Crystal structure of avian carboxypeptidase D domain II: a prototype for the regulatory metallocarboxypeptidase subfamily.

The crystal structure of domain II of duck carboxypeptidase D, a prohormone/propeptide processing enzyme integrated in a three repeat tandem in the natural system, has been solved, constituting a prototype for members of the regulatory metallocarboxypeptidase subfamily. It displays a 300 residue N-terminal alpha/beta-hydrolase subdomain with overall topological similarity to and general coincidence of the key catalytic residues with the archetypal pancreatic carboxypeptidase A. However, numerous significant insertions/deletions in segments forming the funnel-like access to the active site explain differences in specificity towards larger protein substrates or inhibitors. This alpha/beta-hydrolase subdomain is followed by a C-terminal 80 residue beta-sandwich subdomain, unique for these regulatory metalloenzymes and topologically related to transthyretin and sugar-binding proteins. The structure described here establishes the fundamentals for a better understanding of the mechanism ruling events such as prohormone processing and will enable modelling of regulatory carboxypeptidases as well as a more rational design of inhibitors of carboxypeptidase D.     

A Comparative Study of Two Corbicula Morphs (Bivalvia,Corbiculidae) Inhabiting River Danube

In Europe, at least two Corbicula morphs (generally referred as C. fluminea and C. fluminalis) are known. However, their taxonomic and systematic position is controversial. In order to ascertain their taxonomy and systematics, we studied their characteristics in River Danube. Morphometric analysis confirmed the existence of two distinguishable morphs. Based on mtCOI sequence, morph‐1 is identical to C. fluminea, while morph‐2 is an undefined species. According to the median joining analysis, morph‐1 belongs to the C. fluminea clade, whereas morph‐2 forms a distinct clade, which is known only from invasive populations. Differences were recorded in population structure and reproductive biology, too. It is probably correct to treat the two morphs as distinct taxa, but as long as the African and western Asian parts of the genus’ range are poorly studied, taxonomic position of morph‐2 remains unclear. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers

Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.     

Sorption isotherms and kinetics in the primary biodegradation of anionic surfactants by immobilized bacteria:: II. Comamonas terrigena N3H

Comamonas terrigena N3H was immobilized by covalent linking on silanized inorganic supports and by physical entrapment of cells within calcium alginate beads and reticulated polyurethane foam. Both entrapped cells were efficient for the primary biodegradation of the anionic surfactants dihexyl sulphosuccinate (DHSS) and dioctyl sulphosuccinate (DOSS), furthermore, exhibiting, in the case of polyurethane immobilized cells, a positive fractionating effect of the substrate by adsorption onto the polymer matrix. The overall kinetics for the surfactant removal from water were well-fitted to a biphasic process, a rapid passive sorption step of the surfactant onto the cell-loaded support and the intrinsic primary biodegradation slower step, both acting synergically.     

Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans

Here we show that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes unc-51/atg1 and bec-1/atg6/beclin1 results in small body size without affecting cell number. Furthermore, loss-of-function mutations in unc-51 and bec-1 suppress the giant phenotype of mutant animals with aberrant insulin-like growth factor-1 (insulin/IGF-1) or transforming growth factor-beta (TGF-beta) signaling. This function for unc-51 and bec-1 in cell size control and their interaction with these two growth modulatory pathways may represent a link between the hormonal and nutritional regulation of cell growth.     

Anterior cruciate ligament constructs fabricated from human mesenchymal stem cells in a collagen type I hydrogel

BACKGROUND: Disruptions of the anterior cruciate ligament (ACL) of the knee joint are common and are currently treated using ligament or tendon grafts. In this study, we tested the hypothesis that it is possible to fabricate an ACL construct in vitro using mesenchymal stem cells (MSC) in combination with an optimized collagen type I hydrogel, which is in clinical use for autologous chondrocyte transplantation (ACT). METHODS: ACL constructs were molded using a collagen type I hydrogel containing 5 x 10(5) MSC/mL and non-demineralized bone cylinders at each end of the constructs. The constructs were kept in a horizontal position for 10 days to allow the cells and the gel to remodel and attach to the bone cylinders. Thereafter, cyclic stretching with 1 Hz was performed for 14 days (continuously for 8 h/day) in a specially designed bioreactor. RESULTS: Histochemical analysis for H and E, Masson-Goldner and Azan and immunohistochemical analysis for collagen types I and III, fibronectin and elastin showed elongated fibroblast-like cells embedded in a wavy orientated collagenous tissue, together with a ligament-like extracellular matrix in the cyclic stretched constructs. No orientation of collagen fibers and cells, and no formation of a ligament-like matrix, could be seen in the non-stretched control group cultured in a horizontal position without tension. RT-PCR analysis revealed an increased gene expression of collagen types I and III, fibronectin and elastin in the stretched constructs compared with the non-stretched controls. DISCUSSION: In conclusion, ACL-like constructs from a collagen type I hydrogel, optimized for the reconstruction of ligaments, and MSC have been fabricated. As shown by other investigators, who analyzed the influence of cyclic stretching on the differentiation of MSC, our results indicate a ligament-specific increased protein and gene expression and the formation of a ligament-like extracellular matrix. The fabricated constructs are still too weak for animal experiments or clinical application and current investigations are focusing on the development of a construct with an internal augmentation using biodegradable fibers.     

The G protein-coupled receptor GPR162 is widely distributed in the CNS and highly expressed in the hypothalamus and in hedonic feeding areas

The Rhodopsin family is a class of integral membrane proteins belonging to G protein-coupled receptors (GPCRs). To date, several orphan GPCRs are still uncharacterized and in this study we present an anatomical characterization of the GPR162 protein and an attempt to describe its functional role. Our results show that GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in areas related to energy homeostasis and hedonic feeding such as hypothalamus, amygdala and ventral tegmental area, regions known to be involved in the regulation of palatable food consumption.     

Formation of new genes explains lower intron density in mammalian Rhodopsin G protein-coupled receptors

Mammalian G protein-coupled receptor (GPCR) genes are characterised by a large proportion of intronless genes or a lower density of introns when compared with GPCRs of invertebrates. It is unclear which mechanisms have influenced intron density in this protein family, which is one of the largest in the mammalian genomes. We used a combination of Hidden Markov Models (HMM) and BLAST searches to establish the comprehensive repertoire of Rhodopsin GPCRs from seven species and performed overall alignments and phylogenetic analysis using the maximum parsimony method for over 1400 receptors in 12 subgroups. We identified 14 different Ancestral Receptor Groups (ARGs) that have members in both vertebrate and invertebrate species. We found that there exists a remarkable difference in the intron density among ancestral and new Rhodopsin GPCRs. The intron density among ARGs members was more than 3.5-fold higher than that within non-ARG members and more than 2-fold higher when considering only the 7TM region. This suggests that the new GPCR genes have been predominantly formed intronless while the ancestral receptors likely accumulated introns during their evolution. Many of the intron positions found in mammalian ARG receptor sequences were found to be present in orthologue invertebrate receptors suggesting that these intron positions are ancient. This analysis also revealed that one intron position is much more frequent than any other position and it is common for a number of phylogenetically different Rhodopsin GPCR groups. This intron position lies within a functionally important, conserved, DRY motif which may form a proto-splice site that could contribute to positional intron insertion. Moreover, we have found that other receptor motifs, similar to DRY, also contain introns between the second and third nucleotide of the arginine codon which also forms a proto-splice site. Our analysis presents compelling evidence that there was not a major loss of introns in mammalian GPCRs and formation of new GPCRs among mammals explains why these have fewer introns compared to invertebrate GPCRs. We also discuss and speculate about the possible role of different RNA- and DNA-based mechanisms of intron insertion and loss.     

Association of feather colour with constitutively active melanocortin 1 receptors in chicken.

Seven alleles of the chicken melanocortin (MC) 1 receptor were cloned into expression vectors, expressed in mammalian cells and pharmacologically characterized. Four of the clones e(+R), e(+B&D), e(wh)/e(y), E(Rfayoumi) gave receptors to which melanocortin stimulating hormone (alpha-MSH) and NDP-MSH bound with similar IC50 values and responded to alpha-MSH by increasing intracellular cAMP levels in a dose-dependent manner. Three of the cMC1 receptors; e(b), E and E(R), did not show any specific binding to the radioligand, but were found to be constitutively active in the cAMP assay. The E and E(R) alleles are associated with black feather colour in chicken while the eb allele gives rise to brownish pigmentation. The three constitutively active receptors share a mutation of Glu to Lys in position 92. This mutation was previously found in darkly pigmented sombre mice, but constitutively active MC receptors have not previously been shown in any nonmammalian species. We also inserted the Glu to Lys mutation in the human MC1 and MC4 receptors. In contrast with the chicken clones, the hMC1-E94K receptor bound to the ligand, but was still constitutively active independently of ligand concentration. The hMC4-E100K receptor did not bind to the MSH ligand and was not constitutively active. The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a consequence of this E/K mutation, are not conserved within the MC receptors. The results are discussed in relationship to feather colour in chicken, molecular receptor structures and evolution. We suggest that properties for the 'E92K switch' mechanism may have evolved in an ancestor common to chicken and mammals and were maintained over long time periods through evolutionary pressure, probably on closely linked structural features.