Cell surface biotinylation by azaelectrocyclization: easy-handling and versatile approach for living cell labeling

Versatile method for living cell labeling has been established. Cell surfaces are initially biotinylated by azaelectrocyclization, and then treated with the fluorescence-labeled avidin or the anti-biotin antibody.     

CD22 regulates time course of both B cell division and antibody response

Because pathogens induce infectious symptoms in a time-dependent manner, a rapid immune response is beneficial for defending hosts from pathogens, especially those inducing acute infectious diseases. However, it is largely unknown how the time course of immune responses is regulated. In this study, we demonstrate that B cells deficient in the inhibitory coreceptor CD22 undergo accelerated cell division after Ag stimulation, resulting in rapid generation of plasma cells and Ab production. This finding indicates that CD22 regulates the time course of B cell responses and suggests that CD22 is a good target to shorten the time required for Ab production, thereby augmenting host defense against acute infectious diseases as "universal vaccination."     

Topical administration of a multi-targeted kinase inhibitor suppresses choroidal neovascularization and retinal edema

Age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions are complicated by neovascularization and macular edema. Multi-targeted kinase inhibitors that inhibit select growth factor receptor tyrosine kinases and/or components of their down-stream signaling cascades (such as Src kinases) are rationale treatment strategies for these disease processes. We describe the discovery and characterization of two such agents. TG100572, which inhibits Src kinases and selected receptor tyrosine kinases, induced apoptosis of proliferating endothelial cells in vitro. Systemic delivery of TG100572 in a murine model of laser-induced choroidal neovascularization (CNV) caused significant suppression of CNV, but with an associated weight loss suggestive of systemic toxicity. To minimize systemic exposure, topical delivery of TG100572 to the cornea was explored, and while substantial levels of TG100572 were achieved in the retina and choroid, superior exposure levels were achieved using TG100801, an inactive prodrug that generates TG100572 by de-esterification. Neither TG100801 nor TG100572 were detectable in plasma following topical delivery of TG100801, and adverse safety signals (such as weight loss) were not observed even with prolonged dosing schedules. Topical TG100801 significantly suppressed laser-induced CNV in mice, and reduced fluorescein leakage from the vasculature and retinal thickening measured by optical coherence tomography in a rat model of retinal vein occlusion. These data suggest that TG100801 may provide a new topically applied treatment approach for ocular neovascularization and retinal edema.     

Cytotoxicity and apoptosis produced by troglitazone in human hepatoma cells.

Troglitazone is an antidiabetic agent that increases the insulin sensitivity of target tissues in non-insulin-dependent diabetes mellitus. It has been reported that troglitazone causes severe hepatic injury in certain individuals. In the present study, the mechanism for the hepatic injury by troglitazone was investigated with human hepatoma cell lines. HepG2 cells were incubated with troglitazone, its metabolites M-1 (sulfate), M-2 (gulucronide), M-3 (quinone), and other thiazolidinediones (pioglitazone and rosiglitazone). Troglitazone exhibited time- and concentration-dependent cytotoxicity and M-3 also exhibited weak cytotoxicity. Troglitazone induced apoptotic cell death characterized by internucleosomal DNA fragmentation and nuclear condensation. As other thiazolidinediones, pioglitazone and rosiglitazone, did not induce cell death and apoptosis in the present study, the affinity to PPARgamma may not affect the induction of apoptosis by troglitazone. These results suggest that troglitazone induces apoptotic hepatocyte death which it may be one of the factors of liver injury in humans.     

Fluoroquinolones and propionic acid derivatives induce inflammatory responses in vitro

Fluoroquinolones and propionic acid derivatives are widely used antibacterials and non-steroidal anti-inflammatory drugs, respectively, which have been reported to frequently trigger drug hypersensitivity reactions. Such reactions are induced by inflammatory mediators such as cytokines and chemokines. The present study investigated whether levofloxacin, a fluoroquinolone, and loxoprofen, a propionic acid derivative, have the potential to induce immune-related gene expression in dendritic cell-like cell lines such as HL-60, K562, and THP-1, and immortalized keratinocytes such as HaCaT. The expression of IL-8, MCP-1, and TNFα messenger RNA (mRNA) was found to increase following treatment with levofloxacin or loxoprofen in HL-60 cells. In addition, these drugs increased the mRNA content of annexin A1, a factor related to keratinocyte necroptosis in patients with severe cutaneous adverse reactions. Inhibition studies using specific inhibitors of mitogen-activated protein (MAP) kinases and NF-κB suggest that the extracellular signal-regulated kinase (ERK) pathway is the pathway principally involved in the induction of cytokines and annexin A1 by levofloxacin, whereas the involvement of MAP kinases and NF-κB in the loxoprofen-induced gene expression of these factors may be limited. Fluoroquinolones and propionic acid derivatives that are structurally related to levofloxacin and loxoprofen, respectively, were also found to induce immune-related gene expression in HL-60 cells. Collectively, these results suggest that fluoroquinolones and propionic acid derivatives have the potential to induce the expression of immune-related factors and that an in vitro cell-based assay system to detect the immune-stimulating potential of systemic drugs might be useful for assessing the risk of drug hypersensitivity reactions.     

Purification and characterization of the intracellular β-glucosidase from Aureobasidium sp ATCC 20524

β-Glucosidase hydrolyzing cellobiose was extracted from Aureobasidium sp ATCC 20524 and purified to homogeneity. The molecular mass was estimated to be about 331 kDa. The enzyme contained 26.5% (w/w) carbohydrate. The optimum pH and temperature for the enzyme reaction were pH 4 and 80°C, respectively. The enzyme was stable at a wide range of pH, 2.2–9.8, after 3 h and at 75°C for 15 min. The kinetic parameters were determined. The enzyme was relatively stable against typical organic enzyme inhibitors. The enzyme also hydrolyzed gentiobiose, p-nitrophenyl-β-glucoside and salicin. Received 05 November 1998/ Accepted in revised form 14 February 1999     

New cyclic somatostatin analogues containing a pyrazinone ring: importance of Tyr for antiproliferative activity

Novel somatostatin analogues containing a pyrazinone ring, compounds 1 and 2, exhibited good antiproliferative activity on A431 tumor cells. To increase antitumor activity and binding affinity on somatostatin receptors (SSTRs), we substituted Tyr in the critical sequence, Tyr-D-Trp-Lys, with more hydrophobic aromatic residue. The substituted compounds dramatically lost antitumor activity, indicating that Tyr residue was an essential residue.     

Characterization of teleost Mdga1 using a gene‐trap approach in medaka (Oryzias latipes)

MAM domain containing glycosilphosphatidilinositol anchor 1 (MDGA1) is an IgCAM protein present in many vertebrate species including humans. In mammals, MDGA1 is expressed by a subset of neurons in the developing brain and thought to function in neural cell migration. We identified a fish ortholog of mdga1 by a gene‐trap screen utilizing the Frog Prince transposon in medaka (Japanese killifish, Oryzias latipes). The gene‐trap vector was inserted into an intronic region of mdga1 to form a chimeric protein with green fluorescent protein, allowing us to monitor mdga1 expression in vivo. Expression of medaka mdga1 was seen in various types of embryonic brain neurons, and specifically in neurons migrating toward their target sites, supporting the proposed function of MDGA1. We also isolated the closely related mdga2 gene, whose expression partially overlapped with that of mdga1. Despite the fact that the gene‐trap event eliminated most of the functional domains of the Mdga1 protein, homozygous embryos developed normally without any morphological abnormality, suggesting a functional redundancy of Mdga1 with other related proteins. High sequential homology of MDGA proteins between medaka and other vertebrate species suggests an essential role of the MDGA gene family in brain development among the vertebrate phylum. genesis 47:505–513, 2009. © 2009 Wiley‐Liss, Inc.     

Consequences of Lineage-Specific Gene Loss on Functional Evolution of Surviving Paralogs: ALDH1A and Retinoic Acid Signaling in Vertebrate Genomes

Genome duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Little attention, however, has focused on the evolutionary impact of lineage-specific gene loss. Here, we show that identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving functional connectivity among human and model organism genomes. We examine the general principles of gene loss following duplication, coupled with expression analysis of the retinaldehyde dehydrogenase Aldh1a gene family during retinoic acid signaling in eye development as a case study. Humans have three ALDH1A genes, but teleosts have just one or two. We used comparative genomics and conserved syntenies to identify loss of ohnologs (paralogs derived from genome duplication) and to clarify uncertain phylogenies. Analysis showed that Aldh1a1 and Aldh1a2 form a clade that is sister to Aldh1a3-related genes. Genome comparisons showed secondarily loss of aldh1a1 in teleosts, revealing that Aldh1a1 is not a tetrapod innovation and that aldh1a3 was recently lost in medaka, making it the first known vertebrate with a single aldh1a gene. Interestingly, results revealed asymmetric distribution of surviving ohnologs between co-orthologous teleost chromosome segments, suggesting that local genome architecture can influence ohnolog survival. We propose a model that reconstructs the chromosomal history of the Aldh1a family in the ancestral vertebrate genome, coupled with the evolution of gene functions in surviving Aldh1a ohnologs after R1, R2, and R3 genome duplications. Results provide evidence for early subfunctionalization and late subfunction-partitioning and suggest a mechanistic model based on altered regulation leading to heterochronic gene expression to explain the acquisition or modification of subfunctions by surviving ohnologs that preserve unaltered ancestral developmental programs in the face of gene loss.