Studies on the Adjuvant Action of Mineral Oil and Bacterial Endotoxin

Water-in-oil emulsion (WOE) of the Freund's type and a bacterial endotoxin (ET) enhanced the antibody response of mice to bovine _γ-globulin (BGG) in a different manner. WOE even without the antigen revealed an adjuvant action when given prior to or simultaneously with the antigen, while ET was effectual when given simultaneously with or after the antigen. Thus, the concurrent administration of these two adjuvants either before or after the antigen secured enhancement. It was shown that ET facilitated IgM antibody formation. WOE including antigen (BGG-WOE) was found to form an ‘antigen-depot’ at the injected site. Antigen released bit by bit from this depot thus might supply a continuous stimulus for the antibody response. This was mimicked by a divided daily injection of a small amount of antigen without adjuvants. Surgical removal of the hind foot containing the depot resulted in reduction of the circulating antibody. The popliteal lymph node cells from mice given BGG-WOE via hind foot pads could adoptively immunize X-irradiated recipients without the additional administration of antigen, axillary lymph node cells and spleen cells being unable to do so. ET was inadequate for this purpose. The morphological changes of the nodes seemed compatible with these results.     

Design,synthesis and pharmacology of aortic-selective acyl-CoA: Cholesterol O-acyltransferase (ACAT/SOAT) inhibitors

We describe our molecular design of aortic-selective acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also abbreviated as SOAT) inhibitors, their structure–activity relationships (SARs) and their pharmacokinetic (PK) and pharmacological profiles. The connection of two weak ligands—N-(2,6-diisopropylphenyl)acetamide (50% inhibitory concentration [IC₅₀]?=?8.6?μM) and 2-(methylthio)benzo[d]oxazole (IC₅₀?=?31?μM)—via a linker comprising a 6 methylene group chains yielded a highly potent molecule, 9-(benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)nonanamide (3h) that exhibited high potency (IC₅₀?=?0.004?μM) toward aortic ACAT. This head-to-tail design made it possible to markedly enhance the activity to 2150- to 7750-fold and to discriminate the isoform-selectivity based on the double-induced fit mechanism. At doses of 1 and 3?mg/kg, 3h significantly decreased the lipid-accumulation areas in the aortic arch to 74 and 69%, respectively without reducing the plasma total cholesterol level in high fat- and cholesterol-fed F₁B hamsters. Here, we demonstrate the antiatherosclerotic effect of 3hin vivo via its direct action on aortic ACAT and its powerful modulator of cholesterol level. This molecule is a potential therapeutic agent for the treatment of diseases involving ACAT-1 overexpression.     

Current topics in clinical FES in Japan

This paper reviews recent topics of clinical application of functional electrical stimulation (FES) for the paralyzed extremities in Japan. Transcutaneous and percutaneous FES systems have been clinically used in Japan. Candidates of extremity FES arer mostly stroke and spinal cord injury patients. By using percutaneous FES system, all of the joints of the upper extremity including the shoulder have been controlled for activities of daily living in the hemiplegic patient. Simultaneous FES control of the hand and wrist and the bilateral hands have also been achieved in C5 and C6 quadriplegics, respectively. Hybrid FES systems using percutaneous and surface electrodes, where FES is used in combination with orthoses, have been applied to the paraplegics because they are highly practical for assisting their locomotive activities. Percutaneous FES have been also provided the amyotropic lateral sclerosis patients with standing up motion. A total implant FES system with 16 output channels is currently developing as a next generation FES system.     

Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-α-induced cell death

Hsp70 plays an important role in cytoprotection against tumor necrosis factor (TNF) α-mediated cytotoxicity. To investigate the role of Hsp70 in cytoprotein during Salmonella infection, we examined endogenous Hsp70 induction and TNF-α production in a monocyte/macrophage line, J774A.1, after infection with a virulent strain of Salm.choleraesuis RF-1 carrying a 50 kb virulent plasmid or the plasmid-cured avirulent strain 31N-1. Intracellular bacteria progressively increased in J774A.1 cells phagocytosing avirulent 31N-1 bacteria, whereas such progressive growth was not evident in J774A.1 cells phagocytosing avirulent 31N-1 bacteria. On the contrary, J774A.1 cells infected with virulent RF-1 bacteria expressed less Hsp70 than those infected with avirulent 31N-1 bacteria. The level of TNF-α production by J774A.1 infected with virulent RF-1 was much the same as that by J774A.1 infected with avirulent 31N-1. J774A.1 infected with virulent RF-1 died spontaneously; death was inhibited by the addition of anti-TNF-α mAb. Although the frequency of dead J774A.1 with hypodiploid DNA content increased only marginally after infection with avirulent 31N-1, treatment with Hsp70 anti-sense oligonucleotide resulted in a dramatic increase of dead cells in the infected macrophages. Taken together, these results suggest that Hsp70 induced macrophages plays an important role in host defense against Salmonella infection by protecting the macrophages against TNF α-induced cell death. Furthermore, cell death due to impaired endogenous Hsp synthesis in the phagocytes implies a novel pathogenic mechanism for virulence of Salm. choleraesuis RF-1.     

The organic anion transporter SLCO2A1 constitutes the core component of the Maxi‐Cl channel

The maxi‐anion channels (MACs) are expressed in cells from mammals to amphibians with ~60% exhibiting a phenotype called Maxi‐Cl. Maxi‐Cl serves as the most efficient pathway for regulated fluxes of inorganic and organic anions including ATP. However, its molecular entity has long been elusive. By subjecting proteins isolated from bleb membranes rich in Maxi‐Cl activity to LC‐MS/MS combined with targeted siRNA screening, CRISPR/Cas9‐mediated knockout, and heterologous overexpression, we identified the organic anion transporter SLCO2A1, known as a prostaglandin transporter (PGT), as a key component of Maxi‐Cl. Recombinant SLCO2A1 exhibited Maxi‐Cl activity in reconstituted proteoliposomes. When SLCO2A1, but not its two disease‐causing mutants, was heterologously expressed in cells which lack endogenous SLCO2A1 expression and Maxi‐Cl activity, Maxi‐Cl currents became activated. The charge‐neutralized mutant became weakly cation‐selective with exhibiting a smaller single‐channel conductance. Slco2a1 silencing in vitro and in vivo, respectively, suppressed the release of ATP from swollen C127 cells and from Langendorff‐perfused mouse hearts subjected to ischemia–reperfusion. These findings indicate that SLCO2A1 is an essential core component of the ATP‐conductive Maxi‐Cl channel.