Endotoxin Induces Severe Inflammatory Reactions with Necrosis at Sites Primed with Delayed-Type Hypersensitivity Reactions in Guinea Pigs

Guinea pigs immunized with Freund's complete adjuvant received challenge injection of the purified protein derivative of Mycobacterium tuberculosis in the flanks and the corneas to prepare delayed-type hypersensitivity (DTH) reactions. The animals were injected subcutaneously with lipopolysaccharide (LPS) or a synthetic lipid A (LA-15-PP). At the skin site primed with DTH reaction, increased swelling and hemorrhagic reaction followed by a definite necrotic reaction occurred. Severe corneal reactions were also observed in the animals. These findings indicate that bacterial endotoxin modulates DTH reactions and induces severe inflammatory reactions.     

Discovery of AAT-008, a novel,potent, and selective prostaglandin EP4 receptor antagonist

Starting from acylsufonamide HTS hit 2, a novel series of para-N-acylaminomethylbenzoic acids was identified and developed as selective prostaglandin EP4 receptor antagonists. Structural modifications on lead compound 4a were explored with the aim of improving potency, physicochemical properties, and animal PK predictive of QD (once a day) dosing regimen in human. These efforts led to the discovery of the clinical candidate AAT-008 (4j), which exhibited significantly improved pharmacological profiles over grapiprant (1).     

Important role of apoptosis signal-regulating kinase 1 in ischemic acute kidney injury

We investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI). Blood urea nitrogen (BUN) and serum creatinine were significantly higher in ASK1+/+ mice than in ASK1−/− mice after I/R injury. Renal histology of ASK1+/+ mice showed significantly greater tubular necrosis and degradation. In ASK1−/− mice, phosphorylation of ASK1, JNK, and p38K, and the number of TUNEL-positive cells and infiltrated leukocytes decreased after I/R injury. Apoptotic changes were significantly decreased in cultured renal tubular epithelial cells (TECs) from ASK1−/− mice under hypoxic condition. Transfection with dominant-active ASK1 induced apoptosis in TECs. Protein expression of monocyte chemoattractant protein-1 (MCP-1) was significantly weaker in ASK1−/− mice after I/R injury. Transfection with dominant negative-ASK1 significantly decreased MCP-1 production in TECs. These results demonstrated that ASK1 is activated in I/R-induced AKI, and blockage of ASK1 attenuates renal tubular apoptosis, MCP-1 expression, and renal function.     

Regulation of glial development by cystatin C

Cystatin C (CysC) is an endogenous cysteine proteases inhibitor produced by mature astrocytes in the adult brain. Previously we isolated CysC as a factor activating the glial fibrillary acidic protein (GFAP) promoter, and showed that CysC is expressed in astrocyte progenitors during development. Here we show that protease inhibitor activity increased daily in conditioned medium, and that this activity was mainly a result of CysC released from primary cultured cells. Human CysC added to the culture medium of primary brain cells increased the number of GFAP-positive and nestin-positive cells. Human CysC also increased the number of neurospheres formed from embryonic brain, and thus it increases the number of neural stem/precursor cells in a manner similar to glycosylated rat CysC. The addition of a neutralizing antibody, on the other hand, greatly decreased the number of GFAP and glutamate aspartate transporter (GLAST)-positive astrocytes. This decrease was reversed by the addition of CysC but not by another cysteine protease inhibitor. Thus, the promotion of astrocyte development by CysC appears to be independent of its protease inhibitor activity. The antibody increased the number of oligodendrocytes and their precursors. Therefore, CysC modifies glial development in addition to its activity against neural stem/precursor cells.     

Angiotensin II mimics the hypoxic effect on regulating trophoblast proliferation and differentiation in human placental explant cultures

Regulation of cytotrophoblast differentiation toward extravillous trophoblasts (EVTs) is critical for establishing successful pregnancy. Previous studies have focused primarily on the factors promoting the differentiation, while inhibitory regulators except hypoxia have been less documented. In this study, to test our hypothesis that angiotensin II (Ang II) would inhibit EVT differentiation, we investigated the effects of Ang II on trophoblast outgrowth and the expression of molecules associated with the proliferation and invasion of trophoblasts using human first trimester villous explant cultures. Ang II increased EVT outgrowth and the number of cells in cell columns. Moreover, Ang II-treated explants exhibited increased Ki67 and integrin alpha5 immunoreactivity in EVTs as well as matrix metalloproteinase-2 activity in the conditioned media, and decreased alpha1 integrin immunoreactivity, which are compatible with the features of the proliferative phenotype EVTs. These effects of Ang II were similar to those of hypoxia (3% O(2)). Ang II stimulated the expression of hypoxia inducible factor-1alpha at both mRNA and protein levels, and also enhanced the expression of plasminogen activator inhibitor-1 (PAI-1). Data presented herein suggest a possible role for Ang II in impairing trophoblast differentiation toward an invasive phenotype, which might be associated with shallow invasion in preeclamptic placentas.     

Vinculin Is Indispensable for Repopulation by Hematopoietic Stem Cells,Independent of Integrin Function

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function, little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit⁺Sca1⁺Lin⁻ HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages, to migrate toward stromal cell-derived factor 1α, and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However, the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit⁺Sca1⁺Lin⁻ HSCs. In contrast, adhesion to the extracellular matrix was inhibited by silencing of talin-1, but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity, independent of integrin functions.     

Indirect female choice mediated by sex pheromones in the hermit crab Pagurus filholi

Males of the hermit crab Pagurus filholi perform precopulatory guarding behavior, and solitary males often show aggressive behavior to take away guarded females. Males behave coercively while guarding females, so direct mate choice by females seems difficult in such a situation. By performing several experiments we examined possible indirect female choice of hermit crab. Males were attached to a shell by their left cheliped to look like guarding pairs (fake guarding pairs). The shells were filled with cotton containing either seawater or pheromone water. The fake guarding pair with only seawater caused male–male combat in 60% of trials whereas with pheromone water combats occurred in 88% of trials. Mean duration of male–male combat was significantly longer in trials with drops of seawater containing pheromones than in those without pheromones. These results suggest guarding pairs themselves cause male–male combat by visual stimulation, that female sex pheromones have further significant function in the recognition of guarding pairs and intensification of male–male combat, and that females release sex pheromones while they are guarded. As a result of the combat, the larger male ended up guarding a female. This strongly suggests that females choose males indirectly by exploiting male–male competition induced by sex pheromones under male coercive behavior.     

Plasmodium falciparum: Attenuation by irradiation

The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10⁶ parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.     

Detergent-resistant erythrocyte membrane rafts are modified by a Plasmodium falciparum infection

Detergent resistant membranes (DRMs) have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. Flotillins-1 and -2 have recently been shown to be large components of erythrocyte DRMs. In this study, we show that a Plasmodium falciparum infection disrupts the association of flotillins with erythrocyte DRMs. Flotillins are probably released from erythrocyte DRMs through the reduction of cholesterol and sphingomyelin levels during the course of a P. falciparum-infection. Although it is well known that a P. falciparum infection can modify the host erythrocyte membrane, this is the first report that P. falciparum can alter the DRM components of erythrocyte membranes.