Demonstration of a Direct Interaction between β2-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics

Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β₂-adrenergic receptor (β₂AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on β₂AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β₂AR; however, IR reduced β₂AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between β₂AR and IR, we employed quantitative BRET² saturation and competition assays. Saturation assay data suggested constitutive β₂AR and IR homo- and heteromerization. Calculated acceptor/donor (AD₅₀) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET² signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET² data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of β₂AR and IR identified intracellular domains encompassing residues at the end of the 7^th TM domain and C-terminal tail of β₂AR and a cytoplasmic part of the IR β chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order β₂AR:IR oligomer formation, likely comprising heteromers of homodimers.     

Colostrum of Healthy Slovenian Mothers: Microbiota Composition and Bacteriocin Gene Prevalence

Microbial communities inhabiting the breast milk microenvironment are essential in supporting mammary gland health in lactating women and in providing gut-colonizing bacterial ''inoculum'' for their infants’ gastro-intestinal development. Bacterial DNA was extracted from colostrum samples of 45 healthy Slovenian mothers. Characteristics of the communities in the samples were assessed by polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and by quantitative real-time PCR (qPCR). PCR screening for the prevalence of bacteriocin genes was performed on DNA of culturable and total colostrum bacteria. DGGE profiling revealed the presence of Staphylococcus and Gemella in most of the samples and exposed 4 clusters based on the abundance of 3 bands: Staphylococcus epidermidis/Gemella, Streptococcus oralis/pneumonia and Streptococcus salivarius. Bacilli represented the largest proportion of the communities. High prevalence in samples at relatively low quantities was confirmed by qPCR for enterobacteria (100%), Clostridia (95.6%), Bacteroides-Prevotella group (62.2%) and bifidobacteria (53.3%). Bacterial quantities (genome equivalents ml^-1) varied greatly among the samples; Staphylococcus epidermidis and staphylococci varied in the range of 4 logs, streptococci and all bacteria varied in the range of 2 logs, and other researched groups varied in the range of 1 log. The quantity of most bacterial groups was correlated with the amount of all bacteria. The majority of the genus Staphylococcus was represented by the species Staphylococcus epidermidis (on average 61%), and their abundances were linearly correlated. Determinants of salivaricin A, salivaricin B, streptin and cytolysin were found in single samples. This work provides knowledge on the colostrum microbial community composition of healthy lactating Slovenian mothers and reports bacteriocin gene prevalence.     

Liposomal Encapsulation of Oleuropein and an Olive Leaf Extract: Molecular Interactions,Antioxidant Effects and Applications in Model Food Systems

The influence of actively/passively encapsulated oleuropein on DPPC liposomes thermal and structural properties, and its antioxidant capacity against lipid peroxidation were investigated. Also, an oleuropein-rich olive leaf extract was encapsulated in soy phosphatidylcholine (PL-90 g) and incorporated in model and commercial drinks. Oleuropein induced a concentration-dependent broadening and splitting of the gel-to-liquid phase transition temperature. Fluorescence measurements revealed a fluidizing effect on liposomes below their gel-to-liquid phase transition temperature, and a higher lipid ordering above, especially to active encapsulation. Oleuropein also showed an antioxidant effect against lipid peroxidation in PL-90 g liposomes. PL-90 g Liposomes with olive leaf extract showed a mean diameter of 405 ± 4 nm and oleuropein encapsulation efficiency of 34% and delayed oleuropein degradation at pH 2.0 and 2.8 model drinks. In conclusion, greater effects were observed on the structure and fluidity of DPPC liposomes when oleuropein was actively encapsulated, while its incorporation into acidic foods in encapsulated form could enhance its stability.

     

Contributions to molecular systematics of water scavenger beetles (Hydrophilidae,Coleoptera)

Phylogenetic relationships within Hydrophilidae were examined by analyses of separate and combined nuclear and mitochondrial markers (28S rRNA, 18S rRNA, 16S rRNA, 12S rRNA, COI and COII genes). The preferred (Bayesian) tree topology suggests a sister group relationship between Spercheidae and Hydrophilidae, supporting the ‘hydrophilid lineage’; Epimetopidae are placed on the base of the ‘helophorid branch’, the monophyly of Sphaeridiinae is highly supported, nested deeply within Hydrophilidae closest to Enochrus, making Hydrophilinae and Acidocerini paraphyletic; Hydrobius appears as sister taxon to (Hydrochara + Hydrophilus) without a closer relationship to Acidocerini; the hydrophiloid–histeroid sister group relationship is confirmed. The topology of several taxa remains contradictory, and requires further investigations with a larger taxon sampling and additional molecular markers.     

ELLSA based profiling of surface glycosylation in microorganisms reveals that ß-glucan rich yeasts’ surfaces are selectively recognized with recombinant banana lectin

Glycoconjugate Journal - The surface of microorganisms is covered with polysaccharide structures which are in immediate contact with receptor structures on host’s cells and antibodies. The...     

Identification of phosphorylation sites in glycine N-methyltransferase from rat liver

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.