Dye affinity chromatography for fast and simple purification of fucoidan from marine brown algae

Fucoidan is a sulfated polysaccharide with promising pharmacological applications. Due to its medicinal properties, there is a demand for a separation technique that yields a high purification grade. Here, we present a novel purification tool for recovering fucoidan from the marine brown macroalgae Fucus vesiculosus. The developed method is based on amino‐derivatized Sepabeads^® EC‐EA. The beads were modified with toluidine blue (TB), a thiazine derivative, to exploit the strong donor acceptor interactions between the cationic dye and the anionic polysaccharide. The adsorption kinetics and the binding capacity of the resin were analyzed. A Sips model was used to approximate the adsorption isotherm, resulting in a maximum capacity of 127.7 mg fucoidan per g adsorbent. Investigation of the effect of adsorption step's pH on purity and chemical structure was performed by TB and Fourier transform infra‐red spectroscopy assays. Results showed that adsorption at pH 1 and 6 had negligible effects on fucoidan's chemical structure. However, purity was actually improved by 1.55‐ and 1.69‐fold at pH 1 and 6, respectively, with an average yield of 5 g/100 g dried algae powder. In contrast, only a 1.46‐fold increment was observed in fucoidan purified by the traditional method at pH 2, with a yield of 7.5 g/100 g dried algae powder. Furthermore, fucoidan purified by this method at pH 6 complies with, or even exceeds the quality of the commercially available (≥95% pure) fucoidan (Sigma‐Aldrich^®) with respect to molecular weight and sulfur content. Therefore, dye affinity chromatography provides more advantages than the classically used techniques for fucoidan purification.     

Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co-assemble into the same receptor complex

The second beta-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced specific antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to that of the alpha-subunit ALS in the synaptic neuropil. The subunits ALS, D(alpha)2 and SBD can be co-purified by alpha-bungarotoxin affinity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and D(alpha)2 and, vice versa, ALS and D(alpha)2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaffinity chromatography with immobilized antibodies against ALS and D(alpha)2 revealed the existence of nAChR complexes that include ALS, D(alpha)2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional beta-subunit, but neither ALS nor D(alpha)2 nor the third alpha-subunit D(alpha)3. These findings suggest a considerable complexity of the Drosophila nicotinic receptor system.     

A strategy for rapid production and screening of yeast artificial chromosome libraries

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture pools, and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4–5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the 1(I) collagen (Colla1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.     

Bacterial Surface Traits Influence Digestion by Tetrahymena pyriformis and Alter Opportunity to Escape from Food Vacuoles

Endosymbiotic interactions are frequently found in nature, especially in the group of protists. Even though many endosymbioses have been studied in detail, little is known about the mechanistic origins and physiological prerequisites of endosymbiont establishment. A logical step towards the development of endocytobiotic associations is evading digestion and escaping from the host's food vacuoles. Surface properties of bacteria are probably involved in these processes. Therefore, we chemically modified the surface of a transformant strain of Escherichia coli prior to feeding to Tetrahymena pyriformis. N‐(3‐dimethylaminopropyl)‐N’‐ethylcarbodiimide allows any substance carrying amino‐ or carboxyl groups to be bound covalently to the bacterial surface by forming a peptide bond, thus, altering its properties biochemically and biophysically in a predictable manner. The effect of different traits on digestion of T. pyriformis was examined by fluorescence and transmission electron microscopy. The efficiency of digestion differs considerably depending on the coupled substances. Alkaline substances inhibit digestion partially, resulting in incomplete digestion and slightly enhanced escape rates. Increasing hydrophobicity leads to much higher escape frequencies. Both results point to possible mechanisms employed by pathogenic bacteria or potential endosymbionts in evading digestion and transmission to the host's cytoplasm.     

Serotonin Transporters in the Rat Frontal Cortex: Lack of Circadian Rhythmicity but Down-Regulation by Food Restriction

Abstract: Molecular biological findings have indicated that the affinity and the density of presynaptic serotonin transporters may be subject to adaptive regulation, but the physiological conditions that may act to trigger such changes are presently unknown. By means of [³H]paroxetine binding to rat cortical membranes, we studied the influence of two physiological variables that are clearly associated with altered serotonergic activity—circadian rhythm and semistarvation—on K _D and B _max values of the serotonin transporter of the rat frontal cortex. No circadian fluctuations of both parameters were observed. Also, semistarvation (50% reduction of normal voluntary food intake) for 2 days had no effect on either K _D or B _max values of cortical [³H]paroxetine binding. Food restriction for either 7 days or 2 weeks, however, resulted in a significant, ∼30%, reduction of the density of cortical serotonin transporters with unchanged transporter affinity. These findings indicate that long-term changes in the density of cortical serotonin transporters can be induced by long-lasting alterations of certain environmental variables. Because the duration and the radius of action of presynaptically released serotonin are governed by the efficiency of the reuptake mechanism, such adaptive changes of serotonin transporter density must be expected to cause long-term alterations of the modulatory impact of the central serotonin system on certain brain functions.     

Kurze Mitteilungen

Zusammenfassung Die Schlußfolgerungen, dieStegmann (J. Orn. 109, 1968, p. 441–445) für die verwandtschaftliche Stellung der Flughühner auf Grund seiner eigenen Untersuchungen und an Hand anderer Quellen zieht, werden kommentiert. Es wird gezeigt, daß einige dieser Schlußfolgerungen nur auf morphologischen Merkmalen beruhen und mit den zu anderen Resultaten gelangten ethologischen und physiologischen Untersuchungsergebnissen nicht zu vereinbaren sind. Außerdem erscheint es unwahrscheinlich, daß die Flughühner, deren Ursprung von bodenlebenden Nestflüchtern unbestritten ist, auf dem Umweg über die baum- und felsbewohnenden nesthockenden Tauben erst sekundär wieder zu bodenbewohnenden Nestflüchtern geworden sind. Es gibt kein einziges Flughuhn-Merkmal, das diese Hypothese stützen könnte.     

Kurze Mitteilungen

Ohne Zusammenfassung     

Evolutionary algorithms for the selection of single nucleotide polymorphisms

Background  

Large databases of single nucleotide polymorphisms (SNPs) are available for use in genomics studies. Typically, investigators must choose a subset of SNPs from these databases to employ in their studies. The choice of subset is influenced by many factors, including estimated or known reliability of the SNP, biochemical factors, intellectual property, cost, and effectiveness of the subset for mapping genes or identifying disease loci. We present an evolutionary algorithm for multiobjective SNP selection.     

Histone Deacetylase Inhibitors Modulate Interleukin 6-dependent CD4+ T Cell Polarization in Vitro and in Vivo

Histone deacetylase (HDAC) inhibitors have been associated primarily with an anti-proliferative effect in vitro and in vivo. Recent data provide evidence for an anti-inflammatory potency of HDAC inhibitors in models of experimental colitis. Because the balance of T cell subpopulations is critical for the balance of the mucosal immune system, this study explores the regulatory potency of HDAC inhibitors on T cell polarization as a mechanistic explanation for the observed anti-inflammatory effects. Although HDAC inhibition suppressed the polarization toward the pro-inflammatory T helper 17 (Th17) cells, it enhanced forkhead box P3 (FoxP3)⁺ regulatory T cell polarization in vitro and in vivo at the site of inflammation in the lamina propria. This was paralleled by a down-regulation of the interleukin 6 receptor (IL-6R) on naïve CD4⁺ T cells on the mRNA as well as on the protein level and changes in the chromatin acetylation at the IL6R gene and its promoter. Downstream of the IL-6R, HDAC inhibition was followed by a decrease in STAT3 phosphorylation as well as retinoic acid receptor-related orphan receptor γT (RORγT) expression, thus identifying the IL-6/STAT3/IL-17 pathway as an important target of HDAC inhibitors. These results directly translated to experimental colitis, where IL-6R expression was suppressed in naïve T cells, paralleled by a significant reduction of Th17 cells in the lamina propria of ITF2357-treated animals, resulting in the amelioration of disease. This study indicates that, in experimental colitis, inhibition of HDAC exerts an anti-inflammatory potency by directing T helper cell polarization via targeting the IL-6 pathway.