An improved system for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay

A gas exposure system using rotating vessels was improved for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay. This system was composed of 12 square culture vessels, a device for preparation of air containing test gas, and positive and negative control gases at target concentrations and for supplying these gases to the culture vessels, and a roller apparatus in an incubator. Chinese hamster lung cells (CHL/IU) were grown on one side of the inner surface of the square culture vessel in the MEM medium. Immediately prior to exposure, the medium was changed to the modified MEM. Air in the culture vessel was replaced with air containing test gas, positive or negative control gas. Then, the culture vessels were rotated at 1.0 rpm. The monolayered culture cells were exposed to test gas during about 3/4 rotation at upper positions and alternatively immersed into the culture medium during about 1/4 rotation at lower positions. This system allowed the chromosomal aberration assay simultaneously at least at three different concentrations of a test gas together with positive and negative control gases with and without metabolic activations, and duplicate culture at each exposure concentration. Seven gaseous compounds, 1,3-butadiene, chlorodifluoromethane, ethyl chloride, methyl bromide, methyl chloride, propyne, and vinyl chloride, none of which has been tested to date, were tested on CHL/IU for the chromosomal aberration assay using this gas exposure system. All the compounds except chlorodifluoromethane showed positive responses of the structural chromosomal aberrations, whereas polyploidy was not induced by any of these gases. This improved gas exposure system proved to be useful for detecting chromosomal aberrations of gaseous compounds.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes.

BACKGROUND INFORMATION: Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys(189) as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury-insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys(189) of AQP1. Indeed, the osmotic water permeability (P(f)) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl2. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His-tagged rAQP4 [corrected] (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni2+-nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. RESULTS: The water permeability of AQP4 proteoliposomes with or without HgCl2 was measured with a stopped-flow apparatus. Surprisingly, the P(f) of AQP4 proteoliposomes was significantly decreased by 5 microM HgCl2 within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The dose- and time-dependent inhibitory effects of Hg2+ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site-directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys(178), which is located at loop D facing the intracellular side, is a target responding to Hg2+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. CONCLUSIONS: Our results suggest that mercury inhibits the P(f) of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

Phenotypic Characterization of the Komeda Miniature Rat Ishikawa, an Animal Model of Dwarfism Caused by a Mutation in Prkg2

The Komeda miniature rat Ishikawa (KMI) is a spontaneous animal model of dwarfism caused by a mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII). This strain has been maintained as a segregating inbred strain for the mutated allele mri. In this study, we characterized the phenotype of the KMI strain, particularly growth traits, craniofacial measurements, and organ weights. The homozygous mutant (mri/mri) animals were approximately 70% to 80% of the size of normal, heterozygous (mri/+) animals in regard to body length, weight, and naso-occipital length of the calvarium, and the retroperitoneal fat of mri/mri rats was reduced greatly. In addition, among progeny of the (BN×KMI-mri/mri)F1×KMI-mri/mri backcross, animals with the KMI phenotype (mri/mri) were easily distinguished from those showing the wild-type phenotype (mri/+) by using growth traits such as body length and weight. Genetic analysis revealed that all of the backcrossed progeny exhibiting the KMI phenotype were homozygous for the KMI allele in the 1.2-cM region between D14Rat5 and D14Rat80 on chromosome 14, suggesting strongly that mri acts in a completely recessive manner. The KMI strain is the first and only rat model with a confirmed mutation in Prkg2 and is a valuable model for studying dwarfism and longitudinal growth traits in humans and for functional studies of cGKII.Abbreviations: cGKII, cGMP-dependent protein kinase type II; CNP, C-type natriuretic peptide; KMI, Komeda miniature rat IshikawaDwarfism is caused by both endocrinologic and nonendocrinologic defects. Most instances of dwarfism, including normal variants, are nonendocrinologic, and subjects retain growth hormone secretion. Although spontaneous rodent models of dwarfism with confirmed mutations have been reported—Snell dwarf mice with Pou1f1 (Pit1) mutation,¹⁴ Ames dwarf mice with Prop1 mutation,²² little mice with Ghrhr mutation,¹⁵ pygmy mice (also known as mini-mice) with Hmga2 (HMGI-C) mutation,²⁶ spontaneous dwarf rats with Gh mutation,²³ and rdw rats with Tg mutation^9,11—most of these are models of endocrinologic dwarfism. A few models of nonendocrinologic dwarfism have been produced by gene manipulation techniques, such as transgenic and knockout strategies, and include Col2a1-transgenic mice,^7,24 Col10a1-transgenic mice,¹⁰ and Fgfr3-knock-in mice.¹³A novel spontaneous dwarf mutation, miniature rat Ishikawa (mri), was discovered in a closed colony of Wistar rats at Ishikawa Animal Laboratory (Saitama, Japan) and has been maintained on the genetic background of Wistar rats. This mutant strain, previously termed Miniature Rat Ishikawa (MRI), has recently been established as a segregating inbred strain on the Wistar genetic background, designated Komeda Miniature rat Ishikawa (KMI). The breeding record suggested that the mutation was inherited in an autosomal recessive mode. KMI rats show no abnormality in the basal amounts or distribution of several hormones, including growth hormone, luteinizing hormone, follicle-stimulating hormone, prolactin, thyroid-stimulating hormone, and adrenocorticotropic hormone, but growth hormone response to growth hormone releasing hormone is decreased.²¹Using positional candidate cloning of mri, we recently identified a deletion mutation in Prkg2, which encodes cGMP-dependent protein kinase type II (cGKII), and clarified a role of cGKII as a molecular switch that couples cessation of proliferation and the start of hypertrophic differentiation of chondrocytes.² Longitudinal skeletal growth is achieved by endochondral ossification in the growth plate, in which chondrocyte hypertrophic differentiation is an important step. Due to the impaired coupling of proliferation and hypertrophic differentiation in the growth plate chondrocytes, homozygous mutant (mri/mri) animals show longitudinal growth retardation.In this study, we further characterize the phenotype of the KMI strain, including body length, body weight, organ weight, and craniofacial measurements. Furthermore, we describe phenotypic characteristics of the progeny produced from the (BN×KMI-mri/mri)F1×KMI-mri/mri backcross and provide updated genetic, physical, and comparative maps of the mri region.     

Connexin hemichannel composition determines the FGF-1-induced membrane permeability and free [Ca2+]i responses

Cell surface hemichannels (HCs) composed of different connexin (Cx) types are present in diverse cells and their possible role on FGF-1-induced cellular responses remains unknown. Here, we show that FGF-1 transiently (4-14 h, maximal at 7 h) increases the membrane permeability through HCs in HeLa cells expressing Cx43 or Cx45 under physiological extracellular Ca(2+)/Mg(2+) concentrations. The effect does not occur in HeLa cells expressing HCs constituted of Cx26 or Cx43 with its C-terminus truncated at aa 257, or in parental nontransfected HeLa cells. The increase in membrane permeability is associated with a rise in HC levels at the cell surface and a proportional increase in HC unitary events. The response requires an early intracellular free Ca(2+) concentration increase, activation of a p38 MAP kinase-dependent pathway, and a regulatory site of Cx subunit C-terminus. The FGF-1-induced rise in membrane permeability is also associated with a late increase in intracellular free Ca(2+) concentration, suggesting that responsive HCs allow Ca(2+) influx. The cell density of Cx26 and Cx43 HeLa transfectants cultured in serum-free medium was differentially affected by FGF-1. Thus, the FGF-1-induced cell permeabilization and derived consequences depend on the Cx composition of HCs.     

The effects of the preference for music on sleep inertia after a short daytime nap

Sleep and Biological Rhythms - The effects of the preference for excitative music on sleep inertia after a daytime nap were examined. Sixteen young healthy adults took a 20-min nap at 14:00 after...     

Heterogeneous packing in the folding/unfolding intermediate state of bitter gourd trypsin inhibitor

The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2-3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.     

Discovery, synthesis and biological evaluation of isoquinolones as novel and highly selective JNK inhibitors (2)

3-Metoxycarbonyl isoquinolone derivative 1 has been identified as a potent JNK inhibitor and significantly inhibited cardiac hypertrophy in a rat pressure-overload model. Herein, a series of isoquinolones with an imidazolylmethyl or a pyrazolylmethyl group at the 2-position were designed based on X-ray crystallographic analysis of the complex between the isoquinolone compound and JNK3, as wells as the relationship between compound lipophilicity (logD) and activity in a cell-based assay. The compounds prepared showed potent JNK1 inhibitory activities in a cell-based assay. Among them the isoquinolone derivative possessing 5-[(cyclopropylamino)carbonyl]-1-methyl-1H-pyrazole (16e) exhibited significant anti-hypertrophic activity at doses of more than 1mg/kg (po) in a pressure-overload model.     

Selection of a novel class of RNA-RNA interaction motifs based on the ligase ribozyme with defined modular architecture

To develop molecular tools for the detection and control of RNA molecules whose functions rely on their 3D structures, we have devised a selection system to isolate novel RNA motifs that interact with a target RNA structure within a given structural context. In this system, a GAAA tetraloop and its specific receptor motif (11-ntR) from an artificial RNA ligase ribozyme with modular architecture (the DSL ribozyme) were replaced with a target structure and random sequence, respectively. Motifs recognizing the target structure can be identified by in vitro selection based on ribozyme activity. A model selection targeting GAAA-loop successfully identified motifs previously known as GAAA-loop receptors. In addition, a new selection targeting a C-loop motif also generated novel motifs that interact with this structure. Biochemical analysis of one of the C-loop receptor motifs revealed that it could also function as an independent structural unit.