High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.     

Early embryonic development in the spider Achaearanea tepidariorum: Microinjection verifies that cellularization is complete before the blastoderm stage

The spider Achaearanea tepidariorum is emerging as a non-insect model for studying developmental biology. However, the availability of microinjection into early embryos of this spider has not been reported. We defined the early embryonic stages in A. tepidariorum and applied microinjection to its embryos. During the preblastoderm 16- and 32-nucleus stages, the energids were moving toward the egg periphery. When fluorochrome-conjugated dextran was microinjected into the peripheral region of 16-nucleus stage embryos, it was often incorporated into a single energid and inherited in the progeny without leaking out to surrounding energids. This suggested that 16-nucleus stage embryos consisted of compartments, each containing a single energid. These compartments were considered to be separate cells. Fluorochrome-conjugated dextran could be introduced into single cells of 16- to 128-nucleus stage embryos, allowing us to track cell fate and movement. Injection with mRNA encoding a nuclear localization signal/green fluorescent protein fusion construct demonstrated exogenous expression of the protein in live spider embryos. We propose that use of microinjection will facilitate studies of spider development. Furthermore, these data imply that in contrast to the Drosophila syncytial blastoderm embryo, the cell-based structure of the Achaearanea blastoderm embryo restricts diffusion of cytoplasmic gene products.     

Phenotypic plasticity in the range-margin population of the lycaenid butterfly <Emphasis Type="Italic">Zizeeria maha</Emphasis>

Background  

Many butterfly species have been experiencing the northward range expansion and physiological adaptation, probably due to climate warming. Here, we document an extraordinary field case of a species of lycaenid butterfly, Zizeeria maha, for which plastic phenotypes of wing color-patterns were revealed at the population level in the course of range expansion. Furthermore, we examined whether this outbreak of phenotypic changes was able to be reproduced in a laboratory.     

beta1,4-N-Acetylglucosaminyltransferase III potentiates beta1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells

Aspects of the biological significance of the bisecting N-acetylglucosamine (GlcNAc) structure on N-glycans introduced by beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in Neuro2a cell differentiation are demonstrated. The overexpression of GnT-III in the cells led to the induction of axon-like processes with numerous neurites and swellings, in which beta1 integrin was localized, under conditions of serum starvation. This enhancement in neuritogenesis was suppressed by either the addition of a bisecting GlcNAc-containing N-glycan or erythroagglutinating phytohemagglutinin (E(4)-PHA), which preferentially recognizes the bisecting GlcNAc. GnT-III-promoted neuritogenesis was also significantly perturbed by treatment with a functional blocking anti-beta1 integrin antibody. In fact, beta1 integrin was found to be one of the target proteins of GnT-III, as confirmed by a pull-down assay with E(4)-PHA. These data suggest that N-glycans with a bisecting GlcNAc on target molecules, such as beta1 integrin, play important roles in the regulation of neuritogenesis.     

Superheated steam pyrolysis of biomass elemental components and Sugi (Japanese cedar) for fuels and chemicals

To develop a novel noncatalytic biomass refinery process that can be used as a portable process, superheated steam pyrolysis was investigated to produce both carbonized solid fuels and chemicals using a large-scale reactor. Individual biomass components and native biomass (Sugi, Japanese cedar) were pyrolyzed. Between 150 and 400 degrees C, the vaporizing fractions of cellulose, xylan, and kraft lignin were summarized using a numerical model. Cellulose was converted to glycolaldehyde, furfural, 5-hydroxymethyl furfural and levoglucosan, whereas xylan was converted to glycolaldehyde, furfural, and acetic acid. Kraft lignin produced a slight yield of phenol and guaiacol. The total vaporization fraction of Sugi and its vaporizing rate were explained sufficiently using a numerical model based on the weighted average of the vaporizing properties of the individual components. However, the yields of phenol, guaiacol, and acetic acid were underestimated, while the yields of furfurals and levoglucosan were overestimated. Possible synergetic effects among chemicals in the superheated steam pyrolysis of native biomass were also discussed.     

Discovery of heteroaryl sulfonamides as new EP1 receptor selective antagonists

4-({2-[Isobutyl(phenylsulfonyl)amino]-5-(trifluoromethyl)phenoxy}methyl)benzoic acid (1) is a functional PGE2 antagonist selective for EP1 receptor subtype. Analogs of 1, in which the phenyl-sulfonyl moiety has been replaced with more hydrophilic heteroarylsulfonyl moieties, exhibited more optimized antagonist activity, while some of them showed in vivo antagonist activity. Structure-activity relationship (SAR) studies are also presented.     

4'-Fluorinated carbocyclic nucleosides: synthesis and inhibitory activity against S-adenosyl-L-homocysteine hydrolase

4'-Fluorinated analogue of 9-[(1'R,2'S,3'R)-2',3'-dihydroxy-cyclopentan-1'-yl]adenine (DHCaA) and their related analogues were systematically synthesized under the Mitsunobu and palladium(0) coupling conditions followed by fluorination with inversion of the configuration by using diethylaminosulfur trifluoride, respectively. 4'-beta-Fluoro DHCaA and 2-amino-4'-alpha-fluoro DHCaA demonstrated slight inhibitory activity against human and Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase, respectively.     

Optimization of sulfonamide derivatives as highly selective EP1 receptor antagonists

A series of 4-[(2-{isobutyl[(5-methyl-2-furyl)sulfonyl]amino}phenoxy)methyl]benzoic acids and 4-({2-[isobutyl(1,3-thiazol-2-ylsulfonyl)amino]phenoxy}methyl)benzoic acids were synthesized and evaluated for their EP receptor affinities and EP1 receptor antagonist activities. Further structural optimization was carried out to reduce inhibitory activity against hepatic cytochrome P450 isozymes, which could represent a harmful potential drug interaction. Selected compounds were also evaluated for their binding affinities to hTP, hDP, mFP, and hIP, and for their hEP1 receptor antagonist activities. The results of structure-activity relationship studies are also presented.     

Direct assay for alpha-amylase using fluorophore-modified cyclodextrins

A simple assay method for alpha-amylase was developed based on fluorophore-modified cyclodextrins (CDs). Four kinds of CD derivatives bearing a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety were prepared as artificial substrates for the assay method. The fluorescence intensity of all the NBD-amine-modified CDs decreased upon addition of Aspergillus oryzae alpha-amylase, indicating a reduction in hydrophobicity near the NBD-amine moiety induced by hydrolysis of the CD ring. NC4gammaCD, having a gamma-CD and an amino-tetramethylene spacer, was the most sensitive substrate for the alpha-amylase assay. The initial rate of hydrolysis of NC4gammaCD displayed a liner correlation to the concentration of the alpha-amylase. NC4gammaCD was sensitive to the alpha-amylase but was not sensitive to guest compounds that were accommodated by the native CDs.