Monocyte chemotactic protein-1 regulates leukocyte recruitment during gastric ulcer recurrence induced by tumor necrosis factor-alpha

TNF-alpha has numerous biological activities, including the induction of chemokine expression, and is involved in many gastric injuries. C-C chemokines [monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha] and C-X-C chemokines [MIP-2 and cytokine-induced neutrophil chemoattractant (CINC)-2alpha] mediate chemotaxis of monocytes and neutrophils, respectively. We examined the roles of TNF-alpha and dynamics of chemokine expression in gastric ulceration including ulcer recurrence and indomethacin-induced injury. Rats with healed chronic gastric ulcers received intraperitoneal TNF-alpha to induce ulcer recurrence. Some rats were given neutralizing antibodies against neutrophils or MCP-1 together with TNF-alpha. In a separate experiment, rats were orally administered 20 mg/kg indomethacin with or without pretreatment with pentoxifylline (an inhibitor of TNF-alpha synthesis) or anti-MCP-1 antibody. TNF-alpha (1 microg/kg) induced gastric ulcer recurrence after 48 h, which was completely prevented by anti-neutrophil antibody. TNF-alpha increased the number of macrophages and MCP-1 mRNA expression in scarred mucosa from 4 h, whereas it increased MPO activities (marker of neutrophil infiltration) and mRNA expression of MIP-2 and CINC-2alpha from 24 h. Anti-MCP-1 antibody inhibited leukocyte infiltration with reduction of the levels of C-X-C chemokines and prevented ulcer recurrence. Indomethacin treatment increased TNF-alpha/chemokine mRNA expression from 30 min and induced macroscopic erosions after 4 h. Pentoxifylline inhibited the indomethacin-induced gastric injury with reduction of neutrophil infiltration and expression of chemokine (MCP-1, MIP-2, and CINC-2alpha). Anti-MCP-1 antibody also inhibited the injury and these inflammatory responses but did not affect TNF-alpha mRNA expression. In conclusion, increased MCP-1 triggered by TNF-alpha may play a key role in gastric ulceration by regulating leukocyte recruitment and chemokine expression.     

Inhibition of sphingomyelinase activity helps to prevent neuron death caused by ischemic stress

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.     

Orally active anti-proliferation agents: novel diphenylamine derivatives as FGF-R2 autophosphorylation inhibitors

(6,7-Disubstituted-quinolin-4-yloxy-phenyl)(4-substituted-phenyl)amine derivatives were synthesized and evaluated by a cellular autophosphorylation assay for FGF-R2 in the human scirrhous gastric carcinoma cell line, OCUM-2MD3. We also performed metabolic stability studies showing that substitutions at the 7-position of quinoline affect its biological stability. In this study, we achieved a remarkable improvement in the solubility and metabolic stability of the diphenylamine derivative. The most promising compound 15e showed a significant decrease in tumor volume when orally administered.     

Inhibitory effect of water-soluble chitosan on growth of Streptococcus mutans

The present study was undertaken to evaluate the effects of pH and the degree of polymerization of chitosan on the inhibition of growth of Streptococcus mutans. Three types of chitosan, polymer, oligomer and monomer, were used at 4% (W/V) and three different levels of pH: 6.0, 6.5 and 7.4. Bactericidal activity was calculated by the growth ratio. Chitosan oligomer significantly inhibited bacterial growth at a pH value of 6.5. All three types of chitosan strongly inhibited bacterial growth at pH 6.0. Furthermore, nearly complete inhibition was obtained with 2%(W/V) chitosan solution at constant pH 6.5. This study is the first to report that water-soluble chitosan directly suppresses the growth of the typical cariogenic bacterium S. mutans even at pH 6.5, without causing demineralization of the tooth surface.     

Cathepsin L is crucial for a Th1-type immune response during Leishmania major infection

Prior to the activation of CD4+ T cells, exogenous proteins are digested by endo/lysosomal enzymes in antigen-presenting cells (APCs) to produce antigenic peptides that are presented on MHC class II molecules. In the studies described here, the functional significance of cathepsin L for antigen processing and Th1/Th2 differentiation in experimental leishmaniasis was investigated. We first demonstrated that cathepsin L is one of the candidates for endo/lysosomal enzymes in the processing of soluble Leishmania antigen (SLA) by using CLIK148, a specific inhibitor of cathepsin L. Treatment of BALB/c or DBA/2 mice with CLIK148 exacerbated the disease by enhancing an SLA-specific Th2-type response such as IL-4 production. CLIK148 did not exert any direct influence on Leishmania major promastigotes themselves or on the course of L. major infection in SCID mice. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APCs, resulting in the potentiation of Th2-type immune responses and thus leading to exacerbation of the disease. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D.     

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.     

Synthesis and biological activity of novel alpha-substituted beta-phenylpropionic acids having pyridin-2-ylphenyl moiety as antihyperglycemic agents

We previously reported the identification of novel oximes having 5-benzyl-2,4-thiazolidinedione with antihyperglycemic activity. We now report the synthesis and biological activity of a novel series of oximes and amides having alpha-substituted-beta-phenylpropionic acids. In this series, we obtained potent PPAR alpha/gamma dual agonist (S)-9d, with which activation of PPAR alpha and PPAR gamma was considerably more potent than that of the reference compounds GW9578 22 and rosiglitazone 3, respectively. This means (S)-9d is of the strongest class of PPAR alpha/gamma dual agonists. In the course of this study, we also obtained 8h, which indicated potent plasma glucose lowering effect in spite of weak PPAR alpha/gamma agonistic activity.     

Origin of the scaling rule for fundamental living organisms based on thermodynamics

The regular relationships between metabolic energy and body mass M of unicellular organisms, poikilotherms and homeotherms were well known as general equations. The metabolic energy rate and the life span are proportional to M(0.75) and to M(0.25), respectively. As a result, the product of the metabolic energy rate and the life time, namely, life metabolic energy, is proportional to the mass of the living organism. The origin of the scaling rules for environmental organizing systems is as follows: (1) the scaling rules for internal energy, activation energy and free energy as a function of temperature and mass of a mole of molecules. (2) The majority of species of the living organisms have the same molecules such as polysaccharides, lipids, proteins and nucleic acids in nearly same the ratio. (3) The internal energy of reactants in living organisms is equilibrium with the internal energy of water. Then, the integrated metabolic energy over the synthesizing time depends on internal energy of water and is proportional to mass M, despite the synthesizing time of the system depending on reaction rate. The proportional constant is obtained based on the thermodynamics for fundamental living organisms such as unicellular organisms and plants. Information on the environmental organizing system is also discussed.     

Streptococcal preparation OK-432: a new maturation factor of monocyte-derived dendritic cells for clinical use

For vaccinations based on dendritic cells (DCs), maturation of DCs is critical to the induction of T-cell responses. We tested the efficacy of streptococcal preparation OK-432 as a Good Manufacturing Practice (GMP)-grade maturation agent. OK-432 is currently used in Japan as a cancer immunotherapy drug. Immature monocyte-derived dendritic cells (imMo-DCs) isolated from human peripheral blood monocytes stimulated with granulocyte-macrophage colony stimulating factor and interleukin-4 were exposed to maturation factors, i.e., lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha) plus prostaglandin E2 (PGE2), and OK-432 for 2 days. OK-432 increased expression of activation- and maturation-related molecules such as HLA-DR, CD80, CD83, and CD86 in imMo-DCs at levels similar to that of TNF-alpha plus PGE2, and higher than that of LPS. All agents examined induced allogeneic T-cell proliferation at a similar level. Only OK-432 caused significant production of interleukin-12 (IL-12) p70 and interferon gamma (IFN-gamma) at both the mRNA and protein levels in imMo-DCs. Neutralizing antibody against IL-12 p70 blocked IFN-gamma secretion from OK-432-stimulated Mo-DCs. IL-12 p70 produced by OK-432-stimulated imMo-DCs induced secretion of IFN-gamma by CD4+ T cells. OK-432 and LPS activated nuclear factor kappa B (NF-kappaB) in imMo-DCs. Both secretion of IL-12 p70 and IFN-gamma and activation of NF-kappaB induced by OK-432 were suppressed when imMo-DCs were pretreated with cytochalasin B. These results indicate that uptake of OK-432 by imMo-DCs is an early critical event for IL-12 p70 production and that NF-kappaB activation induced by OK-432 also contributes partially to IL-12 p70 production. In conclusion, OK-432 is a GMP-grade maturation agent and may be a potential tool for DC-based vaccine therapies.