Synthesis and biological evaluation of fused oxepinocoumarins as free radicals scavengers

Some fused dihydrooxepino[f]-, [g]-, and [h]coumarins were obtained from the ring-closing metathesis of the corresponding o-allyl-allyloxycoumarins under the treatment with the first generation Grubbs' catalyst. These compounds were tested in vitro for their antioxidant activity, and they present significant scavenging activity. They were also showed to inhibit in vitro soybean lipoxygenase.     

In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells

Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF-MPCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MPCs and RS-AF-MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MPCs. Furthermore, SS-AF-MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MPCs and thus we further evaluated SS-AF-MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MPCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF-MPCs consisted of at least two different MPC populations. In addition, SS-AF-MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications.     

Association between regulator of G protein signaling 9-2 and body weight

Regulator of G protein signaling 9-2 (RGS9-2) is a protein that is highly enriched in the striatum, a brain region that mediates motivation, movement and reward responses. We identified a naturally occurring 5 nucleotide deletion polymorphism in the human RGS9 gene and found that the mean body mass index (BMI) of individuals with the deletion was significantly higher than those without. A splicing reporter minigene assay demonstrated that the deletion had the potential to significantly decrease the levels of correctly spliced RGS9 gene product. We measured the weights of rats after virally transduced overexpression of RGS9-2 or the structurally related RGS proteins, RGS7, or RGS11, in the nucleus accumbens (NAc) and observed a reduction in body weight after overexpression of RGS9-2 but not RGS7 or 11. Conversely, we found that the RGS9 knockout mice were heavier than their wild-type littermates and had significantly higher percentages of abdominal fat. The constituent adipocytes were found to have a mean cross-sectional area that was more than double that of corresponding cells from wild-type mice. However, food intake and locomotion were not significantly different between the two strains. These studies with humans, rats and mice implicate RGS9-2 as a factor in regulating body weight.     

The role of perceived well-being in the family,school and peer context in adolescents’ subjective health complaints: evidence from a Greek cross-sectional study

Background

During adolescence children are usually confronted with an expanding social arena. Apart from families, schools and neighbourhoods, peers, classmates, teachers, and other adult figures gain increasing importance for adolescent socio-emotional adjustment. The aim of the present study was to investigate the extent to which Greek adolescents’ perceived well-being in three main social contexts (family, school and peers) predicted self-reported Subjective Health Complaints.

Methods

Questionnaires were administered to a Greek nation-wide, random, school-based sample of children aged 12–18 years in 2003. Data from 1.087 adolescents were analyzed. A hierarchical regression model with Subjective Health Complaints as the outcome variable was employed in order to i) control for the effects of previously well-established demographic factors (sex, age and subjective economic status) and ii) to identify the unique proportion of variance attributed to each context. Bivariate correlations and multicollinearity were also explored.

Results

As hypothesized, adolescents’ perceived well-being in each of the three social contexts appeared to hold unique proportions of variance in self-reported Subjective Health Complaints, after controlling for the effects of sex, age and subjective economic status. In addition, our final model confirmed that the explained variance in SHC was accumulated from each social context studied. The regression models were statistically significant and explained a total of approximately 24% of the variance in Subjective Health Complaints.

Conclusions

Our study delineated the unique and cumulative contributions of adolescents’ perceived well-being in the family, school and peer setting in the explanation of Subjective Health Complaints. Apart from families, schools, teachers and peers appear to have a salient role in adolescent psychosomatic adjustment. A thorough understanding of the relationship between adolescents’ Subjective Health Complaints and perceived well-being in their social contexts could not only lead to more effective tailored initiatives, but also to promote a multi- and inter-disciplinary culture in adolescent psychosomatic health.

     

Expression of a Highly Antigenic and Native-Like Folded Extracellular Domain of the Human α1 Subunit of Muscle Nicotinic Acetylcholine Receptor,Suitable for Use in Antigen Specific Therapies for Myasthenia Gravis

We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.     

Cannabinoid receptor 1 induces a biphasic ERK activation via multiprotein signaling complex formation of proximal kinases PKCε, Src, and Fyn in primary neurons

Cannabinoid receptors 1 (CB1Rs) play important roles in the regulation of dendritic branching, synapse density, and synaptic transmission through multiple G-protein-coupled signaling systems, including the activation of the extracellular signal-regulated kinases ERK1/2. The proximal signaling interactions leading to ERK1/2 activation by CB1R in CNS remain, however, unclear. Here, we present evidence that the CB1R agonist methanandamide induced a biphasic and sustained activation of ERK1/2 in primary neurons derived from E7 telencephalon. We show that E7 neurons natively express high levels of CB1R message and protein, the majority of which associates with PKC? at basal conditions. We now demonstrate that the first peak of ERK activation by CB1R was mediated by the sequential activation of G(q), PLC, and PKC?, selectively, and that the CB1R-activated PKC? acutely formed transient signaling modules containing activated Src and Fyn. A second pool of CB1Rs, coupled to PTX-sensitive activation of G(i/o), utilized as effectors additional Src and Fyn molecules to generate a second, additional wave of ERK activation at 15 min. Concurrently to these intermolecular signaling interactions, cytoskeleton-associated proteins MARCKS and p120catenin were drastically modified by phosphorylation of PKC and Src, respectively. These receptor-proximal signaling events correlated well with induction of neuritic outgrowth in the long term. Our data provide evidence for multiprotein signaling complex formation in the coupling of CB1R to activation of ERK in CNS neurons, and may elucidate several of the less understood acute effects of cannabinoid drugs.     

Evidence for cellular uptake of recombinant hepatitis C virus non-enveloped capsid-like particles

Although the hepatitis C virus (HCV) is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients, and most recently novel HCV subgenomes with deletions of the envelope proteins have been identified. However the significance of these findings remains unclear. In this study, we used the baculovirus expression system to generate recombinant HCV capsid-like particles, and investigated their possible interactions with cells. We show that expression of HCV core in insect cells can sufficiently direct the formation of capsid-like particles in the absence of the HCV envelope glycoproteins and of the 5' untranslated region. By confocal microscopy analysis, we provide evidence that the naked capsid-like particles could be uptaken by human hepatoma cells. Moreover, our findings suggest that they have the potential to produce cell-signaling effects.     

Trypanosoma brucei encodes a bifunctional capping enzyme essential for cap 4 formation on the spliced leader RNA

The 5' end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.     

Cytokeratin 8 and 18 expression in imprint smears of chronic viral hepatitis, autoimmune hepatitis and hepatocellular carcinoma. A preliminary study

OBJECTIVE: To investigate whether the expression of cytokeratin (CK) 8 and 18 is altered in chronic active viral hepatitis, autoimmune hepatitis and hepatocellular carcinoma. STUDY DESIGN: Cytologic imprint smears were obtained from 53 liver core biopsy specimens and were studied immunocytochemically for the expression of CK8 and 18. RESULTS: CK8-positive expression was observed in 45.5% of chronic active hepatitis B (CH-B), 20% of chronic active hepatitis C (CH-C), 90% of autoimmune hepatitis (AIH) and 83.3% of hepatocellular carcinoma (HCC) cases. CK18-positive expression was observed in 36.4% of CH-B, 26.7% of CH-C, 70% of AIH and 83.3% of HCC cases. A statistically significant association was found between CK8- and CK18-positive expression and the diagnosis of AIH and HCC. In contrast, CH-C and CH-B were associated with negative CK8 and CK18 expression. In addition, a negative [CK8(-)/CK18(-)] or imbalanced [CK8(-)/CK18(+), CK8(+)/CK18(-)] expression pattern was found in 100.0% and 81.18% of CH-C and CH-B cases, respectively, while the relative percentages of AIH and HCC cases were significantly lower (30.0% and 16.7%, respectively) (p < 0.0001). CONCLUSION: Our results indicate that CK8 and 18 expression is maintained in AIH and HCC and altered in CH-B and CH-C. The pathogenetic mechanism of this alteration remains to be clarified.