SOX2-Dependent Transcription in Clock Neurons Promotes the Robustness of the Central Circadian Pacemaker

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Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.     

A prospective study of breakfast consumption and weight gain among U.S. men

Objective: The aim was to investigate the association between breakfast consumption and long‐term weight gain in an adult male population. Research Methods and Procedures: We evaluated prospective data on 20,064 U.S men, 46 to 81 years of age, who participated in the Health Professionals Follow‐up Study. Data on body weight, dietary factors, and lifestyle variables were obtained by validated questionnaires. We examined weight gain during 10 years of follow‐up. Results: Overall, 5857 men had a weight gain of 5 kg or greater during 10 years of follow‐up. Breakfast consumption was inversely associated with the risk of 5‐kg weight gain after adjustment for age [hazard ratio (HR) = 0.77 (95% confidence interval [CI], 0.72 to 0.82)], and this association was independent of lifestyle and BMI at baseline [HR = 0.87 (95% CI, 0.82 to 0.93)]. Fiber and nutrient intakes partially explained the association between breakfast consumption and weight gain. The inverse association between breakfast consumption and weight gain was more pronounced in men with a baseline BMI of 25 kg/m² or lower [multivariate HR = 0.78 (95% CI, 0.70 to 0.87)] than in men who were overweight at baseline [HR = 0.92 (95% CI, 0.85 to 1.00)]. Furthermore, we observed that an increasing number of eating occasions in addition to three standard meals was associated with a higher risk of 5‐kg weight gain [HR = 1.15 (95% CI, 1.06 to 1.25, for ≥2 vs. 0 additional eating occasions)]. Discussion: These findings suggest that the consumption of breakfast may modestly contribute to the prevention of weight gain as compared with skipping breakfast in middle‐aged and older men.     

Validation of an HIV-1 inactivation protocol that is compatible with intracellular drug analysis by mass spectrometry

Mass spectrometry is a powerful tool for studying the intracellular pharmacokinetics of antiretroviral drugs. However, the biohazard of HIV-1 calls for a safety protocol for such analyses. To this end, we extracted HIV-1 producing cells with methanol or ethanol at 4 degrees C. After extraction, no viral infectivity was detected, as shown by a reduction in infectious titers of more than 6log. In addition, this protocol is compatible with the quantitative analysis of antiretroviral drugs in cell extracts using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Thus, using this protocol, infectious HIV-1 is inactivated and antiretroviral drugs are extracted from cells in a single step.     

NMR-based metabolomics at work in phytochemistry

Metabolomics is defined as both the qualitative and quantitative analysis of all metabolites in an organism unraveling correlation with other OMICs data. Many of the technologies used in metabolomics have method-specific advantages and drawbacks in terms of diversity of metabolites detected, sensitivity, or resolution. In this paper, the potential of NMR spectrometry applied to metabolomics is reviewed using examples of Nicotiana tabacum and Catharanthus roseus.     

Role of vacuolar transporter proteins in plant secondary metabolism: Catharanthus roseus cell culture

Here the current status of knowledge on some well-characterized transporters located in the vacuolar membrane is reviewed. As different cellular compartments and even different cells may be involved in certain steps of a biosynthetic pathway, the regulation of the flux is not only dependent on structural genes encoding enzymes catabolizing certain steps but also transport has a major regulatory function. The aim of the present review is to give an overview of the present knowledge of transport of secondary metabolites in plants, and to use this information in the context of our knowledge about Catharanthus roseus alkaloid biosynthesis. This should lead to further insight in the possible role of various transporters in the regulation of the biosynthesis of these alkaloids.     

Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

Exposure of neural crest cells to elevated glucose leads to congenital heart defects, an effect that can be prevented by N-acetylcysteine

BACKGROUND: Diabetes mellitus during pregnancy increases the risk for congenital heart disease in the offspring. The majority of the cardiovascular malformations occur in the outflow tract and pharyngeal arch arteries, where neural crest cells are essential for normal development. We studied the effects of specific exposure of neural crest cells to elevated glucose on heart development. Antioxidants reduce the damaging effect of glucose on neural crest cells in vitro; therefore, we investigated the effect of supplementing N-acetylcysteine in vivo. METHODS: Cardiac neural crest of HH 8-12 chicken embryos was directly exposed by a single injection in the neural tube with 30 mM D-glucose (or 30 mM L-glucose as a control). To examine the effect of a reduction in oxidative stress, we added 2 mM N-acetylcysteine to the injected D-glucose. RESULTS: Exposure of neural crest cells to elevated D-glucose-induced congenital heart malformations in 82% of the embryos. In the embryos injected with L-glucose, only 9% developed a heart malformation. As expected, all malformations were located in the outflow tract and pharyngeal arch arteries. The frequency of heart malformations decreased from 82% to 27% when 2 mM N-acetylcysteine was added to the injected D-glucose. CONCLUSIONS: These data are the first to confirm that the vulnerability of neural crest cells to elevated glucose induces congenital heart malformations. The fact that N-acetylcysteine limits the teratogenicity of glucose implies that its damaging effect is mediated by an increase of oxidative stress in the neural crest cells.