FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase

Bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), are hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase. Moreover, we found another amidohydrolase catalyzing the same reaction only at acidic pH, and we purified it from rat lung (Ueda, N., Yamanaka, K., and Yamamoto, S. (2001) J. Biol. Chem. 276, 35552-35557). Here we report complementary DNA cloning and functional expression of the enzyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)" from human, rat, and mouse. The deduced primary structures revealed that NAAA had no homology to fatty acid amide hydrolase but belonged to the choloylglycine hydrolase family. Human NAAA was essentially identical to a gene product that had been noted to resemble acid ceramidase but lacked ceramide hydrolyzing activity. The recombinant human NAAA overexpressed in HEK293 cells hydrolyzed various N-acylethanolamines with N-palmitoylethanolamine as the most reactive substrate. Most interestingly, a very low ceramide hydrolyzing activity was also detected with NAAA, and N-lauroylethanolamine hydrolyzing activity was observed with acid ceramidase. By the use of tunicamycin and endoglycosidase, NAAA was found to be a glycoprotein. Furthermore, the enzyme was proteolytically processed to a shorter form at pH 4.5 but not at pH 7.4. Expression analysis of a green fluorescent protein-NAAA fusion protein showed a lysosome-like distribution in HEK293 cells. The organ distribution of the messenger RNA in rats revealed its wide distribution with the highest expression in lung. These results demonstrated that NAAA is a novel N-acylethanolamine-hydrolyzing enzyme that shows structural and functional similarity to acid ceramidase.     

Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation.

A translation initiation factor, eIF4E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap-analogue was estimated to be 10(-2)-10(-4) M. These results suggest that the participation of another initiation factor is required for eIF4E to recognize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al., in which eIF4E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.     

Recovery of saltwater crocodiles following unregulated hunting in tidal rivers of the Northern Territory,Australia

Saltwater crocodiles (Crocodylus porosus) in the Northern Territory of Australia were protected in 1971, after a severe population decline resulting from 26 yr of intense commercial hunting. By that time wild saltwater crocodiles were rarely sighted anywhere and they were commercially extinct in areas where they had once been abundant. Standardized monitoring by spotlight surveys started in 1975 and provided relative density indices over time (1975–2009) as a unique record of the post-protection recovery of a wild crocodilian population. We examined the survey data for populations at 12 major tidal rivers, individually and as a single subpopulation. The pattern of recovery in the subpopulation in both abundance and biomass was approximated by logistic curves, predicting 5.26 non-hatchling crocodiles weighing 387.64 kg sighted per kilometer of river in 2010. We predicted potential carrying capacity as 5.58 non-hatchling crocodiles (5.73% higher than 2010) weighing 519.0 kg (25.31% higher than 2010). Individual rivers showed largely different abundance and biomass among rivers. The statistical model that best described the recovery in individual rivers was not always logistic. However, where it was logistic, expected carrying capacity of different rivers showed considerable variation in abundance and biomass. The variation indicates different habitat quality among the rivers. Recovery occurred despite various consumptive uses, particularly a widespread egg-harvest program, which has been an integral part of the incentive-driven conservation program for saltwater crocodiles in the Northern Territory since 1983. We suggest that the saltwater crocodile population of the Northern Territory is achieving full recovery from uncontrolled hunting in 1945–1971. Although saltwater crocodiles are considered an important natural resource, their increase in number, size, and distribution is posing management issues for public safety. Continuation of human–crocodile conflict management through public education and strategic removal of problem crocodiles will be essential. © 2011 The Wildlife Society.     

Resonance Raman studies on xanthine oxidase: observation of Mo<Superscript>VI</Superscript>-ligand vibrations

Resonance Raman spectra were investigated for the sulfo and desulfo forms of cow's milk xanthine oxidase, with various visible excitation lines between 400 and 650 nm, and Mo(VI)-ligand vibrations were observed for the first time. The Mo(VI)=S stretch was identified at 474 and 462 cm(-1 )for the (32)S- and (34)S-sulfo forms, respectively, but was absent in the reduced state and in the desulfo form. The Mo(VI)=O stretch was weakly observed at 899 cm(-1 )for the sulfo form and shifted to 892 cm(-1) with very weak intensity for the dioxo desulfo form. In measurements of an excitation profile, the two bands at 474 and 899 cm(-1) showed maximum intensity at similar excitation wavelengths, suggesting that the Raman intensity of the metal-ligand modes is due to the Mo(VI)<--S charge transfer transition, and that this is the origin of the intrinsically weak features of the Mo(VI)-ligand Raman bands. When the sulfo form was regenerated from the desulfo form, the 899 cm(-1) band reappeared. However, the band at 899 cm(-1) showed no frequency shift when regeneration was conducted in H(2)(18)O, or after several turnovers in the presence of xanthine in H(2)(18)O. When the sulfo form was reduced and reoxidized in H(2)(18)O buffer, the 899 cm(-1) band reappeared without any frequency shift. These observations suggest that the oxo oxygen in the Mo center of xanthine oxidase is not labile. Low-frequency vibrations of the Mo center were observed together with those of the Fe(2)S(2) center with some overlaps, while FAD modes were observed clearly. The absence of dithiolene modes in XO is in contrast to the Mo(VI) centers of DMSO reductase and sulfite oxidase.     

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

Background

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.

Methodology/Principal Findings

Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.

Conclusions/Significance

We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.     

Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: A case study with isoxanthopterin and its derivatives

Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 × 10⁶ M^?1 and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 × 10⁶ M^?1 and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand–nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand–nucleotide interaction, which leads to the improved binding affinity.     

Novel polymer biomaterials and interfaces inspired from cell membrane functions

Background

Materials with excellent biocompatibility on interfaces between artificial system and biological system are needed to develop any equipments and devices in bioscience, bioengineering and medicinal science. Suppression of unfavorable biological response on the interface is most important for understanding real functions of biomolecules on the surface. So, we should design and prepare such biomaterials.

Scoop of review

One of the best ways to design the biomaterials is generated from mimicking a cell membrane structure. It is composed of a phospholipid bilayered membrane and embedded proteins and polysaccharides. The surface of the cell membrane-like structure is constructed artificially by molecular integration of phospholipid polymer as platform and conjugated biomolecules. Here, it is introduced as the effectiveness of biointerface with highly biological functions observed on artificial cell membrane structure.

Major conclusions

Reduction of nonspecific protein adsorption is essential for suppression of unfavorable bioresponse and achievement of versatile biomedical applications. Simultaneously, bioconjugation of biomolecules on the phospholipid polymer platform is crucial for a high-performance interface.

General significance

The biointerfaces with both biocompatibility and biofunctionality based on biomolecules must be installed on advanced devices, which are applied in the fields of nanobioscience and nanomedicine.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.