Control of trophectoderm differentiation by inner cell mass-derived fibroblast growth factor-4 in mouse blastocysts and corrective effect of FGF-4 on high glucose-induced trophoblast disruption

Previous studies have suggested that fibroblast growth factor-4 (FGF-4) may be a paracrine signal used by inner cell mass (ICM) cells to maintain adjacent trophectoderm (TE) cells in an undifferentiated state. In the present work, immunocytochemical analysis of mouse blastocysts confirmed that FGF-4 was predominantly detected in the ICM before and after spreading over a fibronectin-coated culture substrate. Addition of human recombinant FGF-4 did not influence morphological progression, cell allocation and proliferation in ICM and TE lineages or mitosis and karyorhexis frequencies during blastocyst expansion. Addition of FGF-4 to outgrowing blastocysts, in contrast, induced a significant decrease in the surface of the trophoblast outgrowths formed by the TE cells and in the proportion of giant trophoblasts per outgrowth. The fact that blastocysts display excessive trophoblast expansion and spreading over their culture substrate upon pre-exposure to high concentrations of glucose in vitro was used to further assess the regulatory effect of FGF-4. Addition of FGF-4 was indeed found to fully neutralize the disruptive impact of high glucose on trophoblast outgrowths. Altogether, our data indicate that ICM-derived FGF-4 participates actively in the regulation of trophoblast development.     

Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.     

Proteome alterations in gamma-irradiated human T-lymphocyte leukemia cells

Analyses of the protein expression profiles of irradiated cells may be beneficial for identification of new biomolecules of radiation-induced cell damage. Therefore, in this study we exploited the proteomic approach to identify proteins whose expression is significantly altered in gamma-irradiated human T-lymphocyte leukemia cells. MOLT-4 cells were irradiated with 7.5 Gy and the cell lysates were collected at different times after irradiation (2, 5 and 12 h). The proteins were separated by two-dimensional electrophoresis and quantified using an image evaluation system. Proteins exhibiting significant radiation-induced alterations in abundance were identified by peptide mass fingerprinting. We identified 14 proteins that were either up- or down-regulated. Cellular levels of four of the proteins (Rho GDP dissociation inhibitor 1 and 2, Ran binding protein 1, serine/threonine protein kinase PAK2) were further analyzed by two-dimensional immunoblotting to confirm the data obtained from proteome analysis. All identified proteins were classified according to their cellular function, including their participation in biochemical and signaling pathways. Taken together, our results suggest the feasibility of the proteome method for monitoring of cellular radiation responses.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

New culture system for human embryonic stem cells: autologous mesenchymal stem cell feeder without exogenous fibroblast growth factor 2

Human embryonic stem (hES) cells have been successfully maintained using human-cell feeder systems or feeder-free systems. However, despite advances in culture techniques, hES cells require supplementation with fibroblast growth factor 2 (FGF-2), an exogenous stemness factor, which is needed to sustain the authentic undifferentiated status. We developed a new culture system for hES cells; this system does not require supplementation with FGF-2 to obtain hES cells that are suitable for tissue engineering and regenerative medicine. This culture system employed mesenchymal stem cells derived from hES cells (hESC-MSCs) as autologous human feeder cells in the absence of FGF-2. The hES cell line SNUhES3 cultured in this new autologous feeder culture system maintained the typical morphology of hES cells and expression of pluripotency-related proteins, SSEA-4, TRA-1-60, OCT4, and alkaline phosphatase, without development of abnormal karyotypes after more than 30 passages. RNA expression of the pluripotency-related genes OCT4 and NANOG was similar to the expression in SNUhES3 cells maintained on xenofeeder STO cells. To identify the mechanism that enables the cells to be maintained without exogenous FGF-2, we checked the secretion of FGF-2 from the mitomycin-C treated autofeeder hESC-MSCs versus xenofeeder STO cells, and confirmed that hESC-MSCs secreted FGF-2 whereas STO cells did not. The level of FGF-2 in the media from the autofeeder system without exogenous FGF-2 was comparable to that from the xenofeeder system with addition of FGF-2. In conclusion, our new culture system for hES cells, which employs a feeder layer of autologous hESC-MSCs, supplies sufficient amounts of secreted FGF-2 to eliminate the requirement for exogenous FGF-2.     

Treatment of Salmonella enterica serovar Enteritidis with a sublethal concentration of trisodium phosphate or alkaline pH induces thermotolerance

The responses of Salmonella enterica serovar Enteritidis to a sublethal dose of trisodium phosphate (TSP) and its equivalent alkaline pH made with NaOH were examined. Pretreatment of S. enterica serovar Enteritidis cells with 1.5% TSP or pH 10.0 solutions resulted in a significant increase in thermotolerance, resistance to 2.5% TSP, resistance to high pH, and sensitivity to acid and H(2)O(2). Protein inhibition studies with chloramphenicol revealed that thermotolerance, unlike resistance to high pH, was dependent on de novo protein synthesis. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of total cellular proteins from untreated control cells resolved as many as 232 proteins, of which 22 and 15% were absent in TSP- or alkaline pH-pretreated cells, respectively. More than 50% of the proteins that were either up- or down-regulated by TSP pretreatment were also up- or down-regulated by alkaline pH pretreatment. Sodium dodecyl sulfate-PAGE analysis of detergent-insoluble outer membrane proteins revealed the up-regulation of at least four proteins. Mass spectrometric analysis showed the up-regulated proteins to include those involved in the transport of small hydrophilic molecules across the cytoplasmic membrane and those that act as chaperones and aid in the export of newly synthesized proteins by keeping them in open conformation. Other up-regulated proteins included common housekeeping proteins like those involved in amino acid biosynthesis, nucleotide metabolism, and aminoacyl-tRNA biosynthesis. In addition to the differential expression of proteins following TSP or alkaline pH treatment, changes in membrane fatty acid composition were also observed. Alkaline pH- or TSP-pretreated cells showed a higher saturated and cyclic to unsaturated fatty acid ratio than did the untreated control cells. These results suggest that the cytoplasmic membrane could play a significant role in the induction of thermotolerance and resistance to other stresses following TSP or alkaline pH treatment.     

Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition.

FGF signaling uses receptor tyrosine kinases that form high-affinity complexes with FGFs and heparan sulfate (HS) proteoglycans at the cell surface. It is hypothesized that assembly of these complexes requires simultaneous recognition of distinct sulfation patterns within the HS chain by FGF and the FGF receptor (FR), suggesting that tissue-specific HS synthesis may regulate FGF signaling. To address this, FGF-2 and FGF-4, and extracellular domain constructs of FR1-IIIc (FR1c) and FR2-IIIc (FR2c), were used to probe for tissue-specific HS in embryonic day 18 mouse embryos. Whereas FGF-2 binds HS ubiquitously, FGF-4 exhibits a restricted pattern, failing to bind HS in the heart and blood vessels and failing to activate signaling in mouse aortic endothelial cells. This suggests that FGF-4 seeks a specific HS sulfation pattern, distinct from that of FGF-2, which is not expressed in most vascular tissues. Additionally, whereas FR2c binds all FGF-4-HS complexes, FR1c fails to bind FGF-4-HS in most tissues, as well as in Raji-S1 cells expressing syndecan-1. Proliferation assays using BaF3 cells expressing either FR1c or FR2c support these results. This suggests that FGF and FR recognition of specific HS sulfation patterns is critical for the activation of FGF signaling, and that synthesis of these patterns is regulated during embryonic development.     

Fibroblast growth factor-2 interacts with free ribosomal protein S19.

Exogenous FGF-2 added to cells is internalized and part of it translocates to the nucleus of the cells. To get a better understanding of the FGF-2-induced signaling pathway, we looked for proteins associated with FGF-2 in the cytoplasm of the target cells. We first used the GST-FGF-2 to isolate cytoplasmic proteins complexes containing FGF-2 from S100 extract (supernatant 100,000g). Among the retrieved proteins, we focused our studies on RPS19, a protein of the 40S small ribosomal subunit. We showed that FGF-2 interacts directly with RPS19 in vitro. Second, we coimmunoprecipitated RPS19 and FGF-2 from a S240 extract (240,000g supernatant) prepared from FGF-2-stimulated cells and devoid of 40S ribosomal subunit. The result of these experiments suggest that a pool of free RPS19 exists in cells and that FGF-2 interacts in vivo with free RPS19.     

Agitation Down-Regulates Immunoglobulin Binding Protein EibG Expression in Shiga Toxin-Producing Escherichia coli (STEC)

Shiga toxin (Stx)-producing Escherichia coli (STEC) carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG). EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.     

Effects of benzo(a)pyrene on protein expression in Jurkat T-cells

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants of air, water and soil, and are produced by the incomplete combustion of organic materials. The International Agency for Research on Cancer has characterized PAHs as carcinogens. In this study, we investigated the effects of benzo(a)pyrene (B(a)P), which is the most carcinogenic member of the PAHs, on Jurkat cell protein by proteomic analysis. Jurkat cells were treated with various concentrations of B(a)P (0, 2.5, 5, 10, 20 or 40 microM) for 24 or 48 h and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and lactate dehydrogenase assays were carried out to determine cytotoxicity and a Comet assay was used to determinate genotoxicity. The cytotoxicity assays showed that 2.5 microM of B(a)P was the maximal concentration that did not cause any toxicity, but nevertheless, at this level B(a)P produced significant DNA damage in Jurkat cells at 48 h. Proteomic analysis using three different pI ranges and large two-dimensional gel electrophoresis showed 3427 protein spots. A total of 46 (13 up- and 33 down-regulated) proteins were identified as biomarkers of B(a)P and showed dose-dependent expressions in Jurkat T-cell line exposed to B(a)P. Of these, 27 protein spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Two functionally differentiated protein groups were found. The protein group involving apoptosis and tumor suppression were found to be up-regulated, and B(a)P down-regulated enzyme was involved in energy metabolism, DNA synthesis and in cell structure and motility.     

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.     

Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms

Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-I and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis. These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-l and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo.     

mRNA and protein expression of FGF-1, FGF-2 and their receptors in the porcine umbilical cord during pregnancy

The fibroblast growth factors (FGFs) are multifunctional proteins that, among other roles, regulate structural reorganization of uterine and placental vascular bed during pregnancy. Thus, we analyzed mRNA and protein expression and immunohistochemical localization of FGF-1 and FGF-2, and their receptors (FGFR-1 and FGFR-2) in the developing umbilical cord (UC) on days 40, 60, 75 and 90 of pregnancy and after the physiological delivery in the pig (day 114). qPCR analysis demonstrated an increase in FGF-1 and FGF-2 mRNA levels beginning on day 75 and on day 114 of pregnancy, respectively. In addition, significantly increased FGFR-1IIIc mRNA expression was also found on day 114. On the other hand, no significant changes in FGFR-2IIIb mRNA expression were observed. Western Blot analysis revealed a decrease in FGF-1 and FGFR-2 protein expression after day 40. Beside an increased protein expression of FGF-2 on day 60, no significant changes in FGFR-1 protein expression were detected. Immunohistochemical staining enabled detection of FGF-FGFR system, with different intensity of immunoreaction in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium as well as in myofibroblasts. In conclusion, our results show that members of FGF-FGFR system are expressed specifically in UC structures. Furthermore their day of pregnancy-related expression suggest that they may be an important players during UC formation and development.     

Heat and osmotic stress responses of probiotic Lactobacillus rhamnosus HN001 (DR20) in relation to viability after drying

The viability of lactic acid bacteria in frozen, freeze-dried, and air-dried forms is of significant commercial interest to both the dairy and food industries. In this study we observed that when prestressed with either heat (50 degrees C) or salt (0.6 M NaCl), Lactobacillus rhamnosus HN001 (also known as DR20) showed significant (P < 0.05) improvement in viability compared with the nonstressed control culture after storage at 30 degrees C in the dried form. To investigate the mechanisms underlying this stress-related viability improvement in L. rhamnosus HN001, we analyzed protein synthesis in cultures subjected to different growth stages and stress conditions, using two-dimensional gel electrophoresis and N-terminal sequencing. Several proteins were up- or down-regulated after either heat or osmotic shock treatments. Eleven proteins were positively identified, including the classical heat shock proteins GroEL and DnaK and the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, enolase, phosphoglycerate kinase, and triose phosphate isomerase, as well as tagatose 1,6-diphosphate aldolase of the tagatose pathway. The phosphocarrier protein HPr (histidine-containing proteins) was up-regulated in cultures after the log phase irrespective of the stress treatments used. The relative synthesis of an ABC transport-related protein was also up-regulated after shock treatments. Carbohydrate analysis of cytoplasmic contents showed higher levels (20 +/- 3 microg/mg of protein) in cell extracts (CFEs) derived from osmotically stressed cells than in the unstressed control (15 +/- 3 microg/mg of protein). Liquid chromatography of these crude carbohydrate extracts showed significantly different profiles. Electrospray mass spectrometry analysis of CFEs revealed, in addition to normal mono-, di-, tri-, and tetrasaccharides, the presence of saccharides modified with glycerol.     

Overexpression of angiopoietin-like protein 4 alters mitochondria activities and modulates methionine metabolic cycle in the liver tissues of db/db diabetic mice

Angiopoietin-like protein 4 (ANGPTL4) is a circulating protein predominantly produced from fat tissue and liver. Recent data from others and our laboratory have demonstrated this protein to be an important player in energy metabolism and insulin sensitivity. However, the molecular mechanisms underlying its metabolic actions remain elusive. In this study, we have employed a two-dimensional fluorescence difference gel electrophoresis technique to study the protein profiles in the livers of db/db mice treated with or without ANGPTL4. When compared with those of lean mice, 118 proteins were found to be up- or down-regulated in db/db mice. Adenovirus-mediated overexpression of ANGPTL4 could reverse a large portion of the up- or down-regulated proteins to control levels. Especially, a number of mitochondria proteins were down-regulated by ANGPTL4 to a great extent. Chronic treatment with ANGPTL4 resulted in an elevated activity of mitochondria respiratory chain complexes II-III and IV in db/db mice. Additionally, several key enzymes in the methionine/homocysteine metabolic cycle were found to be increased in db/db diabetic mice but decreased by ANGPTL4 treatment. HPLC analysis consistently revealed that ANGPTL4 could significantly restore the augmented S-adenosylmethionine levels and S-adenosylmethionine/S-adenosylhomocysteine ratios in livers of db/db mice. In summary, our results suggest that ANGPTL4 might elicit its metabolic effects through modulating the mitochondria functions and methionine metabolic cycles in the liver tissue.     

Proteome profiling of interleukin-12 treated human T helper cells

Selective activation of T helper subsets 1 (Th1) and 2 (Th2) plays a crucial role in different pathological conditions. Th1 cell response is involved in pathogenesis of autoimmune diseases, such as type II diabetes and multiple sclerosis, and Th2 cell response in pathogenesis of allergy and asthma. Cytokine interleukin-12 (IL-12) is one of the key factors in the differentiation of na?ve CD4(+) T cells into Th1 cells. In this study we used 2-DE and MS to find and identify IL-12 regulated proteins in human CD4(+) T cells. In total, 42 protein spots were found to be differentially expressed following IL-12 stimulation, of which 22 were up- and 20 down-regulated. Among the upregulated proteins there are a multifunctional cytokine macrophage migration inhibitory factor and a known IL-12 target gene Programmed cell death 4. Downregulated proteins include p21-activated kinase 2 and its upstream GTPase Cdc42. Compared to previous reports our analysis provides a new view on the IL-12 induced changes on CD4(+) T cells underscoring the importance of creating and combining the data generated at various levels to build a comprehensive view of a given biological process of the cell.     

Treatment of Salmonella enterica Serovar Enteritidis with a Sublethal Concentration of Trisodium Phosphate or Alkaline pH Induces Thermotolerance

The responses of Salmonella enterica serovar Enteritidis to a sublethal dose of trisodium phosphate (TSP) and its equivalent alkaline pH made with NaOH were examined. Pretreatment of S. enterica serovar Enteritidis cells with 1.5% TSP or pH 10.0 solutions resulted in a significant increase in thermotolerance, resistance to 2.5% TSP, resistance to high pH, and sensitivity to acid and H₂O₂. Protein inhibition studies with chloramphenicol revealed that thermotolerance, unlike resistance to high pH, was dependent on de novo protein synthesis. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of total cellular proteins from untreated control cells resolved as many as 232 proteins, of which 22 and 15% were absent in TSP- or alkaline pH-pretreated cells, respectively. More than 50% of the proteins that were either up- or down-regulated by TSP pretreatment were also up- or down-regulated by alkaline pH pretreatment. Sodium dodecyl sulfate-PAGE analysis of detergent-insoluble outer membrane proteins revealed the up-regulation of at least four proteins. Mass spectrometric analysis showed the up-regulated proteins to include those involved in the transport of small hydrophilic molecules across the cytoplasmic membrane and those that act as chaperones and aid in the export of newly synthesized proteins by keeping them in open conformation. Other up-regulated proteins included common housekeeping proteins like those involved in amino acid biosynthesis, nucleotide metabolism, and aminoacyl-tRNA biosynthesis. In addition to the differential expression of proteins following TSP or alkaline pH treatment, changes in membrane fatty acid composition were also observed. Alkaline pH- or TSP-pretreated cells showed a higher saturated and cyclic to unsaturated fatty acid ratio than did the untreated control cells. These results suggest that the cytoplasmic membrane could play a significant role in the induction of thermotolerance and resistance to other stresses following TSP or alkaline pH treatment.