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.     

Death receptor-induced signaling pathways are differentially regulated by gamma interferon upstream of caspase 8 processing

FasL and gamma interferon (IFN-gamma) are produced by activated T cells and NK cells and synergistically induce apoptosis. Although both cytokines can also elicit proinflammatory responses, a possible cross talk of these ligands with respect to nonapoptotic signaling has been poorly addressed. Here, we show that IFN-gamma sensitizes KB cells for apoptosis induction by facilitating death-inducing signaling complex (DISC)-mediated caspase 8 processing. Moreover, after protection against death receptor-induced apoptosis by caspase inhibition or Bcl2 overexpression, IFN-gamma also sensitized for Fas- and TRAIL death receptor-mediated NF-kappaB activation leading to synergistic upregulation of a variety of proinflammatory genes. In contrast, Fas-mediated activation of JNK, p38, and p42/44 occurred essentially independent from IFN-gamma sensitization, indicating that the apoptosis- and NF-kappaB-related FasL-IFN-gamma cross talk was not due to a simple global enhancement of Fas signaling. Overexpression of FLIP(L) and FLIP(S) inhibited Fas- as well as TRAIL-mediated NF-kappaB activation and apoptosis induction in IFN-gamma-primed cells suggesting that both responses are coregulated at the level of the DISC.     

Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure-activity relationships in the bacterial membrane disruptor betapep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of betapep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel beta-sheet structure is the bioactive conformation of betapep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, betapep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.     

Head and neck anthropometry, vertebral geometry and neck strength in height-matched men and women

Women have an increased incidence of whiplash injury and neck pain compared to men. Physical and numerical models represent one avenue to explore and potentially explain these gender differences, but a valid model of the female neck does not yet exist. A fundamental question in the development of a female neck model is whether female necks are simply scaled versions of male necks, or whether there are significant inter-gender geometrical differences. The goal of this study was to quantify differences in head and neck geometry and neck strength in pairs of male and female subjects matched for standing height and neck length. Based on 14 matched pairs of men and women, we found that most head and neck anthropometric parameters were significantly smaller in females compared to males. Moreover, gender differences in a number of neck anthropometry parameters (an average of 9-16% smaller in females) were larger than differences in head anthropometry parameters (an average of 3-6% smaller in females). Female vertebrae between C3 and C7 were significantly smaller than male vertebrae in the anterior-posterior dimension (p < 0.012) but not in the medial-lateral dimension (p > 0.07). Female necks were also significantly weaker than male necks (32% weaker in flexion and 20% weaker in extension; p < 0.001), and these strength differences corresponded well to those predicted solely from the observed geometric differences. These results demonstrate that male and female necks are not geometrically similar and indicate that a female-specific model will be necessary to study gender differences in neck-related disorders.     

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.     

Effects of repeated (NH4)2SO4 application on sulfur pools in soil,soil microbial biomass,and ground vegetation of two watersheds in the Black Forest/Germany

The effect of repeated (NH₄)₂SO₄ applications (3 × 700 kg ha^–1 in 1988, 1991, and 1994, respectively) on S pools in soil, soil microbial biomass, and ground vegetation was studied at two Norway spruce (Picea abies L. [Karst.]) sites in the Black Forest/Germany. In both eco-systems, most of the total S pool was located in the soil. The soil also was the predominant compartment for retention of applied SO₄ ^2--S. The fractions of organic and inorganic S forms in the initial soil S content, and the retention of experimentally applied S was different for both sites. In the podzol Schluchsee, organic S accounted for 92% of total S. In the cambisol Villingen, the S pool consisted of 33% organic S and 67% inorganic S. The retention of applied S in various compartments of both ecosystems reflected these proportions. Only minor amounts of fertilized S (<1%) was retained in the spruce trees, ground vegetation, and soil microbial biomass. However, between 51% (Villingen) and 72% (Schluchsee) of the applied S was retained in the soil. In the Schluchsee podzol, 75% of retained fertilizer S was accumulated as ester sulfate, whereas SO₄ ^2-adsorption and precipitation of Al hydroxy sulfates were restricted by dissolved organic matter in the soil solution. In the Villingen cambisol, SO₄ ^2- adsorption was the dominant process of S retention, although 20% of the fertilized S again was retained as ester sulfate. The significant relevance of organic S forms in the retention of fertilizer S in both soils emphasizes the need for models which include the formation and re-mineralization of organic S compounds, especially of ester sulfates, for correctly simulating and predicting the retention and remobilization of S in acid forest soils subject to changing atmospheric N and S deposition.