Abstract: Nanoparticles are effective of delivering cargo into cells. Here, sodium borocaptate (BSH) was encapsulated in liposomes composed of nickel lipid, and anti-epidermal growth factor receptor (EGFR) antibodies were conjugated to the liposomes using the antibody affinity motif of protein A (ZZ) as an adaptor (immunoliposomes). The immunoliposomes were used to deliver BSH into EGFR-overexpressing glioma cells. Immunohistochemical analysis using an anti-BSH monoclonal antibody revealed that BSH was delivered effectively into the cells but not into EGFR-deficient glioma or primary astrocytes. In an animal model of brain tumors, both the liposomes and the BSH were only observed in the tumor. Moreover, the efficiency of (10)B's delivery into glioma cells was confirmed by inductively coupled plasma-atomic emission spectrometry (ICP-AES) both in vitro and in vivo. The results suggest that this system utilizing immunoliposomes provides an effective means of delivering (10)B into glioma cells in boron neutron capture therapy (BNCT).

Abstract: ErbB2, which is a member of the epidermal growth factor (erbB) receptor family, is frequently overexpressed in breast and ovarian cancers. Antibody and small molecule anti-tyrosine kinase inhibitors have been developed for targeted therapies for cancers overexpressing erbB2. Internalization and downregulation of erbB2, which is induced by a ligand, may be important for efficacious therapeutic effects. However, ligand-dependent erbB2 internalization has not been well characterized. Here we investigated the internalization of erbB2 in SKBr3 and SKOv3 cells, both overexpressing erbB2, using an EC-1 peptide fused to eGFP (EC-eGFP), which specifically binds to erbB2. ErbB2 was internalized in SKOv3 cells when the cells were treated with EC-eGFP. The accumulation of endosomal erbB2 was EC-eGFP dependent, which colocalized with transferrin implying endocytosis via clathrin-coated pits. In contrast, internalization of erbB2 was not observed in SKBr3 cells. As a result, two different mechanisms, which are cell type dependent for the internalization of erbB2, are proposed.

Abstract: Polyethylenimine (PEI) cationization is a powerful strategy for protein transduction into cells. In this study, we attempted the artificial regulation of cell proliferation by protein transduction of the N-terminal domain (1-132 amino acids) of the simian virus 40 large T-antigen (SVLT-N), which inactivates retinoblastoma family proteins but not p53. To deliver SVLT-N into cells, we employed an indirect cationization method by forming a complex of biotynylated SVLT-N through disulfide bonds (biotin-SS-SVLT-N) and PEI-cationized avidin (PEI600-avidin). Using this complex, SVLT-N was transduced into the nucleus of confluent and quiescent Balb/c 3T3 cells and was found to be complexed with a cellular target protein, pRb. Furthermore, SVLT-N transduction induced cell proliferation in spite of confluent conditions. Because SVLT-N thus transduced into cells gradually degraded and was not detectable after a 4-d incubation, transiently transformed cells were obtained by this method. These results suggest that oncogene protein transduction technology has great potential for in vitro regulation of cell proliferation.

Abstract: NELL1 is an extracellular protein inducing osteogenic differentiation and bone formation of osteoblastic cells. To elucidate the intracellular signaling cascade evoked by NELL1, we have shown that NELL1 protein transiently activates the MAPK signaling cascade, induces the phosphorylation of Runx2, and promotes the rapid intracellular accumulation of Tyr-phosphorylated proteins. Unlike BMP2, NELL1 protein does not activate the Smad signaling cascade. These findings suggest that upon binding to a specific receptor NELL1 transduces an osteogenic signal through activation of certain Tyr-kinases associated with the Ras-MAPK cascade, and finally leads to the osteogenic differentiation.

Abstract: Betacellulin (BTC) is one of the members of the epidermal growth factor (EGF) ligand family of ErbB receptor tyrosine kinases. It is a differentiation factor as well as a potent mitogen. BTC promotes the differentiation of pancreatic acinar-derived AR42J cells into insulin-producing cells. It independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4. However, the physiochemical characteristics of BTC that are responsible for its preferential binding to these two receptors have not been fully defined. In this study, to investigate the essential amino acid residues of BTC for binding to the two receptors, we introduced point mutations into the EGF domain of BTC employing error-prone PCR. The receptor binding abilities of 190 mutants expressed in Escherichia coli were assessed by enzyme immunoassay. Replacement of the glutamic acid residue at position 88 with a lysine residue in BTC was found to produce a significant loss of affinity for binding to ErbB1, while the affinity of binding to ErbB4 was unchanged. In addition, the mutant of BTC-E/88/K showed less growth-promoting activity on BALB/c 3T3 cells compared with that of the wild-type BTC protein. Interestingly, the BTC mutant protein promoted differentiation of pancreatic acinar AR42J cells at a high frequency into insulin-producing cells compared with AR42J cells that were treated with wild-type BTC protein. These results indicate the possibility of designing BTC mutants, which have an activity of inducing differentiation only, without facilitating growth promotion.

Abstract: The present study was conducted to establish a method to induce differentiation of bone marrow (MB)-derived mesenchymal cells into insulin-producing cells. When mouse BM-derived mesenchymal cells were cultured for 60 days in medium containing 10% fetal calf serum and 25 mM glucose, they expressed insulin. Addition of activin A and betacellulin (BTC) accelerated differentiation, and immunoreactive insulin was detected 14 days after the treatment. Insulin-containing secretory granules were observed in differentiated cells by electron microscopy. Treatment of BM-derived mesenchymal cells with conophylline (CnP) and BTC-delta4 further accelerated differentiation, and mRNA for insulin was detected 5 to 7 days after the treatment. Mesencymal cells treated with CnP and BTC-delta4 responded to a high concentration of glucose and secreted mature insulin. When these cells were transplanted into streptozotocin-treated mice, they markedly reduced the plasma glucose concentration, and the effect continued for at least 4 weeks. These results indicate an efficacy of the combination of CnP and BTC-delta4 in inducing differentiation of BM-derived mesenchymal cells into insulin-producing cells.

Abstract: We analyzed gene expression profiles of five tumor cell lines (NB2a, NB41A3, C1300N18, BC3H1, and Neuro2a) derived from a category of nervous system using our originally developed cell surface marker DNA microarray in order to search for tumor-specific cell surface markers common to these cells. To visualize the expression patterns and to extract candidate genes of interest based on the expression profiles of several cell lines, we employed the clustering procedure of spherical self-organizing-map. As the result, three candidates of tumor-specific cell surface markers were picked up when the expression profiles were compared with that from normal brain tissue. RT-qPCR showed the expression of these genes was higher in tumor cells than in normal brain. Here we demonstrated the spherical self-organizing-map analysis should be useful to identify the candidates of cell surface markers common and specific to the group of cells or tissues of interest.

Abstract: The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

Abstract: Previous studies have shown the efficacy of betacellulin (BTC) to promote beta-cell regeneration. Because of its short half-life, however, the effect of BTC may have been underestimated. This study was conducted to assess the effect of continuous administration of BTC on beta-cell regeneration. Adenovirus vectors encoding proBTC (Ad-proBTC) and mature BTC (Ad-mBTC) were prepared, and the efficacy of secretion of BTC was compared in AML12 hepatocytes. When AML12 cells were infected with Ad-proBTC or Ad-mBTC, cells infected with Ad-mBTC secreted considerably larger amount of BTC. We then infused Ad-mBTC into the mouse tail vein. Expression of BTC was detected in the liver for at least 21 days, and serum BTC was maintained at approximately 1 ng/ml for 7 days. When Ad-mBTC was infused immediately after administration of STZ (170 mg/kg), elevation of the plasma glucose induced by STZ was markedly inhibited, and the plasma glucose concentration remained at less than 200 mg/dl for 21 days. The insulin content and the beta-cell mass were significantly increased in Ad-mBTC-infused mice. These results indicate that continuous administration of BTC is quite effective in promoting regeneration of beta-cells.

Abstract: Both canonical Wnt/beta-catenin and TGFbeta/Smad signaling pathways coordinately regulate pattern formation during embryogenesis as well as tumor progression. Evidence of cross-talk between these two pathways has been reported. Here we demonstrated that the Activin-like kinase 4 (Alk4)/Smad2 pathway facilitates the transcriptional activity of the oncogenic Wnt/beta-catenin/Tcf4 pathway through a novel Smad4-independent mechanism. Upon activation, Smad2 physically interacted with Tcf4, beta-catenin and the co-activator p300 to enhance transcriptional activity of beta-catenin/Tcf4 through the histone acetyltransferase activity of p300. Transactivation by Smad2 was independent of a Smad-binding element (SBE) and Smad4. Indeed, the enhancement of beta-catenin/Tcf4 transcriptional activity by activated Smad2 was negatively regulated by the presence of Smad4. Moreover, a tumor-derived missense mutant of Smad2, lacking the ability to bind to Smad4 was still able to enhance the Tcf4 transcriptional reporter in the presence of beta-catenin and Tcf4. Our findings suggest that Smad2 may function as an activator of canonical Wnt/beta-catenin/Tcf4 signaling through a SBE/Smad4-independent pathway.

Abstract: Conophylline and betacellulin-delta4 reproduce differentiation-inducing activity of activin A and betacellulin, respectively. We examined the effect of conophylline and betacellulin-delta4 on beta cell differentiation. In AR42J cells, conophylline and betacellulin-delta4 converted them into insulin-producing cells. Cells treated with conophylline and betacellulin-delta4 continued to grow after differentiation. Thus, cell number and insulin content were much greater compared to cells treated with activin A and betacellulin. Furthermore, cells treated with conophylline and betacellulin-delta4 secreted insulin in response to glucose. Likewise, conophylline and betacellulin-delta4 converted pancreatic ductal cells into insulin-producing cells. Insulin content, cell number and glucose-evoked insulin secretion were significantly greater than those in cells treated with activin A and betacellulin. Transplantation of pseudoislets prepared using ductal cells treated with conophylline and betacellulin-delta4 was able to reduce effectively the plasma glucose concentration in streptozotocin-treated nude mice. Conophylline and betacellulin-delta4 are effective in inducing differentiation of beta cells from progenitors.

Abstract: The development of safe and efficient liver-specific gene delivery approaches offers new perspectives for the treatment of liver disease, in particular, liver cancer. We evaluated the therapeutic potential of hepatotropic nanoparticles for gene therapy of liver tumor. These nanoparticles do not contain a viral genome and display the hepatitis B virus L antigen, which is essential to confer hepatic specificity. It has not been shown whether a therapeutic effect could be obtained using L nanoparticles in a human liver tumor xenograft model. Rats bearing human hepatic (NuE) and non-hepatic tumors were injected with L nanoparticles containing a green fluorescent protein (GFP) expression plasmid. GFP expression was observed only in NuE-derived tumors but not in the non-hepatic tumor. The potential for treatment of liver tumors was analyzed using L nanoparticles containing the herpes simplex virus thymidine kinase gene, in conjunction with ganciclovir pro-drug administration. The growth of NuE-derived tumors in L particle-injected rats was significantly suppressed, but not of the non-hepatic tumor control. In summary, this is the first demonstration that nanoparticles could be used for delivery of therapeutic genes with anti-tumor activity into human liver tumors. This intravenous delivery system may be one of the major advantages as compared to many other viral vector systems.

Abstract: The bio-nanocapsules (BNCs) composed of the recombinant envelope L-protein of hepatitis B virus constitute efficient delivery vectors specifically targeting human hepatocytes. Here, we have tried to enhance the stability of the BNCs because the L-proteins in the BNCs were aggregated due to random disulfide bridging when stored for a long period at 4 degrees C. The envelope protein contains fourteen cysteine residues in the S domain. Aggregation of the envelope proteins might be avoided if unessential cysteine residues are replaced or removed because the irreversible alkylation of the free sulfhydryl group protects against the aggregation and enhances the efficiency of encapsulation. In this study, the possibility of reducing the number of cysteine residues in the S domain to enhance the stability of the BNCs was assessed. The replacement of each cysteine residue by site-directed mutation showed that nine of fourteen cysteine residues were not essential to obtaining BNCs secreted into the culture media. Furthermore, upon evaluating the combination of these mutations, it was found that eight residues of replacement were acceptable. The mutant BNCs with replaced eight cysteine residues were not only more resistant against trypsin, but also more effective in transducing genes into human hepatoma-derived HepG2 cells than the original type BNC. Thus, we demonstrated that the minimized number of cysteine residues in the S domain could enhance the stability of the BNCs.

Abstract: A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

Abstract: Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.

Abstract: Bionanocapsules (BNCs) are hollow nanoparticles that are composed of L protein (the hepatitis B virus surface antigen) and show specific affinity for human hepatocytes. The pre-S1 peptide displayed on the surface of BNCs is the specific ligand for binding to the receptor on human hepatocytes. Therefore, BNCs are not delivered to other tissues, such as the brain. The aim of the present study was to develop a novel drug delivery system (DDS) targeting brain tumors using BNCs that selectively targeted brain tumors. Epidermal growth factor receptor (EGFR), especially a constitutively active genomic sequence deletion variant of EGFR (EGFRvIII), is overexpressed in human glioblastoma. In the present study, we replaced the pre-S1 peptide with the antibody affinity motif of protein A and made hybrid BNCs conjugated with anti-human EGFR antibody recognizing EGFRvIII. The hybrid BNCs were efficiently delivered to glioma cells but not normal glial cells. Moreover, we confirmed the specific delivery of the hybrid BNCs to brain tumors in an in vivo brain tumor model. These results suggest that this new approach using BNCs is a promising system for brain tumor-targeted drug delivery.

Abstract: The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.

Abstract: The multiplications of low level Legionella pneumophila serogroup 1 cells by the coculture procedure with Acanthamoeba castellanii were tested in five strains. The cells in all strains proliferated effectively for isolating. This procedure might be a useful means of improving the successful isolation from environmental and clinical specimens of low level Legionella cells, and pursuing the source of infection.

Abstract: BACKGROUND: We previously showed the usefulness of a fused protein of human pancreatic ribonuclease1 (hRNase1) with human epidermal growth factor (hEGF) for molecular targeting of EGF receptor (EGFR)-overexpressing cells. In this study, the mechanisms underlying the inhibition of cell growth by RNase-EGF fused proteins was confirmed. MATERIALS AND METHODS: Des.1-7 hRNase1 was genetically fused to hEGF. The fused proteins were expressed and isolated from Escherichia coli. The internalization of hRNase1-hEGF was confirmed by confocal fluorescence microscopy. The growth-inhibitory effect of the fused proteins was evaluated by MTT assay. RESULTS: FITC-labelled hRNase1-hEGF was internalized into EGFR-overexpressing A431 cells. The internalization was not observed in A431 cells pre-treated with hEGF and EGFR-deficient H69 cells. The growth-inhibitory effect of des.1-7 hRNase1-hEGF against A431 cells was statistically significantly more pronounced than that of hRNase1-hEGF. CONCLUSION: RNase-EGF fused proteins are internalized through EGFR and inhibit cell growth by exerting their ribonucleolytic activity in the cytosol.

Abstract: The betacellulin precursor (pro-BTC) is a novel substrate for ADAM10-mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G-protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro-BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro-BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro-BTC shedding by H2O2 was blocked by the broad-spectrum metalloprotease inhibitor TAPI-0 but was still functional in ADAM17 (TACE)-deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2-induced pro-BTC shedding was blocked by co-culturing cells in the anti-oxidant N-acetyl-L-cysteine but was unaffected by culture in calcium-deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro-BTC shedding, was sensitive to calcium depletion but was unaffected by co-culture with the anti-oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro-BTC, the GPCR agonist endothelin-1 (ET-1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385A ADAM10. However, overexpression of wild-type ADAM10 or ADAM17 led to an increase in ET-1-induced pro-BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET-1 as two novel inducers of pro-BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli.

Abstract: It is thought that the export of angiogenic fibroblast growth factors (FGF) from tumors may be involved in the onset of tumor angiogenesis. To create a new active targeting drug that inhibits the tumor angiogenic process without toxicities to normal cells, human basic FGF (h-bFGF) was inserted genetically into the Gly89 position of cross-linked RNase1 (the ribonuclease inhibitor protein [RI] binding site of cross-linked human pancreatic RNase) to prevent stereospecific binding to RI. The resultant insertional-fusion protein (CL-RFN89) was active both as h-bFGF and as RNase1. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI binding caused by the fused h-bFGF domain. In the present study, the effect of the resultant protein, CL-RFN89, on the antitumor response though its antiangiogenic properties was investigated in an in vivo model. Continuous systemic treatment with CL-RFN89 significantly inhibited the growth of human A431 squamous cell carcinomas in vivo. Seven days of treatment with CL-RFN89 resulted in a 58.2% inhibition of tumor growth compared with control mice (P < 0.0001). Furthermore, immunohistochemistry using a rat antimouse CD31 antibody showed that treatment with CL-RFN89 reduced tumor vascularization. These findings identify CL-RFN89 as a potent systemic inhibitor of tumor growth as a result of its antiangiogenic properties. This protein appears to be a new systemic antitumor agent.

Abstract: Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host-vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 microg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.

Abstract: Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available.

Abstract: Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.

Abstract: Human pancreatic ribonuclease-1 (RNase1) does not exhibit its cytotoxicity unless it is artificially internalized into the cytosol. Furthermore, once it encounters the cytosolic RNase inhibitor (RI), the activity of RNase1 is seriously reduced. To achieve the cellular targeting of RNase1 and the blocking of RI binding simultaneously, the basic fibroblast growth factor (bFGF) sequence was inserted into RNase1 at the RI binding site using a gene fusion technique. The effect of this fusion protein, CL-RFN89, on the angiogenesis, which was accelerated by FGF-FGF receptor interaction, was investigated. It was shown by using fluorescein-labeled CL-RFN89, that the binding to human umbilical vein endothelial cells (HUVECs) was dependent on the existence of the FGF receptors. In addition, CL-RFN89 inhibited the cellular growth of HUVECs in vitro and also inhibited the tube formation, using a three-dimensional tube formation assay. Furthermore, this fusion protein was shown to prevent in vivo tumor cell-induced angiogenesis, using the mouse dorsal air sac assay. These results demonstrated that CL-RFN89 inhibits angiogenesis in vitro and in vivo and that it can be expected to be a potent antiangiogenic agent.

Abstract: A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

Abstract: To apply cell transplantation for treatment of diabetes mellitus, a sufficient number of beta-cell sources are required. In the present study, we examined whether an epithelial cell line obtained from normal adult rat liver, namely hepatic stem-like (HSL) cells, which can be converted to both hepatocytes and billiary epithelial cells, could be a potential beta-cell source. The growth speed of HSL cells was rapid and these cells were easily expanded in vitro. Bipotential hepatic stem cells, HSL cells, also expressed PGP9.5, which is expressed in neurons, beta-cells, and progenitor cells of the pancreatic endocrine cells as well. Sodium butyrate induced morphological changes in HSL cells and converted them into flattened cells with large cytoplasm. When HSL cells were incubated with a combination of 5 mM sodium butyrate and 1 nM betacellulin, most of the cells were converted into morphologically neuron-like cells. RT-PCR analysis revealed that a series of transcriptional factors involved in differentiation of pancreatic endocrine cells was induced by the treatment with sodium butyrate and betacellulin. mRNAs for insulin, pancreatic polypeptide, and somatostatin were also observed. Immunoreactive pancreatic polypeptide, somatostatin, and insulin were detected in sodium butyrate and betacellulin-treated HSL cells. In conclusion, HSL cells obtained from adult normal liver also have the potential to differentiate into pancreatic endocrine cells in vitro. HSL cells may be one of the potential beta-cell sources for cell transplant therapy for insulin-dependent diabetes.

Abstract: We previously described a novel alternatively spliced mRNA transcript of the betacellulin (BTC) gene. This splice isoform, termed BTC-delta4, lacks the C-loop of the epidermal growth factor motif and the transmembrane domain as a result of exon 4 'skipping'. In this study, we expressed BTC-delta4 recombinantly to explore its biological function. When BTC-delta4 was expressed in COS-7 cells, it was secreted largely into the culture medium, in contrast to BTC. Unlike BTC, highly purified recombinant BTC-delta4 produced in Escherichia coli failed to bind or induce tyrosine phosphorylation of either ErbB1 or ErbB4, nor did it antagonize the binding of BTC to these receptors. Consistent with this, BTC-delta4 failed to stimulate DNA synthesis in Balb/c 3T3 and INS-1 cells. However, BTC-delta4 induced differentiation of pancreatic beta-cells; BTC-delta4 converted AR42J cells to insulin-producing cells. When recombinant BTC-delta4 was administered to streptozotocin-treated neonatal rats, it reduced the plasma glucose concentration and improved glucose tolerance. Importantly, BTC-delta4 significantly increased the insulin content, the beta-cell mass, and the numbers of islet-like cell clusters and PDX-1-positive ductal cells. Thus, BTC-delta4 is a secreted protein that stimulates differentiation of beta-cells in vitro and in vivo in an apparent ErbB1- and ErbB4-independent manner. The mechanism by which BTC-delta4 exerts this action on beta-cells remains to be defined but presumably involves an, as yet, unidentified unique receptor.

Abstract: In the post-genomic era, there is pressing need for development of protein manipulation methodology to analyze functions of proteins in living cells. For this purpose, techniques to deliver functional proteins into living cells are currently being evaluated as alternative approaches to the introduction of transcriptionally active DNA. Here, we describe a novel method for efficient protein transduction into living cells in which a protein is simply cationized with polyethylenimine (PEI) by limited chemical conjugation. PEI-cationized proteins appear to adhere to the cell surface by ionic charge interaction and then internalize into cells in a receptor- and transporter-independent fashion. Since PEI is an organic macromolecule with a high cationic-charge density, limited coupling with PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as ribonuclease (RNase), green fluorescent protein (GFP) and immunoglobulin (IgG), efficiently entered cells and functioned in the cytosol. Our results suggest that protein cationization techniques using PEI will be useful for the development of protein transduction technology.

Abstract: We analyzed gene expression profiles of normal mouse fibroblast BALB/c 3T3 cells and its SV40 transformant SV-T2 cells using our originally developed cell surface marker DNA microarray, which is prepared on a diamond-like carbon-coated glass. As a result, CD62L and IL-6 receptor alpha gene expressions were upregulated in SV-T2 and were thought to be candidates for call surface markers of the cells. The result of microarray analysis was validated by real-time quantitative PCR immunohistochemistry and biological assays. These data show that our cell surface marker DNA microarray should be useful in finding the candidates of cell type-specific surface markers.

Abstract: Activin A and betacellulin (BTC) are thought to regulate differentiation of pancreatic beta-cells during development and regeneration of beta-cells in adults. In the present study, we used neonatal rats treated with streptozotocin (STZ) to investigate the effects of activin A and BTC on regeneration of pancreatic beta-cells. One-day-old Sprague-Dawley rats were injected with STZ (85 micro g/g) and then administered for 7 days with activin A and/or BTC. Treatment with activin A and BTC significantly reduced the plasma glucose concentration and the plasma glucose response to intraperitoneal glucose loading. The pancreatic insulin content and beta-cell mass in rats treated with activin A and BTC were significantly increased compared with the control group on day 8 and at 2 months. Treatment with activin A and BTC significantly increased the DNA synthesis in preexisting beta-cells, ductal cells, and delta-cells. The number of islet cell-like clusters (ICCs) and islets was significantly increased by treatment with activin A and BTC. In addition, the number of insulin/somatostatin-positive cells and pancreatic duodenal homeobox-1/somatostatin-positive cells was significantly increased. These results indicate that, in neonatal STZ-treated rats, a combination of activin A and BTC promoted regeneration of pancreatic beta-cells and improved glucose metabolism in adults.

Abstract: Basic fibroblast growth factor (bFGF) was inserted in the middle of human ribonuclease 1 (RNase1) sequence at an RNase inhibitor (RI)-binding site (Gly89) by a new gene fusion technique, insertional-fusion. The resultant insertional-fusion protein (CL-RFN89) was active both as bFGF and as RNase. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI-binding caused by fused bFGF domain. As a result, CL-RFN89 showed stronger growth inhibition on B16/BL6 melanoma cells than an RI-sensitive tandem fusion protein. Thus, the insertional-fusion technique increases accessible positions for gene fusion on RNase, resulting in construction of a potent cytotoxic RNase.

Abstract: The hepatitis B virus (HBV) surface antigen (HBsAg) L particle is a hollow nano-scale particle. HBsAg L particles have many properties that make them useful for in vivo gene transfer vectors and drug delivery systems. Gene therapy so far has required the in vivo pinpoint delivery of genetic materials into the target organs and cells. Gene transfer by HBsAg L particles might be an attractive method, since their tropism is the same as that of HBV. The HBsAg L particles are able to deliver therapeutic payloads with high specificity to human hepatocytes. In addition, the specificity of L particle can be altered by displaying various cell-binding molecules on the surface. Our results indicate that the L particle is suitable for a cell- and tissue-specific gene/drug transfer vector. In this review, we discuss HBsAg L particles as a gene/drug transfer vector and its potential for the treatment of infectious diseases.

Abstract: The present study was conducted in an attempt to treat streptozotocin (STZ)-induced hyperglycemia by transplanting beta cells derived from pancreatic ductal cells. Ductal cells obtained from neonatal rats were cultured in vitro. Approximately 70% of the cells were converted to insulin-secreting cells by incubating with betacellulin and activin A. Differentiated cells responded to a depolarizing concentration of potassium, tolbutamide and a high concentration of glucose, and insulin secretion increased by 2.5-, 2.3- and 1.6-fold, respectively. We then prepared pseudoislets using the differentiated cells, which exhibited greatly improved glucose-responsiveness, with a high concentration of glucose inducing a 3-fold increase in insulin secretion. We transplanted these pseudoislets into the portal vein of STZ-treated nude mice. Before transplantation, the plasma glucose concentration was above 400 mg/dl, and after transplantation it was markedly reduced, the effect of which persisted for two weeks. These results indicate that STZ-induced hyperglycemia can be treated by transplanting pseudoislets consisting of beta cells derived from ductal cells.

Abstract: Epithelial-mesenchymal transition (EMT) facilitates migration and invasion of epithelial tumor cells. Cripto-1 (CR-1), a member of the epidermal growth factor-CFC protein family increases migration of cells in vitro. Here the expression of molecular markers and signaling molecules characteristic of EMT were assessed in mammary gland hyperplasias and tumors from mice expressing the human CR-1 transgene by the MMTV promoter (MMTV-CR-1) and in mouse mammary epithelial cell line HC-11 overexpressing CR-1 (HC-11/CR-1). Western blot analysis showed decreased expression of E-cadherin in MMTV-CR-1 tumors and in HC-11/CR-1 cells. The expression of N-cadherin, vimentin, cyclin-D1, and of the zinc-finger transcription factor, snail, was increased in MMTV-CR-1 tumors. Increased snail mRNA was also found in HC-11/CR-1 cells. Expression of phosphorylated (P)-c-Src, P-focal adhesion kinase (FAK), P-Akt, P-glycogen synthease kinase 3beta (GSK-3beta), dephosphorylated (DP)-beta-catenin, and various integrins such as, alpha 3, alpha v, beta 1, beta 3, and beta 4 was also increased in MMTV-CR-1 tumors. Immunohistochemistry showed positive staining for vimentin, N-cadherin, cyclin-D1, smooth muscle actin, fibronectin, snail, and beta-catenin in MMTV-CR-1 tumor sections. HC-11/CR-1 cells treated with the c-Src inhibitor PP2 reduced the expression of P-c-Src and of P-FAK, P-Akt, P-GSK-3beta, DP-beta-catenin all known to be activated by c-Src. Migration of HC-11/CR-1 cells was also reduced by PP2 treatment. These results suggest that CR-1 may play a significant role in promoting the increased expression of markers and signaling molecules associated with EMT.

Abstract: Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

Abstract: Betacellulin (BTC) induces differentiation of pancreatic beta-cells and promotes regeneration of beta-cells in experimental diabetes. The present study was conducted to determine if BTC improved glucose metabolism in severe diabetes induced by a high dose of streptozotocin (STZ) in mice. Male ICR mice were injected with 200 microg/g ip STZ, and various doses of BTC were administered daily for 14 days. The plasma glucose concentration increased to a level of >500 mg/dl in STZ-injected mice. BTC (0.2 microg/g) significantly reduced the plasma glucose concentration, but a higher concentration was ineffective. The effect of BTC was marked by day 4 but became smaller on day 6 or later. The plasma insulin concentration and the insulin content were significantly higher in mice treated with 0.1 and 0.2 microg/g BTC. BTC treatment significantly increased the number of beta-cells in each islet as well as the number of insulin-positive islets. Within islets, the numbers of 5-bromo-2-deoxyuridine/somatostatin-positive cells and pancreatic duodenal homeobox-1/somatostatin-positive cells were significantly increased by BTC. These results indicate that BTC improved hyperglycemia induced by a high dose of STZ by promoting neoformation of beta-cells, mainly from somatostatin-positive islet cells.

Abstract: Cripto-1 (CR-1), an epidermal growth factor-CFC (EGF-CFC) family member, has a demonstrated role in embryogenesis and mammary gland development and is overexpressed in several human tumors. Recently, EGF-CFC proteins were implicated as essential signaling cofactors for Nodal, a transforming growth factor beta family member whose expression has previously been defined as embryo specific. To identify a receptor for CR-1, a human brain cDNA phage display library was screened using CR-1 protein as bait. Phage inserts with identity to ALK4, a type I serine/threonine kinase receptor for Activin, were identified. CR-1 binds to cell surface ALK4 expressed on mammalian epithelial cells in fluorescence-activated cell sorter analysis, as well as by coimmunoprecipitation. Nodal is coexpressed with mouse Cr-1 in the mammary gland, and CR-1 can phosphorylate the transcription factor Smad-2 in EpH-4 mammary epithelial cells only in the presence of Nodal and ALK4. In contrast, CR-1 stimulation of mitogen-activated protein kinase and AKT in these cells is independent of Nodal and ALK4, suggesting that CR-1 may modulate different signaling pathways to mediate its different functional roles.

Abstract: Human Cripto-1 (CR-1), a member of the epidermal growth factor-CFC (EGF-CFC) family of peptides, is expressed in the developing mouse mammary gland and can modulate mammary epithelial cell migration, branching morphogenesis and milk protein expression in vitro. In order to screen for a CR-1 receptor and to identify potential CR-1 target tissues, we constructed a fusion protein comprising the EGF-like domain of CR-1 and the Fc domain of a human IgG1. The recombinant CR-1 fusion protein (CR-1-Fc) was biologically active as it was able to activate the ras/raf/mitogen activated protein kinase (MAPK) pathway and to inhibit transcription of the milk protein beta-casein in NMuMG and HC-11 mouse mammary epithelial cells. By using immunocytochemistry and by an in situ enzyme-linked immunosorbent assay (ELISA), CR-1-Fc was found to specifically bind to NMuMG and HC-11 cells. Finally, immunohistochemical analysis using CR-1-Fc showed a specific localization of CR-1 binding to tissue sections from mouse mammary gland. In particular, more than 60% of the epithelial cells were intensely stained with the CR-1-Fc fusion protein in the lactating mouse mammary gland, whereas approximately 25% of the mammary epithelial cells were stained in the gland from pregnant mouse. Since expression of mouse cripto-1 (Cr-1) in the pregnant and lactating mouse mammary gland as well as its presence in milk has been previously demonstrated, these data strongly suggest that an autocrine pathway involving Cr-1 and its putative receptor is operating in the mouse mammary gland during pregnancy and lactation.

Abstract: Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC(50) values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were estimated to be approximately 1-5 microm. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic.

Abstract: Immature rat intestinal stem cells (IEC-6) given the ability to express the transcription factor, pancreatic duodenal homeobox 1 (Pdx-1), yielded YK cells. Although these cells produced multiple enteroendocrine hormones, they did not produce insulin. Exposure of YK cells to 2 nmol/l betacellulin yielded BYK cells that showed the presence of insulin expression in cytoplasm and that secreted insulin into culture media. By examining the mechanism of differentiation in BYK cells, we found that another transcription factor, islet factor 1 (Isl-1) was newly expressed with the disappearance of Pax-6 expression in those cells after exposure to betacellulin. These results indicated that combined expression of Pdx-1 and Isl-1 in IEC-6 cells was required for the production of insulin. In fact, overexpression of both Pdx-1 and Isl-1 in IEC-6 cells (Isl-YK-12, -14, and -15 cells) gave them the ability to express insulin without exposure to betacellulin. Furthermore, implantation of the Isl-YK-14 cells into diabetic rats reduced the animals' plasma glucose levels; glucose levels dropped from 19.4 to 16.9 mmol/l 1 day after the injection of cells. As expected, the plasma insulin concentrations were 2.7 times higher in the diabetic rats injected with Isl-YK-14 cells compared to in controls. In summary, our results indicated that immature intestinal stem cells can differentiate into insulin-producing cells given the ability to express the transcription factors Pdx-1 and Isl-1.

Abstract: BACKGROUND: Conventional targeted therapy with foreign proteins is highly immunogenic. In this study, we developed targeted therapy composed of human endogenous proteins and evaluated its efficacy in vitro. MATERIALS AND METHODS: Human epidermal growth factor (EGF) was chemically linked to human eosinophil cationic protein (ECP). The cytotoxicity of the EGF-ECP conjugate was evaluated by MTT assay. RESULTS: The conjugate showed dose-dependent cytotoxicity on EGF receptor (EGFR)-overexpressing BT-20 cells with an IC50 of 1.5 x 10(-7) M, whereas the IC50 of ECP alone was almost 10(-4) M. The conjugate had no detectable cytotoxicity against EGF receptor-deficient H69 cells. Excess EGF protected BT-20 cells from the cytotoxicity of the conjugate. Comparing the cytotoxicity and the level of EGFR expression, the cytotoxicity of the conjugate was positively correlated with the level of EGFR expression of each cell line. CONCLUSION: Conjugates composed solely of human proteins might be useful with less immunogenicity and less toxicity than the conventional immunotoxins for targeted therapy.

Abstract: There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25 kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNase-based immunotoxins.

Abstract: The solution structure of the EGF-like domain of betacellulin (BTCe), a newly discovered member of the epidermal growth factor (EGF) family, has been determined using two-dimensional nuclear magnetic resonance spectroscopy. This is the first report to identify the solution structure of the EGF-family ligand monomers that interact with both ErbB-1 and ErbB-4. The solution structure of BTCe was calculated using 538 NMR-derived restraints. The overall structure of BTCe was stabilized by three disulfide bonds, a hydrophobic core, and 23 hydrogen bonds. It appears that BTCe is comprised of five beta-strands and one short 3(10) helical turn. The secondary structural elements of BTCe are basically similar to those of the other EGF-family proteins, except that several significant variations of the structural properties were found. It is suggested that the structural variations between BTCe and the other EGF-family ligands may affect the specific receptor-recognition properties of EGF-family ligands.

Abstract: Cationization of a protein is considered to be a powerful strategy for internalizing a functional protein into cells. Cationized proteins appear to adsorb to the cell surface by electrostatic interactions, then enter the cell in a receptor- and transporter-independent fashion. Thus, in principle, all cell types appear to take up cationized proteins. Since ribonucleases (RNases) have a latent cytotoxic potential, cationized RNases could be useful cancer chemotherapeutics. In this study, we investigated the effect of the degree of cationization on the cytotoxicity of RNase A by modifying carboxyl groups with ethylenediamine. We found that there is an optimum degree of modification for cytotoxicity, in which 5 to 7 out of 11 carboxyl groups in RNase A are modified, toward MCF-7 and 3T3-SV-40 cells. More interestingly, the cytotoxicity of cationized RNase As correlates well with the value of [RNase activity] x [estimated concentration of RNase free from RNase inhibitor], mimicking the practical enzymatic activity of cationized RNase As in cytosol. The results indicate that cationization of a protein to an optimum level is important for maintaining protein function in the cytosol. Sophisticated protein cationization techniques will help to advance protein transduction technology.

Abstract: We have modeled betacellulin (BTC) to gain insight into the structural elements that can explain its properties. The epidermal growth factor (EGF) signal transduction pathway, a significant mediator of several cell functions, is based on four closely related tyrosine kinase receptors. The ErbB receptors are transmembrane glycoproteins and signal transduction is initiated by ligand binding that induces receptor homo- or heterodimerization to form a complex containing two molecules of ligand and two molecules of receptor. The EGF family of ligands can be divided into three groups based on their ability to bind and activate distinct ErbB receptor homo- and heterodimers. Each member of the group formed by BTC, heparin binding EGF (HB-EGF) and epiregulin (EP) can interact with both the EGF receptor (EGFR) and heregulin receptors (ErbB-3 and ErbB-4), and are hence called "bispecific" ligands. BTC and EP also present the distinctive feature that they activate all possible heterodimeric ErbB receptors. BTC has been modeled with the program MODELLER, using human EGF, human transforming growth factor alpha (hTGFalpha), human HB-EGF and human heregulin one alpha (hHRG-1alpha) as templates. The structure of the model as well as that of the templates were optimized and a simulation of 100 ps was run for all. The main structural properties of the model and the templates were compared and in conclusion the hBTC conformation was closely similar to that of hTGFalpha.

Abstract: Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1 Delta N7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 A resolution. The molecule displays the alpha + beta folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1 Delta N7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1 Delta N7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

Abstract: The hepatitis B virus (HBV) envelope (env) protein is composed of three regions; the 108- or 119-residue pre-S1 region involved in the direct interaction with hepatocytes, the 55-residue pre-S2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Thus, to improve the immunogenic potency of conventional HB vaccines, development of a new vaccine containing the entire pre-S1 region in addition to pre-S2 and S is desired. We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells [J Biol Chem 267 (1992) 1953]. In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized. By equilibrium sedimentation, an average molecular weight of L particle was estimated to be approximately 6.4 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle. By atomic force microscopy in a moist atmosphere, the L particles were observed as large spherical particles with a diameter of 50-500 nm. The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol. When immunized in mice, L particles elicited efficiently and simultaneously the anti-S, anti-pre-S2, and anti-pre-S1 antibodies. The ED(50) values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles. Furthermore, the anti-pre-S1 rabbit antibodies were found to recognize various segments of the pre-S1 region, including the pre-S1 (21-47) segment. These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HB vaccine.

Abstract: Betacellulin is thought to promote growth and differentiation of pancreatic beta-cells. We investigated the effect of betacellulin on regeneration of pancreatic beta-cells in 90%-pancreatectomized rats. Ninety percent pancreatectomy was performed in male Wistar rats and betacellulin (0.5 microg/g body weight) or saline was administered daily for 10 d starting immediately after pancreatectomy. In pancreatectomized rats, the morning-fed plasma glucose was significantly lower and the plasma insulin concentration was significantly higher in betacellulin-treated rats than those in control rats for up to 4 wk. Thirty days after pancreatectomy, a glucose tolerance test was performed. Betacellulin reduced the plasma glucose response to ip glucose loading. In control rats, the plasma insulin concentration was significantly lower and did not respond to glucose. In contrast, the plasma insulin concentration increased slightly but significantly in betacellulin-treated rats. Thirty days after pancreatectomy, the beta-cell mass was greater and the insulin content was significantly higher in betacellulin-treated rats than those in control rats. The numbers of islet cell-like cluster and bromodeoxy uridine/insulin double- positive cells in both islet cell-like cluster and islets were significantly higher in betacellulin-treated rats. These results indicate that administration of betacellulin improves glucose metabolism by promoting beta-cell regeneration in 90%-pancreatectomized rats.

Abstract: Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives, cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.

Abstract: A hybrid human protein was produced in E. coli by fusing the genes encoding human pancreatic RNase1 (hpRNase1) and human IL-2 (hIL-2). The recombinant hpRNase1-hIL-2 inhibited protein synthesis in HTLV-1-infected, malignant T cells, which hyperproduce high affinity IL-2 receptors, with an IC(50)of 2x10(-8) M, whereas no inhibition was detectable in control cells with lower affinity receptors. HpRNase1 alone had an IC(50)of almost 10(-3) M. A molar excess of hIL-2 blocked the protein synthesis inhibition dose-dependently. In a human mixed lymphocyte culture, hpRNase1-hIL-2 inhibited the proliferation of responder cells with potency comparable to that of cyclosporine, while non-effective doses of FK506 importantly improved its potency. Despite its short half-life in animals, hpRNase1-hIL-2 rapidly enters cells in a few minutes and arrests the protein translation in less than 10 h. Thus, hpRNase1-hIL-2 may be useful to selectively eliminate activated lymphocytes hyperproducing high affinity IL-2 receptors, as in allograft rejection, graft-versus-host disease, autoimmune disorders, adult T cell leukaemia and other lymphoproliferative or retroviral malignancies including HIV infection, without inducing general immunosuppression. As an entirely human "immunotoxin analogue" it may alleviate the dose limiting toxicity and immunogenicity of conventional immunotoxins.

Abstract: We investigated how the folding yield of disulfide-containing globular proteins having positive net charges from crude bacterial inclusion bodies was affected by additives in the folding buffer. In screening folding conditions for human ribonucleases and its derivative, we found that addition of salt (about 0.4 M) to a folding buffer increased the folding yield. This suggested that electrostatic interaction between polyanionic impurities such as nucleic acids and cationic unfolded protein led to the formation of aggregates under the low-salt conditions. Since inclusion bodies were found to contain nucleic acids regardless of the electrostatic nature of the expressed protein, the electrostatic interaction between phosphate moieties of nucleic acids and basic amino acid residues of a denatured protein may be large enough to cause aggregation, and therefore the addition of salt in a folding buffer may generally be useful for promotion of protein folding from crude inclusion bodies. We further systematically investigated additives such as glycerol, guanidium chloride, and urea that are known to act as chemical chaperons, and found that these additives, together with salt, synergistically improved folding yield. This study, suggesting that the addition of salt into the folding buffer is one of the crucial points to be considered, may pave the way for a systematic investigation of the folding conditions of disulfide-containing foreign proteins from crude bacterial inclusion bodies.

Abstract: Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption.

Abstract: Cripto-1 (CR-1) is an epidermal growth factor (EGF)-related protein. CR-1 can inhibit beta-casein and whey acidic protein expression in mouse mammary epithelial cells. The present study demonstrates that CR-1 can induce apoptosis in HC-11 mouse mammary epithelial cells, as measured by bis-benzimide stained nuclei, TUNEL assay and cell death ELISA. Apoptosis could be observed after 2 days of exposure of confluent HC-11 cells to CR-1 in the absence of the survival factors EGF and insulin, with maximum apoptosis occurring at 3 days. A reduction in poly(ADP-ribose) polymerase (PARP) expression and an increase in beta-catenin cleavage was found after 18 h of exposure to CR-1 suggesting that apoptosis was preceded by the induction of a caspase activity since the caspase inhibitor ZFAD.FMK could block the CR-1-induced reduction in PARP expression and CR-1-induced apoptosis. CR-1 was found to increase the expression of caspase-3-like protease. Although, the levels of p27kip1 and p21Bax did not change after exposure to CR-1 for 18 h, the levels of Bcl-xL became undetectable. These studies suggest that CR-1 promotes apoptosis by mediating the induction of caspase-3-like protease and downregulating the expression of Bcl-xL.

Abstract: In order to stabilize human RNase 1 by introduction of an intramolecular cross-link, a mutant protein (4-118CL RNase 1), in which Arg4 and Val118 are replaced with cysteine residues and linked by a disulfide bond, was designed and expressed in Escherichia coli as inclusion bodies. The 4-118CL RNase 1 that refolded under redox conditions was a monomer without free SH groups and retained 11% of the activity of the wild-type recombinant RNase 1, indicating that the mutant enzyme was correctly folded with the formation of an additional disulfide bond between Cys4 and Cys118. From guanidium chloride denaturation experiments based on the assumption of a two-state transition for unfolding, it was demonstrated that the introduction of the present cross-link increased the thermodynamic stability of RNase 1 by 2.0 kcal/mol. This value was lower than that, 5.4 kcal/mol, theoretically calculated from the reduction of chain entropy of the unfolded state due to the introduction of the cross-link. These results suggest that the present cross-link also destabilized the folded state of RNase 1 by 3.4 kcal/mol. Along with the increase in the thermodynamic stability, the stability of RNase 1 against trypsin digestion was also significantly increased by the introduction of this cross-link. It is likely, although not proven, that stabilized human RNases are favorable for clinical use, because human RNase-based immunotoxins should have long half-lives as to proteolytic degradation after endocytosis.

Abstract: The EGF-CFC gene family encodes a group of structurally related proteins that serve as important competence factors during early embryogenesis in Xenopus, zebrafish, mice and humans. This multigene family consists of Xenopus FRL-1, zebrafish one-eyed-pinhead (oep), mouse cripto (Cr-1) and cryptic, and human cripto (CR-1) and criptin. FRL-1, oep and mouse cripto are essential for the formation of mesoderm and endoderm and for correct establishment of the anterior/posterior axis. In addition, oep and cryptic are important for the establishment of left-right (L/R) asymmetry. In zebrafish, there is strong genetic evidence that oep functions as an obligatory co-factor for the correct signaling of a transforming growth factor-beta (TGFbeta)-related gene, nodal, during gastrulation and during L/R asymmetry development. Expression of Cr-1 and cryptic is extinguished in the embryo after day 8 of gestation except for the developing heart where Cr-1 expression is necessary for myocardial development. In the mouse, cryptic is not expressed in adult tissues whereas Cr-1 is expressed at a low level in several different tissues including the mammary gland. In the mammary gland, expression of Cr-1 in the ductal epithelial cells increases during pregnancy and lactation and immunoreactive and biologically active Cr-1 protein can be detected in human milk. Overexpression of Cr-1 in mouse mammary epithelial cells can facilitate their in vitro transformation and in vivo these Cr-1-transduced cells produce ductal hyperplasias in the mammary gland. Recombinant mouse or human cripto can enhance cell motility and branching morphogenesis in mammary epithelial cells and in some human tumor cells. These effects are accompanied by an epithelial-mesenchymal transition which is associated with a decrease in beta-catenin function and an increase in vimentin expression. Expression of cripto is increased several-fold in human colon, gastric, pancreatic and lung carcinomas and in a variety of different types of mouse and human breast carcinomas. More importantly, this increase can first be detected in premalignant lesions in some of these tissues. Although a specific receptor for the EGF-CFC proteins has not yet been identified, oep depends upon an activin-type RIIB and RIB receptor system that functions through Smad-2. Mouse and human cripto have been shown to activate a ras/raf/MAP kinase signaling pathway in mammary epithelial cells. Activation of phosphatidylinositol 3-kinase and Akt are also important for the ability of CR-1 to stimulate cell migration and to block lactogenic hormone-induced expression of beta-casein and whey acidic protein. In mammary epithelial cells, part of these responses may depend on the ability of CR-1 to transactivate erb B-4 and/or fibroblast growth factor receptor 1 through an src-like tyrosine kinase.

Abstract: The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney. Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media. Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently.

Abstract: Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that fails to directly bind to any of the four known erb B type 1 receptor tyrosine kinases. The present study demonstrates that CR-1 indirectly induces tyrosine phosphorylation of erb B-4 but not of the epidermal growth factor-related receptors erb B-2 and erb B-3 in different mouse and human mammary epithelial cell lines. In addition, down-regulation of erb B-4 in NMuMG mouse mammary epithelial cells and in T47D human breast cancer cells, using an anti-erb B-4 blocking antibody or a hammerhead ribozyme vector targeted to erb B-4 mRNA, impairs the ability of CR-1 to fully activate mitogen-activated protein kinase. Finally, chemical cross-linking of 125I-CR-1 to mouse and human mammary epithelial cell membranes results in the labeling of two specific bands with a molecular weight of 130 and 60 kDa, suggesting that the CR-1 receptor represents a novel receptor structurally unrelated to any of the known type I receptor tyrosine kinases. In conclusion, these data demonstrate that CR-1, upon binding to an unknown receptor, can enhance the tyrosine kinase activity of erb B-4 and that a functional erb B-4 receptor is required for CR-1-induced MAPK activation.

Abstract: The RNase 4 family is unique among RNase enzymes, displaying the highest level of sequence similarity and encompassing the shortest polypeptide chain. It is the only one showing high specificity. The human representative is an intracellular and plasma enzyme, first isolated from colon adenocarcinoma cell line HT-29. The crystal structures of human recombinant RNase 4, unliganded and in complex with d(Up), have been determined, revealing in the unique active site an explanation for the uridine specificity. Arg101, at a position not involved in catalysis in the other RNase enzymes, penetrates the enzyme moiety shaping the recognition pocket, a flip that is mediated by the interaction with the (shorter chain) C-terminal carboxylate group, providing an anchoring point for the O4 atom of the substrate uridine. The bulky Phe42 side-chain forces Asp80 to be in the chi1=-72.49 degrees rotamer, accepting a hydrogen bond from Thr44, further converting the latter into a hydrogen bond acceptor. This favours an interaction with the -NH-donor group of uridine at position 3 over that with the =N-acceptor of cytidine. The two chemical groups that distinguish uracyl from cytosine are used by the enzyme to discriminate between these two bases.

Abstract: Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic beta-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells.

Abstract: Pancreatic AR42J cells have the feature of pluripotency of the common precursor cells of the pancreas. Dexamethasone (Dx) converts them to exocrine cells, whereas activin A (Act) converts them into endocrine cells expressing pancreatic polypeptide. A combination of Act and betacellulin (BTC) converts them further into insulin-secreting cells. The present study identifies some of the genes involved in the process of differentiation that is induced by these factors, using the mRNA differential display and screening of the cDNA expression array. The expression levels of 7 genes were increased by Act alone, and a combination of Act and BTC increased the expression of 25 more genes. Of these, 16 represented known genes or homologues of genes characterized previously. Nine of the identified genes were unrelated to any other sequences in the database. An inhibitor of the mitogen-activated protein kinase pathway, PD098059, which blocks the differentiation into insulin-secreting cells, inhibited the expression of 18 of the 25 genes, suggesting that the proteins encoded by these genes are associated with the differentiation into insulin-producing cells. These include known genes encoding extracellular signaling molecules, such as parathyroid hormone-related peptide, cytoskeletal proteins, and intracellular signaling molecules. Identification and characterization of these differentially expressed genes should help to clarify the molecular mechanism of differentiation of pancreatic cells and the gene products that enable the beta-cells to produce insulin.

Abstract: Betacellulin (BTC) purified from mouse beta cell tumor (betaTC-3) is a new member of the epidermal growth factor (EGF) family which can bind receptor tyrosine kinase, EGF receptor (erbB1) and erbB4. It has been demonstrated that proBTC mRNA was abundantly expressed in human pancreas tissue, and that BTC converted amylase-secreting rat acinar cell line (AR42J) into insulin-secreting cells, suggesting that BTC might be important for the growth and/or differentiation of islet cells. However, the cell type producing BTC in the pancreas has not been clarified. In this study, we examined the localization of BTC in human pancreas and islet cell tumors. Immunohistochemistry using specific antibodies to human BTC revealed that this protein was produced in alpha cells and duct cells, and probably in beta cells in normal adult pancreas. Furthermore, strong immunoreactivity to BTC was detected in primitive duct cells of the fetal pancreas, and both insulinoma and glucagonoma cells also showed positive immunoreactivity to BTC. EGF receptor (erbB1) and erbB4 were expressed mainly in islet and duct cells, and duct cells, respectively. These results demonstrate the localization of BTC and its receptors, and suggest that BTC may be one of the factors that have physiologically important roles such as growth and differentiation of islet cells in the human pancreas.

Abstract: Pancreatic-type RNases are considered to have cytotoxic potential due to their ability to degrade RNA molecules when they enter the cytosol. However, most of these RNases show little cytotoxicity because cells have no active uptake mechanism for these RNases and because the ubiquitous cytoplasmic RNase inhibitor is considered to play a protective role against the endocytotic leak of RNases from the outside of cells. To study the cytotoxic potential of RNase toward malignant cells targeting growth factor receptors, the C-terminus of human RNase 1 was fused to the N-terminus of human basic fibroblast growth factor (bFGF). This RNase-FGF fused protein effectively inhibited the growth of mouse melanoma cell line B16/BL6 with high levels of cell surface FGF receptor. This effect appeared to result from prolongation of the overall cell cycle rather than the killing of cells or specific arrest in a particular phase of the cell cycle. Thus, human RNase 1 fused to a ligand of cell surface molecules, such as the FGF receptor, is shown to be an effective candidate for a selective cell targeting agent with low toxic effects on normal cell types.

Abstract: Cripto-1 (CR-1), a member of the epidermal growth factor-CFC peptide family, activates the ras/raf/mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In the present study, the role of CR-1 in the phosphatidylinositol 3'-kinase (PI3K)/AKT (protein kinase B)/glycogen synthase kinase 3beta (GSK-3beta)-dependent signaling pathway was evaluated in human SiHa cervical carcinoma cells. Our data demonstrate that CR-1 can enhance the tyrosine phosphorylation of the p85 regulatory subunit of PI3K and transiently induce the phosphorylation of AKT in a time- and dose-dependent manner. In addition, CR-1 was found to induce the phosphorylation of GSK-3beta. Phosphorylation of AKT and GSK-3beta by CR-1 can be blocked by LY294002, a specific inhibitor of PI3K, thus leading to apoptosis. Finally, the apoptotic effect of LY294002 can be partially rescued by exogenous CR-1. In summary, our data suggest that human CR-1 may function as a survival factor through a PI3K-dependent signaling pathway involving AKT and GSK-3beta.

Abstract: Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors. To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E. coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein. We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites. Expression of the modified rhCR-1 protein in E. coli was limited to the inclusion bodies. The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence. Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation. By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethylammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent. This modification facilitates solubilization of the protein when rhCR-1 is denatured. The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer. The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit beta-casein expression, to stimulate the tyrosine phosphorylation of Shc and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.

Abstract: The physiological significance of the soluble fibroblast growth factor (FGF) receptors is not clear yet although they are present in blood, vitreous fluid and in the extracellular matrix of vascular endothelial cells. A hypothesis that they might help FGF-2 release from cells is very interesting because FGF-2 does not have clear secretion signal and the mechanism of the secretion of FGF-2 is still unclear. Single overexpression of FGF-2 is related neither to the secretion potential of the molecule nor to the tumorigenicity of the cells. In this report, BALB/c 3T3 cells transformed with the full length of human FGF-2 cDNA are further transformed with the cDNA coding the extracellular domain of human FGF receptor 1. The obtained transformants co-expressing FGF-2 and soluble FGF receptor are highly tumorigenic in nude mice, while the parental cells do not show any tumorigenicity. In the conditioned medium of the double-transformants, FGF-2 is immunologically detected. These results suggest that naturally produced soluble form of FGF receptor supports the release of FGF-2 from the cells and that over-expression of these two molecules leads to induce the malignant tumours in vivo.

Abstract: The gene encoding human pancreatic ribonuclease 1 (hpRNasel) was fused with a gene encoding human epidermal growth factor (hEGF). The hybrid human protein was isolated from Escherichia coli inclusion bodies, refolded and purified to homogeneity. The fusion protein competed with 125I-hEGF for binding to hEGF receptors (EGFR) and had ribonucleolytic activities approaching those of hpRNase1. Several conformations having different enzymatic activities could be detected after reversed-phase high-performance liquid chromatographic analysis, the less hydrophobic molecules being the most active. The hybrid protein was specifically cytotoxic to A431, an EGFR overexpressing squamous carcinoma cell line, with an IC50 of approximately 10(-7) M. In contrast, recombinant hpRNase1 had an IC50 higher than 10(-4) M. A mixture of free hEGF and free hpRNasel was not more cytotoxic than hpRNasel alone and no cytotoxicity was detected in EGFR-deficient control cells. Taken together, these data suggest that this construct might be useful for targeted therapy of esophageal, lung and other squamous cell carcinomas and also breast cancers overexpressing EGFR, which correlate with a poor prognosis and cannot be cured by surgery alone. Engineering hybrid molecules with endogenous human proteins for targeted therapy may alleviate the dose-limiting immunogenicity and toxicity of conventional immunotoxins.

Abstract: Betacellulin is a member of the epidermal growth factor family and converts pancreatic AR42J cells into insulin-producing cells. This study was conducted to characterise the receptor for betacellulin in AR42J cells. AR42J cells expressed two classes of binding sites for radioactive iodine labelled betacellulin, with Kd values of 4.6 x 10(-11) mol/l and 3.0 x 10(-10) mol/l. The binding of [125I]betacellulin was inhibited by unlabelled betacellulin in a dose-dependent manner, but epidermal growth factor was 50 fold less effective than betacellulin. Affinity cross-linking showed a [125I]betacellulin-binding protein with a molecular weight of approximately 180 KDa. When this protein was immunoprecipitated with antibody against epidermal growth factor receptors ErbB-1, ErbB-2, ErbB-3 or ErbB-4, it was immunoprecipitated only by the anti-ErbB-1 antibody. When the [125I]betacellulin-labelled proteins were immunoprecipitated with a combination of the four ErbB antibodies, and the unprecipitated proteins were then immunoprecipitated with anti-phosphotyrosine antibody, a 190 KDa protein was observed. Betacellulin induced the tyrosine phosphorylation of ErbB-1, ErbB-2 and ErbB-4. Finally, while 100 pmol/1 betacellulin converted all of the AR42J into insulin-producing cells in the presence of activin A, 10 nmol/l epidermal growth factor induced differentiation in only about 30 % of the cells. Higher concentrations of epidermal growth factor were less effective. Neu differentiation factor in the presence or absence of epidermal growth factor was ineffective. These results indicate that betacellulin binds to ErbB-1 and possibly another protein with a molecular weight of 190 KDa. The latter betacellulin-binding protein may be involved in the differentiation-inducing activity of betacellulin.

Abstract: The tissue-specific expression of five human pancreatic-type RNases and RNase inhibitor was analyzed by Northern hybridization against poly(A)+ RNA prepared from 16 normal tissues. The widespread expression of RNase 1 was observed in almost all of the tissues. RNase 4 and angiogenin showed a similar distribution of expression abundantly present in the liver. This suggested the identity of the cell types producing these two molecules. However, no relativity appeared to be present between the vascularization of the tissues and the angiogenin expression. A narrow range of expression of the eosinophil-derived neurotoxin gene was observed. This localization seems related to the phagocytic cells in the tissues. The undetectable level of the eosinophil cationic protein mRNA in normal tissues suggests that the differentiation of eosinophils, triggered by inflammation and/or atopy, is required. The expression of RNase inhibitor was found to be ubiquitous. The regulatory function of inhibitor against RNases in the cell should be considered in studying the physiological significance of the pancreatic-type RNase family.

Abstract: Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor alpha, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin beta1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42(erk-2) mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Abstract: Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that does not directly activate any of the known erbB type 1 tyrosine kinase receptors. Also, CR-1 stimulates the growth of HC-11 mouse mammary epithelial cells. We found that prior treatment of HC-11 cells with exogenous CR-1 induced a competency response to the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) with respect to the induction of the milk protein beta-casein. In contrast, simultaneous treatment of mouse HC-11 cells with CR-1 in the presence of DIP inhibited beta-casein expression. The inhibitory effects of CR-1 on beta-casein expression in response to DIP were not unique to this mouse mammary epithelial cell line, because beta-casein and whey acidic protein expression in primary mouse mammary explant cultures established from midpregnant mice were also differentially inhibited by several epidermal growth factor-related peptides including CR-1. The mitogenic and differentiation effects of CR-1 are mediated by the binding of CR-1 to a cell surface receptor that is known to activate the ras/raf/mitogen-activated protein kinase (MAPK)/MAPK kinase pathway. The inhibitory response of CR-1 in HC-11 cells on beta-casein expression after treatment with DIP can be attenuated by B581, a peptidomimetic farnesyltransferase inhibitor that blocks p21ras farnesylation and activation, and by the phosphatidylinositol 3'-kinase (PI3k) inhibitor LY 294002 but not by PD 98059, a MAPK kinase inhibitor that blocks MAPK activation. These data suggest that the ability of CR-1 to block lactogenic hormone-induced expression of beta-casein is mediated through a p21ras-dependent, PI3k-mediated pathway. This is further substantiated by the observation that CR-1 is able to stimulate the tyrosine phosphorylation of the p85 PI3k regulatory subunit and to increase the activity of PI3k in HC-11 cells.

Abstract: Recombinant human ribonuclease 1 (RNase 1) was chemically linked to recombinant human epidermal growth factor (EGF). The cytotoxicity of this conjugate was assayed using MTT assay. The EGF-RNase conjugate showed dose-dependent cytotoxicity against breast and squamous cell carcinomas overexpressing the EGF receptor (EGFR). The cytotoxicity of the conjugate correlated positively with the level of EGFR expression by each cell line. These results suggest that the EGF-RNase conjugate is a more effective anticancer agent with less immunogenicity and toxicity than conventional chimeric breast cancer toxins.

Abstract: Recombinant human ribonuclease 1 (RNase 1) was chemically linked to recombinant human epidermal growth factor (EGF). The EGF-RNase conjugate showed dose-dependent cytotoxicity for EGF receptor-overexpressing A431 and TE-8 human squamous carcinoma cells with an IC50 of 2 x 10(-7)M and 10(-6)M, respectively, whereas the IC50 of RNase alone was almost 10(-4)M. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone. The conjugate showed no detectable cytotoxicity against EGF receptor-deficient small cell lung cancer cells (H69). Addition of excess EGF in the medium protected A431 cells from the EGF-RNase conjugate cytotoxicity. The cytotoxicity of the EGF-RNase conjugate was positively correlated with the EGF receptor numbers of each cell line. The chimeric toxin composed of only human proteins might be a more useful anti-cancer agent with less immunogenicity than the conventional chimeric toxins.

Abstract: Rat pancreatic AR42J cells possess exocrine and neuroendocrine properties. Activin A induces morphological changes and converts them into neuron-like cells. In activin-treated cells, mRNA for pancreatic polypeptide (PP) but not that for either insulin or glucagon was detected by reverse transcription-PCR. About 25% of the cells were stained by anti-PP antibody. When AR42J cells were incubated with betacellulin, a small portion of the cells were stained positively with antiinsulin and anti-PP antibodies. The effect of betacellulin was dose dependent, being maximal at 2 nM. Approximately 4% of the cells became insulin positive at this concentration, and mRNAs for insulin and PP were detected. When AR42J cells were incubated with a combination of betacellulin and activin A, approximately 10% of the cells became insulin positive. Morphologically, the insulin-positive cells were composed of two types of cells: neuron-like and round-shaped cells. Immunoreactive PP was found in the latter type of cells. The mRNAs for insulin, PP, glucose transporter 2, and glucokinase, but not glucagon, were detected. Depolarizing concentration of potassium, tolbutamide, carbachol, and glucagon-like peptide-1 stimulated the release of immunoreactive insulin. These results indicate that betacellulin and activin A convert amylase-secreting AR42J cells into cells secreting insulin. AR42J cells provide a model system to study the formation of pancreatic endocrine cells.

Abstract: Betacellulin (BTC) was found to be expressed mainly in human pancreas and small intestine. This finding suggests that BTC possesses some specific function distinguished from the other members of epidermal growth factor (EGF) family. To clarify this function, the released form of human BTC has been expressed in E.coli, purified, and characterized. The recombinant human BTC was produced as an inclusion body. This material was dissolved in guanidine-HCl under reducing conditions, refolded, and purified through sequential liquid chromatography. Purified BTC was electrophoresed under reducing conditions and a molecular size of 18 kDa was determined, which is the supposed size of a dimer of the peptide. However, chemical analysis failed to show a covalently linked dimer. The molecular mass of BTC analyzed by mass spectrometry revealed it to be 9 kDa, which is consistent with theoretical value for a monomer. Recombinant BTC showed growth promoting activity for mouse fibroblasts and rat aortic smooth muscle cells which was equivalent to EGF On the other hand, BTC was found to exhibit a growth inhibitory effect on the cells overexpressing EGF receptor.

Abstract: A cDNA coding for human ribonuclease 4 was isolated from a pancreas cDNA library and sequenced. This cDNA (996 bp) includes an entire open reading frame encoding mature protein (119 aa) following signal peptide (28 aa). Expression of mature protein in Escherichia coli showed an apparent molecular mass of about 16 kDa, which was slightly lower than the mature form of human RNase 1, in SDS-PAGE.

Abstract: Human pancreatic ribonuclease 1 (hRNase 1) in the mature form has been produced in E. coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3',5'-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase 1 to the characteristics of the protein.

Abstract: A novel S-alkylating reagent, N-(3-bromopropyl)-N,N,N',N',N'-pentamethyl-1,3-propanedi(ammonium bromide) (TAP2-Br) which carries two positive charges in the molecule, was prepared to increase the solubility or to decrease the hydrophobicity of cysteine-containing denatured proteins (or peptides). S-Alkylation with TAP2-Br introduces two positive charges per cysteine residue, which will effectively shift the net charge of a protein in the positive direction. Disulfide-containing proteins, such as hen egg-white lysozyme, RNase A, BSA, and soybean trypsin inhibitor (Kunitz type), were reduced and S-alkylated with TAP2-Br to evaluate the potential of this reagent compared with other S-alkylating reagents such as monoiodoacetic acid, bromosuccinic acid and (3-bromopropyl)trimethylammonium bromide. The solubilities of these denatured proteins in the pH range of 2-10 indicated that S-alkylation with TAP2-Br effectively solubilized not only basic proteins (lysozyme and RNase) but also an acidic protein containing a fairly large number of cysteine residues (BSA). Moreover, the retentions of cysteine-containing tryptic peptides derived from lysozyme on reversed-phase HPLC were greatly reduced by S-alkylation with TAP2-Br. These results indicate that TAP2-Br is very useful to increase the solubility of some cysteine-containing denatured proteins and to decrease the hydrophobicity of peptides containing cysteine residue(s).

Abstract: A cDNA coding for human pancreatic ribonuclease was isolated from a pancreas cDNA library and sequenced. This cDNA (1620 bp) includes an entire open reading frame encoding mature protein (128 aa) following a signal peptide (28 aa) as well as 5'- and 3'-untranslated regions.

Abstract: Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibroblast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four MAbs designated AF1-52, 81, 114 and 1C10 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AF1-52, 114, and 1C10 MAbs recognize epitopes within the 1-5, 44-132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF1-81 MAb could not be determined. The sandwich EIA method constructed with these MAbs was sensitive to 1.5 pg/well of haFGF and had no cross-reactivity with human basic FGF, bovine aFGF or the hst-1 gene product.

Abstract:

Abstract: A sensitive sandwich enzyme immunoassay for human basic fibroblast growth factor (HbFGF) was developed employing three monoclonal antibodies (MAb3H3, MAb98 and MAb52). The Fab' fragment of MAb3H3 which inhibits HbFGF biological activity was conjugated to horseradish peroxidase. A mixture of MAb52 and MAb98 was used in the solid phase. Neither human acidic fibroblast growth factor, hst-1/KS3 product nor acid denatured HbFGF was cross-reactive in this assay system. The detection limit of this assay system was 1 pg/well. Using this assay, some tumor cell lines were revealed to produce a higher level of bFGF than a normal one. Serum samples from normal volunteers were also assayed, and immuno-reactive HbFGF could be detected in 16 out of 57 samples at range 30 approximately 206 pg/ml.

Abstract: Two types of cDNAs encoding novel human FGF receptors were isolated. These two cDNAs were found to be closely related to the oncogene bek. Products from these genes were membrane-bound when their cDNAs were transiently expressed in COS cells, whereas products from the regions coding extracellular domains were free of membrane attachment and found in the culture medium.

Abstract: A murine monoclonal antibody (M31-15) was identified using the penetration-inhibiting assay of a human lung adenocarcinoma cell line (MAC10) and remarkably inhibited the phagokinetic tract motility of various cancer cell lines. The antigen, motility-related protein (MRP-1), recognized by M31-15, was 25- and 28-kD proteins, and M31-15 was used to isolate a cDNA clone from a human breast carcinoma cDNA library. Sequence analysis revealed that MRP-1 had strong similarity with a B cell surface antigen (CD37), a melanoma-associated antigen (ME491), the target of an antiproliferative antibody (TAPA-1), a human tumor-associated antigen (CO-029), and the Sm23 antigen of the trematode parasite Schistosoma mansoni.

Abstract: The carboxyl-terminal sequence of basic fibroblast growth factor (bFGF) is rich in basic amino acid residues, a common characteristic amongst fibroblast growth factors, and is considered to contribute greatly to the binding to negatively charged extracellular matrixes such as heparin. To study the relationship between the affinity for heparin and the carboxyl-terminal structure of bFGF, amino- or carboxyl-terminal truncated molecules were produced in Escherichia coli using recombinant DNA techniques. These terminally truncated bFGFs were applied to a heparin-affinity HPLC column. Truncation of more than six amino acid residues from the carboxyl-terminal made the bFGF produced in E. coli markedly difficult to solubilize and weakened its affinity for heparin, though bFGF having up to 46 amino acids removed showed significant stimulation of the DNA synthesis of BALB/c3T3 cells. This stimulation of the DNA synthesis was also recognized by the bFGF having 40 amino acids removed from its amino-terminal, while the affinity of this peptide for heparin has been shown to be equal to that of the mature bFGF (146 amino acids). These results show that the affinity of bFGF for heparin depends significantly on its carboxyl-terminal structure and that the essential part for receptor binding is present between Asp41 and Ser100. Moreover, it suggests that the Phe139Leu140Pro141, present in all members of the FGF family, contributes greatly to the stable structure of the intact molecule.

Abstract: Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.

Abstract: Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF. Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b. All these MAbs bound the complete form of hbFGF produced in E.coli. Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E. coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs. MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF. MAb52 and MAb98 recognized the one located between the amino acid residue no. 14 and 40. None of MAbs bound bovine acidic FGF (aFGF). Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed. This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF. Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column.

Abstract: Hepatitis B virus core antigen (HBcAg) gene was deleted at some unique restriction enzyme sites, or at random, and inserted into the expression plasmids of E. coli which had the tryptophan promoter. E. coli transformants with the plasmids, synthesized materials with many kinds of antigenicity of HBcAg, HBeAg, or both HBcAg and HBeAg. HBeAg-specific material smaller than native HBeAg was produced in a stable condition.

Abstract: Using site directed mutagenesis, each of the four cysteines present at amino acid residues 26, 70, 88, and 93 of the mature protein of human basic fibroblast growth factor (bFGF) was individually changed to serine. The biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the bFGF protein. This finding indicates that the cysteines at these positions are not essential for expressing biological activity. The substitution of the residues at these positions, especially at position 88, reduced the heterogeneity recognized as several peaks of bFGF eluted from a heparin affinity column, even after oxidation with hydrogen peroxide, suggesting that the cysteines at these positions are exposed to the surface of the molecule to form disulfide bonds that induce heterologous conformations. Furthermore, under acidic conditions, these modified bFGFs are revealed to be more stable in maintaining their activity. These facts suggest that this protein has been successfully modified by protein engineering.

Abstract:

Abstract: The cDNA encoding human basic fibroblast growth factor was expressed in E. coli under the control of trp promoter. Bacterially synthesized hbFGF was highly purified using a heparin affinity HPLC column. By this chromatography, hbFGF was eluted as four distinct forms, which were indistinguishable by SDS polyacrylamide gel electrophoresis, amino acid composition, and partial terminal sequence analysis. These molecules stimulated the growth of fibroblasts and endothelial cells although their specific activities varied. The angiogenesis activity of these molecules was also confirmed.

Abstract: The complementary DNAs encoding human interferon-gamma (IFN-gamma) and human interleukin-2 (IL-2), two different proteins involved in the same immune system, were fused to code a hybrid protein, which was expressed in E. coli to investigate the interactions of these two proteins at the molecular level. Through immunoprecipitation analysis, this protein was revealed to be of about 31 kDa, which was expected from nucleotide sequencing, and to have the antigenicities of both IFN-gamma and IL-2. The extract from bacteria expressing this hybrid protein showed at least two biological activities: an antiviral activity derived from IFN-gamma and the ability to support the growth of natural killer (NK) cells derived from IL-2. Comparing the enhancement of NK cell activity of this hybrid protein with IFN-gamma and IL-2, this hybrid protein appears to conserve each activity almost completely without diminishing the other.

Abstract: DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2.

Abstract: Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed. These epsilon chain peptides were synthesized in E. coli under the control of the trp promoter-operator. The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids.