Facile conversion of RNA aptamers to modular fluorescent sensors with tunable detection wavelengths

A GTP aptamer was converted to a modular fluorescent GTP sensor by conjugation of RRE (Rev responsive element) RNA and successive complex formation with a fluorophore-modified Rev peptide. Structural changes associated with substrate binding in the RNA aptamer were successfully transduced into changes in fluorescence intensity because of the modular structure of ribonucleopeptides. A simple modular strategy involving conjugation of a fluorophore-modified ribonucleopeptide to the stem region of an RNA aptamer deduced from secondary structural information helps produce fluorescent sensors, which allow tuning of excitation and detection wavelengths through the replacement of the fluorophore at the N-terminal of the Rev peptide.     

Ultrafine cellulose fibers produced by Asaia bogorensis, an acetic acid bacterium

The ability to synthesize cellulose by Asaia bogorensis, a member of the acetic acid bacteria, was studied in two substrains, AJ and JCM. Although both strains have identical 16S rDNA sequence, only the AJ strain formed a solid pellicle at the air-liquid interface in static culture medium, and we analyzed this pellicle using a variety of techniques. In the presence of cellulase, glucose and cellobiose were released from the pellicle suggesting that it is made of cellulose. Field emission electron microscopy allowed the visualization of a 3D knitted structure with ultrafine microfibrils (approximately 5-20 nm in width) in cellulose from A. bogorensis compared with the 40-100 nm wide microfibrils observed in cellulose isolated from Gluconacetobacter xylinus, suggesting differences in the mechanism of cellulose biosynthesis or organization of cellulose synthesizing sites in these two related bacterial species. Identifying these differences will lead to a better understanding of cellulose biosynthesis in bacteria.     

A trial of production of the plant-derived high-value protein in a plant factory: Photosynthetic photon fluxes affect the accumulation of recombinant miraculin in transgenic tomato fruits

One of the ultimate goals of plant science is to test a hypothesis obtained by basic science and to apply it to agriculture and industry. A plant factory is one of the ideal systems for this trial. Environmental factors affect both plant yield and the accumulation of recombinant proteins for industrial applications within transgenic plants. However, there have been few reports studying plant productivity for recombinant protein in closed cultivation systems called plant factories.To investigate the effects of photosynthetic photon flux (PPF) on tomato fruit yield and the accumulation of recombinant miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits, plants were cultivated at various PPFs from 100 to 400 (µmol m⁻² s⁻¹) in a plant factory. Miraculin production per unit of energy used was highest at PPF100, although miraculin production per unit area was highest at PPF300. The commercial productivity of recombinant miraculin in transgenic tomato fruits largely depended on light conditions in the plant factory. Our trial will be useful to consider the trade-offs between the profits from production of high-value materials in plants and the costs of electricity.Key words: light, molecular farming, recombinant miraculin, taste-modifying protein, transgenic tomato     

Rapid identification of protein-protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements

Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an (15)N-labeled amino acid and several amino acids (13)C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω(1)-TROSY-type backbone (1)H-(15)N and aromatic (1)H-(13)C shift correlations together with single quantum methyl (1)H-(13)C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development.     

<Emphasis Type="Italic">Ophiocordyceps pulvinata</Emphasis> sp. nov., a pathogen of ants with a reduced stroma

Ophiocordyceps pulvinata, a pathogen of ants, is formally described as a new species. Genus level designation of this species is difficult due to several apparently conflicting morphological and ecological characters. Affinity with Ophiocordyceps is suggested by the dark color stroma and ascospore morphology. However, the species was included in a book of entomopathogenic fungi of Japan as Torrubiella sp. due to the production of perithecia on an astipitate stroma. Phylogenetic analyses of molecular data support a close relationship with O. unilateralis, a finding consistent with morphological characteristics of the color, asci and ascospores and ecological traits of host affiliation. Thus, O. pulvinata represents another example of the loss of stipe for the hypocrealean arthropod pathogenic fungi and highlights the utility of asci and ascospore morphology as taxonomically informative characters of closely related taxa.     

Localized cysteine sulfenic acid formation by vascular endothelial growth factor: role in endothelial cell migration and angiogenesis

Reactive oxygen species (ROS) are important mediators for VEGF receptor 2 (VEGFR2) signalling involved in angiogenesis. The initial product of Cys oxidation, cysteine sulfenic acid (Cys-OH), is a key intermediate in redox signal transduction; however, its role in VEGF signalling is unknown. We have previously demonstrated IQGAP1 as a VEGFR2 binding scaffold protein involved in ROS-dependent EC migration and post-ischemic angiogenesis. Using a biotin-labelled Cys-OH trapping reagent, we show that VEGF increases protein-Cys-OH formation at the lamellipodial leading edge where it co-localizes with NADPH oxidase and IQGAP1 in migrating ECs, which is prevented by IQGAP1 siRNA or trapping of Cys-OH with dimedone. VEGF increases IQGAP1-Cys-OH formation, which is prevented by N-acetyl cysteine or dimedone, which inhibits VEGF-induced EC migration and capillary network formation. In vivo, hindlimb ischemia in mice increases Cys-OH formation in small vessels and IQGAP1 in ischemic tissues. In summary, VEGF stimulates localized formation of Cys-OH-IQGAP1 at the leading edge, thereby promoting directional EC migration, which may contribute to post-natal angiogenesis in vivo. Thus, targeting Cys-oxidized proteins at specific compartments may be the potential therapeutic strategy for various angiogenesis-dependent diseases.     

Plasma L-cystine/L-glutamate imbalance increases tumor necrosis factor-alpha from CD14+ circulating monocytes in patients with advanced cirrhosis

Background and Aims

The innate immune cells can not normally respond to the pathogen in patients with decompensated cirrhosis. Previous studies reported that antigen-presenting cells take up L-Cystine (L-Cys) and secrete substantial amounts of L-Glutamate (L-Glu) via the transport system Xc- (4F2hc+xCT), and that this exchange influences the immune responses. The aim of this study is to investigate the influence of the plasma L-Cys/L-Glu imbalance observed in patients with advanced cirrhosis on the function of circulating monocytes.

Methods

We used a serum-free culture medium consistent with the average concentrations of plasma amino acids from patients with advanced cirrhosis (ACM), and examined the function of CD14+ monocytes or THP-1 under ACM that contained 0–300 nmol/mL L-Cys with LPS. In patients with advanced cirrhosis, we actually determined the TNF-alpha and xCT mRNA of monocytes, and evaluated the correlation between the plasma L-Cys/L-Glu ratio and TNF-alpha.

Results

The addition of L-Cys significantly increased the production of TNF alpha from monocytes under ACM. Monocytes with LPS and THP-1 expressed xCT and a high level of extracellular L-Cys enhanced L-Cys/L-Glu antiport, and the intracellular GSH/GSSG ratio was decreased. The L-Cys transport was inhibited by excess L-Glu. In patients with advanced cirrhosis (n = 19), the TNF-alpha and xCT mRNA of monocytes were increased according to the Child-Pugh grade. The TNF-alpha mRNA of monocytes was significantly higher in the high L-Cys/L-Glu ratio group than in the low ratio group, and the plasma TNF-alpha was significantly correlated with the L-Cys/L-Glu ratio.

Conclusions

A plasma L-Cys/L-Glu imbalance, which appears in patients with advanced cirrhosis, increased the TNF-alpha from circulating monocytes via increasing the intracellular oxidative stress. These results may reflect the immune abnormality that appears in patients with decompensated cirrhosis.     

Engagement of overexpressed Her2 with GEP100 induces autonomous invasive activities and provides a biomarker for metastases of lung adenocarcinoma

Overexpression of Her2/ErbB2/Neu in cancer is often correlated with recurrent distant metastasis, although the mechanism still remains largely elusive. We have previously shown that EGFR, when tyrosine-phosphorylated, binds to GEP100/BRAG2 to activate Arf6, which induces cancer invasion and metastasis. We now show that overexpressed Her2 in lung adenocarcinoma cells also employs GEP100. Like EGFR-GEP100 binding, this association is primarily mediated by the pleckstrin homology (PH) domain of GEP100 and Tyr1139/Tyr1196 of Her2. Tyr1139/Tyr1196 are autonomously phosphorylated, when Her2 is overexpressed. Accordingly, invasive activities mediated by the Her2-GEP100 pathway are not dependent on external factors. Blocking Her2-GEP100 binding, as well as its signaling pathway all inhibit cancer invasive activities. Moreover, our clinical study indicates that co-overexpression of Her2 with GEP100 in primary lung adenocarcinomas of patients is correlated with the presence of their node-metastasis with a statistical significance. Since the GEP100 PH domain interacts with both Her2 and EGFR, targeting this domain may provide novel cancer therapeutics.     

A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.