Benzalkonium Chloride Accelerates the Formation of the Amyloid Fibrils of Corneal Dystrophy-associated Peptides

Corneal dystrophies are genetic disorders resulting in progressive corneal clouding due to the deposition of amyloid fibrils derived from keratoepithelin, also called transforming growth factor β-induced protein (TGFBI). The formation of amyloid fibrils is often accelerated by surfactants such as sodium dodecyl sulfate (SDS). Most eye drops contain benzalkonium chloride (BAC), a cationic surfactant, as a preservative substance. In the present study, we aimed to reveal the role of BAC in the amyloid fibrillation of keratoepithelin-derived peptides in vitro. We used three types of 22-residue synthetic peptides covering Leu110-Glu131 of the keratoepithelin sequence: an R-type peptide with wild-type R124, a C-type peptide with C124 associated with lattice corneal dystrophy type I, and a H-type peptide with H124 associated with granular corneal dystrophy type II. The time courses of spontaneous amyloid fibrillation and seed-dependent fibril elongation were monitored in the presence of various concentrations of BAC or SDS using thioflavin T fluorescence. BAC and SDS accelerated the fibrillation of all synthetic peptides in the absence and presence of seeds. Optimal acceleration occurred near the CMC, which suggests that the unstable and dynamic interactions of keratoepithelin peptides with amphipathic surfactants led to the formation of fibrils. These results suggest that eye drops containing BAC may deteriorate corneal dystrophies and that those without BAC are preferred especially for patients with corneal dystrophies.     

IQGAP1 Protein Regulates Nuclear Localization of β-Catenin via Importin-β5 Protein in Wnt Signaling

In the canonical Wnt signaling pathway, the translocation of β-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of β-catenin function via importin-β5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of β-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-β5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and β-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of β-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of β-catenin in the canonical Wnt signaling pathway.     

The Genome-Packaging Signal of the Influenza A Virus Genome Comprises a Genome Incorporation Signal and a Genome-Bundling Signal

The influenza A virus genome comprises eight single-stranded negative-sense RNA segments (vRNAs). All eight vRNAs are selectively packaged into each progeny virion via so-called segment-specific genome-packaging signal sequences that are located in the noncoding and terminal coding regions of both the 3′ and the 5′ ends of the vRNAs. However, it remains unclear how these signals ensure that eight different vRNAs are packaged. Here, by using a reverse genetics system, we demonstrated that, in the absence of the other seven vRNAs, a recombinant NP vRNA bearing only a reporter gene flanked by the noncoding NP regions was incorporated into virus-like particles (VLPs) as efficiently as a recombinant NP vRNA bearing the reporter gene flanked by the complete NP packaging signals (i.e., the noncoding sequences and the terminal coding regions). Viruses that comprised a recombinant NP vRNA whose packaging signal was disrupted, and the remaining seven authentic vRNAs, did not undergo multiple cycles of replication; however, a recombinant NP vRNA with only the noncoding regions was readily incorporated into VLPs, suggesting that the packaging signal as currently defined is not necessarily essential for the packaging of the vRNA in which it resides; rather, it is required for the packaging of the full set of vRNAs. We propose that the 3′ and 5′ noncoding regions of each vRNA bear a virion incorporation signal for that vRNA and that the terminal coding regions serve as a bundling signal that ensures the incorporation of the complete set of eight vRNAs into the virion.     

A Major Latex-Like Protein Is a Key Factor in Crop Contamination by Persistent Organic Pollutants

This is the first report, to our knowledge, to reveal important factors by which members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), squash (C. pepo), and zucchini (C. pepo), are selectively polluted with highly toxic hydrophobic contaminants, including organochlorine insecticides and dioxins. Xylem sap of C. pepo ssp. pepo, which is a high accumulator of hydrophobic compounds, solubilized the hydrophobic compound pyrene into the aqueous phase via some protein(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of xylem sap of two C. pepo subspecies revealed that the amount of 17-kD proteins in C. pepo ssp. pepo was larger than that in C. pepo ssp. ovifera, a low accumulator, suggesting that these proteins may be related to the translocation of hydrophobic compounds. The protein bands at 17 kD contained major latex-like proteins (MLPs), and the corresponding genes MLP-PG1, MLP-GR1, and MLP-GR3 were cloned from the C. pepo cultivars Patty Green and Gold Rush. Expression of the MLP-GR3 gene in C. pepo cultivars was positively correlated with the band intensity of 17-kD proteins and bioconcentration factors toward dioxins and dioxin-like compounds. Recombinant MLP-GR3 bound polychlorinated biphenyls immobilized on magnetic beads, whereas recombinant MLP-PG1 and MLP-GR1 did not. These results indicate that the high expression of MLP-GR3 in C. pepo ssp. pepo plants and the existence of MLP-GR3 in their xylem sap are related to the efficient translocation of hydrophobic contaminants. These findings should be useful for decreasing the contamination of fruit of the Cucurbitaceae family as well as the phytoremediation of hydrophobic contaminants.Numerous agricultural fields and crops have been contaminated with persistent organic pollutants (POPs), including dioxins, such as polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs); dioxin-like compounds, such as coplanar polychlorinated biphenyls (PCBs) and the insecticide dichlorodiphenyltrichloroethane; drins, such as aldrin, dieldrin, and endrin; and chlordane (Hashimoto, 2005; Uegaki et al., 2006; Hilber et al., 2008). POPs show carcinogenicity, teratogenicity, immunotoxicity, and estrogenicity toward humans and wildlife after accumulation through the food chain. Despite the fact that the use of PCBs and these insecticides was prohibited several decades ago, environmental and crop contamination remains a problem due to their high hydrophobicity and chemical stability.Members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullas lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), and zucchini (C. pepo), are some of the major crops in the world. Previous studies reported that members of the Cucurbitaceae family, particularly C. pepo, which includes pumpkin and zucchini, accumulated higher levels of PCDDs and PCDFs (Hülster et al., 1994; Inui et al., 2008), 2,2-bis(p-chlorophenyl) 1,1-dichloroethylene (p,p′-DDE; White et al., 2003), PCBs (Aslund et al., 2008; Inui et al., 2008), chlordane (Mattina et al., 2004), and drins (Otani et al., 2007) compared with the levels in other plant species. Thus, it appears that the Cucurbitaceae family has unique mechanisms of POP uptake and translocation. Lunney et al. (2004) reported that the shoots of pumpkin and zucchini plants showed much higher concentrations of dichlorodiphenyltrichloroethane than those of tall fescue (Festuca arundinacea), alfalfa (Medicago sativa), and ryegrass (Lolium multiflorum), whereas concentrations in roots were similar among these plants. Likewise, significant differences were found between C. pepo ssp. pepo and ssp. ovifera in concentrations of dioxins and dioxin-like compounds in the aerial parts, whereas the concentrations in their roots were similar (Inui et al., 2011). These results suggest that the mechanisms causing the high accumulation of POPs in C. pepo plants mainly occur during translocation from the roots to the aerial parts.The transport of substances such as nutrients and signal molecules over long distances in higher land plants is mediated by the vascular bundles, which consist of phloem and xylem strands. In addition to inorganic salts, organic nutrients such as amino acids, sugars, and organic acids are translocated through the xylem from the roots to the aerial parts (Satoh, 2006). Furthermore, the fact that POPs such as chlordane, dieldrin, and PCBs were detected in xylem sap of C. pepo suggests that their accumulation in the aerial parts of plants occurs during the translocation from roots to aerial parts in xylem sap (Mattina et al., 2004; Murano et al., 2010b; Greenwood et al., 2011). A recent study revealed that there were protein-like materials with the ability to dissolve dieldrin in xylem sap (Murano et al., 2010a). However, these materials have yet to be identified, and the mechanisms underlying the high transport ability and high accumulation of POPs in C. pepo plants are not fully understood.In this study, to clarify the molecular mechanisms of the efficient uptake and high accumulation of POPs by C. pepo plants, xylem sap proteins related to the transport of POPs in xylem sap were identified. The aim of this research is to provide a means of preventing cucumber, melon, watermelon, pumpkin, and zucchini fruits from being contaminated by POPs.     

Modulation of cytochrome P450 gene expression in primary hepatocytes on various artificial extracellular matrices

We studied effect of artificial extracellular matrices (ECMs), such as collagen I, poly (N-p-vinylbenzyl-4-O-β-d-galactopyranosyl-d-gluconamide)(PVLA) and E-cadherin–IgG Fc (E-cad-Fc) on hepatic metabolism to identify the mechanism of in vivo hepatocellular functional and metabolic integrity. mRNA expression of liver function marker, cytochrome P450 (CYP) and transporter genes in hepatocytes were compared among used ECMs using real-time RT-PCR. mRNA expressions of Cyp2c29 and Cyp2d22 among CYP genes in hepatocytes on PVLA were recovered after 3 days due to enhanced liver-specific function by the spheroid formation of hepatocytes whereas mRNA expressions of CYP genes in hepatocytes on collagen and E-cad-Fc drastically decreased with time. mRNA expressions of the Cyp2c29 and Cyp2d22 in hepatocytes on PVLA were more recovered in the presence of epidermal growth factor (EGF) due to the more and bigger spheroid formation of hepatocytes. Multidrug resistance-associated protein 2 (Mrp2) protein was accumulated at intracellular lumen as similar to bile duct in hepatocyte spheroid formed on PVLA, indicating that spheroid formation of hepatocytes is very important for maintaining liver functions.     

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces intimal thickening after vascular injury

Glucagon-like peptide-1 is a hormone secreted by L cells of the small intestine and stimulates glucose-dependent insulin response. Glucagon-like peptide-1 receptor agonists such as exendin-4 are currently used in type 2 diabetes, and considered to have beneficial effects on the cardiovascular system. To further elucidate the effect of glucagon-like peptide-1 receptor agonists on cardiovascular diseases, we investigated the effects of exendin-4 on intimal thickening after endothelial injury. Under continuous infusion of exendin-4 at 24 nmol/kg/day, C57BL/6 mice were subjected to endothelial denudation injury of the femoral artery. Treatment of mice with exendin-4 reduced neointimal formation at 4 weeks after arterial injury without altering body weight or various metabolic parameters. In addition, in vitro studies of isolated murine, rat and human aortic vascular smooth muscle cells showed the expression of GLP-1 receptor. The addition of 10 nM exendin-4 to cultured smooth muscle cells significantly reduced their proliferation induced by platelet-derived growth factor. Our results suggested that exendin-4 reduced intimal thickening after vascular injury at least in part by the suppression of platelet-derived growth factor-induced smooth muscle cells proliferation.     

Inherited genetic variants associated with glucocorticoid sensitivity in leukaemia cells

Identification of genetic variants associated with glucocorticoids (GC) sensitivity of leukaemia cells may provide insight into potential drug targets and tailored therapy. In the present study, within 72 leukaemic cell lines derived from Japanese patients with B‐cell precursor acute lymphoblastic leukaemia (ALL), we conducted genome‐wide genotyping of single nucleotide polymorphisms (SNP) and attempted to identify genetic variants associated with GC sensitivity and NR3C1 (GC receptor) gene expression. IC50 measures for prednisolone (Pred) and dexamethasone (Dex) were available using an alamarBlue cell viability assay. IC50 values of Pred showed the strongest association with rs904419 (P = 4.34 × 10⁻⁸), located between the FRMD4B and MITF genes. The median IC50 values of prednisolone for cell lines with rs904419 AA (n = 13), AG (n = 31) and GG (n = 28) genotypes were 0.089, 0.139 and 297 µmol/L, respectively. For dexamethasone sensitivity, suggestive association was observed for SNP rs2306888 (P = 1.43 × 10⁻⁶), a synonymous SNP of the TGFBR3 gene. For NR3C1 gene expression, suggestive association was observed for SNP rs11982167 (P = 6.44 × 10⁻⁸), located in the PLEKHA8 gene. These genetic variants may affect GC sensitivity of ALL cells and may give rise to opportunities in personalized medicine for effective and safe chemotherapy in ALL patients.