Tyrosine phosphorylation of caveolin-2 at residue 27: differences in the spatial and temporal behavior of phospho-Cav-2 (pY19 and pY27)

Caveolin-2 is an accessory molecule and the binding partner of caveolin-1. Previously, we showed that c-Src expression leads to the tyrosine phosphorylation of Cav-2 at position 19. To further investigate the tyrosine phosphorylation of Cav-2, we have now generated a novel phospho-specific antibody directed against phospho-Cav-2 (pY27). Here, we show that Cav-2 is phosphorylated at both tyrosines 19 and 27. We reconstituted this phosphorylation event by recombinantly coexpressing c-Src and Cav-2. We generated a series of Cav-2 constructs harboring the mutation of each tyrosine to alanine, singly or in combination, i.e., Cav-2 Y19A, Y27A, and Y19A/Y27A. Recombinant expression of these mutants in Cos-7 cells demonstrated that neither tyrosine is the unique phosphorylation site, and that double mutation of tyrosines 19 and 27 to alanine abrogates Cav-2 tyrosine phosphorylation. Immunofluorescence analysis of NIH 3T3 cells revealed that the two tyrosine-phosphorylated forms of Cav-2 exhibited some distinct properties. Phospho-Cav-2 (pY19) is concentrated at cell edges and at cell-cell contacts, whereas phospho-Cav-2 (pY27) is distributed in a dotlike pattern throughout the cell surface and cytoplasm. Further functional analysis revealed that tyrosine phosphorylation of Cav-2 has no effect on its targeting to lipid rafts, but clearly disrupts the hetero-oligomerization of Cav-2 with Cav-1. In an attempt to identify upstream mediators, we investigated Cav-2 tyrosine phosphorylation in an endogenous setting. We found that in A431 cells, EGF stimulation is sufficient to induce Cav-2 phosphorylation at tyrosines 19 and 27. However, the behavior of the two phosphorylated forms of Cav-2 diverges upon EGF stimulation. First, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) display different localization patterns. In addition, the temporal response to EGF stimulation appears to be different. Cav-2 is phosphorylated at tyrosine 19 in a rapid and transient fashion, whereas phosphorylation at tyrosine 27 is sustained over time. Three SH2 domain-containing proteins, c-Src, Nck, and Ras-GAP, were found to associate with Cav-2 in a phosphorylation-dependent manner. However, phosphorylation at tyrosine 27 appears to be more critical than phosphorylation at tyrosine 19 for this binding to occur. Taken together, these results suggest that, in addition to the common characteristics that these two sites appear to share, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) may each possess a set of unique functional roles.     

Solution structure and backbone dynamics of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin mutant: a molecular analysis of its reduced thermal stability

No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.     

Mitochondrial respiratory chain dysfunction, a non-receptor-mediated effect of synthetic PPAR-ligands: biochemical and pharmacological implications

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation, and cell differentiation. Many of their biological activities have been studied by using selective synthetic activators (mainly fibrates and thiazolidinediones) which have been already employed in therapeutic protocols. Both kinds of drugs, however, showed pharmacotoxicological profiles, which cannot be ascribed by any means to receptor activation. To better understand these non-receptorial or extrareceptorial aspects, the effect of different PPAR-ligands on the metabolic status of human HL-60 cell line has been investigated. At this regard, NMR analysis of cell culture supernatants was accomplished in order to monitor modifications at the level of cell metabolism. Cell growth and chemiluminescence assays were employed to verify cell differentiation. Results showed that all the considered PPAR-ligands, although with different potencies and independently from their PPAR binding specificity, induced a significant derangement of the mitochondrial respiratory chain consisting in a strong inhibition of NADH-cytochrome c reductase activity. This derangement has been shown to be strictly correlated to the adaptive metabolic modifications, as evidenced by the increased formation of lactate and acetate, due to the stimulation of anaerobic glycolysis and fatty acid beta-oxidation. It is worthy noting that the mitochondrial dysfunction appeared also linked to the capacity of any given PPAR-ligand to induce cell differentiation. These data could afford an explanation of biochemical and toxicological aspects related to the therapeutic use of synthetic PPAR-ligands and suggest a revision of PPAR pathophysiologic mechanisms.     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.     

Receptor crosstalk: haloperidol treatment enhances A2A adenosine receptor functioning in a transfected cell model

A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.     

Effect of monomer treatment and polymerisation methods on the bond strength of resin teeth to denture base material

Background: The fracture between acrylic denture base material and artificial teeth is a common clinical occurrence in dental prosthodontic practice. Objective: To evaluate the bond strength between acrylic resins and resin denture teeth when submitted by two protocols of monomer liquid application on the tooth surface and using different polymerisation methods. Material and methods: Microwave‐polymerised (Onda‐Cryl), heat‐polymerised (Clássico) and autopolymerising (Jet) acrylic resins and a brand of resin denture teeth (Biotone) were used. The acrylic resins were polymerised according to the cycles: (A) microwave – fast cycle, Onda‐Cryl; (B) microwave – long cycle, Onda‐Cryl; (C) microwave – manufacturer’s cycle, Onda‐Cryl; (T) water bath – long cycle, Clássico and (Q) bench polymerisation cycle, Jet. Thirty specimens were prepared for each polymerisation method; 10 were packed with acrylic resin after 60 s of monomer liquid application on the tooth surface, 10 after 180 s and 10 without any monomer liquid application. For the purpose of the study, a shear test was used. anova and Tukey tests were performed to identify significant differences (α = 0.05). Results: The highest bond strength values were found for monomer surface treatments, regardless of the polymerisation cycles. The highest significant values were found for cycles B (15.4 ± 1.8 MPa), C (11.9 ± 4.9 MPa) and T (15.4 ± 2.6 MPa) for non‐treated and 60 s methylmethacrylate treated groups. Comparing the monomer liquid treatment, they did not differ significantly (p > 0.05), except for cycle A (p < 0.05). Conclusion: Chemical treatment using monomer on the tooth surface prior to the acrylic resin packing improved the bond strength between resin denture tooth and acrylic resin, regardless of monomer liquid treatment protocols. The microwavable resin, polymerised by fast cycle and autopolymerising resin should be avoided for processing denture and denture repairs, respectively.     

High Genetic Diversity Detected in Olives beyond the Boundaries of the Mediterranean Sea

Background

Olive trees (Olea europaea subsp. europaea var. europaea) naturally grow in areas spanning the Mediterranean basin and towards the East, including the Middle East. In the Iranian plateau, the presence of olives has been documented since very ancient times, though the early history of the crop in this area is shrouded in uncertainty.

Methods

The varieties presently cultivated in Iran and trees of an unknown cultivation status, surviving under extreme climate and soil conditions, were sampled from different provinces and compared with a set of Mediterranean cultivars. All samples were analyzed using SSR and chloroplast markers to establish the relationships between Iranian olives and Mediterranean varieties, to shed light on the origins of Iranian olives and to verify their contribution to the development of the current global olive variation.

Results

Iranian cultivars and ecotypes, when analyzed using SSR markers, clustered separately from Mediterranean cultivars and showed a high number of private alleles, on the contrary, they shared the same single chlorotype with the most widespread varieties cultivated in the Mediterranean.

Conclusion

We hypothesized that Iranian and Mediterranean olive trees may have had a common origin from a unique center in the Near East region, possibly including the western Iranian area. The present pattern of variation may have derived from different environmental conditions, distinct levels and selection criteria, and divergent breeding opportunities found by Mediterranean and Iranian olives.These unexpected findings emphasize the importance of studying the Iranian olive germplasm as a promising but endangered source of variation.     

SPI-23 of S. Derby: Role in Adherence and Invasion of Porcine Tissues

Salmonella enterica serovars Derby and Mbandaka are isolated from different groups of livestock species in the UK. S. Derby is predominantly isolated from pigs and turkeys and S. Mbandaka is predominantly isolated from cattle and chickens. Alignment of the genome sequences of two isolates of each serovar led to the discovery of a new putative Salmonella pathogenicity island, SPI-23, in the chromosome sequence of S. Derby isolates. SPI-23 is 37 kb in length and contains 42 ORFs, ten of which are putative type III effector proteins. In this study we use porcine jejunum derived cell line IPEC-J2 and in vitro organ culture of porcine jejunum and colon, to characterise the association and invasion rates of S. Derby and S. Mbandaka, and tissue tropism of S. Derby respectively. We show that S. Derby invades and associates to an IPEC-J2 monolayer in significantly greater numbers than S. Mbandaka, and that S. Derby preferentially attaches to porcine jejunum over colon explants. We also show that nine genes across SPI-23 are up-regulated to a greater degree in the jejunum compared to the colon explants. Furthermore, we constructed a mutant of the highly up-regulated, pilV-like gene, potR, and find that it produces an excess of surface pili compared to the parent strain which form a strong agglutinating phenotype interfering with association and invasion of IPEC-J2 monolayers. We suggest that potR may play a role in tissue tropism.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.