Role of the promyelocytic leukemia body in the dynamic interaction between the androgen receptor and steroid receptor coactivator-1 in living cells

The dynamic interaction between the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) was explored in living cells expressing chimeric forms of the receptor and the coactivator containing two spectral variants of jellyfish fluorescent protein. Laser scanning confocal imaging of transfected cells expressing fluorescently labeled SRC-1 revealed that in an unsynchronized cell population, the coactivator is distributed in approximately 40% cells as nuclear bodies of 0.2-1.0 microm in diameter. Immunostaining of cyan fluorescent protein-labeled SRC-1 (CFP-SRC1)-expressing cells with antibody to promyelocytic leukemia (PML) protein showed significant overlap of the CFP fluorescence with the antibody stain. Cotransfection of cells with a plasmid expressing the CFP conjugate of Sp100 (another marker protein for the PML nuclear body) also showed colocalization of the yellow fluorescent protein (YFP)-SRC1 containing nuclear foci with the PML bodies in living cells. Analysis of the three-dimensional structure revealed that the PML bodies are round to elliptical in shape with multiple satellite bodies on their surface. Some of these satellite bodies contain the SRC-1. Activation and nuclear import of CFP-AR by the agonistic ligand 5alpha-dihydrotestosterone, but not by the antagonist casodex, transferred YFP-SRC1 from the PML bodies to an interlacing filamentous structure. In a single living cell, agonist-activated AR caused a time-dependent movement of YFP-SRC1 from the PML bodies to this filamentous structure. Additionally, coexpression of a constitutively active mutant of AR (AR-deltaligand binding domain) also displaced YFP-SRC1 from the PML bodies to this intranuclear filamentous structure. The fluorescence recovery after photobleaching approach was used to examine changes in the kinetics of movement of YFP-SRC1 during its mobilization from the PML bodies to the intranuclear filamentous structure by the agonist-activated AR. Results of the relative half-times (t(1/2)) of replacement of YFP-SRC1 within the photobleached region of a single PML body from its surrounding nuclear space supported the conclusion that SRC-1 is actively transported from the PML bodies to the intranuclear filamentous structure by the ligand-activated AR. This observation also suggests an interaction between AR and SRC-1 before its binding to the target gene. The PML bodies have been implicated as a cross-road for multiple regulatory pathways that control cell proliferation, cellular senescence, and apoptosis. Our present results along with other recent reports expand the role of this subnuclear structure to include the regulation of steroid hormone action.     

Structure of human aldose reductase holoenzyme in complex with statil: an approach to structure-based inhibitor design of the enzyme

Aldose reductase, a monomeric NADPH-dependent oxidoreductase, catalyzes the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The X-ray structure of human aldose reductase holoenzyme in complex with statil was determined at a resolution of 2.1 A. The carboxylate group of statil interacted with the conserved anion binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Statil's hydrophobic phthalazinyl ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group penetrating the "specificity" pocket. The interactions between the inhibitor's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding to residues Leu300 and Thr113. Based on the model of the ternary complex, the program GRID was used in an attempt to design novel potential inhibitors of human aldose reductase with enhanced binding energies of the complex. Molecular modeling calculations suggested that the replacement of the fluorine atom of statil with a carboxylate functional group may enhance the binding energies of the complex by 33%.     

Deciphering a novel thioredoxin-like fold family

Sequence--and structure-based searching strategies have proven useful in the identification of remote homologs and have facilitated both structural and functional predictions of many uncharacterized protein families. We implement these strategies to predict the structure of and to classify a previously uncharacterized cluster of orthologs (COG3019) in the thioredoxin-like fold superfamily. The results of each searching method indicate that thioltransferases are the closest structural family to COG3019. We substantiate this conclusion using the ab initio structure prediction method rosetta, which generates a thioredoxin-like fold similar to that of the glutaredoxin-like thioltransferase (NrdH) for a COG3019 target sequence. This structural model contains the thiol-redox functional motif CYS-X-X-CYS in close proximity to other absolutely conserved COG3019 residues, defining a novel thioredoxin-like active site that potentially binds metal ions. Finally, the rosetta-derived model structure assists us in assembling a global multiple-sequence alignment of COG3019 with two other thioredoxin-like fold families, the thioltransferases and the bacterial arsenate reductases (ArsC).     

Changes in lignin content of leaf litters during mulching

Alkaline nitrobenzene oxidation, ozonation and methoxyl content determinations were applied to decomposing leaf litter of Ginkgo biloba L., Cinnamomum camphora sieb., Zelkova serrata Makino and Firmiana simplex W. F. Wight, respectively, during mulching to investigate the properties and estimate changes in lignin composition and content. Since the Klason lignin residue originated from components highly resistant to degradation by acid, the methoxyl content of Klason residue was used to estimate the lignin content of leaf litter. Quantitative analysis of presumed lignin-derived fragments, by use of alkaline nitrobenzene oxidation and ozonation methods, suggested that the estimated lignin content approximates that of the real lignin content of leaves, which is greatly overestimated by the Klason procedure. The estimated lignin contents ranged from 3.9 to 10.0% while the Klason lignan residue varied from 37.1 to 46.7% in un-mulched leaf litter. The absolute amounts of the measured lignin somewhat decreased during mulching, while the structure of lignin remaining in leaf litters after mulching was considered not to be very different from its original structure.     

The absolute configuration of prunioside A from Spiraea prunifolia and biological activities of related compounds

The stereochemistry of prunioside A isolated from Spiraea prunifolia was determined by chemical transformations and NMR spectral data analysis. The configurations at C-5 and C-6 were determined to be 5S and 6R by application of the modified Mosher's method, CD analysis, and 13C NMR spectroscopic data analysis of an acetonide derivative. Other compounds related to prunioside A have inhibitory effects on the synthesis of nitric oxide in LPS-stimulated macrophage-like RAW 264.7 cells.     

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.     

Vascularized acellular dermal matrix island flaps for the repair of abdominal muscle defects

The potential widespread use of tissue-engineered matrices in soft-tissue reconstruction has been limited by the difficulty in fabricating and confirming a functional microcirculation. Acellular dermal matrix placed in a soft-tissue pocket acts as a scaffold to be incorporated by the host's fibrovascular tissue. A new method for noninvasive real-time observation of functional microvascular networks using orthogonal polarization spectral (OPS) imaging has recently been reported. Arterioles, venules, and capillaries can be directly visualized, and the movement of individual blood cells through them can be observed. The present study was performed to investigate the use of prefabricated acellular dermal matrix with an arteriovenous unit for the repair of abdominal muscle defects. OPS imaging was used to determine the presence of a functional microcirculation in the neovascularized matrix. In Sprague-Dawley rats, vascularized matrix was prefabricated by placing the superficial epigastric artery and vein on a 2-cm x 2-cm implant-type acellular dermal matrix in the thigh. Three weeks after implantation, the matrix-arteriovenous unit was elevated as an axial-type flap and a 2-cm x 2-cm full-thickness block of abdominal muscle immediately superior to the inguinal ligament was resected. Additional procedures were performed according to group: no repair (group 1, n = 20); repair with nonvascularized acellular dermal matrix (group 2, n = 20); repair with devascularized acellular dermal matrix (group 3, = 20); and repair with vascularized acellular dermal matrix (group 4, n = 20). OPS imaging (field of view, 1 mm in diameter; scan depth range, 0.2 mm) was performed on both sides of each flap on a total of 10 random distal regions before and after pedicle transection in group 3 and with the pedicle preserved in group 4. Hernia rate and duration of survival were compared for 21 days. OPS imaging showed directional blood cell movement through the capillary network in all areas scanned in group 4. No microvascular perfusion was observed after pedicle transection in group 3. Hernia rates of 100, 80, 90, and 0 percent were seen in groups 1, 2, 3, and 4, respectively. Median survival times of 9, 11.5, 9, and 21 postoperative days were noted in groups 1, 2, 3, and 4, respectively. Histopathologic analysis with factor VIII revealed full-thickness infiltration of the matrix by endothelial cells, signifying newly formed blood vessels. Repair of abdominal muscle defects using vascularized acellular dermal matrix resulted in no hernia and survival of all animals for the duration of study. However, repairs using avascular or devascularized matrix resulted in significant rates of hernia and decreased survival. Acellular dermal matrix can be prefabricated into vascularized tissue using an arteriovenous unit and used successfully to repair abdominal muscle defects. OPS imaging allowed for high-contrast direct visualization of microcirculation in previously acellular tissue following prefabrication with an arteriovenous unit.     

RNA/DNA ratio as an index of physiological condition of Colossoma macropomum and Piaractus branchypomus (Pisces: Characiformes) during embryonic development

We evaluated RNA/DNA ratio as an index of physiological condition during larval development of a hybrid between the fishes Colossoma macropomum (cachama) and Piaractus brachypomus (morocoto). The samples were obtained by induced reproductive technology and the eggs were maintained in acrylic conical incubator with a continuous waterflow. Embryonic development, from egg fertilization to cell division and hatch out, took 12 hours 20 minutes at 29.5 degrees C, dissolved oxygen contents of 6.0 ppm and pH 7.5. Nucleic acids quantification was determined by fluorometry with ethidium bromide and Hoechst 33258 dyes. We observed significant changes of RNA/DNA ratios during all stages of the embryonic larval development. Therefore, RNA/DNA relation is an useful technique to evaluate physiological condition in short period and could be utilized as nutritional condition and/or instantaneous growth for routine check to verify the health status in early life of cultivated species.     

The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers