Detection and genetic characterization of porcine circovirus 2 isolates from the first cases of postweaning multisystemic and wasting syndrome in wild boars in Greece

In 2002, postweaning multisystemic wasting syndrome (PMWS) was diagnosed in a European female wild boar (Sus scrofa), based on the detection of porcine circovirus 2 (PCV2) DNA in various organs, including the uterus, and on histopathologic lesions. This is the first detection of PCV2 DNA in the uterus of a wild boar. Three years later (2005), a wild boar < 6-8 mo of age was found moribund. It presented wasting and dyspnea and finally died. PCV2 DNA was detected in tissue samples, and histopathologic lesions consistent with PMWS were observed. Both wild boars were from neighboring hunting areas in central Greece. Two PCV2 strains from the wild boars were genetically characterized and compared to other reported PCV2 sequences from wild boars and domestic pigs. The PCV-2 sequences from the wild boars in this study were closely related to each other and were grouped with two isolates from wild boars from Hungary. The phylogenetic analysis revealed that the virus might be transmitted between hunting areas. In addition, PCV2 may spread from domestic pigs to wild boars and vice versa.     

Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity

Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand-gated ion-channel superfamily, which has precursors in the prokaryotic world. They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (alpha1-10, beta1-4, gamma, delta, and epsilon). There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions. They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia. nAChRs are also found in non-neuronal/nonmuscle cells (keratinocytes, epithelia, macrophages, etc.). Extensive research has determined the specific function of several nAChR subtypes. nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, and schizophrenia, as well as for the cessation of smoking. However, knowledge is still incomplete, largely because of a lack of high-resolution X-ray structures for these molecules. Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high-resolution X-ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype-specific drugs. Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology.     

Synthesis and Biological Evaluation of New GnRH Analogues on Pituitary and Breast Cancer Cells

GnRH analogues have been extensively used in oncology to induce reversible chemical castration due to their hypophysiotropic action. In addition to that, it has recently been shown that many malignant cells, such as breast cancer cells, locally produce GnRH and express the GnRH receptor/s. In order to investigate the structure-activity relationships in both pituitary and extrapituitary biological systems, we synthesized eight new GnRH analogues with modifications in the N-terminal part and/or in position 6 and studied their pituitary binding affinity (in αT3-1 cell membranes) and effect on breast cancer (MCF-7) cell proliferation. 2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (ATPC) was incorporated instead of pGlu¹-His²- and/or Gly⁶ was substituted by α-aminoisobutyric acid, D-Leu and D-Lys (alone or covalently linked to Gly, Ala, Sar, ATPC). Most GnRH analogues lacked the carboxy-terminal Gly¹⁰-amide of GnRH and an ethylamide residue was added to Pro⁹, a modification common in many potent GnRH agonists, such as leuprolide ([D-Leu⁶, des-Gly¹⁰]-GnRH-NHEt. Results show differential impact of these modifications on the binding affinity to the GnRH receptor in mouse pituitary cells and on the inhibition of human breast cancer cell proliferation. ATPC in the N-terminus resulted in analogues with low binding affinity but high antiproliferative effect. Substitutions in position 6 always resulted in high binding affinities. In particular, [D-Lys⁶(Gly), desGly¹⁰]-GnRH-NHEt and [D-Lys⁶(Sar), desGly¹⁰]-GnRH-NHEt have higher pituitary binding affinity than leuprolide, but only the latter had significant antiproliferative effect on both MCF-7 and MDA-MB-231 cells. These results contribute to the on-going research for more potent GnRH analogues. Abbreviations of common amino acids are in accordance with the recommendations of IUPAC-IUB Joint Commission on Biochemical Nomenclature: Arch. Biochem. Biophys. 206, pp.v-xxii (1988), J. Biol. Chem. 264, 668–673 (1989) or J. Peptide Sci. 9, 1–8 (2003).     

Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids

Amino Acids - Neurotensin (NT) (pGlu–Leu–Tyr–Glu–Asn–Lys–Pro–Arg–Arg–Pro–Tyr–Ile–Leu) exerts a dual function as a...     

Proximate composition, fatty acids, cholesterol, minerals in frozen red porgy

The proximate composition of frozen red porgy (Pagrus pagrus) was determined. The moisture, ash, protein and total lipids (45.5+/-1.4% PL of which 90.4+/-2.0% PhL) were found to be 71.7+/-1.0%, 1.73+/-0.12%, 21.5+/-0.8% and 0.81+/-0.09% of the wet muscle tissue, respectively. 16:0 and 18:0 were the main SFA, 18:1 (omega-9 and omega-7) the main MUFA while DHA, EPA and arachidonic acid were the main polyunsaturated fatty acids (PUFA). The SFA/PUFA ratio was 1.5 and the omega-3/omega-6 ratio was 3.02. The cholesterol content was found to be 8.18+/-0.34 mg/100 g of the wet muscle tissue. Ni, Cr, Mn, Cu, Zn, Fe and Mg were determined in the muscles, skin, hepatopancreas and head of the fish. The covering percentage of the recommended daily allowance/intake (RDA/RDI) for each mineral, in the muscle tissue, has been calculated to 14.2% (males) and 7.89% (females) for Fe, 2.87% for Cu, 4.07% for Zn 0.4% for Mn, 13.9% for Ni, 20.2% for Cr and 10.4% for Mg.     

The Shepherds’ Tale: A Genome-Wide Study across 9 Dog Breeds Implicates Two Loci in the Regulation of Fructosamine Serum Concentration in Belgian Shepherds

Diabetes mellitus is a serious health problem in both dogs and humans. Certain dog breeds show high prevalence of the disease, whereas other breeds are at low risk. Fructosamine and glycated haemoglobin (HbA1c) are two major biomarkers of glycaemia, where serum concentrations reflect glucose turnover over the past few weeks to months. In this study, we searched for genetic factors influencing variation in serum fructosamine concentration in healthy dogs using data from nine dog breeds. Considering all breeds together, we did not find any genome-wide significant associations to fructosamine serum concentration. However, by performing breed-specific analyses we revealed an association on chromosome 3 (p_corrected ≈ 1:68 × 10^-6) in Belgian shepherd dogs of the Malinois subtype. The associated region and its close neighbourhood harbours interesting candidate genes such as LETM1 and GAPDH that are important in glucose metabolism and have previously been implicated in the aetiology of diabetes mellitus. To further explore the genetics of this breed specificity, we screened the genome for reduced heterozygosity stretches private to the Belgian shepherd breed. This revealed a region with reduced heterozygosity that shows a statistically significant interaction (p = 0.025) with the association region on chromosome 3. This region also harbours some interesting candidate genes and regulatory regions but the exact mechanisms underlying the interaction are still unknown. Nevertheless, this finding provides a plausible explanation for breed-specific genetic effects for complex traits in dogs. Shepherd breeds are at low risk of developing diabetes mellitus. The findings in Belgian shepherds could be connected to a protective mechanism against the disease. Further insight into the regulation of glucose metabolism could improve diagnostic and therapeutic methods for diabetes mellitus.     

Comparative proteomic analysis of hypertrophic chondrocytes in osteoarthritis

Background

Osteoarthritis (OA) is a multi-factorial disease leading progressively to loss of articular cartilage and subsequently to loss of joint function. While hypertrophy of chondrocytes is a physiological process implicated in the longitudinal growth of long bones, hypertrophy-like alterations in chondrocytes play a major role in OA. We performed a quantitative proteomic analysis in osteoarthritic and normal chondrocytes followed by functional analyses to investigate proteome changes and molecular pathways involved in OA pathogenesis.

Methods

Chondrocytes were isolated from articular cartilage of ten patients with primary OA undergoing knee replacement surgery and six normal donors undergoing fracture repair surgery without history of joint disease and no OA clinical manifestations. We analyzed the proteome of chondrocytes using high resolution mass spectrometry and quantified it by label-free quantification and western blot analysis. We also used WebGestalt, a web-based enrichment tool for the functional annotation and pathway analysis of the differentially synthesized proteins, using the Wikipathways database. ClueGO, a Cytoscape plug-in, is also used to compare groups of proteins and to visualize the functionally organized Gene Ontology (GO) terms and pathways in the form of dynamical network structures.

Results

The proteomic analysis led to the identification of a total of ~2400 proteins. 269 of them showed differential synthesis levels between the two groups. Using functional annotation, we found that proteins belonging to pathways associated with regulation of the actin cytoskeleton, EGF/EGFR, TGF-β, MAPK signaling, integrin-mediated cell adhesion, and lipid metabolism were significantly enriched in the OA samples (p ≤10⁻⁵). We also observed that the proteins GSTP1, PLS3, MYOF, HSD17B12, PRDX2, APCS, PLA2G2A SERPINH1/HSP47 and MVP, show distinct synthesis levels, characteristic for OA or control chondrocytes.

Conclusion

In this study we compared the quantitative changes in proteins synthesized in osteoarthritic compared to normal chondrocytes. We identified several pathways and proteins to be associated with OA chondrocytes. This study provides evidence for further testing on the molecular mechanism of the disease and also propose proteins as candidate markers of OA chondrocyte phenotype.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9085-6) contains supplementary material, which is available to authorized users.