Human dignity, bioethics, and human rights

The authors analyse and assess the Universal Draft Declaration on Bioethics and Human Rights published by UNESCO. They argue that the Draft has two main weaknesses. It unnecessarily confines the scope of bioethics to life sciences and their practical applications. And it fails to spell out the intended role of human dignity in international ethical regulation.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

Stochastic boundary molecular dynamics simulation of L-ribose in the active site of Actinoplanes missouriensis xylose isomerase and its Val135Asn mutant with improved reaction rate

We used molecular dynamics simulations to study how a non-natural substrate, L-ribose, interacts with the active site of Actinoplanes missouriensis xylose isomerase. The simulations showed that L-ribose does not stay liganded in the active site in the same way as D-xylose, in which the oxygens O2 and O4 are liganded to the metal M1. The oxygen O4 of L-ribose moved away from the metal M1 to an upside down position. Furthermore, the distances of the carbons C1 and C2 of L-ribose to the catalytic metal M2 were higher than in the case of D-xylose. These findings explain the extremely low reaction rate of xylose isomerase with L-ribose. The mutation V135N close to the C5-OH of the substrate increased the reaction efficiency 2- to 4-fold with L-ribose. V135N did not affect the reaction with D-xylose and L-arabinose, whereas the reaction with D-glucose was impaired, probably due to a hydrogen bond between Asn-135 and the substrate. When L-ribose was the substrate, Asn-135 formed a hydrogen bond to Glu-181. As a consequence, O4 of L-ribose stayed liganded to the metal M1 in the V135N mutant in molecular dynamics simulations. This explains the decreased K(m) of the V135N mutant with L-ribose.     

Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.     

Secretion of sterols and the NPC2 protein from primary astrocytes

Astrocytes secrete cholesterol in lipoprotein particles. Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1-/-) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1-/- astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors.     

Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threo-hexarate

Microorganisms use different pathways for D-galacturonate catabolism. In the known microbial oxidative pathway, D-galacturonate is oxidized to D-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and α-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes D-galacturonic acid to D-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the D-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on D-galactarolactone and D-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from D-galactaro-1,4-lactone and D-glucaro-1,4-lactone conversion is in both cases the L-threo form of 3-deoxy-2-keto-hexarate.     

Spontaneous remodeling of HDL particles at acidic pH enhances their capacity to induce cholesterol efflux from human macrophage foam cells

HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL(2) and HDL(3) subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL(3) than in HDL(2). Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects.     

Genetic effects of supportive stockings on native pikeperch populations in boreal lakes--cases, three different outcomes

The genetic consequences and gene flow of pikeperch (Sander lucioperca) stocking were assessed in three boreal lakes based on admixture model analysis and comparison of the pre- and post-release patterns of genetic variability at 9 DNA microsatellite loci in the recipient populations. In two out of the three cases, the releases of fish from foreign populations caused significant changes in the genetic structure of the recipient population. The largest changes were observed in Lake Ouluj?rvi, where the post-release sample was almost identical to the released Lake Vanajanselk? population, and about 90% of the catch was composed of the released population. The genetic composition of Lake Lohjanj?rvi pikeperch also shifted markedly towards that of the released Lake Vanajanselk? population, and about half of the later catch was of released Vanajanselk? origin. In Lake Vanajanselk?, in contrast, releases of pikeperch from lakes Painio and Averia had only a small impact on the genetic structure of the pikeperch population. These results indicate that the current stocking practices create an effective artificial gene flow that may strongly shape and reduce the genetic differentiation among the remaining native pikeperch populations. A common feature of all three cases was the lack of prior appraisal of the potential genetic and ecological risks in relation to the expected benefits of the release programmes.