JNK, as well as increased IL-1beta-induced IGFBP-1 mRNA levels, suggesting that age-related oxidative stress underlies the upregulated JNK activation and IGFBP-1 expression.     

Insulin receptor substrates form high-molecular-mass complexes that modulate their availability to insulin/insulin-like growth factor-I receptor tyrosine kinases

Insulin receptor substrates (IRSs) are phosphorylated by activated insulin/insulin-like growth factor (IGF)-I receptor tyrosine kinases. Phosphotyrosyl IRSs are recognized by signaling molecules possessing src homology region 2 (SH2) domains, which mediate various insulin/IGF bioactivities. However, we have shown that IRSs are also associated with other proteins by a phosphotyrosine-independent mechanism. Here, we demonstrated that IRSs form high-molecular-mass complexes (we named these complexes IRSomes) with various proteins and we elucidated their possible roles. Blue native-polyacrylamide gel electrophoresis of cell lysates revealed IRSome formation. Some proteins associated with IRSs in IRS-isoform-, cell-type-, or stimulus-specific manners. Results of the in vitro tyrosine phosphorylation assay indicated that tyrosine phosphorylation of IRS-1 by insulin receptor was decreased when IRS-1 was contained in IRSomes prepared from 3T3-L1 adipocytes treated with TNF-α. Also, tyrosine phosphorylation of IRS-2 by IGF-I receptor was increased when IRS-2 was contained in IRSomes prepared from FRTL-5 thyrocytes treated with dibutyryl cAMP. These results demonstrated that cytokine/hormone-induced formation of IRSomes modulates availability of IRSs to receptor tyrosine kinases.     

Chitosan stabilizes platelet growth factors and modulates stem cell differentiation toward tissue regeneration

The idea of using chitosan as a functional delivery aid to support simultaneously PRP, stem cells and growth factors (GF) is associated with the intention to use morphogenic biomaterials to modulate the natural healing sequence in bone and other tissues. For example, chitosan–chondroitin sulfate loaded with platelet lysate was included in a poly(d,l-lactate) foam that was then seeded with human adipose-derived stem cells and cultured in vitro under osteogenic stimulus: the platelet lysate provided to the bone tissue the most suitable assortment of GF which induces the osteogenic differentiation of the mesenchymal stem cells. PDGF, FGF, IGF and TGF-β were protagonists in the repair of callus fractures. The release of GF from the composites of chitosan–PRP and either nano-hydroxyapatite or tricalcium phosphate was highly beneficial for enhancing MSC proliferation and differentiation, thus qualifying chitosan as an excellent vehicle. A number of biochemical characteristics of chitosan exert synergism with stem cells in the regeneration of soft tissues.     

Fibroblast growth factor 2 induces apoptosis in the early primary culture of rat cortical neurons

In the central nervous system, fibroblast growth factor 2 (FGF2) is known to have important functions in cell survival and differentiation. In addition to its roles as a neurotrophic factor, we found that FGF2 caused cell death in the early primary culture of cortical neurons. FGF2-induced neuronal cell death showed apoptotic characters, e.g., chromatin condensation and DNA fragmentation. The ultrastructural morphology of FGF2-treated neurons indicated apoptotic features such as progressive cell shrinkage, blebbing of the plasma membrane, loss of cytosolic organelles, clumping of chromatin, and fragmentation of DNA. Tyrosine kinase inhibitors significantly rescued neurons from FGF2-induced apoptosis. FGF2 potentiated a marked influx of Ca²⁺ into neurons before apoptosis. Both a calcium chelator and L-type voltage-sensitive Ca²⁺ channel (L-VSCC) blockers attenuated FGF2-induced apoptosis, whereas other blockers of VSCCs such as N-type and P/Q-types did not. Blockers of L-VSCCs significantly suppressed FGF2-enhanced Ca²⁺ influx into neurons. Moreover, FGF2 also generated reactive oxygen species (ROS) before apoptosis. Radical scavengers reduced not only the FGF2-generated ROS, but also the FGF2-induced Ca²⁺ influx and apoptosis. In conclusion, we demonstrated that FGF2 caused apoptosis via L-VSCCs in the early neuronal culture.     

Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Extracellular adenosine activates P1 receptors (A₁, A_2A, A_2B, A₃) on cellular membranes. Here, we investigated the involvement of P1 receptor-mediated signaling in differentiation to regulatory T cells (Treg). Treg were induced in vitro by incubating isolated CD4⁺CD62L⁺ naïve murine T cells under Treg-skewing conditions. Antagonists of A₁ and A_2B receptors suppressed the expression of Foxp3, a specific marker of Treg, and the production of IL-10, suggesting the involvement of A₁ and A_2B receptors in differentiation to Treg. We also investigated the effect of these antagonists on T cell activation, which is essential for differentiation to Treg, and found that A₁ antagonist, but not A_2B antagonist, suppressed T cell activation. We conclude that A₁ and A_2B receptors are both involved in differentiation to Treg, but through different mechanisms. Since A_2B antagonist blocked differentiation to Treg without suppressing T cell activation, it is possible that blockade of A_2B receptor would facilitate tumor immunity.     

Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: Capturing transient ternary complexes by HDXMS

cAMP signaling is a fundamental cellular process necessary for mediating responses to hormonal stimuli. In contrast to cAMP-dependent activation of protein kinase A (PKA), an important cellular target, far less is known on termination in cAMP signaling, specifically how phosphodiesterases (PDEs) facilitate dissociation and hydrolysis of bound cAMP. In this study, we have probed the dynamics of a ternary complex of PKA and a PDE–RegA with an excess of a PDE-nonhydrolyzable cAMP analog, Sp-cAMPS by amide hydrogen/deuterium exchange mass spectrometry (HDXMS). Our results highlight how HDXMS can be used to monitor reactions together with mapping conformational dynamics of transient signaling complexes. Our results confirm a two-state model for active RegA-mediated dissociation of bound cAMP. Further, our results reveal that Sp-cAMPS and RegA mediate mutually exclusive interactions with the same region of PKA and at specific concentrations of Sp-cAMPS, RegA is capable of blocking Sp-cAMPS reassociation to PKA. This provides a molecular basis for how PDEs modulate levels of intracellular cAMP so that PKA is better suited to responding to fluxes rather than constant levels of cAMP. This study underscores how HDXMS can be a powerful tool for monitoring reactions together with mapping conformational dynamics in signaling proteins. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Cellular interplay in pulmonary arterial hypertension: Implications for new therapies

Pulmonary arterial hypertension (PAH) is a complex and multifactorial disease characterized by vascular remodeling, vasoconstriction, inflammation and thrombosis. Although the available therapies have resulted in improvements in morbidity and survival, PAH remains a severe and devastating disease with a poor prognosis and a high mortality, justifying the need of novel therapeutic targets. An increasing number of studies have demonstrated that endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts of the pulmonary vessel wall, as well as platelets and inflammatory cells have a role in PAH pathogenesis. This review aims to integrate the interplay among different types of cells, during PAH development and progression, and the impact of current therapies in cellular modulation. The interplay among endothelial cells, smooth muscle cells and fibroblasts present in pulmonary vessels wall, platelets and inflammatory cells is regulated by several mediators produced by these cells, contributing to the pathophysiologic features of PAH. Current therapies are mainly focused in the pulmonary vascular tone and in the endothelial dysfunction. However, once they have not been effective, novel therapies targeting other PAH features, such as inflammation and platelet dysfunction are emerging. Further understanding of the interplay among different vascular cell types involved in PAH development and progression can contribute to find novel therapeutic targets, decreasing PAH mortality and morbidity in the future.     

Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.     

Isolation and characterization of lipids strictly associated to PSII complexes: Focus on cardiolipin structural and functional role

In this work, lipid extracts from spinach membrane fragments enriched in Photosystem II (PSII) and from spinach PSII dimers were analyzed, by means of Thin Layer Chromatography (TLC) and Electro-Spray Ionization Mass Spectrometry. Cardiolipin found in association with PSII was isolated and purified by preparative TLC, then characterized by mass and mass-mass analyses. Cardiolipin structures with four unsaturated C18 acyl chains and variable saturation degrees were evidenced. Structural and functional effects of different phospholipids on PSII complexes were investigated by Fluorescence, Resonance Light Scattering and Oxygen Evolution Rate measurements. An increment of PSII thermal stability was observed in the presence of cardiolipin and phosphatidylglycerol.     

Quantitative analysis of EGFR affinity to immobilized glycolipids by surface plasmon resonance

EGF-induced activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, its dimer, and other mimetics. However, details of the interaction, such as kinetic properties, have not yet been clarified. The direct interaction is now defined by the surface plasmon resonance (SPR) technique. To determine the affinity of EGFR for lyso-GM3 or lyso-GM3 mimetic, these glycolipid ligands were covalently immobilized onto a sensor chip, and binding affinities were investigated. Results of these studies confirmed the direct interaction of lyso-GM3 or its mimetic with EGFR. A strong interaction between EGFR and lyso-GM3 or its mimetic was indicated by increased binding of EGFR to glycolipid-immobilized surface, in an EGFR dose-dependent manner.     

Microsatellite markers uncover cryptic species of Odontotermes (Termitoidae: Termitidae) from Peninsular Malaysia

Termites from the genus Odontotermes are known to contain numerous species complexes that are difficult to tell apart morphologically or with mitochondrial DNA sequences. We developed markers for one such cryptic species complex, that is, Odontotermes srinakarinensis sp. nov. from Maxwell Hill Forest Reserve (Perak, Malaysia), and characterised them using a sample of 41 termite workers from three voucher samples from the same area. We then genotyped 150 termite individuals from 23 voucher samples/colonies of this species complex from several sites in Peninsular Malaysia. We analysed their population by constructing dendograms from the proportion of shared-alleles between individuals and genetic distances between colonies; additionally, we examined the Bayesian clustering pattern of their genotype data. All methods of analysis indicated that there were two distinct clusters within our data set. After the morphologies of specimens from each cluster were reexamined, we were able to separate the two species morphologically and found that a single diagnostic character found on the mandibles of its soldiers could be used to separate the two species quite accurately. The additional species in the clade was identified as Odontotermes denticulatus after it was matched to type specimens at the NHM London and Cambridge Museum of Zoology.     

Fibroblast growth factor 2 and protein kinase C alpha are involved in syndecan-4 cytoplasmic domain modulation of turkey myogenic satellite cell proliferation

Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain functions in transmitting signals into the cell through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain influence cell proliferation. The current study investigated the function of syndecan-4 cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Syndecan-4 or syndecan-4 without the cytoplasmic domain and with or without the GAG and N-glycosylated chains were transfected or co-transfected with a small interfering RNA targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The overexpression of syndecan-4 mutants increased cell proliferation but did not change differentiation. Syndecan-4 mutants had increased cellular responsiveness to FGF2 during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas the syndecan-4 mutants decreased PKCα cell membrane localization compared to syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants increased cell membrane localization of PKCα. These data indicated that the syndecan‐4 cytoplasmic domain and the GAG and N-glycosylated chains are critical in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα cell membrane localization.     

Effect of selenium-enriched malt on hepatocarcinogenesis, paraneoplastic syndrome and the hormones regulating blood glucose in rats treated by diethylnitrosamine

233 SD rats weighing 100 approximately 120 g were divided randomly into 6 groups. The animals in group I and group II received 0.1 mg/kg selenium in the form of sodium selenite only and served as the negative control and positive control, respectively. Animals in groups III, IV and V were fed with selenium as Se-enriched malt supplemented diets (0.3, 1 and 3 mg/kg), and group VI with selenium by using sodium selenite supplemented diets (3 mg/kg). Animals of groups II approximately VI were induced hepatoma by diethylnitrosamine (100 mg/l) for 16 weeks, then drunk with sterilized water for 2 more weeks. Subsequently, the effects of Se-enriched malt and sodium selenite on hepatoma nodules, relative liver weight, the liver function indices including alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), total bilirubin (TBIL), and the tumor markers, named as gamma-glutamyltranspeptidase (GGT), alpha-fetoprotein (AFP), insulin-like growth factor-II (IGF-II) were recorded. The calcium concentration, glucose content in plasma and values of the hormones regulating blood glucose, such as insulin, glucagons and thyroid hormones (3,5,3'-tetraiodothyronine, T(3); 3,5,3'5'-tetraiodothyronine, T(4)) were observed as well. At the same time, the correlations between the concentration of plasma glucose and related hormones were also analyzed. The results indicated that Se-enriched malt showed a better chemopreventive efficiency in decreasing the number of hepatoma nodules, relative liver weight and the contents of AFP, GGT, IGF-II, ALT, ALP and TBIL in the plasma, and delaying the descent of hormones in the serum, names as insulin, glucagons, T(3) and T(4) than those feeding with sodium selenite. Effect of Se-enriched malt excelled sodium selenite in the aspects of deadening the descent of glucose concentration in the plasma and the rise of calcium concentration in the serum of the rats with hepatoma induced by diethylnitrosamine. The values of glucose and calcium were significantly related to those items fore-named. In conclusion, the function of Se-enriched malt in deadening the lesion and delaying the development of hepatoma of rats induced by diethylnitrosamine was better than that of sodium selenite. Hypoglycemia and hypercalcemia were significantly correlated with the multifactors mentioned above.     

Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism

Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b₅₆₁ family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at g_max = 3.7 corresponding to a highly anisotropic species, and another at g_max = 3.18; these species are similar to those observed in other cytochromes of the b₅₆₁ family, and were reducible by ascorbate. In addition another signal was observed in some preparations at g_max = 2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of + 80 mV ± 30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b₅₆₁ of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.     

Genomic structure, polymorphism and expression analysis of the growth hormone (GH) gene in female and male Half-smooth tongue sole (Cynoglossus semilaevis)

Growth hormone (GH) is a polypeptide which is an important regulator of development and somatic growth in teleosts, and may be associated with the mechanisms which drive sexual growth dimorphism in the Half-smooth tongue sole (Cynoglossus semilaevis). In this study, the full length gh cDNA was cloned from C. semilaevis by homology cloning and the rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length gh cDNA is 826 bp and contains an open reading frame (ORF) of 603 bp encoding a protein of 200 amino acids (AA). The precursor of gh consists of a 17 amino-acid signal peptide followed by a 183 amino-acid mature polypeptide. GH gene sequences obtained from female and male adults consist of 3428 bp and 3371 bp, respectively, each of which includes six exons and five introns, and the difference in the GH gene size was mainly caused by the microsatellites. When 14 tissues from females, normal males and extra-large male adults were analyzed for sex-specific tissue expression, the gh mRNA was found to be predominantly expressed in the pituitary, and the expression levels in females were 3.6 times as much as those in normal males, while the mRNA expression in extra-large males was 1.7 times as much as those in normal males. Sex differences in gh mRNA expression during development were also examined by using a full-sib family of C. semilaevis, and the gh mRNA was detected at all of the 12 time points sampled from 10 to 380 days-old. A significant increase in gh mRNA was detected starting in 80 day old fish and was then followed by a drop to very low levels starting at 230 day old fish. Differential expression indicated that the gh expression level in females was significantly higher than males (P < 0.01) at all of the stages except for 10 days-old. Two microsatellite loci were identified in the second intron of the GH gene. Using these two polymorphic markers to genotype 224 individuals, there was no significant difference between the females and males in the Bohai Sea, the Yellow Sea and the hatchery samples.