Conserved Role of unc-79 in Ethanol Responses in Lightweight Mutant Mice

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.     

Phellinus Linteus Extract Sensitizes Advanced Prostate Cancer Cells to Apoptosis in Athymic Nude Mice

Phellinus linteus (PL) mushroom possesses anti-tumor property. We previously reported that the treatment with PL caused cultured human prostate cancer cells to undergo apoptosis. To further studying the mechanisms of PL-mediated apoptosis, we performed xenograft assay, together with in vitro assays, to evaluate the effect of PL on the genesis and progression of the tumors formed from the inoculation of prostate cancer PC3 or DU145 cells. After the inoculation, nude mice were injected with PL every two days for 12 days. Although PL treatment did not prevent the formation of the inoculated tumors, the growth rate of the tumors after PL treatment was dramatically attenuated. We then tested the effect of PL on the tumors 12 days after the inoculation. After inoculated tumors reached a certain size, PL was administrated to the mice by subcutaneous injection. The histochemistry or immunochemistry analysis showed that apoptosis occurred with the activation of caspase 3 in the tumors formed by inoculating prostate cancer DU145 or PC3 cells. The data was in a good agreement with that from cultured cells. Thus, our in vivo study suggests that PL not only is able to attenuate tumor growth, but also to cause tumor regression by inducing apoptosis.     

Diversity of the pufferfish Takifugu rubripes fast skeletal myosin heavy chain genes

Myosin is a highly conserved, ubiquitous actin-based molecular motor that is distributed as diverse as from prokaryotes to mammalian tissues. Among various types in the myosin family proteins, class II, also called sarcomeric, myosin is a classical, conventional molecule that has been extensively studies on its functional and structural properties. It consists of two heavy chains (MYH) of about 200 kDa and four light chains of about 20 kDa. The exon–intron organization was determined for the major subunit of MYH, which contains ATP-hydrolysis and actin-binding sites, from torafugu (tiger pufferfish) Takifugu rubripes fast skeletal muscles. Comprehensive investigation for fast skeletal MYHs based on the fugu (torafugu) genome database and subsequent construction of their physical map revealed that torafugu contains at least 8 putative skeletal MYHs. Furthermore, genomic structural analysis revealed that skeletal MYHs are not clustered in a single locus, but rather spread to at least four loci, with two of them locating at the mammalian syntenic regions. Such arrangement of torafugu MYHs are in a marked contrast to mammalian fast skeletal MYHs that are clustered in a single locus. These data suggest that an ancient segmental duplication or whole-genome duplication occurred in fish lineage as in many other reported torafugu genes.     

Spo5/Mug12, a putative meiosis-specific RNA-binding protein, is essential for meiotic progression and forms Mei2 dot-like nuclear foci

We report here a functional analysis of spo5(+)(mug12(+)) of Schizosaccharomyces pombe, which encodes a putative RNA-binding protein. The disruption of spo5(+) caused abnormal sporulation, generating inviable spores due to failed forespore membrane formation and the absence of a spore wall, as determined by electron microscopy. Spo5 regulates the progression of meiosis I because spo5 mutant cells display normal premeiotic DNA synthesis and the timely initiation of meiosis I but they show a delay in the peaking of cells with two nuclei, abnormal tyrosine 15 dephosphorylation of Cdc2, incomplete degradation of Cdc13, retarded formation and repair of double strand breaks, and a reduced frequency of intragenic recombination. Immunostaining showed that Spo5-green fluorescent protein (GFP) appeared in the cytoplasm at the horsetail phase, peaked around the metaphase I to anaphase I transition, and suddenly disappeared after anaphase II. Images of Spo5-GFP in living cells revealed that Spo5 forms a dot in the nucleus at prophase I that colocalized with the Mei2 dot. Unlike the Mei2 dot, however, the Spo5 dot was observed even in sme2Delta cells. Taken together, we conclude that Spo5 is a novel regulator of meiosis I and that it may function in the vicinity of the Mei2 dot.     

Preparation of enzymatically active human Myc-tagged-NCre recombinase exhibiting immunoreactivity with anti-Myc antibody

The Cre-loxP system has been recognized as a tool for conditional gene targeting in mice. However, most anti-Cre antibodies fail to react with Cre expressed in vivo. In an attempt to directly detect Cre by antibodies in vivo, we constructed the tagged-NCre (NCreMH) gene by connecting the human Myc and His tag sequences to the 3' end of the NCre gene carrying a nuclear localizing signal (NLS) sequence. The production of NCre protein and the recombinase activity were detected after co-transfection with pCMV-NCreMH and pCETZ-17 carrying the loxP-flanked lacZ gene into NIH3T3 cells. This activity was also confirmed in vivo after gene transfer of pCMV-NCreMH and pCRTEIL-6 carrying loxP-flanked HcRed1 and EGFP cDNAs, into oviductal epithelium by electroporation. Immunohistochemical staining using anti-Myc antibody demonstrated that the area positive for enhanced green fluorescent protein (EGFP) fluorescence was immunostained with the antibody. These findings indicate that NCreMH is useful as an alternative to NCre for gene targeting.     

Suramin prevents fulminant hepatic failure resulting in reduction of lethality through the suppression of NF-kappaB activity

AIM: Suramin is a symmetrical polysulfonated naphthylamine derivative of urea. There have been few studies on the effect of suramin on cytokines. We examined the effects of suramin on production of inflammatory cytokines. METHODS: We made an acute liver injury model treated with d-galactosamine (GalN) and lipopolysaccharide (LPS). Plasma AST, ALT, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 levels were measured. We compared with survival rate, histological found and NF-kappaB activity between with and without treatment of suramin. In macrophage like cell line, TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity was measured. RESULTS: The lethality of mice administered suramin with GalN/LPS was significantly decreased compared with that in mice without suramin. Changes of hepatic necrosis and apoptosis were slight in suramin-treated mice. Serum AST, ALT, TNF-alpha, IL-6 levels and NF-kappaB activity in the liver were significantly lower in mice administered suramin. In an in vitro model, suramin preincubation inhibited TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity. CONCLUSIONS: Suramin inhibits TNF-alpha and IL-6 production through the suppression of NF-kappaB activity from macrophages and shows therapeutic effects on acute liver damage.     

Recognition of oxidized thymine base on the single-stranded DNA by replication protein A

Replication protein A (RAP) is a eukaryotic single-stranded DNA binding protein involved in DNA replication, repair, and recombination. Recent studies indicate that RPA preferentially binds the damaged sites rather than the undamaged sites. Therefore, RPA is thought to be a member ofrepair factories or a sensor of lesion on DNA. To obtain further information of behavior of RPA against the oxidized lesion, we studied the binding affinity of RPA for the single-stranded DNA containing 5-formyluracil, a major lesion of thymine base yielded by the oxidation, using several synthetic oligonucleotides. The affinity of RPA for oligonucleotides was determined by gel shift assay. Results suggest that the surrounding sequence of 5-formyluracil may affect the affinity for RPA, and that the 5-formyluracil on the purine stretch but not the pyrimidine stretch increases the affinity for RPA. Results of affinity labeling experiment of RPA with the oligonucleotides containing 5-formyluracil indicate that RPA1 subunit may directly recognize and bind to the 5-formyluracil on the single-stranded DNA.     

Characterization of aggregate structure in mercerized cellulose/LiCl.DMAc solution using light scattering and rheological measurements

The structure of a semidilute solution of mercerized cellulose (CC1m) in 8% (w/w) LiCl.DMAc, which contained some aggregates, was investigated using static and dynamic light scattering measurements. The static scattering function of the polymer solution containing a small amount of aggregates can be separated into fast- and slow-mode components by combining static and dynamic light scattering measurements. The osmotic modulus was identical for the fast-mode component of the CC1m solutions and the native cellulose (CC1) solutions, in which cellulose is dispersed molecularly. This indicates that the molecularly dispersed component of the CC1m solutions has an identical conformation with the cellulose molecules in the CC1 solutions. The correlation length was also identical for the fast-mode components of CC1m solutions and the CC1 solutions, indicating that these solutions have the same mesh size of the polymer entanglement. These observations for the fast-mode components are consistent with the concentration dependence of the zero shear rate viscosity and the plateau modulus estimated in the rheological measurements. The slow-mode component, on the other hand, gave information on the aggregate structure in the CC1m solution. The radius of gyration of the aggregate structure estimated from the slow-mode component was about 70 nm, which is independent of the concentration of the solution. The plots for particle scattering factor of the slow-mode component lay between the theoretical curve of a sphere and a Gaussian chain, implying that the structure of the aggregate in the CC1m solution is like a multiarm polymer. A characteristic time of the slow-mode component calculated with the translational diffusion coefficient and the radius of gyration were almost identical with the relaxation time of the long-time relaxation observed in the rheological measurements. This indicates that the long-time relaxation of CC1m solutions originates in the translational diffusion of the aggregate structure in the solution.