Structural analysis and biochemical properties of laccase enzymes from two Pediococcus species

Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6-DMP, and non-conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well-studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.     

Prostaglandin E2 Exerts Multiple Regulatory Actions on Human Obese Adipose Tissue Remodeling,Inflammation, Adaptive Thermogenesis and Lipolysis

Obesity induces white adipose tissue (WAT) dysfunction characterized by unremitting inflammation and fibrosis, impaired adaptive thermogenesis and increased lipolysis. Prostaglandins (PGs) are powerful lipid mediators that influence the homeostasis of several organs and tissues. The aim of the current study was to explore the regulatory actions of PGs in human omental WAT collected from obese patients undergoing laparoscopic bariatric surgery. In addition to adipocyte hypertrophy, obese WAT showed remarkable inflammation and total and pericellular fibrosis. In this tissue, a unique molecular signature characterized by altered expression of genes involved in inflammation, fibrosis and WAT browning was identified by microarray analysis. Targeted LC-MS/MS lipidomic analysis identified increased PGE₂ levels in obese fat in the context of a remarkable COX-2 induction and in the absence of changes in the expression of terminal prostaglandin E synthases (i.e. mPGES-1, mPGES-2 and cPGES). IPA analysis established PGE₂ as a common top regulator of the fibrogenic/inflammatory process present in this tissue. Exogenous addition of PGE₂ significantly reduced the expression of fibrogenic genes in human WAT explants and significantly down-regulated Col1α1, Col1α2 and αSMA in differentiated 3T3 adipocytes exposed to TGF-β. In addition, PGE₂ inhibited the expression of inflammatory genes (i.e. IL-6 and MCP-1) in WAT explants as well as in adipocytes challenged with LPS. PGE₂ anti-inflammatory actions were confirmed by microarray analysis of human pre-adipocytes incubated with this prostanoid. Moreover, PGE₂ induced expression of brown markers (UCP1 and PRDM16) in WAT and adipocytes, but not in pre-adipocytes, suggesting that PGE₂ might induce the trans-differentiation of adipocytes towards beige/brite cells. Finally, PGE₂ inhibited isoproterenol-induced adipocyte lipolysis. Taken together, these findings identify PGE₂ as a regulator of the complex network of interactions driving uncontrolled inflammation and fibrosis and impaired adaptive thermogenesis and lipolysis in human obese visceral WAT.     

Unusual viral ligand with alternative interactions is presented by HLA-Cw4 in human respiratory syncytial virus-infected cells

Short viral antigens bound to human major histocompatibility complex (HLA) class I molecules are presented on infected cells. Vaccine development frequently relies on synthetic peptides to identify optimal HLA class I ligands. However, when natural peptides are analyzed, more complex mixtures are found. By immunoproteomics analysis, we identify in this study a physiologically processed HLA ligand derived from the human respiratory syncytial virus matrix protein that is very different from what was expected from studies with synthetic peptides. This natural HLA-Cw4 class I ligand uses alternative interactions to the anchor motifs previously described for its presenting HLA-Cw4 class I molecule. Finally, this octameric peptide shares its C-terminal core with the H-2D(b) nonamer ligand previously identified in the mouse model. These data have implications for the identification of antiviral cytotoxic T lymphocyte responses and for vaccine development.     

Interleukin-1 beta-induced anorexia is reversed by ghrelin

Interleukins, in particular interleukin-1β (IL-1β), reduce food intake after peripheral and central administration, which suggests that they contribute to anorexia during various infectious, neoplastic, and autoimmune diseases. On the other hand, ghrelin stimulates food intake by acting on the central nervous system (CNS) and is considered an important regulator of food intake in both rodents and humans. In the present study, we investigated if ghrelin could reverse IL-1β-induced anorexia. Intracerebroventricular (i.c.v.) injection of 15, 30 or 45 ng/μl of IL-1β caused significant suppression of food intake in 20 h fasting animals. This effect lasted for a 24 h period. Ghrelin (0.15 nmol or 1.5 nmol/μl) produced a significant increase in cumulative food intake in normally fed animals. However, it did not alter food intake in 20 h fasting animals. Central administration of ghrelin reduced the anorexic effect of IL-1β (15 ng/μl). The effect was observed 30 min after injection and lasted for the next 24 h. This study provides evidence that ghrelin is an orexigenic peptide capable of antagonizing IL-1β-induced anorexia.     

M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.     

Chromosomal evolution of the PKD1 gene family in primates

Correction to Kirsch S, Pasantes J, Wolf A, Bogdanova N, Münch C, Pennekamp P, Krawczak M, Dworniczak B, Schempp W: Chromosomal evolution of the PKD1 gene family in primates. BMC Evolutionary Biology 2008, 8 :263 (doi:10.1186/1471-2148-8-263)     

The zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals

Background  

Aquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown.     

Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin

Ghrelin is a peptide hormone produced and secreted from the stomach. Hypothalamic injection of the peptide increases food intake but it is not known if the peptide affects other brain regions. We measured several behavioral parameters such as anxiety (elevated plus maze), memory retention (step down test), and food intake after injections of different doses of the peptide in the hippocampus, amygdala, and dorsal raphe nucleus (DRN). The injection of ghrelin in the hippocampus and DRN significantly and dose dependently increased food intake in relation to controls rats, while injections into the amygdala did not affect the food intake. We also show for the first time that ghrelin clearly and dose dependently increases memory retention in the hippocampus, amygdala, and DRN. Moreover, ghrelin at different potencies induced anxiogenesis in these brain structures while the highest dose of 3 nmol/microl was effective in all of them. The comparison of sensitivity of each brain structure indicates a specific role of them for each of the behaviors studied. The results provide new insight in to the anatomical substrate and the functional role of extrahypothalamic ghrelin targets in the CNS.