Hirotoshi Hayasawa

p56dok-2 as a cytokine-inducible inhibitor of cell proliferation and signal transduction

p56dok‐2 acts as a multiple docking protein downstream of receptor or non‐receptor tyrosine kinases. However, the role of p56dok‐2 in biological functions of cells is not clear. We found that transcription of the p56dok‐2 gene in macrophages was increased markedly in response to cytokines such as macrophage colony‐stimulating factor (M‐CSF), granulocyte/macrophage‐CSF and interleukin‐3 (IL‐3). Forced expression of p56dok–2 inhibited M‐CSF‐, granulocyte‐CSF‐, IL‐3‐ and stem cell factor‐induced proliferation of myeloid leukemia cells, M‐NFS‐60. The p56dok‐2‐overexpressing cells showed an impaired induction of c‐myc but not of c‐jun, junB or c‐fos when stimulated with M‐CSF. Consistent with these results, the peritoneal cavity of the hairless (hr/hr) strain of mutant mice, whose cells expressed less p56dok‐2 than wild‐type mice, contained more macrophages than that of +/hr mice. Moreover, the inhibition of endogenous p56dok‐2 expression in macrophage‐like tumor cells, J774A.1, by stable expression of antisense p56dok‐2 mRNA accelerated cell proliferation. The study identifies a novel role for p56dok‐2 as a molecule that negatively regulates signal transduction and cell proliferation mediated by cytokines in a feedback loop.

New Biological Function of Bovine α-Lactalbumin : Protective Effect against Ethanol- and Stress-induced Gastric Mucosal Injury in Rats

Although several studies have shown that milk protein components have a wide range of biological activities, the potential role of these proteins in the gastrointestinal mucosal defense system is less well elucidated. In this study, we investigated the effect of the major proteins in cow’s milk on gastric mucosal injury by using two acute ulcer models in Wistar rats. Gastric mucosal injury was induced by either intragastric 60% ethanol-HCl or water-immersion restraint stress (23°C, 7 h). Each test milk protein was orally administered 30 min before the induction of gastric injury. Among the major milk proteins, α-lactalbumin (α-LA) is demonstrated to have a marked protective effect against ethanol-induced gastric injury, with the same potency as that of the typical antiulcer agent, Selbex. Whey protein isolate (WPI), which contained 25% α-LA, also protected against gastric injury, while casein showed no effect. Comparative studies on the protective effect of the four major components of WPI, β-lactoglobulin, α-LA, bovine serum albumin and γ-globulins (immunoglobulins), on the basis of their contents in WPI revealed that α-LA was responsible for the protective effect of WPI, being about 4-fold more effective than WPI itself. α-LA showed dose-dependent protection against gastric injury induced by stress as well as ethanol. Pretreatment with indomethacin (10 mg/kg body weight, s.c.), which is a potent inhibitor of endogenous prostaglandin synthesis, resulted in a significant reduction in the protective effect of α-LA. These results indicate that α-LA has marked antiulcer activity as an active component of cow’s milk protein, and suggest that α-LA intake may serve to protect against gastric mucosal injury, in part through endogenous prostaglandin synthesis.

Molecular cloning of a novel immunoglobulin superfamily gene preferentially expressed by brain and testis.

We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system.

Inhibition of Escherichia coli respiratory enzymes by the lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system.

Aims: The lactoperoxidase‐hydrogen peroxide‐thiocyanate antimicrobial system (LPAS) is known to inhibit bacterial respiration. In the present study, several respiratory enzymes of Escherichia coli were compared in terms of their susceptibility to the LPAS.

A new apoptotic pathway for the complement factor B-derived fragment Bb.

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis‐inducing factor from HL‐60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B‐derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti‐Bb antibody inhibited Bb‐induced apoptosis, and not the anti‐Ba antibody. The apoptosis‐inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B‐induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B‐derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage. J. Cell. Physiol. 185:280–292, 2000.

Effects of Bovine α-Lactalbumin on Gastric Defense Mechanisms in Naive Rats

We recently investigated the effects of the major proteins in cows milk on gastric mucosal injuries in rat ulcer models. We found that α-lactalbumin (α-LA) has marked preventive effects against gastric mucosal injuries and that prostaglandin (PG) synthesis may contribute to these effects [Matsumoto et al., Biosci. Biotechnol. Biochem., 65, 1104-1111, 2001]. In this study, we investigated the effects of α-LA on several defense mechanisms of gastric mucosa by evaluating gastric PGE2 content, gastric mucin content, gastric luminal pH, gastric fluid volume, and gastric emptying in naive rats. Oral administration of α-LA (200, 500, and 1000 mg/kg) elevated endogenous PGE2 levels in gastric tissue and increased the gastric mucin contents of both the gastric fluid and the adherent mucus gel layer. In addition to these PG-related responses, α-LA also caused PG-independent responses such as elevation of gastric luminal pH, increase in gastric fluid volume, and delay in gastric emptying. These responses were observed to be dose-dependent (200-1000 mg/kg of α-LA). Thus, we demonstrated that α-LA enhances both PG-dependent and PG-independent gastric defense mechanisms in naive rats. Both of these mechanisms are probably involved in its gastroprotective action.

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma‐initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony‐stimulating factor (M‐CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma‐inducing activity of a number of cytokines. Neither granulocyte‐CSF, stem cell factor, interleukin (IL)‐1, IL‐6, transforming growth factor, epidermal growth factor, insulin‐like growth factor, platelet‐derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma‐inducing activity, but the factor inhibited the stromal colony formation induced by M‐CSF. In this study, we found that granulocyte/macrophage‐CSF (GM‐CSF) and IL‐3, as well as M‐CSF had the stroma‐inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma‐inducing activity of M‐CSF, GM‐CSF and IL‐3 was observed even if lineage‐negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM‐CSF and IL‐3 as well as M‐CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells. J. Cell. Physiol. 184:351–355, 2000.

Expression of Jagged1 gene in macrophages and its regulation by hematopoietic growth factors

OBJECTIVE Serrate/Jagged and Delta are cell surface ligands for Notch receptors that may influence hematopoietic cell fate decisions and are known to be expressed in bone marrow stromal cells. In a series of screenings of cDNAs constructed by a cDNA library subtraction technique, we identified Jagged1, one of the Notch ligands, as a gene up-regulated by macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Therefore, we compared stromal cells and macrophages for expression of Notch ligands including Jagged1 and analyzed the regulation of their expression by cytokines. MATERIALS AND METHODS Murine bone marrow macrophages were prepared by culturing femoral bone marrow cells with M-CSF. Primary bone marrow fibroblastic stromal cells were prepared by a culture system that we recently developed. The expression of Notch ligands was analyzed by either Northern blot analysis or reverse transcriptase polymerase chain reaction. RESULTS The bone marrow macrophages expressed Jagged1 but not Jagged2 and Delta1 at a level that was detectable by Northern blot analysis. Expression of the Jagged1 gene was markedly up-regulated by growth factors for the cells, i.e., M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. Expression of Jagged2 and Delta1 seldom was affected by the stimuli. The primary bone marrow fibroblastic stromal cells, and murine stromal cell lines, such as PA6 and ST2, also expressed Jagged1 transcript, at levels comparable to the steady-state level in macrophages. However, expression of the Jagged1 gene was little affected when these cells were stimulated with fibroblastic growth factor and platelet-derived growth factor. CONCLUSIONS We demonstrated that bone marrow macrophages as well as stromal cells constitutively produced Jagged1 and that the expression was markedly up-regulated by hematopoietic growth factors, M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. The results highlight the involvement of macrophages and these growth factors in hematopoietic cell fate decisions via the production of Jagged1.

LACTOFERRIN AS A SUPPRESSOR OF CELL MIGRATION OF GASTROINTESTINAL CELL LINES

The effects of lactoferrin (Lf), an iron‐binding glycoprotein, on cell migration were investigated. Lf inhibited the cell migration of three gastrointestinal cell lines (Caco‐2 cells, AGS cells, and IEC‐18 cells) in vitro. Both iron‐saturated (holo) and iron‐depleted (apo) Lf showed this inhibitory effect. Chelation of iron in the culture medium by desferrioxamine did not affect the activity of either form of Lf. A pepsin hydrolysate of Lf exhibited effectiveness similar to that of intact Lf. These results demonstrate a novel activity of Lf and suggest a potential role for this molecule in gastrointestinal wound healing, which is independent of its iron‐binding capacity. J. Cell. Physiol. 170:101–105, 1997.

The Mechanism of in Vivo Bacteriostasis of Bovine Lactoferrin

Recently we have reported that orally administered bovine Lf(bLf) exerts bacteriostatic effects against bacterial overgrowth in the intestine of specific-pathogen-free (SPF) mice fed milk. In this animal model, the in vivo bacteriostatic effect of bLf against the proliferation of intestinal Enterobacteriaceae, the bacteria most sensitive to bLf, was independent of the iron-chelating ability of bLf. In addition various proteolytic hydrolysates of bLf (with differing antibacterial activities in vitro) showed the same bacteriostatic effect as undigested bLf. These results suggest that the mechanism of in vivo bacteriostasis of Lf differs from the in vitro mechanism reported. In SPF mice fed milk differing in concentrations of lactose, glucose and galactose, the proliferation of intestinal Enterobacteriaceae was dependent on the carbohydrate concentration in the diet. The addition of 2% bLf to the diets significantly suppressed this carbohydrate-dependent proliferation of bacteria except in the case of diets containing excess carbohydrate. In germ-free mice fed sterile milk, the addition of 2% bLf to milk resulted in a significant decrease in concentrations of lactose, glucose and galactose in the cecal contents. In an in vitro assay system using everted sacs of the small intestine of SPF mice, both bLf and its pepsin hydrolysate apparently stimulated glucose absorption. Based on these findings, we propose that the in vivo mechanism of action of ingested bLf involves the stimulation of carbohydrate absorption resulting in a bacteriostatic effect against Enterobacteriaceae in the intestine of mice fed milk.