SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients

Introduction

Agonistic autoantibodies (Aabs) against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in patients with systemic sclerosis (SSc). In our present study, we examined the expression of the AT1R and the ETAR in human immune cells and the pathological effects mediated through these receptors by their corresponding Aabs.

Methods

Protein expression of AT1R and ETAR on peripheral blood mononuclear cells (PBMCs) from healthy individuals and SSc patients was analyzed using flow cytometry, and mRNA expression of both receptors in PBMCs from healthy donors was examined by real-time PCR. In addition, PBMCs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin G (IgG) fractions from SSc patients positive for AT1R and ETAR Aabs, as well as with IgG from healthy donors serving as controls. Alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, enzyme-linked immunosorbent assays and chemotaxis assays, respectively. The results were correlated with the characteristics and clinical findings of the IgG donors.

Results

Both AT1R and ETAR were expressed on PBMCs in humans. Protein expression of both receptors was decreased in SSc patients compared with that of healthy donors and declined during the course of disease. IgG fractions of SSc patients positive for AT1R and ETAR Aabs induced T-cell migration in an Aab level–dependent manner. Moreover, IgG of SSc patients stimulated PBMCs to produce more interleukin 8 (IL-8) and chemokine (C-C motif) ligand 18 (CCL18) than did the IgG of healthy donors. All effects were significantly reduced by selective AT1R and ETAR antagonists. Statistical analysis revealed an association of SSc-IgG induced high IL-8 concentrations with an early disease stage and of high CCL18 concentrations with lung fibrosis onset and vascular complications in the respective IgG donors.

Conclusion

In our present study, we could demonstrate the expression of both AT1R and ETAR on human peripheral T cells, B cells and monocytes. The decreased receptor expression in SSc patients, the inflammatory and profibrotic effects upon Aab stimulation of PBMCs in vitro and the associations with clinical findings suggest a role for Aab-induced activation of immune cells mediated by the AT1R and the ETAR in the pathogenesis or even the onset of the disease.     

ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats

Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.     

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 from human plasma

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 human plasma is described. Plasma is fractionated by affinity chromatography on chicken hemoglobin-Sepharose using the full capacity of the column; then after washing the column thoroughly, haptoglobin is eluted with 8 M urea and the eluate is collected in fractions to separate active and denatured haptoglobin. The urea-free, active fractions of haptoglobin are fractionated by affinity chromatography on Affi-Gel Con A to remove nonglycoproteins, principally apolipoprotein A-I, and the haptoglobin is eluted with 0.5 M glucose. Then the haptoglobin-containing fractions are fractionated by negative immunoadsorption chromatography on anti-chicken hemoglobin-protein A-Sepharose to remove chicken hemoglobin-human haptoglobin complexes. Haptoglobin prepared by this three-step procedure is biologically active and nearly homogeneous. The recovery is approximately 70%, irrespective of phenotype. The procedure can be completed in 3 days. A partial purification of apolipoprotein A-I is obtained simultaneously by this method.     

Conformational transitions in N-linked oligosaccharides

An assignment strategy involving 1H-1H correlated spectroscopy (COSY), relayed correlation spectroscopy (RECSY), nuclear Overhauser effect spectroscopy (NOESY), and triple quantum filtered correlated spectroscopy (TQCOSY) is described for six related N-linked oligosaccharides. These are of three "types", i.e., complex, bisected complex, and oligomannose. Using spin-spin coupling constant data derived from these assignments, together with semiempirical quantum mechanical energy calculations, we have examined the rotamer distributions at the Man alpha 1-6Man beta-linkage in each structure, and additionally at the Man alpha 1-6Man alpha-linkage in oligomannose oligosaccharides. We show that while several primary sequence differences are "passive", certain key residues modulate the orientation of the alpha 1-6 arms. These residues may be proximal or distal to the site of the conformational change. There is no direct correlation between these perturbations and the oligosaccharide type. These data are discussed in terms of the proposed recognition function of oligosaccharides in biological systems.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Synthesis of 1-N-glycyl beta-oligosaccharide derivatives. Reactivity of Lens culinaris lectin with a fluorescent labeled streptavidin pseudoglycoprotein and immobilized neoglycolipid.

The lectin from Lens culinaris (lentil) has a binding specificity for glycopeptides bearing 6-O-linked fucose on the reducing terminus on complex-type N-linked oligosaccharides. Lentil lectin therefore provides an excellent example of a carbohydrate binding protein in which high-affinity interactions are dependent on the integrity of the oligosaccharide core structure. We report here the synthesis of the 1-N-glycyl beta-derivative of Gal beta 4GlcNAc beta 2Man alpha 6(Gal beta 4GlcNAc beta 2Man alpha 3)Man beta 4GlcNAc beta 4(Fuc alpha 6)-GlcNAc (Gal-2F) and its subsequent biotinylation and palmitoylation. The biotin derivative when bound to a streptavidin-fluorescein isothiocyanate (FITC) conjugate was able to bind to both concanavalin A (ConA) and lentil lectin affinity columns. In contrast, synthesis of the biotin derivative of the glycamine derivative of Gal-2F and subsequent binding to streptavidin-FITC afforded reactivity to a ConA affinity column but not to a lentil lectin affinity column. Lentil lectin also bound to plastic microtiter plates containing the adsorbed palmitoyl-1-N-glycyl beta-derivative. No binding occurred when the homologous glycamine neoglycolipid was used. These results suggest the 1-N-glycyl beta-derivative of oligosaccharides may have general utility as an intermediate in the synthesis of novel glycoconjugate probes.     

Structures of the N-linked oligosaccharides of Gp63, the major surface glycoprotein, from Leishmania mexicana amazonensis

The extent of protein N-glycosylation in Leishmania mexicana amazonensis has been proposed to be a factor in the virulence of the parasite. The N-linked oligosaccharides of gp63, the major surface glycoprotein of L. mexicana amazonensis, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P-4 yielded four fractions, and the oligosaccharides present were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis, and methylation analysis. Four major structures were found and were biantennary oligomannose type with compositions of Glc1Man6GlcNAc2 (La), Man6GlcNAc2 (Lb), Man5GlcNAc2 (Lc), and Man4GlcNAc2 (Ld). The largest oligosaccharide (La) was shown to contain a terminal glucopyranosyl residue on the alpha (1----3) arm. The biantennary oligomannose structures (Lb and Lc) and the glucosylated structure Glc1Man6GlcNAc2 (La) have not previously been reported as a component of a mature glycoprotein from any source.     

Spatial distribution of growth rates and of epidermal cell lengths in the elongation zone during leaf development in Lolium perenne L.

Relative elemental growth rates (REGR) and lengths of epidermal cells along the elongation zone of Lolium perenne L. leaves were determined at four developmental stages ranging from shortly after emergence of the leaf tip to shortly before cessation of leaf growth. Plants were grown at constant light and temperature. At all developmental stages the length of epidermal cells in the elongation zone of both the blade and sheath increased from 12 m at the leaf base to about 550 m at the distal end of the elongation zone, whereas the length of epidermal cells within the joint region only increased from 12 to 40 m. Throughout the developmental stages elongation was confined to the basal 20 to 30 mm of the leaf with maximum REGR occurring near the center of the elongation zone. Leaf elongation rate (LER) and the spatial distributions of REGR and epidermal cell lengths were steady to a first approximation between emergence of the leaf tip and transition from blade to sheath growth. Elongation of epidermal cells in the sheath started immediately after the onset of elongation of the most proximal blade epidermal cells. During transition from blade to sheath growth the length of the blade and sheath portion of the elongation zone decreased and increased, respectively, with the total length of the elongation zone and the spatial distribution of REGR staying near constant, with exception of the joint region which elongated little during displacement through the elongation zone. Leaf elongation rate decreased rapidly during the phase when only the sheath was growing. This was associated with decreasing REGR and only a small decrease in the length of the elongation zone. Data on the spatial distributions of growth rates and of epidermal cell lengths during blade elongation were used to derive the temporal pattern of epidermal cell elongation. These data demonstrate that the elongation rate of an epidermal cell increased for days and that cessation of epidermal cell elongation was an abrupt event with cell elongation rate declining from maximum to zero within less than 10 h.Abbreviations LER leaf elongation rate - REGR relative elemental growth rates     

High affinity sugar ligands of C-type lectin receptor langerin

Background

Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application.