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The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Staphylococcal α-hemolysin (Hla) is a principal small β-barrel pore forming toxin. It targets a variety of mammalian cells including immune cells; however little is known about its effects on mast cells. In this study, we examined whether Hla affects the degranulation of mast cells. Although Hla bound to the surface of bone marrow-derived mast cells (BMMCs) and formed SDS-stable oligomers on the cells, Hla alone induced neither cytotoxicity nor obvious release of a granule enzyme, β-hexosaminidase. However, Hla more than doubled the releases of β-hexosaminidase from BMMCs induced by FcεRI cross-linking or treatment with ionomycin. The augmentation of the enzyme release by rHla was impaired in the presence of 130 mM of extracellular KCl. The mutants of Hla that lacked pore-formation did not augment the release of the enzyme. These findings demonstrate that Hla is able to enhance the degranulation of mast cells induced by FcεRI cross-linking and ionomycin, although it alone does not induce the degranulation, and the pore-formation of Hla followed by potassium efflux is involved in the augmentation. These findings propose a previously unrecognized role for Hla in S. aureus-associated allergic and inflammatory processes via augmentation of mast cell responses., (Copyright © 2018. Published by Elsevier Inc.)

We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, β1-3 region ( 25 SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK 60 ) in N-terminal half and α4β9 region ( 138 KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD 170 ) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved β1-3 but not α4β9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted β1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored β1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the β1-3 region is responsible for the inhibition of proteolytic activity of MMP-9., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulation factors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulation factors via their γ-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulation factors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulation factors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10β1-β3 ( 23 MEMKN ISALK HGKNN LRFKF RGIKI QVL 60 ) bound to immobilized prothrombin, and mutants that contained SSL10β10-β12 ( 174 SFYNL DLRSK LKFKY MGEVI ESKQI KDIEV NLK 207 ) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to prothrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Staphylococcal superantigen-like protein (SSL), a family of exotoxins composed of 14 SSLs, exhibits no superantigenic activity despite of its structural similarity with superantigens. Several SSLs have been revealed to bind to host immune molecules such as IgA, IgG, complement and cell surface molecules expressed on immune cells, but the physiological function of SSL family has not been fully identified. In this study we attempted to isolate host target proteins of SSLs from human breast milk using SSLs-conjugated Sepharose. SSL8-conjugated Sepharose specifically recovered tenascin C (TNC), a multimodular and multifunctional extracellular matrix protein. Pull down analysis using SSL8-conjugated Sepharose and recombinant truncated fragments of TNC revealed that SSL8 interacts with fibronectin (FN) type III repeats 1-5 of TNC. The interaction of TNC with immobilized FN was attenuated, the scratch wound closure by HaCaT human keratinocytes was delayed and the inhibition of cell spreading on FN by TNC was recovered in the presence of SSL8. These findings suggest that SSL8 binds to TNC, thereby inhibits the TNC-FN interaction and motility of keratinocytes. The present study added a novel role of SSL family protein as an interrupting molecule against the function of extracellular matrix., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0Å. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the number and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Glycosylation is a widespread post-translational modification found in glycoproteins. Glycans play key roles in protein folding, quality control in the endoplasmic reticulum (ER) and protein trafficking within cells. However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles. Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present on inducible costimulator (ICOS), a T-cell costimulatory molecule, in proper protein folding and intracellular trafficking to the cell surface membrane. We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89), but not proteins lacking either N23 or N110, were retained within the cell and were not detected on the cell surface membrane. Additional evidence suggested that N89 glycosylation was indirectly involved in ICOS ligand binding. These data suggest that amongst the three putative ICOS glycosylation sites, N89 is required for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins., (Copyright 2009 Elsevier Inc. All rights reserved.)

To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor alpha ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity. The results of our in vitro simulation imply that alternative splicing may have contributed substantially to the diversification of protein function during evolution.

Long bone abnormality (lbab/lbab) is a spontaneous mutant mouse characterized by dwarfism with shorter long bones. A missense mutation was reported in the Nppc gene, which encodes C-type natriuretic peptide (CNP), but it has not been confirmed whether this mutation is responsible for the dwarf phenotype. To verify that the mutation causes the dwarfism of lbab/lbab mice, we first investigated the effect of CNP in lbab/lbab mice. By transgenic rescue with chondrocyte-specific expression of CNP, the dwarf phenotype in lbab/lbab mice was completely compensated. Next, we revealed that CNP derived from the lbab allele retained only slight activity to induce cGMP production through its receptor. Histological analysis showed that both proliferative and hypertrophic zones of chondrocytes in the growth plate of lbab/lbab mice were markedly reduced. Our results demonstrate that lbab/lbab mice have a hypomorphic mutation in the Nppc gene that is responsible for dwarfism caused by impaired endochondral ossification.

In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.

We investigated the effects of both cytokines and extracellular matrices on the proliferation and differentiation of immature adult rat incisor dental pulp cells. These immature cells, which have a high-proliferative potency in vitro and do not express mRNAs for dentin non-collagenous proteins such as dentin sialoprotein (DSP), bone sialoprotein (BSP), and osteocalcin, exist in the root regions of adult rat incisors. Fibroblast growth factor-2 (FGF-2) stimulated the proliferation of these immature cells and the subsequent production of mineralized calcium was induced by beta-glycerophosphate treatment. Additionally, FGF-2 dramatically induced the expression of DSP and BSP mRNAs, but only in collagen type I gel cultures, whereas neither plate-coated collagen type I nor fibronectin, laminin or collagen type IV cultures could produce this effect and generate sufficient physiological levels of these transcripts. Although bone morphogenetic protein-4 could not induce the proliferation of immature dental pulp cells nor upregulate DSP mRNA expression, it had a synergistic effect upon DSP transcript levels in conjunction with FGF-2. These results suggest that both the presence of FGF-2 and the three-dimensional formation of immature dental pulp cells in collagen type I gel cultures are essential for both DSP expression and odontoblast differentiation. These observations provide valuable information concerning the study of the commitment and differentiation of odontoblast lineages, and also provide a basis for the rational design of cytokine and extracellular matrix based compounds for regenerative therapies in new dental treatments.

The production of eotaxin, which is a critical mediator for airway inflammation, is inhibited by IFN-gamma. Here, we investigated the precise mechanisms underlying IFN-gamma-dependent inhibition of eotaxin production using mouse embryonic fibroblasts (MEF). MEF produced high levels of eotaxin in STAT6-dependent manner when they were cultured with both IL-4 and TNF-alpha. However, the eotaxin production by MEF was strongly inhibited by addition of IFN-gamma. Western-blotting analysis demonstrated that IFN-gamma downmodulated STAT6 phosphorylation induced by IL-4 and TNF-alpha. Moreover, IFN-gamma did not exhibit its inhibitory effect on both STAT6-phosphorylation and eotaxin production in MEF obtained from deficient mice in STAT1, a key molecule of IFN-gamma signaling. We also demonstrated that SOCS-1, a potent inhibitory molecule of IL-4 signaling, was induced by IFN-gamma in STAT1-dependent manner. This indicated that SOCS-1 might be involved in IFN-gamma-mediated STAT1-dependent inhibition of eotaxin production. In SOCS-1(-/-) MEF, IFN-gamma inhibited neither STAT6 phosphorylation nor eotaxin production induced by IL-4 and TNF-alpha. Conversely, retroviral transduction of SOCS-1 into MEF inhibited STAT6 phosphorylation and eotaxin production induced by IL-4 and TNF-alpha, in the absence of IFN-gamma. Thus, we demonstrated that IFN-gamma-induced inhibition of STAT6 phosphorylation and eotaxin production were mediated by SOCS-1 induced in STAT1-dependent manner.

P-selectin, a carbohydrate-binding cell adhesion molecule expressed on activated endothelial cells and platelets, plays a key role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. It simultaneously recognizes a sialic acid-containing carbohydrate chain and the sulfated tyrosine residues of a specific counter-receptor expressed on the leukocyte surface. We examined the inhibitory effects of a synthetic sulfated derivative of sialic acid (NMSO3) on P-selectin-mediated cell adhesion and found the following: (1) P-selectin/IgG chimera bound to immobilized NMSO3. (2) The binding of P-selectin/IgG chimera to purified P-selectin glycoprotein ligand-1 was inhibited by soluble NMSO3. (3) The adhesion of HL60 cells to P-selectin-expressing CHO cells was inhibited by NMSO3. (4) NMSO3 inhibited P-selectin-induced tumor necrosis factor-alpha production in monocytes and activated platelet-induced generation of reactive oxygen species in neutrophils. In conclusion, NMSO3 acts as a specific inhibitor for P-selectin-mediated cell adhesion and for adhesion-dependent leukocyte activation.

Both AILIM/ICOS and CD28 provide positive costimulatory signals for T-cell activation, resulting in proliferation and cytokine production. In this study, we attempted to clarify the key signaling molecules in T-cell proliferation, and also IL-2 and IL-10 production, during T-cell activation by CD3 induced by costimulation with either AILIM/ICOS or CD28. We examined the role of both the PI3-kinase/Akt pathway and MAP kinase family members such as ERK1/2, JNK, and p38 kinase in this process. PI3-kinase and Erk1/2 were shown to potentially regulate primary T-cell activation and subsequent proliferation via both AILIM/ICOS- or CD28-mediated costimulation and the Erk signaling cascade was essential for this proliferation induction and also for IL-2 production. The JAK inhibitor, AG490, inhibited this induction. Our studies indicate that IL-2 is necessary for induction of T-cell proliferation and that the quantities of IL-2 produced by AILIM/ICOS ligation are also sufficient for T-cells to proliferate. In contrast, inhibition of Akt and p38, that are phosphorylated by both AILIM/ICOS and CD28-ligation, could downregulate IL-10 production but not T-cell proliferation. These data raise the interesting possibility that the signaling cascades between T-cell proliferation and IL-10 production are regulated by different molecules in AILIM/ICOS- and CD28-costimulated T-cells.

The administration of branched-chain amino acids (BCAAs) to cirrhosis patients increases serum albumin levels and improves the blood Fischer's ratio. Although it has been reported that albumin synthesis in rat primary hepatocytes is diminished under lower Fisher's ratio conditions compared to normal Fischer's ratio conditions, the mode of action at the molecular level for these effects is still uncertain. It has been reported recently that the triggering signal for protein synthesis is transmitted through mTOR (mammalian target of rapamycin). We have had an interest in the mTOR signal transduction system. In the present study, we analyzed the mode of action of BCAA-induced albumin synthesis using rat primary hepatocytes. The BCAA mixture dose-dependently promoted the production of albumin, with leucine being the major effector half of which was inhibited by the mTOR inhibitor rapamycin. We also showed that only leucine induces P70 S6 kinase activation and 4E-BP1 phosphorylation which are mTOR's downstream translational effectors. These activations were completely inhibited by rapamycin. Our results suggest that BCAAs, especially leucine, promote the production of albumin in rat primary hepatocytes through an mTOR signal transduction system.

Angiogenin is a potent angiogenic protein whose inhibition is known to prevent human tumor growth in athymic mice. It is secreted by both tumor and normal cells; and interacts with endothelial and smooth muscle cells to induce a wide range of cellular responses including cell migration and invasion, proliferation, and formation of tubular structures. Angiogenin is rapidly endocytosed and translocated to the cell nucleus where it accumulates in the nucleolus and binds to DNA. Although nuclear translocation is necessary for its angiogenic activity, the nuclear function of angiogenin is unclear. Here we report that exogenous angiogenin enhances the production of 45S rRNA in endothelial cells, and reduction of endogenous angiogenin inhibits its transcription. In a nuclear run-on assay, angiogenin stimulates RNA synthesis including that containing the initiation site sequences of 45S rRNA. This suggests that the nuclear function of angiogenin relates to its capacity to induce rRNA synthesis. Because rRNA transcription is essential for the synthesis of new ribosomes that are necessary for protein translation and cell growth, inhibition of angiogenin-stimulated transcription of rRNA may inhibit angiogenesis and therefore, would serve as a molecular target for therapeutic intervention., ((c) 2002 Elsevier Science (USA).)

We recently reported the cloning of the REIC/Dkk-3 gene, whose expression was shown to be down-regulated in many human immortalized and tumor-derived cell lines [T. Tsuji et al. (2000) Biochem. Biophys. Res. Commun. 268, 20-24]. In the present study, we demonstrated that expression of the exogenous REIC/Dkk-3 gene in tumor cells inhibited cell growth. Furthermore, the level of REIC/Dkk-3 mRNA in normal human cells was lowest in the late G(1) phase during the cell cycle. Then we found that the expression of REIC/Dkk-3 was significantly down-regulated in surgically resected non-small-cell lung carcinomas. We determined the REIC/Dkk-3 locus on chromosome 11p15, where loss of heterozygosity has frequently been observed in human tumors. These findings indicate that REIC/Dkk-3 may function as a tumor suppressor., (Copyright 2001 Academic Press.)

Cardiotrophin-1 (CT-1) is a new member of the interleukin (IL)-6 family of cytokines and one of the endogenous ligands for gp130 signaling pathways in the heart, which has potent hypertrophic and survival effects on cardiac myocytes. However, the clinical significance of CT-1 is poorly understood, mainly because there is no widely applicable specific and sensitive assay system for measuring plasma levels of circulating CT-1. We therefore developed a competitive radioimmunoassay (RIA) for human CT-1 with rabbit antiserum recognizing the N-terminus region of human CT-1 and using recombinant human CT-1 as a calibrator. The assay displays no cross-reactivities with any of the IL-6 family of cytokines including IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. The lower detection limit in buffer was found to be 43 fmol/ml, and the working range was 120-8300 fmol/ml (CV < 15%). This RIA directly recognizes CT-1-like immunoreactivity in human plasma with a mean value of 571 +/- 75 fmol/ml (mean +/- SD) in healthy volunteers. The RIA coupled with gel filtration chromatographic analyses showed that the major molecular form of circulating CT-1 corresponds to recombinant full-length human CT-1. Moreover, there is a significant increase in the plasma CT-1 concentration from the aorta and coronary sinus, which clearly indicates that the heart secretes CT-1 via the coronary sinus into the peripheral circulation. This RIA should serve as a powerful tool for investigating the clinical significance of CT-1., (Copyright 2000 Academic Press.)

Bone marrow (BM) cells originally include alpha-fetoprotein (AFP)- and c-Met [a receptor for hepatocyte growth factor (HGF)]-expressing cells. In vitro treatment of BM cells with HGF induced albumin-expressing hepatocyte-like cells. Furthermore, those hepatocyte-like cells expressed cytokeratins 8 and 18, which are typically expressed in normal adult hepatocytes. These findings demonstrate that BM cells include AFP-expressing hepatic progenitor cells that can be differentiated into hepatocytes by HGF in culture, indicating that such cultures are useful resources for cell transplantation therapy for liver diseases., (Copyright 2000 Academic Press.)

To improve the efficacy of interferon (IFN) therapy for chronic hepatitis C, we proposed a therapy with twice-a-day injection of IFNbeta as the induction. To assess its biological enhancement, we compared antiviral activities in vitro using intermittent treatment schedules simulating the clinical condition. FL cells were treated with IFNbeta twice in 12 h interval (Single treatment, 1000 and 0 IU/ml; Double treatment, 500 IU/ml each) and challenged with Sindbis virus. Antiviral activities were determined with 50% cytopathic effect. Activities and mRNA expressions of 2'5'oligoadenylate synthetase (2'5'AS) were also examined. Single treatment showed its peak activity at 9 h, while Double treatment was at 3 h after the second treatment. Double treatment had a significantly higher peak activity. The up-regulated activities of 2'5'AS lasted much longer with Double treatment. The present findings demonstrated Double treatment could induce efficient biological enhancement, which is thought based on the priming effect., (Copyright 2000 Academic Press.)

Connective tissue growth factor (CTGF) is up-regulated by TGF-beta1 during wound healing. The present study examined the expression of CTGF during regeneration after 70% partial hepatectomy (PH) or d-galactosamine (GalN)-injured liver in rats. CTGF, TGF-beta1, and type I collagen mRNAs were semiquantified by a ribonuclease protection assay. After PH, TGF-beta1 and type I collagen were increased at 2-6 h and at 12-48 h. CTGF increased at 6 h and returned to the control level thereafter. The ribonuclease protection assay of cultured hepatic stellate cells (HSC) and in situ hybridization suggest that the cells express CTGF along sinusoid might be HSCs. After GalN administration, CTGF increased at 2-96 h with a shoulder peak at 6-12 h followed by a main peak at 24 h. TGF-beta1 and type I collagen were up-regulated with kinetics similar to those of CTGF. The different kinetics between PH and GalN regenerations indicate that regulation of CTGF in the two processes is different. Higher TGF-beta1 expression after inflammatory/necrotic process in the GalN regeneration may caused the prolonged CTGF expression., (Copyright 2000 Academic Press.)

Activation-inducible lymphocyte immuno-mediatory molecule (AILIM) is an inducible cell surface glycoprotein expressed on thymocytes and activated lymphocytes. Specific monoclonal antibody to rat AILIM induced the cell aggregation of a rat thymoma cell line and ConA-activated splenocytes. In the present study, we identified the primary structure of two species of rat AILIM by expression cloning. We also cloned mouse and human AILIM homologues and the predicted amino acid sequences were identical to those of the inducible costimulator ICOS/CRP-1, which belongs to the CD28/CTLA4 family. Although the human and mouse AILIM/ICOS molecule is localized on T-cells, the major population of AILIM/ICOS-positive cells in rat splenocyte was CD45RA-positive B-cells. The expression level of AILIM/ICOS on T-cells was relatively low; however, its expression was drastically induced by the treatment with PMA plus Ca-ionophore or the engagement of CD3 and these costimulatory molecules. Almost all T-cells exhibited potency as to its expression. Functional analysis of AILIM/ICOS demonstrated that AILIM-mediated costimulation was relatively weak compared to that of human., (Copyright 2000 Academic Press.)

Normal human cells stop proliferation after a certain number of cell divisions. This phenomenon is called cellular aging. The fact that the senescence phenotype is dominant and the immortal one is recessive indicates that immortalization of human cells may be caused by loss of functions of certain genes in normal cells. Based on this evidence, several cDNA clones whose expression was down-regulated during the immortalization process of human cells were isolated by the representative difference analysis (RDA) system in our laboratory. One of them, which was named REIC, was expressed to a lower degree in three human immortalized cell lines as compared with their parental normal counterparts. In addition, the expression of REIC was markedly lower in eight human tumor-derived cell lines (Hep3B and HuH-7 hepatocellular carcinomas, HuH-6 Clone 5 hepatoblastoma, HuCCT-1 cholangiocarcinoma, A549 lung cancer, HaCaT immortalized keratinocyte, HeLa cervical carcinoma, and Saos-2 osteosarcoma). In contrast, among the human tissues examined, the heart and brain, which contain a large number of post-mitotic cells, showed the highest expression of REIC. The full-length REIC cDNA revealed that the predicted protein is 350 amino acids in length and possesses coiled-coil tertiary structures in each of the amino- and carboxyl-termini. Furthermore, a search of the protein database revealed a match of this gene product with Dkk-3, which is a novel inhibitor of Wnt oncogene. These results indicate that the REIC cloned by us may function as a tumor suppressor., (Copyright 2000 Academic Press.)

Targeting a specific DNA sequence to the desired tissues is an important step in gene therapy. The hepatitis B virus (HBV) is the only DNA virus that has hepatocyte specificity. We attempted to construct an HBV-based vector for targeting the liver. We observed the replication and secretion of virus particles in an HBV construct that lacks X gene and carries an extra 63 bp DNA fragment in vitro. Replication was observed in the cell line HuH-7 but not HepG2. From this construct, we designed an HBV-based vector that could carry foreign DNA. HBV based vectors provide for the possibilities of generating therapeutic agents for individual patients. Our host vector system may be used to clear out the HBV from the HBV carrier or chronic hepatitis B patients by introducing a genetically engineered HBV into these patients., (Copyright 1999 Academic Press.)

The tumor suppressor gene MEN1 and several oncogenes including CCND1/cyclin D1/PRAD1 map to chromosome 11q13. However, molecular and cytogenetic analysis suggests the presence of a second tumor suppressor locus at this chromosome region. We have identified a novel gene from chromosome 11q13, which encodes a protein of 126 amino acids sharing an overall 57% identity with the p12(DOC-1) protein encoded by the DOC-1 gene, the human homolog of hamster putative tumor suppressor doc-1 (deleted in oral cancer-1). We therefore designated the novel gene as DOC-1R for DOC-1-related. The cytogenetic location was confirmed by chromosome fluorescent in situ hybridization. Northern blot analysis indicated that it was expressed in all the tissues examined. DOC-1R protein showed heterogeneous subcellular localization. RT-PCR-SSCP analysis failed to detect deleterious mutations of the DOC-1R transcript in four premalignant oral keratinocyte lines and 20 different cancer cell lines from tumor types which frequently harbor LOH at chromosome 11q13., (Copyright 1999 Academic Press.)

Transforming growth factor-beta (TGF-beta) regulates cell proliferation positively or negatively. The mitoinhibition by TGF-beta has been attributed to induction of cyclin-dependent kinase (CDK) inhibitors, such as p15/ Ink4B, p27/Kip1, and p21/Waf1 also known as Cip1 and Sdi1. However, the biological process by which TGF-beta exerts the stimulatory effects on cell growth remains poorly understood. Here we report that TGF-beta 1 stimulates DNA synthesis of IMR-90 human embryonic lung fibroblasts but inhibits that of HuCCT1 human cholangiocarcinoma cells, via down- or up-regulation of p21/Waf1, respectively. TGF-beta 1 markedly suppresses IMR-90 cells to express two different kinds of the p21/Waf1 gene transcription factors, the p53 tumor suppressor and the interferon regulatory factor-1 (IRF-1). This is followed by a marked decrease in expression of p21/Waf1 in a manner consistent with the timing of activation of cyclin E-associated kinase, which normally accompanies the G1-S transition in the cell cycle. Contrarily, TGF-beta 1-induced inhibition of DNA synthesis in HuCCT1 cells is preceded by IRF-1-dependent but p53-independent up-regulation of p21/Waf1 expression followed by inactivation of cyclin E-associated kinase. Thus the cell growth stimulation or inhibition by TGF-beta 1 are mediated by the down- or up-regulation of p21/ Waf1, respectively.

The 3' terminal region (3'-X tail) of hepatitis C virus (HCV) genomic RNA forms a stable stem-loop structure. The 3'-X tail consists of 98 nucleotides (nt) that are highly conserved among the HCV strains and supposed to function as a cis-acting region for replication of negative strand RNA and/or viral encapsidation. In the present study, by UV cross-linking assay we found two kinds of cellular proteins of approximately 87 and 130 kDa, which specifically bind to the full-length 3'-X tail (nt 1 to 98), but not the 3'- or 5'-truncated 3'-X tail, consisting of nt 1 to 50 or nt 51 to 98, respectively. These proteins were detected in human cell lines such as hepatic tumor cell lines and a T-lymphocyte cell line and also in a human embryonic lung fibroblast cell strain. In addition, human hepatocellular carcinoma tissues expressed these proteins regardless of infection or uninfection of HCV. Furthermore, these proteins were also detected in normal human tissues derived from the lung, heart, kidney, stomach, intestine, and colon. Thus, these cellular proteins, which are ubiquitously present in human tissues, might be involved in viral replication and/or encapsidation., (Copyright 1998 Academic Press.)

We examined a relationship between p21WAF1/CIP1/SDI1 and cell-cycle-related proteins in 12 human liver tumor cell lines (JHH-1, -2, -4, -5, -6, -7; HLE; HuH-7; Hep3B; PLC/PRF/5; HuH-6; HepG2). Seven (JHH-1, -2, -5, -6, -7; Hep3B; HepG2) out of eight cell lines having p21WAF1/CIP1/SDI1 protein overexpressed cyclin E protein, although one of them (JHH-5) overexpressed a reduced size of cyclin E. The rest (HuH-6) of the 8 cell lines with p21WAF1/CIP1/SDI1 showed a decreased expression of cyclin E. Four cell lines (JHH-4; HLE; HuH-7; PLC/PRF/5) deficient of p21WAF1/CIP1/SDI1 protein did not overexpress cyclin E protein. As to expression of the other cell-cycle-related proteins, cyclin A, cyclin D1, CDK2 or CDK4, no significant difference was detected among the 12 cell lines. These findings indicate that the human liver tumor cell lines which have the p21WAF1/CIP1/SDI1-inducible barriers of the cell cycle progression can go through the G1/S checkpoint by overexpressing cyclin E.

Malignant rodent cells transformed by human adenovirus 12 produce a potent cell growth inhibitory factor. The cell growth inhibitory factor inhibits the growth of and DNA synthesis in normal fibroblasts in vitro. Extent of the production of the cell growth inhibitory factor appears to be proportional to that of the malignancy of the transformed cells. C57AT1-AB cells, an adenovirus 12-transformant of C57BL/6 mouse origin, are highly tumorigenic in the syngeneic and allogeneic mice. The cell growth inhibitory factor produced by these cells was characterized for the physicochemical properties; the cell growth-inhibitory activity was quantitatively recovered in the filtrates of YM-2 membrane (M(r) less than 1,000), resistant to the heat treatments at 56 degrees C for 30 min and 100 degrees C for 5 min, and extractable by ethyl acetate under acid-condition. These results suggest that the cell growth inhibitory factor may be lipid or oligopeptides.

Mouse mammary carcinoma mutant cell line FUA169, characterized by high GM3 ganglioside content, was established from parent cell line FM3A/F28-7, which has high LacCer content but no GM3. Although both cell lines showed the same quantity and quality of integrin receptors, FUA169 showed much stronger adhesion to fibronectin (FN)-coated plates than did F28-7. Liposomes containing phosphatidylcholine, cholesterol, alpha 5 beta 1, and a moderate amount of GM3 showed greatly enhanced adhesion to FN-coated plates, but adhesion of similar liposomes containing a large amount of GM3, or no GM3, was much lower. Our results suggest that GM3 regulates integrin receptor function essential for cell adhesion to FN.

Human embryonic lung fibroblasts (MRC5) produced scatter factor which enhanced motility of Madin-Darby canine kidney (MDCK) epithelial cells and a factor which stimulates DNA synthesis of adult rat hepatocytes in primary culture. These activities were both completely neutralized by antibody against human hepatocyte growth factor (HGF). Human recombinant HGF induced a marked scattering of MDCK cells. Moreover, MRC5 cells highly expressed 6kb mRNA which hybridized with HGF cDNA probe and scatter factor cDNA cloned from the MRC5 cDNA library had the same sequence as that of HGF cDNA from human leukocytes. These results indicate that HGF possesses scatter factor activity and the scatter factor derived from the MRC5 cells is probably identical to HGF.

We have examined the effect of inflammatory cytokines on the platelet activation. IL-1 beta and IFN-gamma were found to enhance the adhesion of thrombin-treated platelets to monocytic leukemia cells (U937), when the adhesion was assayed by platelet-mediated cell agglutination. The agglutination was inhibited by a monoclonal anti-GMP140 antibody or EDTA, suggesting that the enhanced platelet adhesion to the leukemic cells was mediated by GMP140. In addition, these cytokines also increased the release of 5-HT from platelets in the presence of a low concentration of thrombin. These data suggest that platelet functions are regulated by the cytokines and that activated platelets participate in inflammatory process.

Adult rat hepatocytes formed floating multicellular spheroids, when they were cultured with proteoglycan fraction isolated from rat liver reticulin fibers. Cells in the spheroid showed only low growth activity. Albumin production by the spheroids increased up to 1.5 micrograms/micrograms DNA/day (180 micrograms/mg Protein/day) during the first 6 days and remained constant thereafter. In contrast, the albumin production by the monolayer markedly decreased after 4 days. The spheroid culture appears to be more suitable than the monolayer in studying differentiated functions of adult hepatocytes.

We isolated a protein, from a cell line of human origin, which exhibits extensive differentiation inducing activity toward Friend leukemia cells. The protein, called Erythroid Differentiation Factor (EDF), was found in a 4 day culture of THP-1 cells performed in the presence of 4 beta-phorbol 12-myristate 13-acetate(PMA). EDF is a homodimer of a molecular weight of 25,000, with an NH2-terminal sequence identical to that of the beta A-chain of porcine Inhibin. It was suggested that a single protein species is responsible for the activities of both EDF and FRP, a FSH releasing protein isolated from porcine ovarian follicular fluid.

The band 3 glycoprotein from human erythrocytes was found to be phosphorylated on tyrosine residues by the purified EGF receptor kinase and the purified src kinase in vitro. Kinetic analysis revealed that Km of the band 3 protein phosphorylation by the EGF receptor kinase was 0.17 microM and 0.65 microM in the absence and presence of EGF (3 X 10(-7)M), respectively, and that in the case of the src kinase it was 0.4 microM. From these data the band 3 protein can be regarded as one of the best substrates common for the EGF receptor kinase and the src kinase in vitro.