Synthesis and characterization of a new bioactivated paramagnetic gadolinium(III) complex [Gd(DOTA-FPG)(H2O)] for tracing gene expression

A smart contrast agent for magnetic resonance imaging (MRI) can be used to exploit an enzymatic activity specific to the tissue or disease state signified by converting an MRI-inactivated agent to an activated MRI agent. In this study, a beta-galactopyranose-containing gadolinium(III) complex [Gd(DOTA-FPG)(H 2O)] was designed, synthesized, and characterized as being potentially suitable for a bioactivated MRI contrast agent. The (17)O NMR experiments were conducted to estimate the water exchange rate k e x 298 and rotational correlation time tau R 298 . The k ex 298 value of [Gd(DOTA-FPG)(H 2O)] is similar to that of [Gd(DO3A-bz-NO 2)(H 2O)]. The rotational correlation time value of [Gd(DOTA-FPG)(H 2O)] is dramatically longer than that of [Gd(DOTA)(H 2O)] (-) Relaxometric studies show that the percentage change in the T 1 value of [Gd(DOTA-FPG)(H 2O)] decreases dramatically in the presence of beta-galactosidase and human serum albumin. The T(1) change percentage of [Gd(DOTA-FPG)(H 2O)] (60%) is significantly higher than those of Egad and gadolinium(III)-1-(4-(2-(1-(4,7,10-triscarboxymethyl-(1,4,7,10-tetraazacyclododecyl)))-ethylcarbamoyloxymethyl)-2-nitrophenyl)-beta- d-glucopyronuronate. The signal intensity of the MR image for [Gd(DOTA-FPG)(H 2O)] in the presence of human serum albumin and beta-galactosidase (2670 +/- 210) is significantly higher than that of [Gd(DOTA-FPG)(H 2O)] in the sodium phosphate buffer solution (1490 +/- 160). In addition, the MR images show a higher-intensity enhancement in CT26/beta-gal tumor with beta-galactosidase gene expression but not for the CT26 tumor without beta-galactosidase gene expression. We conclude that [Gd(DOTA-FPG)(H 2O)] is a suitable candidate for a bioactivated MRI contrast agent in tracing gene expression.     

Quantitative profiling of histone post-translational modifications by stable isotope labeling

Methods for accurately quantitating changes in histone post-translational modifications are necessary for developing an understanding of how their dynamic nature influences nuclear events involving access to genomic DNA. This article describes methods for the use of in vivo stable isotope label incorporation for quantitating the levels of modification at specific residues in histone proteins. These methods are applicable to a wide variety of model systems and examples of their use in both mammalian cells and Saccharomyces cerevisiae are presented.     

Transplantation of engineered bone tissue using a rotary three-dimensional culture system

Bone is a complex, highly structured, mechanically active, three-dimensional (3-D) tissue composed of cellular and matrix elements. We previously published a report on in situ collagen gelation using a rotary 3-D culture system (CG–RC system) for the construction of large tissue specimens. The objective of the current study was to evaluate the feasibility of bone tissue engineering using our CG–RC system. Osteoblasts from the calvaria of newborn Wistar rats were cultured in the CG–RC system for up to 3 wk. The engineered 3-D tissues were implanted into the backs of nude mice and calvarial round bone defects in Wistar rats. Cell metabolic activity, mineralization, and bone-related proteins were measured in vitro in the engineered 3-D tissues. Also, the in vivo histological features of the transplanted, engineered 3-D tissues were evaluated in the animal models. We found that metabolic activity increased in the engineered 3-D tissues during cultivation, and that sufficient mineralization occurred during the 3 wk in the CG–RC system in vitro. One mo posttransplantation, the transplants to nude mice remained mineralized and were well invaded by host vasculature. Of particular interest, 2 mo posttransplantation, the transplants into the calvarial bone defects of rats were replaced by new mature bone. Thus, this study shows that large 3-D osseous tissue could be produced in vitro and that the engineered 3-D tissue had in vivo osteoinductive potential when transplanted into ectopic locations and into bone defects. Therefore, this system should be a useful model for bone tissue engineering.     

Isolation of a novel strain of Aeromonas media producing high levels of DOPA-melanin and assessment of the photoprotective role of the melanin in bioinsecticide applications

AIMS: To isolate a bacterium that produces high yield of melanin and to examine the effect of this bacterial pigment on the efficacy of a bioinsecticide. METHODS AND RESULTS: A novel melanin-producing bacterium, designated as strain WS, was isolated from the East Lake, Wuhan, China. Taxonomic studies of this strain indicate that it belongs to Aeromonas media. Physicochemical analysis of the pigment produced by strain WS (melanin WS) suggests that it is the authentic 3,4-dihydroxyphenylalanine (DOPA)-melanin. This melanin and that produced by Pseudomonas maltophilia P28 (melanin P28) share many biophysical properties, but the yield of the melanin WS is significantly higher than that of the melanin P28. In addition, the melanin WS appears to be more effective in the protection of a bioinsecticide against ultraviolet (UV) or solar radiation. At the concentration of 10 ppm, the melanin P28 exhibited no photoprotective effect on the bioinsecticide against UV radiation; in contrast, 5 ppm of melanin WS displayed an obvious protective effect. Similarly, the melanin WS displayed more protective effect on the bioinsecticide against solar radiation than the melanin P28 did over a 4-day period, with the effect being more dramatic for the last 2 days. CONCLUSIONS: We have isolated a novel bacterial strain of A. media that produces high levels of melanin. The melanin produced by this strain offers effective photoprotection of a commercial bioinsecticide BTI against UV and solar radiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study suggests that the melanin produced by our newly isolated A. media strain has the potential to be used as a general photoprotective agent for bioinsecticides.     

Modular evolution and increase of functional complexity in replicating RNA molecules

At early stages of biochemical evolution, the complexity of replicating molecules was limited by unavoidably high mutation rates. In an RNA world, prior to the appearance of cellular life, an increase in molecular length, and thus in functional complexity, could have been mediated by modular evolution. We describe here a scenario in which short, replicating RNA sequences are selected to perform a simple function. Molecular function is represented through the secondary structure corresponding to each sequence, and a given target secondary structure yields the optimal function in the environment where the population evolves. The combination of independently evolved populations may have facilitated the emergence of larger molecules able to perform more complex functions (including RNA replication) that could arise as a combination of simpler ones. We quantitatively show that modular evolution has relevant advantages with respect to the direct evolution of large functional molecules, among them the allowance of higher mutation rates, the shortening of evolutionary times, and the very possibility of finding complex structures that could not be otherwise directly selected.     

Anti-Bcl-2 family members,zfBcl-x<Subscript>L</Subscript> and zfMcl-1a,prevent cytochrome <Emphasis Type="Italic">c</Emphasis> release from cells undergoing betanodavirus-induced secondary necrotic cell death

Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 μg/ml) was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 μg/ml) completely blocked viral protein B2 expression, and partly blocked protein A, protein α, and a pro-apoptotic death protein (Bad) expression. Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2) may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins, zfBcl-x_L and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members. J.-L. Wu and J.-R. Hong contributed equally to the research.     

Satellite-DNA evolutionary patterns under a complex evolutionary scenario: the case of Acrolophus subgroup (Centaurea L., Compositae) from the western Mediterranean

Within the genus Centaurea (subtribe Centaureinae, tribe Cardueae, Compositae) hybridizations and reticulate-evolution phenomena have widely been recognized. This is especially true in the taxa included in the subgroup Acrolophus from the western Mediterranean area, in which recurrent hybridizations of parapatric ("microallopatric") lineages within the geographical range of a primary radiation have been suggested. The subgroup Acrolophus includes taxa from three sections (i.e. Acrolophus, Phalolepis and Willkommia), and, together with other subgroups, forms the named Jacea group (one of the three main groups into which Centaurea is divided). In this paper, we have studied the influence that the complex evolutionary scenario described for the Acrolophus subgroup from the western Mediterranean exerts on the evolutionary pattern of a satellite-DNA family, the HinfI family, which exists within the genomes of these taxa. To this end, we have analyzed the evolution of this satellite-DNA family in taxa from different taxonomic comparative levels: i) seven subspecies of the C. boissieri complex (one of which with two varieties) of the sect. Willkommia; ii) species of the sections Willkommia (10 species, 19 taxa), Acrolophus (two species), and Phalolepis (two species), all in the Acrolophus subgroup; iii) one external species to the Jacea group, C. granatensis from the group Acrocentron; iv) and species from other related genera from the Centaureinae subtribe (Phonus and Carthamus, both belonging to the Carthamus group). The influence of the suggested model for the origin and diversification of the Acrolophus subgroup is evidenced by the existence of three different HinfI satellite-DNA subfamilies coexisting in some genomes, and by the analysis that we have made by comparing site-by-site the transition stages in the process of concerted evolution between the sequences of the each subfamily. From this analysis, we can deduce that the HinfI repeated subfamilies evolved in a gradual manner, and that the different stages of concerted evolution fit quite well with the combined nuclear-chloroplast-DNA-deduced divergences and phylogeny of the subtribe Centaureinae. The HinfI satellite-DNA from the Carthamus species group (genera Carthamus and Phonus) and from the Acrocentron group (Centaurea granatensis) shows a high intraspecific conservation of the repeats, suggesting that the mechanisms producing concerted evolution have been efficient in these taxa. In addition, the comparison of individual nucleotide positions between related species shows a paucity in the spreading of variants in each subfamily with satellite-DNA divergence, an indication of a constant rate of homogenization of the repeated cluster. On the contrary, this trend is absent in the comparisons of the HinfI sequences from taxa of the subgroup Acrolophus. In this subgroup, we have found in this repetitive family similar representative average sequences for each taxon analyzed, polymorphic sites in each taxon being scant, most of them autapomorphic, representing early stages of genetic differentiation between taxa in the process of concerted evolution. The absence of concerted evolution was visualized by similar levels of intraspecific variation and interspecific divergence and by the lack of fixed species-diagnostic nucleotide sites. These facts might reflect the reticulate mode of evolution of Acrolophus.     

Fusion expression of DDR2 extracellular domain in insect cells and its purification and function characterization

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, a ligand for DDR2, up-regulates matrix metalloproteinase 1 (MMP-1) and MMP-2 expression in extracellular matrix (ECM). To investigate the role of DDR2 in cartilage destruction in rheumatoid arthritis (RA), we expressed the extracellular domain (ECD) of DDR2 (without signal peptide and transmembrane domain, designated DR) in insect cells, purified and characterized DR, hoping to use it as a specific antagonist of DDR2. By using Bac-To-Bac Expression System with a His tag, we successfully obtained the recombinant bacularvirus containing DDR2 ECD, purified it and characterized its function. The soluble fraction of DR was about 12% of the total fused protein. After chromatographic purification, DR with 92% purity was obtained. Competitive inhibition assay demonstrated that DR blocked the binding between DDR2 and natural DDR2 receptors on NIH3T3 and synovial cells. Results of RT-PCR, Western blotting, and gelatinase zymography showed that DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIH3T3 and RA synoviocytes stimulated by collagen II. For MMP-1, inhibition was displayed at the levels of mRNA and protein, whereas for MMP-2 it was at the level of protein. These findings suggested that the expressed DR inhibited the activity of natural DDR2 and relevant MMP-1 and MMP-2 expression in RA synoviocytes and NIH3T3 cells provoked by collagen II.