Simple Method of Zygosity Identification in Transgenic Mice by Real-time Quantitative PCR

To determine zygosity in transgenic (Tg) mice, a new technology, real-time quantitative PCR, has recently been introduced in transgenic research to overcome several drawbacks (time-consuming, specialized techniques and/or ambiguity in the results) of previously established methods, for example, Southern blot hybridization, dot blot hybridization, fluorescence in situ hybridization (FISH), etc. However, the previous real-time quantitative PCR method still possesses several drawbacks, for example, it needs two sets of primers/probes and the complicated setting up of appropriate conditions, both of which are expensive and remain time-consuming. We therefore developed an improved real-time quantitative PCR system for determination of zygosity, which is easy, rapid and less expensive, because the technique needs only two experimental processes: estimation of DNA concentration and CYBR Green PCR. We found that homozygous, hemizygous and non-Tg animals could easily be distinguished among F1 littermates in crosses of hemizygous EGFP- and DsRed2-Tg mice. Our improved method will be applicable to any Tg mouse strains, when a primer set is matched to the corresponding transgene.     

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.     

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL)

BACKGROUND: Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. METHODS: A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. RESULTS: We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. CONCLUSIONS: PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity.     

Addition and reexamination of Japanese species belonging to the genus <Emphasis Type="Italic">Cercospora</Emphasis> and allied genera. VI. Four <Emphasis Type="Italic">Pseudocercospora</Emphasis> species from Ohshima Island,Tokyo

Four fungi belonging to the genus Pseudocercospora were collected from Ohshima Island, which has commonly been called Izu-ohshima, Tokyo. Among them, two on Helwingia japonica and on Stachyurus praecox var. matsuzakii are recognized as new species, and named P. izuohshimensis and P. hachijokibushi, respectively. Styrax japonica var. jippei-kawamurae was newly added to the host list of P. fukuokaensis. Presence of P. myrticola in Japan was reconfirmed in examining a fresh diseased material on Myrtus communis.     

Biological and inherited characteristics of a newly identified <Emphasis Type="Italic">Lentinula edodes</Emphasis> strain capable of forming a fruiting body without a shift to low temperature

A strain of the basidiomycete Lentinula edodes (Shiitake) was newly identified from the mushroom library of Mori Sangyo Co., Ltd., Japan. This strain, named MIL-LEW-M13-1, is capable of forming the fruiting body on sawdust-based medium without a reduction in temperature. Mating experiments with a monokaryotic mycelium of L. edodes strain that does require low temperature for fruiting–body formation suggest that the unique property of the MIL-LEW-M13-1 strain is a dominant trait that can be inherited by its progeny in a nucleus-dependent manner.     

A major determinant quantitative-trait locus responsible for atopic dermatitis-like skin lesions in NC/Nga mice is located on Chromosome 9

NC/Nga (NC) is a newly discovered model mouse for human atopic dermatitis, NC mice showing specific symptoms such as dermatitis and overproduction of IgE. To detect the loci responsible for the onset of dermatitis in the mice, backcross (N2) progeny between (NCxMSM/MS)F1 and NC were generated, where MSM/MS is an inbred strain from Japanese wild mice, Mus musculus molossinus. Linkage disequilibrium between dermatitis and various chromosome-specific microsatellite markers was then examined in the N2 segregants with severe dermatitis. The analysis revealed that the locus of the major determinant (designated here as derm1) was tightly linked to D9Mit163, D9Mit72, D9Mit143, D9Mit103, D9Mit207, and D9Mit209, because these markers showed the highest and most significant chi2 values. Since no recombination was observed among the markers in our linkage map, a radiation hybrid (RH) panel was applied to locate the derm1 locus more precisely. The markers were separated on the RH map, and their order was D9Mit163-D9Mit72-D9Mit143-D9Mit103-D9Mit207-D9Mit209 from the centromere. Several functional candidate genes are located near the locus derm1. These candidates are Thy1, Cd3d, Cd3e, Cd3g, Il10ra, 1118, and Csk, all of which could be involved in allergic responses through effects on T-cell function. Of these candidates, Csk is the strongest for NC dermatitis, since its map position was most tightly linked to the derm1 locus.     

Global tyrosine kinome profiling of human thyroid tumors identifies Src as a promising target for invasive cancers

Background: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using a bead-based, high-throughput system.Methods: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells.Results: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p < 0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation.Conclusion: Global kinome analysis enables the discovery of novel targets for thyroid cancer therapy. Further investigation of Src targeted therapy for advanced thyroid cancer is warranted.     

Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer

Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells.     

Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection

Salmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogen-activated protein kinases (MAPK) through β-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti-inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.     

Mutants of Ficus pumila produced by ion beam irradiation with an improved ability to uptake and assimilate atmospheric nitrogen dioxide

Production of novel mutants with a high ability to mitigate pollutants is important for phytoremediation. We investigated the use of ion beam irradiation to produce mutants of Ficus pumila L. with an improved ability to mitigate atmospheric nitrogen dioxide (NO2). More than 25,000 shoot explants were irradiated with an ion beam (12C5+, 12C6+, or 4He2+), from which 263 independent plant lines were obtained. The plants were analyzed for NO2 uptake by fumigation with 1 ppm 15N-labeled NO2 for 8 h in light, followed by mass spectrometric analysis. The mean NO2 uptake values of each of the 263 lines differed over a 110-fold range. Propagation was attempted using cuttings from 44 lines showing the greatest NO2 uptake; in total, 15 lines were propagated. Two of the 15 lines showed a mean NO2 uptake 1.7- to 1.8-fold greater than that of the wild-type. This increase in NO2 uptake was heritable in both lines; their progenies showed a significantly greater ability to take up and assimilate NO2 than did the wild-type. RAPD analysis demonstrated DNA variation between the progeny plants and the wild type, suggesting that the progeny were true mutants. These mutants of F. pumila may prove useful in mitigating atmospheric NO2.