[Classification of self-organizing peptides].

Amino acid sequences of known natural and synthetic self-assembling peptides were searched and analyzed for their characteristic patterns. The attempted formal numerical description of the repeating motifs, which have been revealed, resulted in building of general classification system embracing core-sequences of the peptides capable of nanostructure formation. Advantages and potency of the proposed rational classification were demonstrated via its comparison with the output from the earlier system described by the others.

A nucleoprotein complex containing CCAAT/enhancer-binding protein beta interacts with an insulin response sequence in the insulin-like growth factor-binding protein-1 gene and contributes to insulin-regulated gene expression.

Highly related insulin response sequences (IRSs) mediate effects of insulin on the expression of multiple genes in the liver, including insulin-like growth factor binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK). Gel shift studies reveal that oligonucleotide probes containing an IRS from the IGFBP-1 or PEPCK gene form a similar complex with hepatic nuclear proteins. Unlabeled competitors containing the IGFBP-1 or PEPCK IRS or a binding site for C/EBP proteins inhibit the formation of this complex. Antibody against C/EBPbeta (but not other C/EBP proteins) supershifts this complex, and Western blotting of affinity purified proteins confirms that C/EBPbeta is present in this complex. Studies with affinity purified and recombinant protein indicate that C/EBPbeta does not interact directly with the IRS, but that other factors are required. Gel shift assays and reporter gene studies with constructs containing point mutations within the IRS reveal that the ability to interact with factors required for the formation of this complex correlates well with the ability of insulin to regulate promoter activity via this IRS (r = 0.849, p < 0.01). Replacing the IRS in reporter gene constructs with a C/EBP-binding site (but not an HNF-3/forkhead site or cAMP response element) maintains the effect of insulin on promoter activity. Together, these findings indicate that a nucleoprotein complex containing C/EBPbeta interacts with IRSs from the IGFBP-1 and PEPCK genes in a sequence-specific fashion and may contribute to the ability of insulin to regulate gene expression.

Galanin-1 receptor up-regulation mediates the excess colonic fluid production caused by infection with enteric pathogens.

Galanin is widely distributed in enteric nerve terminals lining the gastrointestinal tract. We previously showed that pathogenic Escherichia coli, but not normal commensal organisms, increase galanin-1 receptor expression by epithelial cells lining the colon (i.e., colonocytes). When present, galanin-1 receptor activation by ligand causes colonocyte Cl- secretion. We herein demonstrate that disparate pathogens including Salmonella typhimurium and Shigella flexerii also increase colonocyte galanin-1 receptor expression, whose activation is responsible for a principal component of the increased colonic fluid secretion observed. Although eliminating the GAL1R gene by homologous recombination does not alter basal colonic fluid secretion, removal of one or both alleles completely attenuates the increase in fluid secretion due to infection with enteric pathogens. Galanin-1 receptor up-regulation therefore represents a novel mechanism accounting for the increased colonic fluid secretion observed in infectious diarrhea due to several different pathogens.

Characterization of gastrin-releasing peptide and its receptor aberrantly expressed by human colon cancer cell lines.

Gastrin-releasing peptide (GRP) is a mitogen and morphogen important in the development of human colon cancers. Although epithelial cells lining the colon do not normally express GRP or its receptor (GRP-R), most human tumors express GRP-R mRNA. Yet functional protein has only been detected in 24 to 40% of colon cancers. To elucidate the reason for the difference between the expression of GRP/GRP-R mRNA and protein, we studied nine human colon cancer cell lines. Quantitative polymerase chain reaction revealed that all colon cancer cell lines expressed similar amounts of mRNA for both GRP as well as GRP-R. Yet binding studies using (125)I-Tyr(4)-bombesin detected functional receptors on only five of the nine cell lines studied. Conformational fragment-length polymorphism analysis indicated that although mRNA for the ligand GRP was never mutated, mRNA for the GRP-R was always mutated. Sequencing revealed that the message for GRP-R contained between two and seven separate mutations at the nucleotide level. This resulted in 14 separate coding mutations, 2 of which were observed in more than one cell line. Each mutation was individually recreated by site-directed mutagenesis and studied in transiently transfected Chinese hamster ovary-K1 cells. Alteration of Pro(145) into a tyrosine, of Val(317) into a glutamic acid, and insertion of a 32-nucleotide segment resulting in a frameshift distal to Asp(137) all resulted in GRP receptors incapable of binding ligand. Thus, these data indicate that human colon cancers commonly express GRP and GRP-R mRNA but that receptor mutations account for the failure of functional protein to be generated.

Pathogenic Escherichia coli increase Cl- secretion from intestinal epithelia by upregulating galanin-1 receptor expression.

Galanin is widely distributed in enteric nerve terminals lining the human gastrointestinal (GI) tract. We have shown previously that galanin-1 receptors (Gal1-R) are expressed by epithelial cells lining the human GI tract, and upon activation cause Cl- secretion. Because expression of this receptor is transcriptionally regulated by nuclear factor-kappa B (NF-kappa B), which is activated by enteric pathogens as a part of the host epithelial response to infection, we investigated whether such bacterial pathogens could directly increase Gal1-R expression in the T84-cell model system. Pathogenic Escherichia coli, but not nonpathogenic E. coli, activate a p50/p65 NF-kappa B complex that binds to oligonucleotides corresponding to a recognition site located within the 5' flanking region of the human GAL1R gene. Pathogenic E. coli, but not normal commensal organisms, increase Gal1-R mRNA synthesis and [(125)I]galanin binding sites. Whereas galanin increases short-circuit current (Isc) approximately 5-fold in uninfected T84 cells, exposure to pathogenic, but not nonpathogenic, E. coli results in galanin increasing Isc approximately 20-fold. To confirm the validity of these in vitro observations, we also studied C57BL/6J mice infected with enterohemorrhagic E. coli (EHEC) by gavage. Infection caused a progressive increase in both NF-kappa B activation and Gal1-R expression, with maximal levels of both observed 3 days after gavage. Ussing chamber studies revealed that colons infected with EHEC, but not those exposed to normal colonic flora, markedly increased Isc in response to galanin. These data indicate that pathogen-induced increases in Gal1-R expression by epithelial cells lining the colon may represent a novel unifying pathway responsible for at least a portion of the excessive fluid secretion observed during infectious diarrhea.

Azoxymethane-induced fulminant hepatic failure in C57BL/6J mice: characterization of a new animal model.

Without transplantation, approximately 50-90% of all patients with fulminant hepatic failure (FHF) die. This poor outcome is due in part to the absence of an appropriate animal model, which would allow for a greater understanding of the pathophysiology of this syndrome. Given the reports of liver injury in humans and livestock fed cycad palm nuts on the island of Guam, we hypothesized that the active ingredient azoxymethane (AOM) could cause FHF. We therefore evaluated AOM in C57BL/6J mice. Histologically, we observed microvesicular steatosis 2 h, sinusoidal dilatation 4 h, and centrilobular necrosis 20 h after AOM administration, and transmission electron microscopy showed that this agent causes mitochondrial injury. FHF was associated with all four stages of encephalopathy, as well as by a prodromal period of decreased eating and drinking lasting approximately 15 h before the development of stage I encephalopathy (i.e., loss of scatter reflex). Late encephalopathy was associated with increased arterial ammonia, decreased serum glucose, and evidence of brain edema (astrocyte swelling). We show that AOM-induced FHF is highly reproducible, without evidence of lot-to-lot variability, and is dose dependent. These findings therefore suggest that AOM is an excellent agent for the study of FHF, as well as indicate that Guamanian FHF may be due to AOM found in unwashed cycad palm nuts.

Protein kinase B/Akt mediates effects of insulin on hepatic insulin-like growth factor-binding protein-1 gene expression through a conserved insulin response sequence.

Insulin regulates the expression of multiple hepatic genes through a conserved insulin response sequence (IRS) (CAAAAC/TAA) by an as yet undetermined mechanism. Protein kinase B/Akt (PKB/Akt), a member of the PKA/PKC serine/threonine kinase family, functions downstream from phosphatidylinositol 3'-kinase (PI3K) in mediating effects of insulin on glucose transport and glycogen synthesis. We asked whether PKB/Akt mediates sequence-specific effects of insulin on hepatic gene expression using the model of the insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Insulin lowers IGFBP-1 mRNA levels, inhibits IGFBP-1 promoter activity, and activates PKB/Akt in HepG2 hepatoma cells through a PI3K-dependent, rapamycin-insensitive mechanism. Constitutively active PI3K and PKB/Akt are each sufficient to mediate effects of insulin on the IGFBP-1 promoter in a nonadditive fashion. Dominant negative K179 PKB/Akt disrupts the ability of insulin and PI3K to activate PKB/Akt and to inhibit promoter activity. The IGFBP-1 promoter contains two IRSs each of which is sufficient to mediate sequence-specific effects of insulin, PI3K, and PKB/Akt on promoter activity. Highly related IRSs from the phosphoenolpyruvate carboxykinase and apolipoprotein CIII genes also are effective in this setting. These results indicate that PKB/Akt functions downstream from PI3K in mediating sequence-specific effects of insulin on the expression of IGFBP-1 and perhaps multiple hepatic genes through a conserved IRS.

Galanin causes Cl- secretion in the human colon. Potential significance of inflammation-associated NF-kappa B activation on galanin-1 receptor expression and function.

Galanin is widely distributed in enteric nerves and nerve terminals throughout the gastrointestinal (GI) tract. Within the GI tract galanin is best known for its ability to alter smooth muscle contractility and regulate intestinal motility. However, recent studies also indicate that galanin can modulate epithelial ion transport. We previously showed that epithelial cells lining the human GI tract, including those of colonic origin, express Gal1 galanin receptors (Gal1-R). We herein demonstrate that epithelial cells lining the human colon only express Gal1-R receptors and do not express other galanin receptor subtypes. We previously showed that Gal1-R expression was transcriptionally regulated by the transcription factor NF-kappa B. Consistent with this transcription factor being activated in a number of inflammatory conditions, we show increased colonic Gal1-R expression in patients with colitis due to a variety of causes. To further evaluate the physiology of Gal1-R activation, we studied this receptor expressed by the human colon epithelial cell line T84. Gal1-R activation resulted in a dose-dependent increase in Cl- secretion; whereas infection of T84 cells with pathogens known to activate NF-kappa B augmented Gal1-R expression and Cl- secretion. Thus, galanin acts as a secretagogue in epithelial cells lining the human colon, with alterations in Gal1-R expression possibly playing an important role in the diarrhea associated with various inflammatory processes affecting the GI tract.

[Interaction of cytokines with cellular receptors]. / Vzaimodeistvie tsitokinov s kletochnymi retseptorami.

The cytokinin family includes biologically active polypeptide molecules secreted by haemopoietic and immunocompetent cells which control cell proliferation and differentiation. Cytokinin interactions with specific receptors of the cell surface results in oligomerization of these receptors, i.e. in association of two or more membrane molecules. It is becoming obvious that oligomerization of receptors is an indispensable stage in the manifestation by cytokinins of their biological activity. In this context, studies of regularities of cytokinin receptor interactions resulting in receptor oligomerization is important for both elucidation of molecular mechanisms underlying kinin action and construction of compounds having the properties of agonists (or antagonists) of cytokinin-induced oligomerization of membrane receptors. A conclusion is draw about the important role of polyvalent cytokinin interactions with cell receptors in the initiation of oligomerization and subsequent formation of functionally active receptor complex.

Influence of synthetic peptides, corresponding to fragments of the human alpha-2 interferon molecule, on the proliferation of lymphoblastoid cells in vitro. Growth inhibition and receptor binding.

Influence of nine synthetic peptides from the C-terminal part of human alpha-2 interferon (IFN) molecule on growth of a human T-lymphoblastoid cell line MT-4 was investigated. It was shown that some peptides inhibited the proliferation of MT-4 cells. It was also found that MT-4 cells expressed specific receptors on the outer surface of the plasma membrane. All studied peptides competed for binding to a common binding site. Antiproliferative activity of the peptides correlated with their length and affinity to receptors on the outer membrane of MT-4 cells.

The alternative splicing of human Fc gamma RII mRNA is regulated by activation of B cells with mIgM cross-linking, interleukin-4, or phorbolester.

The human type two IgG binding receptors (Fc gamma RII) are encoded by three genes (Fc gamma RIIA, -B and C) resulting in at least six protein isoforms generated by alternative mRNA splicing. Surface expression of Fc gamma RII has been shown to be modulated during B cell activation, although data characterizing the isoform(s) expressed are not available. The extracellular as well as the transmembrane domains of various Fc gamma RII are highly homologous. Only the intracellular domains vary between the different Fc gamma RII isoforms, suggesting differences in signal transduction. Using reverse transcriptase and polymerase chain reaction of mRNA obtained from resting tonsil B cells, we show that the majority of Fc gamma RII mRNA species to be of b2 type, although b1 type and a low level of Fc gamma RIIa type are also present. Culturing the cells for 18 h in the presence of 2.5 U/ml interleukin-4 or 10 micrograms/ml affinity-purified anti-IgM F(ab')2 fragments induced a switch in alternative splicing, resulting in a significant increase of Fc gamma RIIb1 mRNA expression, while the synthesis of Fc gamma RIIb2 mRNA was down-regulated. Stimulation of B cells with 100 ng/ml phorbol 12-myristate 13-acetate induced similar alteration, although only after 48-h treatment. The accumulation of Fc gamma RIIb1 and the reduction of both Fc gamma RIIb2 and Fc gamma RIIa mRNA in activated cells is accompanied by the enhanced expression of Fc gamma RII on the cell surface, representing most probably the Fc gamma RIIb1 isoform. Heat-aggregated IgG inhibited the anti-IgM-induced proliferation of resting but not that of activated B cells, suggesting that aggregation of Fc gamma RIIb2 constitutively expressed on resting B cells might be responsible for the prevention of inadequate activation of resting B cells.

Early lymphocyte activation events are inhibited by antiproliferative synthetic peptide 2438, a fragment of human interferon alpha-2, and immunosuppressant cyclosporine A.

A synthetic peptide (designated 2438) corresponding to the human interferon alpha-2 amino acid sequence 124-138 inhibits proliferation of T-lymphocytes in vitro. Time-course experiments suggest that peptide 2438 affects early stages of lymphocyte activation. Molecular mechanisms of peptide 2438 action were studied. By western-blotting with monoclonal antibodies against phosphotyrosine peptide 2438 was shown to decrease the phosphotyrosine content of an endogenous protein substrate (M.M. = 36 kDa) in human lymphocytes activated with concanavalin A (ConA). Similar effect on tyrosine-specific phosphorylation in mitogen-stimulated lymphocytes was observed with the native interferon or Cyclosporine A (CsA). Calcium fluxes induced by ConA in human lymphocytes were measured using a fluorescent calcium chelator Fura-2. In contrast to CsA, peptide 2438 did not affect the ConA-induced calcium influx in lymphocytes.

Synthetic peptide with antiproliferative activity: a short C-terminal fragment of the human interferon alpha-2 molecule.

The biological activity of six synthetic peptides of the 124-144 region of the human interferon alpha-2 (IFN alpha-2) molecule was studied. Peptides were examined for their ability to inhibit mitogen induced proliferation of human blood cells in vitro. Only the peptide corresponding to the amino acid sequence 124-138 (2438) possessed IFN-like antiproliferative activity. Other tested synthetic peptides did not affect cell proliferation in this experimental system. As with the native IFN alpha-2 molecule, the inhibitory effect of the peptide 2438 was dose-dependent. On simultaneous addition of peptide 2438, antiproliferative activity of IFN alpha-2 was enhanced. Direct cytotoxic effects of synthetic peptide 2438 were not revealed. These results suggest that a synthetic peptide corresponding to the 124-138-amino acid sequence of the human IFN alpha-2 molecule serves as a cytostatic agent.

Interaction of a synthetic peptide of the interferon alpha 2 C-terminal part with human blood leukocytes. II. Effect on protein tyrosine phosphorylation.

Tyrosine phosphorylation in human blood lymphocytes was studied as a function of stimulation with concanavalin A (ConA) and treatment of the cells with interferon alpha 2 (IFN alpha 2) and/or an IFN-derived C-terminal synthetic peptide 2438 (amino acid residues 124-138). Both IFN alpha 2 and the peptide 2438 decreased the level of protein tyrosine phosphorylation in the ConA-stimulated cells. In unstimulated cells, IFN alpha 2 increased, and the peptide 2438 decreased the level of the tyrosine phosphorylation. A possible correlation of these effects with stimulation of cell proliferation is discussed.