Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis

TNF-α, IFN-γ, IL-4, and MIP-2 are known to be involved in Con A-induced hepatitis. Although Kupffer cells are reportedly involved in TNF-α production, it is largely unknown whether or not Kupffer cells also play a role in the production of other cytokines, such as IFN-γ, IL-4, and MIP-2. In this study we examined the liver injury and the levels of plasma cytokines, including above four cytokines, KC, and IL-10 in Kupffer cell-depleted mice obtained through administration of liposome-encapsulated dichloromethylene bisphosphonate. The liver injury was significantly suppressed in Kupffer cell-depleted mice, as assessed as to the plasma ALT level and histochemistry. The cytokine levels were also significantly suppressed in such mice except for those of IFN-γ, which was slightly suppressed at 12 h, and IL-10, which was not significantly suppressed at any time. Apoptosis was also significantly suppressed in such mice, as found immunohistochemically with anti-ssDNA Ab. Taken together, these results suggest that Kupffer cells are involved in the production of MIP-2, KC, IL-4, and TNF-α in Con A-induced hepatitis, thereby contributing to the liver injury either directly or indirectly.     

Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below −40 °C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2007) 158–166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to −25 °C, holding them 10 min, rapidly cooling them to −70 °C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at −56 °C during warming and was complete by −46 °C. The present study differs in that the oocytes were warmed rapidly from −70 °C to temperatures between −65 and −50 °C and held for 3–60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (E_a) for the blackening process; namely, 27.5 kcal/mol. This translates to about a quadrupling of the blackening rate for every 5 °C rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 °C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 °C. These findings have significant implications both for cryobiology and cryo-electron microscopy.     

A strategy for screening an inhibitor of viral silencing suppressors, which attenuates symptom development of plant viruses

To find out whether we can control plant virus diseases by blocking viral RNA silencing suppressors (RSSs), we developed a strategy to screen inhibitors that block the association of RSSs with siRNAs using a surface plasmon resonance assay. The screened chemicals were tested in competition with RSSs for binding to siRNAs using a mobility shift assay. We then confirmed that tested chemicals actually inhibited the RSS activity in vivo using a protoplast assay which was developed for this purpose. This entire system can be adapted to screening inhibitors of not only plant viruses but also some animal viruses possessing RSSs.     

A lysophosphatidic acid receptor lacking the PDZ-binding domain is constitutively active and stimulates cell proliferation

Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein-coupled LPA receptors, LPA(1) to LPA(5). We generated an LPA(1) mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA(1) showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G(betagamma), G(alphai) and G(alphaq) or G(alpha12/13), or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA(1) in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA(1) constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation.     

Identification of novel non-peptide CXCR4 antagonists by ligand-based design approach

The design and synthesis of novel non-peptide CXCR4 antagonists is described. The peptide backbone of highly potent cyclic peptide-based CXCR4 antagonists was entirely replaced by an indole framework, which was expected to reproduce the disposition of the key pharmacophores consistent with those of potential bioactive conformations of the original peptides. A structure–activity relationship study on a series of modified indoles identified novel small-molecule antagonists having three pharmacophore functional groups through the appropriate linkers.     

Sequence Analysis of Amplified DNA Fragments Containing the Region Encoding the Putative Lipase Substrate-Binding Domain and Genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

DISC1-kendrin interaction is involved in centrosomal microtubule network formation

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation segregating with schizophrenia, bipolar disorder and other major mental illnesses in a Scottish family. We previously identified 446-533 amino acids of DISC1 as the kendrin-binding region by means of a directed yeast two-hybrid interaction assay and showed that the DISC1-kendrin interaction is indispensable for the centrosomal localization of DISC1. In this study, to confirm the DISC1-kendrin interaction, we examined the interaction between deletion mutants of DISC1 and kendrin. Then, we demonstrated that the carboxy-terminus of DISC1 is indispensable for the interaction with kendrin. Furthermore, the immunocytochemistry revealed that the carboxy-terminus of DISC1 is also required for the centrosomal targeting of DISC1. Overexpression of the DISC1-binding region of kendrin or the DISC1 deletion mutant lacking the kendrin-binding region impairs the microtubule organization. These findings suggest that the DISC1-kendrin interaction plays a key role in the microtubule dynamics.     

Development of high-throughput screening system for osteogenic drugs using a cell-based sensor

To effectively treat osteoporosis and other bone-loss disorders, small compounds that potently induce bone formation are needed. The present study initially attempted to establish a monitoring system that could detect osteogenic differentiation easily, precisely, and noninvasively. For this purpose, we established pre-osteoblastic MC3T3E1 cells stably transfected with the GFP reporter gene driven by a 2.3 kb fragment of rat type I collagen promoter (Col1a1GFP-MC3T3E1). Among these cells, we selected a clone that fluoresced upon osteogenic stimulation by BMP2. The GFP fluorescence intensity corresponded well to the intensity of alkaline phosphatase (ALP) staining and to the level of osteocalcin (Oc) mRNA. Using this system, we screened natural and synthetic compound libraries and thus identified an isoflavone derivative, glabrisoflavone (GI). GI induced ALP staining and Oc mRNA in a dose-dependent manner. The Col1a1GFP-MC3T3E1 system may be useful for identifying novel osteogenic drugs.     

Further insight into the mechanism of stereoselective proton abstraction by bacterial copper amine oxidase

During the catalytic reaction of copper amine oxidase, one of the two prochiral hydrogen atoms at the C1 position of substrate amine is stereoselectively abstracted by a conserved Asp residue serving as a general base. Using stereospecifically deuterium-labeled enantiomers of 2-phenylethylamine, we previously showed that the pro-S alpha-proton is abstracted by the enzyme from Arthrobacter globiformis (AGAO) [Uchida, M., et al. (2003) Biosci. Biotechnol. Biochem. 67, 2664-2667]. More recently, we have also demonstrated that the pro-S selectivity of alpha-proton abstraction is fully retained even in the reaction of a mutant AGAO lacking the catalytic base [Chiu, Y.-C., et al. (2006) Biochemistry 45, 4105-4120]. On the basis of these findings, we have proposed that the stereoselectivity of alpha-proton abstraction is primarily determined by the conformation of the Schiff base intermediate formed between the substrate and the topa quinone cofactor (TPQ), stabilized by the binding of the distal part of the substrate to a hydrophobic pocket of the enzyme. In this conformation, the pro-S hydrogen atom to be abstracted is nearly perpendicular to the plane of the Schiff base-TPQ conjugate system, achieving the maximum overlap of sigma- and pi-orbitals. To further elucidate the stereochemical details, we have synthesized stereospecifically deuterium-labeled enantiomers of ethylamine, a very poor substrate for AGAO, in addition to those structurally related to the preferred substrate, 2-phenylethylamine. In marked contrast to the nearly complete pro-S selectivity of alpha-proton abstraction for most substrates that have been examined, the stereoselectivity for ethylamine decreased significantly to as little as 88%. The crystal structure of AGAO soaked with ethylamine showed very poor electron densities for the substrate Schiff base intermediate, showing that its conformation is not defined uniquely. Thus, the stereoselectivity of alpha-proton abstraction during the copper amine oxidase reaction is closely associated with the conformational flexibility of the substrate Schiff base intermediate.