A murine model for the assessment of placental and fetal development in teratogenicity studies

During normal pregnancy in the mouse, maternal serum levels of the analogues to human schwangerschaftsprotein-1 and alpha-fetoprotein correlate significantly with the growth of the placenta and fetus respectively. This relationship has been utilized in the analysis of the effect of sodium selenite on placental and fetal growth in mice. Moderate doses of sodium selenite did not affect the growth of the placenta and fetus significantly, whereas high doses of selenite resulted in a large percentage of abortions. The protein markers were found to be useful in the prediction of placental and fetal growth, and they are suggested to be of general use in the study of the impact of teratogenic substances, since they reflect the status of the fetoplacental mass during gestation.     

Simultaneous and sympatric occurrence of early juveniles of Acanthopagruslatus and A.schlegelii (Sparidae) in the estuary of northern Vietnam

Limnology - Acanthopagruslatus and A.schlegelii (Sparidae) are closely related species distributed in northern tropical to temperate coastal waters of eastern Asia. Early life histories of the two...     

Effect of photoperiod on incubation period in a wild passerine,Sylvia atricapilla

Time required for avian embryos to develop is influenced by incubation temperature and the amount of time adults incubate eggs. Experiments on poultry indicate that photoacceleration, the light‐induced stimulation of embryonic development, decreases the length of the incubation period as embryos receive more light. We hypothesized that eggs of wild birds exposed to longer periods of light should also have shorter incubation periods. We tested whether photoacceleration would occur in a species of open‐cup nesting passerine, the blackcap Sylvia atricapilla. We artificially incubated blackcap eggs under four different photoperiods, four hours of light (4L) and 20 h of dark (20D), 12L:12D, 20L:4D, and a skeleton photoperiod (1 h light, 2 times per day) that framed a 20 h day. While incubation periods were accelerated with increasing photoperiod length, the differences among photoperiods of 4, 12 and 20L were weak. Embryos exposed to skeleton photoperiods developed as fast as those exposed to 20L and significantly faster than those exposed to 4L and 12L treatments. Skeleton photoperiods may most closely approximate natural patterns of light exposure that embryos experience during dawn and dusk incubation recesses typically associated with adult foraging. If our results from this species also occur in other wild birds, exposure to different day lengths may help explain some of the variation in the observed seasonal and latitudinal trends in avian incubation period.     

Simultaneous Protein Expression and Modification: An Efficient Approach for Production of Unphosphorylated and Biotinylated Receptor Tyrosine Kinases by Triple Infection in the Baculovirus Expression System

Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin–biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and site-specific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for enzymatic and biophysical studies.     

New Aspects of an Old Drug – Diclofenac Targets MYC and Glucose Metabolism in Tumor Cells

Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies.     

Different Genetic Associations of the IgE Production among Fetus,Infancy and Childhood

Elevation of serum IgE levels has long been associated with allergic diseases. Many genes have been linked to IgE production, but few have been linked to the developmental aspects of genetic association with IgE production. To clarify developmental genetic association, we investigated what genes and gene-gene interactions affect IgE levels among fetus, infancy and childhood in Taiwan individuals. A birth cohort of 571 children with completion of IgE measurements from newborn to 1.5, 3, and 6 years of age was subject to genetic association analysis on the 384-customized SNPs of 159 allergy candidate genes. Fifty-three SNPs in 37 genes on innate and adaptive immunity, and stress and response were associated with IgE production. Polymorphisms of the IL13, and the HLA-DPA1 and HLA-DQA1 were, respectively, the most significantly associated with the IgE production at newborn and 6 years of age. Analyses of gene-gene interactions indentified that the combination of NPSR1, rs324981 TT with FGF1, rs2282797 CC had the highest risk (85.7%) of IgE elevation at 1.5 years of age (P = 1.46×10⁻⁴). The combination of IL13, CYFIP2 and PDE2A was significantly associated with IgE elevation at 3 years of age (P = 5.98×10⁻⁷), and the combination of CLEC2D, COLEC11 and CCL2 was significantly associated with IgE elevation at 6 years of age (P = 6.65×10⁻⁷). Our study showed that the genetic association profiles of the IgE production among fetus, infancy and childhood are different. Genetic markers for early prediction and prevention of allergic sensitization may rely on age-based genetic association profiles.     

Mice Do Not Habituate to Metabolism Cage Housing–A Three Week Study of Male BALB/c Mice

The metabolism cage is a barren, non-enriched, environment, combining a number of recognized environmental stressors. We investigated the ability of male BALB/c mice to acclimatize to this form of housing. For three weeks markers of acute and oxidative stress, as well as clinical signs of abnormality were monitored. Forced swim tests were conducted to determine whether the animals experienced behavioral despair and the serotonergic integrity was tested using an 8-OH-DPAT challenge. The metabolism cage housed mice excreted approximately tenfold higher amounts of corticosterone metabolites in feces throughout the study when compared to controls. Urinary biomarkers confirmed that these mice suffered from elevated levels of oxidative stress, and increased creatinine excretions indicated increased muscle catabolism. Changes in the core body temperature (stress-induced hyperthermia) and the fur state of the mice also indicated impaired well-being in the metabolism cage housed mice. However, monitoring body weight and feed intake was found misleading in assessing the wellbeing of mice over a longer time course, and the forced swim test was found poorly suited for studying chronic stress in mice in the present setup. In conclusion, the mice were found not to acclimatize to the metabolism cages whereby concern for animal welfare would dictate that mice should be housed in this way for as short periods as possible. The elevated degree of HPA axis activity, oxidative stress, and increased overall metabolism warrant caution when interpreting data obtained from metabolism cage housed mice, as their condition cannot be considered representative of a normal physiology.     

Efficacy of Acibenzolar‐S‐Methyl and β‐Aminobutyric Acid for Control of Downy Mildew in Greenhouse Grown Basil and Peroxidase Activity in Response to Treatment with these Compounds

Downy mildew, caused by the oomycete pathogen Peronospora belbahrii, is a devastating foliar disease of basil in the United States and worldwide. Currently there are very few chemistries or organic choices registered to control this disease. In this study, two systemic acquired resistance (SAR) inducers, acibenzolar‐S‐methyl (ASM) and β‐aminobutyric acid (BABA), were evaluated for their in vitro effects on the pathogen, for their potential to control basil downy mildew in greenhouses, and for changes in peroxidase activity in basil plants treated with these two SAR inducers. No significant inhibition of sporangial germination was detected in water agar amended with ASM at concentrations lower than 100 mg/l or with BABA at concentrations lower than 500 mg/l. Efficacy of ASM and BABA in greenhouses varied depending on the rate, method and timing of application. The area under the disease progress curve (AUDPC) of disease severity was significantly reduced compared to the non‐treated control when ASM was sprayed (in all experiments) or drenched (in one out of two experiments) pre‐, or pre‐ + post‐inoculation at rates of 25–400 mg/l. Three weekly post‐inoculation sprays of ASM at the rate of 50 mg/l reduced AUDPC by 93.0 and 47.2% when started 3 and 7 days after inoculation (DAI), respectively. The AUDPC of disease severity was also significantly reduced when BABA was sprayed pre‐ + post‐inoculation at rates of 125–500 mg/l. According to the prediction using a log‐logistic function, 50% maximum disease protection was achieved at a concentration of 27.5 mg/l of ASM. Basil plants treated with these two SAR inducers and challenged with the pathogen showed significantly higher peroxidase activity than the non‐treated control at 8 DAI. Temporally, the highest activity of peroxidase was detected at 8 DAI, decreased at 15 DAI and waned further at 23 DAI.