Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene

Various liver micronucleus assay methods, such as those involving partial hepatectomy, treatment with mitogens, and the use of juvenile animals, have been developed. These assays have been proven to be of high sensitivity and specificity to predict hepatocarcinogenicity of compounds that cannot be detected by bone marrow micronucleus assays. On the contrary, the existing assays have only been evaluated for their use in detecting micronucleus induction in the settings of relatively short-term cell proliferation. However, the integration of in vivo genotoxicity endpoints into routine toxicity studies is increasingly desired from the viewpoint of animal welfare to reduce the number of animals used. In the present study, the rodent hepatocarcinogens diethylnitrosamine (DEN) and 2,4-diaminotoluene (2,4-DAT) were repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old at the beginning of administration) for 5, 14, and 28 days, and changes in the frequency of hepatocytes with micronuclei in liver tissues that had undergone no artificial treatment to accelerate cell proliferation were evaluated. At the same time, a new method of hepatocyte isolation involving the treatment of a portion of the liver with collagenase in a centrifuge tube, without the use of in situ perfusion, was established. The induction of micronucleated hepatocytes was achieved after the repeated administration of DEN for 5 days or longer and of 2,4-DAT for 14 days or longer. Micronucleus frequencies were increased depending on the number of administrations, indicating that micronucleated hepatocytes had possibly remained for a long period of time and accumulated additively. It therefore appears that even in adult rat liver with low mitotic activity, a repeated-dose of a chemical substance for 14 days or longer enables the detection of micronucleus induction. In addition, the establishment of a method to isolate hepatocytes without perfusion using only a part of the liver enables the integration of liver micronucleus assays into general toxicity studies.     

Simultaneous screening of multiple mutations by invader assay improves molecular diagnosis of hereditary hearing loss: a multicenter study

Although etiological studies have shown genetic disorders to be a common cause of congenital/early-onset sensorineural hearing loss, there have been no detailed multicenter studies based on genetic testing. In the present report, 264 Japanese patients with bilateral sensorineural hearing loss from 33 ENT departments nationwide participated. For these patients, we first applied the Invader assay for screening 47 known mutations of 13 known deafness genes, followed by direct sequencing as necessary. A total of 78 (29.5%) subjects had at least one deafness gene mutation. Mutations were more frequently found in the patients with congenital or early-onset hearing loss, i.e., in those with an awareness age of 0-6 years, mutations were significantly higher (41.8%) than in patients with an older age of awareness (16.0%). Among the 13 genes, mutations in GJB2 and SLC26A4 were mainly found in congenital or early-onset patients, in contrast with mitochondrial mutations (12S rRNA m.1555A>G, tRNA(Leu(UUR)) m.3243A>G), which were predominantly found in older-onset patients. The present method of simultaneous screening of multiple deafness mutations by Invader assay followed by direct sequencing will enable us to detect deafness mutations in an efficient and practical manner for clinical use.     

A putative tyrosine phosphorylation site of the cell surface receptor Golden goal is involved in synaptic layer selection in the visual system

Golden goal (Gogo) is a cell surface protein that is crucial for proper synaptic layer targeting of photoreceptors (R cells) in the Drosophila visual system. In collaboration with the seven-transmembrane cadherin Flamingo (Fmi), Gogo mediates both temporary and final layer targeting of R-cell axons through its cytoplasmic activity. However, it is not known how Gogo activity is regulated. Here, we show that a conserved Tyr-Tyr-Asp (YYD) tripeptide motif in the Gogo cytoplasmic domain is required for photoreceptor axon targeting. Deleting the YYD motif is sufficient to abolish Gogo function. We demonstrate that the YYD motif is a phosphorylation site and that mutations in the YYD tripeptide impair synaptic layer targeting. Gogo phosphorylation results in axon stopping at the temporary targeting layer, and dephosphorylation is crucial for final layer targeting in collaboration with Fmi. Therefore, both temporary and final layer targeting strongly depend on the Gogo phosphorylation status. Drosophila Insulin-like receptor (DInR) has been reported to regulate the wiring of photoreceptors. We show that insulin signaling is a positive regulator, directly or indirectly, of YYD motif phosphorylation. Our findings indicate a novel mechanism for the regulation of Gogo activity by insulin signaling-mediated phosphorylation. We propose the model that a constant phosphorylation signal is antagonized by a presumably temporal dephosphorylation signal, which creates a permissive signal that controls developmental timing in axon targeting.     

Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration

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Highlights? Not all hepatocytes undergo cell division during liver regeneration ? Hypertrophy precedes proliferation in the regenerating liver ? Hepatocytes infrequently enter into M phase during liver regeneration ? Binuclear hepatocytes undergo cell division to generate mononuclear hepatocytes     

Caveolin-1 hydrophobic segment peptides insertion into membrane mimetic systems: role of proline residue

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122 that are anchored to the membrane with cholesterol-rich domains. Previously, we reported that changing the Pro(110) residue to Ala (the P110A mutant) prevents not only the localization of the protein into lipid rafts but also the formation and functioning of caveolae. The conformational state of caveolin-1 can be shifted toward the transmembrane arrangement by this single amino acid mutation. To model the conformation, and extent of membrane insertion of this segment into membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of caveolin-1 having the sequence KKKKLSTIFGIPMALIWGIYFAILKKKKK-amide and the mutated version, KKKKLSTIFGIAMALIWGIYFAILKKKKK-amide. These peptides contain flanking Lys residues to facilitate purification and handling of the peptide. Circular dichroism measurements demonstrated that the mutated peptide has increased helical content compared with the wild type both in the presence and absence of lipid. The fluorescence emission from the Trp residues in the peptide showed significant blue shifts in the presence of liposomes, however the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. Our overall findings support our studies with the intact protein in cells and suggest that the peptide of WT caveolin-1 hydrophobic segment has an intrinsic preference not to maintain its conformation as a rigid transmembrane helix. Substituting the Pro residue with an Ala allows the peptide to exist in a more hydrophobic environment likely as a consequence of a change in its conformation to a straight hydrophobic helix that traverses the membrane.