Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10′-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

The Contribution of Diet and Genotype to Iron Status in Women: A Classical Twin Study

This is the first published report examining the combined effect of diet and genotype on body iron content using a classical twin study design. The aim of this study was to determine the relative contribution of genetic and environmental factors in determining iron status. The population was comprised of 200 BMI- and age-matched pairs of MZ and DZ healthy twins, characterised for habitual diet and 15 iron-related candidate genetic markers. Variance components analysis demonstrated that the heritability of serum ferritin (SF) and soluble transferrin receptor was 44% and 54% respectively. Measured single nucleotide polymorphisms explained 5% and selected dietary factors 6% of the variance in iron status; there was a negative association between calcium intake and body iron (p = 0.02) and SF (p = 0.04).     

Presynaptic to postsynaptic relationships of the neuromuscular junction are held constant across age and muscle fiber type

The neuromuscular junction (NMJ) displays considerable morphological plasticity as a result of differences in activity level, as well as aging. This is true of both presynaptic and postsynaptic components of the NMJ. Yet, despite these variations in NMJ structure, proper presynaptic to postsynaptic coupling must be maintained in order for effective cell‐to‐cell communication to occur. Here, we examined the NMJs of muscles with different activity profiles (soleus and EDL), on both slow‐ and fast‐twitch fibers in those muscles, and among young adult and aged animals. We used immunofluorescent techniques to stain nerve terminal branching, presynaptic vesicles, postsynaptic receptors, as well as fast/slow myosin heavy chain. Confocal microscopy was used to capture images of NMJs for later quantitative analysis. Data were subjected to a two‐way ANOVA (main effects for myofiber type and age), and in the event of a significant (p < 0.05) F ratio, a post hoc analysis was performed to identify pairwise differences. Results showed that the NMJs of different myofiber types routinely displayed differences in presynaptic and postsynaptic morphology (although the effect on NMJ size was reversed in the soleus and the EDL), but presynaptic to postsynaptic relationships were tightly maintained. Moreover, the ratio of presynaptic vesicles relative to nerve terminal branch length also was similar despite differences in muscles, their fiber type, and age. Thus, in the face of considerable overall structural differences of the NMJ, presynaptic to postsynaptic coupling remains constant, as does the relationship between presynaptic vesicles and the nerve terminal branches that support them. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 744–753, 2013     

Intraspecific Relationships among Wood Density,Leaf Structural Traits and Environment in Four Co-Occurring Species of Nothofagus in New Zealand

Plant functional traits capture important variation in plant strategy and function. Recent literature has revealed that within-species variation in traits is greater than previously supposed. However, we still have a poor understanding of how intraspecific variation is coordinated among different traits, and how it is driven by environment. We quantified intraspecific variation in wood density and five leaf traits underpinning the leaf economics spectrum (leaf dry matter content, leaf mass per unit area, size, thickness and density) within and among four widespread Nothofagus tree species in southern New Zealand. We tested whether intraspecific relationships between wood density and leaf traits followed widely reported interspecific relationships, and whether variation in these traits was coordinated through shared responses to environmental factors. Sample sites varied widely in environmental variables, including soil fertility (25–900 mg kg^–1 total P), precipitation (668–4875 mm yr^–1), temperature (5.2–12.4 °C mean annual temperature) and latitude (41–46 °S). Leaf traits were strongly correlated with one another within species, but not with wood density. There was some evidence for a positive relationship between wood density and leaf tissue density and dry matter content, but no evidence that leaf mass or leaf size were correlated with wood density; this highlights that leaf mass per unit area cannot be used as a surrogate for component leaf traits such as tissue density. Trait variation was predicted by environmental factors, but not consistently among different traits; e.g., only leaf thickness and leaf density responded to the same environmental cues as wood density. We conclude that although intraspecific variation in wood density and leaf traits is strongly driven by environmental factors, these responses are not strongly coordinated among functional traits even across co-occurring, closely-related plant species.     

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF^Cdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF^Cdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF^Cdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF^Cdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.     

Synthesis,characterization, and biological assessment of the four stereoisomers of the H3 receptor antagonist 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3′]bipyrrolidinyl-1′-yl)phenyl]benzamide

This Letter describes the asymmetric synthesis of the four stereoisomers (8a–8d) of a potent and highly selective histamine H₃ receptor (H₃R) antagonist, 5-fluoro-2-methyl-N-[2-methyl-4-(2-methyl[1,3′]bipyrrolidinyl-1′-yl) phenyl]benzamide (1). The physico-chemical properties, in vitro H₃R affinities and ADME of 8a–8d were determined. Stereoisomer 8c (2S,3′S) displayed superior in vitro H₃R affinity over other three stereoisomers and was selected for further profiling in in vivo PK and drug safety. Compound 8c exhibited excellent PK properties with high exposure, desired brain to plasma ratio and reasonable brain half life. However, all stereoisomers showed similar unwanted hERG affinities.     

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (Part I)

A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective histamine H₃ receptor (H₃R) antagonists. The SAR was explored and the data obtained set up the starting point and foundation for further optimization. The most potent tool compounds, as exemplified by compounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-H₃R FLIPR assays and rhesus monkey H₃R binding assays.