Stability and nickel binding properties of peptides designed as scaffolds for the stabilization of NiII-Fe4S4 bridged assemblies

Helix-loop-helix peptides containing 63 residues (HC(4)H(2), HC(4)HC, HC(5)H), designated by their sequence and content of histidyl (H) and cysteinyl (C) residues, have been previously synthesized for the purpose of stabilizing certain bridged metal sites in proteins. These peptides bind one Fe(4)S(4) cluster by means of a ferredoxin tricysteinyl consensus sequence and an additional Cys residue, and one Ni(II) atom (HC(4)H(2), HC(5)H) in predesigned binding sites. In this investigation, the apopeptides and their Fe(4)S(4) derivatives are shown to be relatively stable to unfolding by guanidine hydrochloride, indicating stability of secondary structure. With this property demonstrated, Ni(II) binding equilibria have been evaluated in the terms of site-specific (Scatchard model) and stepwise (stoichiometric) binding constants. Two peptides were designed to have preformed CysHis(3) (HC(4)H(2)) and Cys(2)His(2) (HC(5)H) binding sites. The data indicate one strong binding site in each peptide with preferred binding constants k(1)=4.4x10(5) M(-1)(HC(4)H(2)) and 2.7x10(5) M(-1)(HC(5)H). Based on X-ray absorption spectroscopic data, these binding steps are associated with the formation of the desired coordination units Ni(II)CysHis(3) and Ni(II)Cys(2)His(2). For peptide HC(4)HC, k(1)=2.5x10(5) M(-1), but the binding site could not be fully identified. Collective evidence from this and prior investigations supports the presence of the bridged assemblies Ni(II)-(mu(2)-S x Cys)-[Fe(4)S(4)], stabilized by a scaffolding effect in peptides HC(4)H(2) and HC(5)H. The assembly Ni(II)-X-[Fe(4)S(4)] is the minimal structure of the A-Cluster of carbon monoxide dehydrogenase adduced from spectroscopic evidence; bridge X is currently unidentified. These results suggest that de novo designed peptides may serve as scaffolds for the construction of native bridged sites in proteins. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0320-4.     

Regeneration of the barley zygote in ovule culture

An ovule culture technique has been established for barley that allows the regeneration of plants from zygotes. An average of 1.3 plantlets per ovule could be regenerated from more than 60% of the cultured ovules and about 75% of the regenerated plantlets developed into normal, fertile plants. The same regeneration frequencies were obtained in intact ovules and in ovules where the two integuments had been removed from the micropylar region. Unfertilized ovules and ovules where the fertilized eggs had been destroyed by a microinjection needle did not give rise to embryo-like structures. Plants could be regenerated from the zygote at the same frequency at developmental stages from immediately after fertilization until the formation of bicellular embryos. This tissue culture system appeared to be largely independent of genotype since similar regeneration frequencies were obtained in two different barley cultivars, Igri and Alexis, that in anther and microspore culture behave differently. The same technique has also been applied successfully in the wheat cultivar Walter.     

Association between the AGTR1 polymorphism +1166A>C and serum levels of high-sensitivity C-reactive protein

Genetic factors have been shown to influence high-sensitivity C-reactive protein (hsCRP) levels, however, which genes that are involved in this process remains to be clarified. The renin–angiotensin system (RAS) is of importance for the regulation of inflammation, and blockade of angiotensin II type 1 receptors (AGTR1) influences hsCRP levels. These findings prompted us to investigate whether a polymorphism in the AGTR1 gene may influence hsCRP levels. Additionally, a polymorphism in the CRP gene that has previously been shown to influence hsCRP levels was genotyped. Serum levels of hsCRP were measured in 270 42-year-old women recruited from the population registry. Two single nucleotide polymorphisms were analysed: +1166A>C and +1444C>T of the AGTR1 and CRP gene, respectively. The A allele of the AGTR1 polymorphism +1166A>C was dose-dependently associated with higher hsCRP levels (p = 0.014, adjusted for confounding factors and multiple comparisons). hsCRP levels were not significantly influenced by the CRP +1444C>T genotype; however, an interaction between the two studied polymorphisms with respect to hsCRP levels was observed (p = 0.018). The significant association between the AGTR1 polymorphism and hsCRP levels, which appears to be independent of anthropometric and metabolic traits, is yet another indication of a direct influence of RAS on inflammation.     

Iron transport, deposition and bioavailability in the wheat and barley grain

It has been reported that ground-penetrating radar (GPR) is a nondestructive tool that can be used to detect coarse roots in forest soils. However, successful GPR application for root detection has been site-specific and numerous factors can interfere with the resolution of the roots. We evaluated the effects of root diameter, root volumetric water content, and vertical and horizontal intervals between roots on the root detection of Cryptomeria japonica in sand using 900-MHz GPR. We found that roots greater than 19 mm in diameter were clearly detected. Roots having high volumetric water content were easily detected, but roots with less than 20% water content were not detected. Two roots that were located closely together were not individually distinguished. These results confirm that root diameter, root water content, and intervals between roots are important factors when using GPR for root detection and that these factors lead to an underestimation of root biomass.     

Site identity and moss species as determinants of soil microbial community structure in Norway spruce forests across three vegetation zones

Soil microbial community structure was investigated by PLFA-analysis in four spruce forests in Norway. The maximum latitudinal distance between the sites was approximately 350 km. Bilberry Vaccinium myrtillus dominated the forest floor vegetation in the study sites, which were selected because of the vegetation type. Soil samples were taken from all four sites under close to 100% homogeneous ground cover of each of two feathermoss species, i.e. Hylocomium splendens or Pleurozium schreberi, respectively. These mosses are ubiquitous in the boreal forest and constitute an abundant component of the forest floor vegetation over vast areas. Since there are no studies on how these mosses affect soil microbial community structure, our first aim was to investigate the effect of moss species on soil microbial communities. Our second aim was to investigate whether microbial communities differ among geographically separated forest sites with similar vegetation across vegetation zones. Soil microbial community structure differed between the study sites, although they appeared similar in terms of vegetation and abiotic soil conditions. Study site was the most important predictor of the variation in the PLFAs, more important than moss species, although there was a tendency for separation of microbial community structure between the two moss species.     

Porous Magnesium Aluminometasilicate Tablets as Carrier of a Cyclosporine Self-Emulsifying Formulation

The aim of this study was to investigate the ability of liquid loadable tablets (LLT) to be loaded with a self-microemulsifying drug delivery system (SMEDDS) containing cyclosporine (CyA). LLT were prepared by direct compression of the porous carrier magnesium aluminometasilicate and subsequently loaded with SMEDDS by a simple absorption method. SMEDDS was evaluated regarding visual appearance and droplet size distribution after dispersion in aqueous media. The developed SMEDDS was found to be similar to Neoral®. LLT were characterized before and after loading regarding weight variation, tablet hardness, disintegration time, and in vitro drug release. It was found that LLT with high porosities suitable for liquid loading and further processing could be prepared. Adding a tablet disintegrant was found to improve in vitro drug release. Additionally, the volume-based loading capacity of LLT was evaluated and found to be comparable to soft gelatin and hard two-piece capsules. Furthermore, the pharmacokinetic performance of CyA from loaded LLT was tested in two PK-studies in dogs. Absorption of CyA from SMEDDS loaded into LLT was found in the first study to be significantly lower than the absorption of CyA from SMEDDS filled into a capsule. However, addition of a superdisintegrant improved the absorption markedly. The bioavailability of CyA from SMEDDS loaded into disintegrating LLT was found in the second study to be at the same level as from capsule formulation. In conclusion, the LLT technology is therefore seen as a promising alternative way of achieving a solid dosage form from liquid drug delivery systems.