Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism

Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 microM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants.     

Nano-mechanical properties of living cells expressing constitutively active RhoA effectors

Filamentous actin and myosin-II are major determinants of cell mechanics and are tightly regulated by a small guanosine triphosphatase, RhoA, and its downstream effectors. We examined the effects of constitutively active mutants of RhoA effectors, which have not been reported before, on cortical stiffness of living cells by using scanning probe microscopy, fluorescence microscopy, and truncated mutants of RhoA effectors labeled with a fluorescent protein. Our data indicated that expression of a constitutively active mutant of Dia1, a formin-family actin polymerizer, enhanced cortical stiffness and increased actin filament quantity in cells. Furthermore, expression of a constitutively active mutant of Rho-associated coiled-coil kinase, a myosin-II activator, softened the cell cortex but increased myosin-II activity. Our findings provide new insights into anomalous mechanics of cells, which is a topic of current interest in a variety of biological research fields.     

NF-κB–inducing kinase (NIK) promotes hyperglycemia and glucose intolerance in obesity by augmenting glucagon action

The canonical inhibitor of nuclear factor κB kinase subunit β (IKK-β)–nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB1) pathway has been well documented to promote insulin resistance; however, the noncanonical NF-κB–inducing kinase (NIK)–NF-κB2 pathway is not well understood in obesity. Additionally, the contribution of counter-regulatory hormones, particularly glucagon, to hyperglycemia in obesity is unclear. Here we show that NIK promotes glucagon responses in obesity. Hepatic NIK was abnormally activated in mice with dietary or genetic obesity. Systemic deletion of Map3k14, encoding NIK, resulted in reduced glucagon responses and hepatic glucose production (HGP). Obesity is associated with high glucagon responses, and liver-specific inhibition of NIK led to lower glucagon responses and HGP and protected against hyperglycemia and glucose intolerance in obese mice. Conversely, hepatocyte-specific overexpression of NIK resulted in higher glucagon responses and HGP. In isolated mouse livers and primary hepatocytes, NIK also promoted glucagon action and glucose production, at least in part by increasing cAMP response element-binding (CREB) stability. Therefore, overactivation of liver NIK in obesity promotes hyperglycemia and glucose intolerance by increasing the hyperglycemic response to glucagon and other factors that activate CREB.     

Damage to photosystem II due to heat stress without light-driven electron flow: involvement of enhanced introduction of reducing power into thylakoid membranes

Under a moderately heat-stressed condition, the photosystems of higher plants are damaged in the dark more easily than they are in the presence of light. To obtain a better understanding of this heat-derived damage mechanism that occurs in the dark, we focused on the involvement of the light-independent electron flow that occurs at 40 °C during the damage. In various plant species, the maximal photochemical quantum yield of photosystem (PS) II (Fv/Fm) decreased as a result of heat treatment in the dark. In the case of wheat, the most sensitive plant species tested, both Fv/Fm and oxygen evolution rapidly decreased by heat treatment at 40 °C for 30 min in the dark. In the damage, specific degradation of D1 protein was involved, as shown by immunochemical analysis of major proteins in the photosystem. Because light canceled the damage to PSII, the light-driven electron flow may play a protective role against PSII damage without light. Light-independent incorporation of reducing power from stroma was enhanced at 40 °C but not below 35 °C. Arabidopsis mutants that have a deficit of enzymes which mediate the incorporation of stromal reducing power into thylakoid membranes were tolerant against heat treatment at 40 °C in the dark, suggesting that the reduction of the plastoquinone pool may be involved in the damage. In conclusion, the enhanced introduction of reducing power from stroma into thylakoid membranes that occurs around 40 °C causes over-reduction of plastoquinone, resulting in the damage to D1 protein under heat stress without linear electron flow.     

Magnetic resonance imaging of a microvascular-interstitium model on a microfluidic device

We report a microvascular-interstitium model on microfluidic devices to study leakage of drugs from blood vessels under in vivo-like flow conditions. We employed magnetic resonance imaging to demonstrate the compatibility of the model for experimental animals and humans. We observed transport of two types of different molecular-weight contrast agents into the model interstitium. The ratio of the transport rates of agents agreed with the ratio calculated from diffusion coefficients of the agents. We expect that the model will be useful for the estimation and evaluation of leakage of many kinds of agents in vivo.