Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

A comparative study of fluorescent singlet oxygen probes in plant leaves

Four fluorescent singlet oxygen sensors: DanePy, its oxalate salt, Singlet Oxygen Sensor Green and MVP, were infiltrated into tobacco leaves and tested for toxicity, subcellular localization, light sensitivity and capacity to trap the singlet oxygen produced in photoinhibition. For reference, a broad sensitivity free radical probe, TEMPO-9-AC, was also included. Photochemical yield was approximately 15% and 10% inhibited by Singlet Oxygen Sensor Green and MVP, respectively, but was not significantly affected by the other probes. Under photoinhibitory conditions, brought about by irradiating lincomycin-treated leaves with strong photosynthetically active radiation, DanePy and Singlet Oxygen Sensor Green were responsive. Singlet Oxygen Sensor Green was also reactive to low, non-photoinhibitory light exposure of the leaf, which was not characteristic to the other probes. MVP did not respond to singlet oxygen which can partly be explained by a possible attenuation of its blue emission in the leaf, as shown by the example TEMPO-9-AC. DanePy-oxalate did not respond to photosynthetic singlet oxygen due to lack of its penetration into photosynthetic tissue and hence could be useful in detecting any singlet oxygen which escapes from a chloroplast initiation site. DanePy was localized in the chloroplasts, while Singlet Oxygen Sensor Green was mainly found in the epidermal cells preferentially associated with the nucleus.      

Sex steroid binding proteins in the plasma of hatchling <Emphasis Type="Italic">Chelonia mydas</Emphasis>

Sex steroid binding proteins were identified in hatchling female and male Chelonia mydas by dialysis and steady-state gel electrophoresis when examined at 4 degrees C. A testosterone binding protein with high binding affinity (K (a) = 0.98 +/- 0.5 x 10(8) M(-1)) and low to moderate binding capacity (B (max) = 7.58 +/- 4.2 x 10(-5) M) was observed in male hatchlings. An oestradiol binding protein with high affinity (K (a) = 0.35 +/- 1.8 x 10(8) M(-1)) and low to moderate binding capacity (B (max) = 0.16 +/- 0.5 x 10(-4) M) was identified in female hatchlings. This study confirmed that sex steroid binding proteins (SSBPs) become inactivate in both sexes at 36 degrees C, the maximum body temperature of sea turtle hatchlings at emergence. The inactivation of SSBPs at this temperature indicates that sex steroid hormones circulate freely in the body of the green turtles and are biologically available in the blood plasma. This observation is consistent with female and male hatchling C. mydas having different physiological (hormonal) and developmental requirements around the time of emergence. Moreover, concurrently conducted competition studies showed that sex steroids including testosterone and oestradiol do compete for binding sites in both male and female C. mydas hatchling plasma. Competition also occurred between testosterone and dihydrotestosterone for binding sites in the male C. mydas plasma. However, competition studies in the plasma of female hatchling C. mydas demonstrate that oestrone does not compete with oestradiol for binding sites.     

Effect of quinones on formation and properties of bacteriochlorophyll c aggregates

Chlorosomes of green photosynthetic bacterium Chlorobium tepidum contain aggregates of bacteriochlorophyll c (BChl c) with carotenoids and isoprenoid quinones. BChl aggregates with very similar optical properties can be prepared also in vitro either in non-polar solvents or in aqueous buffers with addition of lipids and/or carotenoids. In this work, we show that the aggregation of BChl c in aqueous buffer can be induced also by quinones (vitamin K₁ and K₂), provided they are non-polar due to a hydrophobic side-chain. Polar vitamin K_3, which possess the same functional group as K₁ and K₂, does not induce the aggregation. The results confirm a principal role of the hydrophobic interactions as a driving force for the aggregation of chlorosomal BChls. The chlorosomal quinones play an important role in a redox-dependent excitation quenching, which may protect the cells against damage under oxygenic conditions. We found that aggregates of BChl c with vitamin K₁ and K₂ exhibit an excitation quenching as well. The amplitude of the quenching depends on quinone concentration, as determined from fluorescence measurements. No lipid is necessary to induce the quenching, which therefore originates mainly from interactions of BChl c with quinones incorporated in the aggregate structure. In contrast, only a weak quenching was observed for dimers of BChl c in buffer (either with or without vitamin K₃) and also for BChl c aggregates prepared with a lipid (lecithin). Thus, the weak quenching seems to be a property of BChl c itself.     

Investigation on chlorosomal antenna geometries: tube,lamella and spiral-type self-aggregates

Molecular mechanics calculations and exciton theory have been used to study pigment organization in chlorosomes of green bacteria. Single and double rod, multiple concentric rod, lamella, and Archimedean spiral macrostructures of bacteriochlorophyll c molecules were created and their spectral properties evaluated. The effects of length, width, diameter, and curvature of the macrostructures as well as orientations of monomeric transition dipole moment vectors on the spectral properties of the aggregates were studied. Calculated absorption, linear dichroism, and polarization dependent fluorescence-excitation spectra of the studied long macrostructures were practically identical, but circular dichroism spectra turned out to be very sensitive to geometry and monomeric transition dipole moment orientations of the aggregates. The simulations for long multiple rod and spiral-type macrostructures, observed in recent high-resolution electron microscopy images (Oostergetel et al., FEBS Lett 581:5435–5439, 2007) gave shapes of circular dichroism spectra observed experimentally for chlorosomes. It was shown that the ratio of total circular dichroism intensity to integrated absorption of the Q _y transition is a good measure of degree of tubular structures in the chlorosomes. Calculations suggest that the broad Q _y line width of chlorosomes of sulfur bacteria could be due to (1) different orientations of the transition moment vectors in multi-walled rod structures or (2) a variety of Bchl-aggregate structures in the chlorosomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation

Epigallocatechin-3-gallate (EGCG), a tea polyphenol, inhibits the proliferation of many cancer cell lines; however, the antiproliferative mechanism(s) are not well-characterized. The objective of this study is to identify the cellular signaling mechanism(s) responsible for the antiproliferative effects of EGCG in the PC-3 prostate cancer cell line. EGCG inhibited PC-3 cell proliferation in a concentration-dependent manner with an IC(50) value of 39.0 microM, but had no effect on the proliferation of a nontumorigenic prostate epithelial cell line (RWPE-1). Treatment of PC-3 cells with EGCG (0-50 microM) resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells. The activation of ERK1/2 by EGCG was not inhibited using PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK), the immediate upstream kinase responsible for ERK1/2 activation; suggesting a MEK-independent signaling mechanism. Pretreatment of PC-3 cells with a phosphoinositide-3 kinase (PI3K) inhibitor partially reduced both EGCG-induced ERK1/2 activation and the antiproliferative effects of this polyphenol. These results suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signaling pathway is partially responsible for the antiproliferative effects of EGCG in PC-3 cells.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

Colonization behavior of bacterium Burkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter

Colonization behavior of endophytic bacteria Burkholderia cepacia strains RRE-3 and RRE-5 was studied in the seedlings of rice variety NDR97 using confocal laser scanning microscopy under controlled laboratory and greenhouse conditions. For studying colonization pattern, bacterial strains were tagged with pHRGFPGUS plasmid. The role of bacterial strains (both gfp/gus-tagged and untagged) in growth promotion was also studied. After coming into contact with the host root system the bacteria showed an irregular spreading. Dense colonization was observed on the primary and secondary roots and also on the junction of emergence of the lateral roots. Results showed that the colonization pattern of Burkholderia cepacia strains was similar to that of other endophytic bacteria isolated from non-legumes. Burkholderia cepacia got entry inside the root at the sites of emergence of lateral roots, without formation of infection threads as in the case of symbiotic rhizobacteria. Observations suggested that the endophytic bacterial strains RRE-3 and RRE-5 entered inside the rice roots in a progressive manner. Bacteria were found to line up along the intercellular spaces of adjoining epidermal cells adjacent to the lateral root junction, indicating endophytic colonization pattern of Burkholderia cepacia strains. Experiments with the rice seedlings inoculated with RRE-3 and RRE-5 strains revealed that both strains enhanced plant growth considerably when observed under laboratory and greenhouse conditions and produced significantly higher plant biomass. No considerable difference was observed between the gfp/gus-tagged and non-gfp/gus-tagged strains in the plant growth experiments both in the laboratory and greenhouse conditions.     

Transgenic Christmas Trees Regenerated from Agrobacterium tumefaciens-mediated Transformation of Zygotic Embryos Using the Green Fluorescence Protein as a Reporter

Mature zygotic embryos of recalcitrant Christmas tree species Fraser fir [Abies fraseri (Pursh) Poir], and Nordmann fir (Abies nordmanniana L.k.), and Virginia pine (Pinus virginiana Mill.) were used as explants for Agrobacterium tumefaciens strain GV3850-mediated transformation using the gfp (green fluorescent protein) gene as a reporter. Factors including media used for inoculation and co-cultivation, concentrations of acetosyringone, and antibiotics in tissue culture media have been evaluated. A high transformation frequency was obtained on TE medium containing 50μM acetosyringone and using 500 mg/l timentin to eliminate bacteria. Transient gene expression was observed in all three Christmas tree species, but transgenic plants were only produced from Virginia pine. Stable integration and expression of transgenes in the plant genome of Virginia pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable transformation system has been established in Virginia pine and this system would provide an opportunity to transfer economically important genes into Christmas tree species.     

Structural features of acetylated galactomannans from green Coffea arabica beans

Polysaccharides were extracted from green Coffea arabica beans with water (90 °C, 1 h). Galactomannans were isolated from the water extract using preparative anion-exchange chromatography. Almost all of the galactomannans eluted in two neutral populations, while almost all of the arabinogalactans bound to the column, indicating that these arabinogalactans contain charged groups. Analysis of the molecular weight distribution of the two neutral populations showed that they differ in their molecular weight. Further characterization of these neutral populations by NMR and by MALDI-TOF MS after enzymatic degradation with an endo-mannanase, showed the presence of acetyl groups linked to the galactomannans, a feature not previously described for this type of polysaccharides from coffee beans. It was found that the high molecular weight (ca. 2000 kDa) neutral fraction was highly substituted both with galactose residues and acetyl groups, while the low molecular weight (ca. 20 kDa) population was much less substituted. Based on these results it can be concluded that at least two distinctly different populations of galactomannans are present in green coffee beans. It was also shown that the degradation of the galactomannans from green coffee beans with an endo-mannanase from A. niger is hindered by the presence of acetyl groups.